premedication with clarithromycin is effective against...
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1521-01033583457ndash463$2500 httpdxdoiorg101124jpet116233932THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 358457ndash463 September 2016Copyright ordf 2016 by The American Society for Pharmacology and Experimental Therapeutics
Premedication with Clarithromycin Is Effective against SecondaryBacterial Pneumonia during Influenza Virus Infection in aPulmonary Emphysema Mouse Model
Tatsuhiko Harada Yuji Ishimatsu Atsuko Hara Towako Morita Shota NakashimaTomoyuki Kakugawa Noriho Sakamoto Kosuke Kosai Koichi IzumikawaKatsunori Yanagihara Hiroshi Mukae and Shigeru KohnoDepartment of Infectious Diseases Unit of Molecular Microbiology and Immunology (TH KI) Department of CardiopulmonaryRehabilitation Sciences (YI) Department of Respiratory Medicine (NS HM) and Department of Laboratory Medicine (KK KY)Nagasaki University Graduate School of Biomedical Sciences Nagasaki Japan Second Department of Internal Medicine NagasakiUniversity School of Medicine Nagasaki Japan (TH AH TM SN TK NS HM SK)
Received March 28 2016 accepted July 6 2016
ABSTRACTSecondary bacterial pneumonia (SBP) during influenza increasesthe severity of chronic obstructive pulmonary disease (COPD)and its associated mortality Macrolide antibiotics includingclarithromycin (CAM) are potential treatments for a variety ofchronic respiratory diseases owing to their pharmacologicalactivities in addition to antimicrobial action We examined theefficacy of CAM for the treatment of SBP after influenza infec-tion in COPD Specifically we evaluated the effect of CAM inelastase-induced emphysema mice that were inoculated withinfluenza virus (strain APR834) and subsequently infected withmacrolide-resistant Streptococcus pneumoniae CAM was ad-ministered to the emphysema mice 4 days prior to influenzavirus inoculation Premedication with CAM improved patho-logic responses and bacterial load 2 days after S pneumoniae
inoculation Survival rates were higher in emphysema micethan control mice While CAM premedication did not affectviral titers or exert antibacterial activity against S pneumoniaein the lungs it enhanced host defense and reduced inflam-mation as evidenced by the significant reductions in total celland neutrophil counts and interferon (IFN)-g levels in bron-choalveolar lavage fluid and lung homogenates These resultssuggest that CAM protects against SBP during influenza inelastase-induced emphysema mice by reducing IFN-g pro-duction thus enhancing immunity to SBP and by decreasingneutrophil infiltration into the lung to prevent injury Accord-ingly CAM may be an effective strategy to prevent secondarybacterial pneumonia in COPD patients in areas in whichvaccines are inaccessible or limited
IntroductionChronic obstructive pulmonary disease (COPD) is the third-
leading cause of death globally (World Health Organizationhttpwwwwhointmediacentrefactsheetsfs310en (AccessedMarch 14 2016) and is of particular concern in middle- andlow-income countries (Mathers and Loncar 2006) Secondaryinfections by viral or bacterial pathogens often trigger acuteexacerbations of COPD resulting in morbidity and mortality(Bautista et al 2010) The influenza virus is associated with5ndash26 of cases of COPD exacerbation (Rohde et al 2003Kurai et al 2013 Dai et al 2015) and predisposes the host tosecondary bacterial pneumonia (SBP) Streptococcus pneumo-niae is associated with 10ndash25 of all cases of acute exacerba-tion of COPD (Sethi et al 2002 Sapey and Stockley 2006)
Coinfection with influenza virus and S pneumoniae contrib-utes substantially to the increased morbidity and mortalityassociated with seasonal and pandemic influenza (Murataet al 2007 Morens et al 2008 van der Sluijs et al 2010Cilloniz et al 2012) In fatal cases of influenza virus infectionthe time to death from the onset of illness is significantlyshorter in patients with bacterial coinfections (Brundage andShanks 2008)Owing to the significant increase inmorbidity andmortality
associated with influenza and secondary bacterial pneumoniainfection in COPD patients the Global Initiative for ChronicObstructive Lung Disease (GOLD) recommends the annualadministration of the influenza vaccine and the pneumococcalpolysaccharide vaccine to high-risk COPD patients Howeverthese vaccines are often not readily accessible in low-incomecountries and in low-resource settings Accordingly alterna-tive preventive and prophylactic measures are necessaryLong-term treatment with macrolide antibiotics decreases
the risk of acute exacerbation of COPD according to some
This work was supported by Japan Society for the Promotion of Science(JSPS) Grants-in-Aid for Scientific Research (KAKENHI) (Grant No23791139]
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ABBREVIATIONS ATCC American Type Culture Collection BALF bronchoalveolar lavage fluid CAM clarithromycin COPD chronic obstructivepulmonary disease ELISA enzyme-linked immunosorbent assay IFN interferon IL interleukin PBS phosphate-buffered saline TCID50 50tissue culture infective dose
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studies (Albert et al 2011 Yamaya et al 2012 Donath et al2013 Spagnolo et al 2013) A variety of pharmacologicalactivities including direct antibacterial antiviral (eg againstinfluenza virus and rhinovirus) anti-inflammatory andimmunomodulatory activity as well as inhibitory effectson mucus secretion bacterial virulence and biofilm forma-tion (Kanoh and Rubin 2010 Zarogoulidis et al 2012Spagnolo et al 2013) are thought to contribute to thisprotective effect but the precise mechanisms of action andthe effects of long-termmacrolide therapy on influenza virusand S pneumoniae coinfection in COPD patients have notbeen clarifiedIn the present study we investigated the effects and mech-
anism of action of clarithromycin (CAM) in a mouse model ofCOPD with secondary infection by influenza virus and subse-quent coinfection by CAM-resistant S pneumoniae
Materials and MethodsMicroorganisms Influenza virus (strain APR834 H1N1 type)
was purchased from the American Type Culture Collection (ATCCManassas VA) The virus was cultured inMadin-Darby canine kidneycells for 3 days Cells were then centrifuged at 2000g for 15 minutesand the supernatant was stored at ndash80degC as a primary virus stockA mouse-adapted influenza virus was prepared Briefly primaryvirus stock solutions were inoculated intranasally to anesthetizedC57BL6J mouse After 3 days lung homogenates were collected andcentrifuged at 2000g for 15 minutes The supernatant was termed thefirst-generation mouse-adapted influenza virus a third-generationmouse-adapted influenza virus was prepared by repeating the abovemethods and storing the stock solutions at ndash80degC Viral stocks werethawed and diluted with phosphate-buffered saline (PBS) to thedesired concentration prior to use
The macrolide-susceptible S pneumoniae strain ATCC49619and a clinical isolate of macrolide-resistant S pneumoniae strainNU4471 were used ATCC49619 (minimum inhibitory concentra-tion of clarithromycin 0125 mgml serotype 19F) was purchased fromATCC NU4471 (minimum inhibitory concentration of clarithromycin256 mgml serotype 19) was obtained from the Nagasaki UniversitySchool of Medicine (Nagasaki Japan) The presence of both erm B andmef EA resistance genes in NU4471 was confirmed by polymerasechain reaction as previously reported (Fukuda et al 2006) Bacteriawere stored at ndash80degC until use
Viral Titer and 50 Tissue Culture Infective Dose The virustiter in Madin-Darby canine kidney cells was determined bymeasuring the 50 tissue culture infective dose (TCID50) followingthe methods described by Reed and Muench (1938) Briefly whencells reached 80 confluence in a 96-well plate they were infectedwith 50 ml of 10-fold serial virus dilutions (four replicates)Minimal essential medium supplemented with 05 bovine serumalbumin and 25 mgml trypsin was used for the virus dilutionsAfter 2 hours the solutions were removed and the cells wereincubated at 37degC Seventy-two hours later the cytopathic effectwas monitored using an inverted microscope and TCID50 wasdetermined
Emphysema Mouse Model with Influenza and SecondaryBacterial Pneumonia Themouse model is described in Fig 1A Allexperimental protocols and procedures were carried out in accordancewith theGuide for the Care andUse of Laboratory Animals as adoptedand promulgated by the US National Institutes of Health andperformed in compliance with Nagasaki University guidelines foranimal experiments Specific-pathogenndashfree male C57BL6J mice
Fig 1 Experimental schedule of treatment Mice were treated withclarithromycin (CAM 200 mgkg per day) or vehicle once daily Thepostexposure medication group was treated from Day 1 and the pre-medication group was treated from Day ndash4
Fig 2 Survival curve of secondary bacterial pneumonia mice treatedwith or without CAM (A) Survival curve of the postexposure medicationgroup Open triangle mice treated with vehicle from Day 0 (control) (n =6) solid triangle mice treated with CAM from Day 0 (n = 10) (B) Survivalcurve of the premedication group Open circle mice treated with vehiclefrom Day ndash4 (control) (n = 9) solid circle mice treated with CAM from Dayndash4 (n = 9) NS no significant difference versus control P 005significant difference versus control SP Streptococcus pneumoniae
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(8ndash9 weeks old Charles River Laboratories Japan Inc YokohamaJapan) were anesthetized by intraperitoneal injection of 125 (wv)pentobarbital Pulmonary emphysema was induced by injecting3 units of porcine pancreatic elastase (Wako Pure Chemical IndustriesLtd Osaka Japan) dissolved in 50 ml of sterile PBS into the trachea(Day ndash21) Pulmonary emphysema was verified by microscopic exam-ination 14 days later in a preliminary evaluation Twenty-one daysafter the porcine pancreatic elastase injection (Day 0) mice wereinoculated intranasally with 1 104 TCID50mouse influenza virusFour days later (Day 4) a subgroup of mice were also inoculated with1 106 colony-forming units of S pneumoniae NU4471 to inducesecondary bacterial pneumonia Eight to ten mice were used in eachexperimental group
Clarithromycin Medication and Survival Analysis CAMwas obtained from Taisho Toyama Pharmaceutical (Tokyo Japan)
CAM powder was dissolved in 5 gum arabic solution and diluted toa concentration of 25 mgml This solution was stored at 4degC for nomore than 7 days CAM was administered orally once daily(200 mgkg per day) either from Day ndash4 (premedication group) orfrom Day 1 (postexposure medication group) until Day 14 In thepremedication and postexposure medication groups sets of micewere administered 5 gum arabic solution (200 ml) orally once dailyas the vehicle control group A survival analysis was performedusing the log-rank test and the Kaplan-Meier method was used toestimate short-term (14 days) survival for comparisons between theCAM premedication and postexposure medication groups and therespective vehicle controls
Pathologic and Bacteriological Examination of LungTissues On Day 6 (ie 6 days after inoculation with influenzavirus and 2 days after coinfection with S pneumoniae NU4471) asubset of mice from the CAM premedication group were sacri-ficed and lung tissue was taken for pathologic (n 5 3 mice) and
Fig 3 Representative hematoxylin and eosinndashstained tissue sections of the vehicle controlgroup (A B) and CAM premedication group (CD) showing pathologic inflammatory changes andbacterial proliferation on Day 6 Magnification40 (A C) and 200 (B D)
Fig 4 Bacterial counts in lung homogenates (n = 7 micegroup) Data arepresented as means6 SE P 005 significant difference versus control
Fig 5 Local antibacterial activity of CAM in the lung Agar platesinoculated with (A) NU4471 (macrolide-resistant S pneumoniae) and (B)ATCC49619 (macrolide-susceptible S pneumoniae) Four paper discswere arranged as follows upper left lung homogenates of vehicle-treatedmice lower left lung homogenates of CAM-treated mice upper rightgarenoxacin and lower right CAM
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bacteriological (n 5 7 mice) examinations For the pathologic exam-ination the tissue sample was fixed in 10 buffered formalin andstained with hematoxylin and eosin For the bacteriological examina-tion the number of bacteria in the lungs was determined as describedpreviously (Otsu et al 2003) Briefly the lungs were dissected underaseptic conditions and suspended in 1 ml of sterile saline at Day 6Organs were homogenized quantitatively inoculated onto blood agarplates by serial dilution and incubated at 37degC for 24 hours Colonynumbers were counted and the number of bacteria was calculated ascolony-forming units per milliliter
Local Antibacterial Activity and Viral Titers in LungTissue On Day 4 (ie 4 days after inoculation with influenzavirus and immediately prior to coinfection with S pneumoniaeNU4471) a subset of mice from the CAM premedication group weresacrificed Local antibacterial activity in the lung was determinedusing the paper disc agar diffusion method Petri dishes containingblood agar (TSA II 5 Sheep Blood Agar M Japan BectonDickinson and Company Tokyo Japan) were prepared and eachplate was inoculated with cultures of macrolide-susceptible Spneumoniae (ATCC49619) or macrolide-resistant S pneumoniae(NU4471) adjusted to McFarland No 05 by swabbing on the wholeagar surface with cotton wool Lungs were homogenized andcentrifuged at 3000 rpm for 10 minutes and the supernatant wasevenly spread on both sides of filter paper discs (6-mm diameterpaper disc for antibiotic assay Toyo Roshi Kaisha Ltd TokyoJapan) The discs were aseptically placed on the plates CAM discs(15 mg Eiken Chemical Co Ltd Tokyo Japan) and garenoxacindiscs (5 mg Eiken Chemical Co Ltd) were used as positive controlsThe Petri dishes were incubated at 370degC for 24 hours At the endof this period inhibition zones that formed on the media wererecorded
The influenza viral titer in the lung was determined using thesupernatant of the lung homogenates as viral solutions as describedpreviously (50 Tissue Culture Infective Dose TCID50ml)
Bronchoalveolar Lavage Cell Counts and Cytokine Analy-sis On Day 4 (ie 4 days after inoculation with influenza virus andimmediately prior to coinfection with S pneumoniae NU4471) asubset of mice from the CAM premedication group and vehicle controlgroup were sacrificed for a bronchoalveolar lavage fluid (BALF)analysis Additional control mice that were inoculated with PBSinstead of influenza virus onDay 0were also investigated The effect ofCAM premedication on inflammation in the lung was examined
The anesthetized mice were sacrificed by incision of the axillaryartery and vein The lung blood was perfused by injection of 3 ml of
sterile saline into the right ventricle BAL was performed bywashing the lungs and airways with 1 ml of sterile saline threetimes per mouse The number of live cells in the BALF wasdetermined using a LUNA-FL Dual Fluorescence Cell Counter(Logos Biosystems Inc Annandale VA) BALF was centrifuged at1100 rpm for 2 minutes using the Cytospin III (Thermo FisherScientific KK Kanagawa Japan) and differential cell counts wereanalyzed using Diff Quick staining (Sysmex Kobe Japan) TheBALF supernatants were analyzed for cytokines and chemokinesassociated with the immune response to viral infections [interleu-kin (IL)-4 IL-10 IL-12 macrophage inflammatory protein-2 (MIP-2) IFN-a IFN-b and IFN-g] by enzyme-linked immunosorbentassay (ELISA RampD Systems Inc Minneapolis MN) according tothe manufacturerrsquos instructions
Cytokine and Chemokine Analysis in Lung Homogenatesand Serum On the basis of ELISA results from the BALF analysisthe levels of IL-10 and IFN-gwere examined in lung homogenates andserum on Day 4 using ELISA
Statistical Analysis Data are presented as means 6 SE Asurvival analysis was performed using the log-rank test and survivalrates were calculated using the Kaplan-Meier method Comparisonsbetween two groups were performed using Mann-Whitney U testsComparisons among four groups were performed using the Tukey-Kramer method A P-value of 005 was considered statisticallysignificant
Fig 6 Influenza viral titers of lung homogenates at Day 4 Data arepresented as means6 SE n = 4mice NS no significant difference betweentwo groups P 005 significant difference versus the vehicle control group
Fig 7 Anti-inflammatory effect of CAM on BALF on the basis of thenumber of (A) total cells and (B) neutrophils in each group Data arepresented as means 6 SE n = 5ndash9 mice NS no significant differencebetween two groups P 005 significant difference between two groups
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ResultsPremedication with CAM Improved Survival Rates
Postexposure treatment with CAM did not improve the sur-vival rate compared with that of the vehicle control Howeverpremedication with CAM resulted in a significantly highersurvival rate than that of the vehicle control (P 005 Fig 2A and B)CAM Premedication Suppressed Lung Inflammation
and Bacterial Counts Figure 3 shows representative im-ages of hematoxylin and eosinndashstained lung sections from thepremedication group at Day 6 Vehicle control mice exhibitedbronchopneumonia characterized by abundant neutrophilsparticularly in peribronchiolar lesions as well as in airwaysalveolar spaces and the interstitium (Fig 3 A and B) Incontrast the premedication group showed significantly lessneutrophilic inflammation than that of the control group (Fig3 C and D) on the basis of a quantitative analysis of histologicimages (control group 102 6 6 neutrophilsmm2 premedica-tion group 656 4 neutrophilsmm2P 00001) The bacterialcount was significantly lower in the lungs of the premedicationgroup than those of the control group (P 005 Fig 4)CAM Did Not Exhibit Antibacterial Activity against
NU4471 in the Lungs The discs with lung homogenates(Fig 5A lower left) and thosewith 15mg of CAM (Fig 5A lowerright) did not form inhibition zones on the macrolide-resistant
NU4471 plate but both discs formed inhibition zones on themacrolide-susceptible ATCC49619 plate (Fig 5B)CAM Did Not Affect Viral Titers at the Time of
Inoculation with NU4471 There was no significant differ-ence between the premedication and control groups in theviral titers of the lung homogenates on Day 4 (Fig 6)CAM Reduced the Number of Neutrophils in BALF
The total number of cells and neutrophils in BALF from theCAM-negativeinfluenza virusndashpositive group was significantlyhigher than that observed in the BALF from the CAM-negativeinfluenza virusndashnegative group Although no significant dif-ferences in cell number were observed after CAM medicationthere was a significant reduction in the number of neutrophilscompared with the control group (Fig 7) No significant differ-ences between groups were observed for other cell subsets(lymphocytes basophils and eosinophils data not shown)Effects of CAM on Cytokines and Chemokines The
levels of IFN-a IFN-b and INF-g were significantly elevatedin BALF at Day 4 of influenza infection compared with thelevels observed in mice without influenza (Fig 8) There wereno significant differences in the levels of IL-4 IL-12 andmacrophage inflammatory protein-2 (data not shown) CAMmedication significantly reduced IFN-g levels in BALFwhereas there were no significant changes in IFN-a IFN-band IL-10 levels after CAM medication (Fig 8) Further
Fig 8 Anti-inflammatory effect of CAM on cytokine levels in BALF (A) IFN-a (B) IFN-b (C) IFN-g and (D) IL-10 Data are presented asmeans6 SEn = 5ndash9 mice NS no significant difference between two groups P 005 significant difference between two groups
Effect of CAM on SBP during Influenza in Emphysema Mice 461
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investigation of IFN-g levels in the serumand lung revealed thatINF-g levels were significantly higher in the lung and serumduring influenza infection than in the controls (Fig 9) CAMmedication significantly reduced IFN-g levels in lung homoge-nates but did not significant influence its serum levels (Fig 9)
DiscussionOn the basis of our results premedication with CAM
initiated 4 days before influenza virus inoculation in COPD-model mice but not postexposure medication improved thesurvival of mice against subsequent coinfection with theinfluenza virus and S pneumoniae Mice with improvedsurvival also had a less severe inflammatory response in lungtissues than those of mice in the control group Although viraltiters were not affected by CAM premedication viable bacte-rial counts decreased in lung tissues even though S pneumo-niae NU4471 is macrolide-resistant CAM accumulates tohigher concentrations in the bronchial epithelial fluid than inthe serum (McCarty 2000 Kikuchi et al 2008) accordinglywe examined the local antimicrobial activity of CAM in lungtissues (Fig 5) and confirmed that the lung homogenatesdid not possess antibacterial activity against S pneumoniae
NU4471 Further investigation revealed that CAM premed-ication significantly reduced the number of total inflammatorycells including neutrophils in BALF and significantly atten-uated IFN-g levels in BALF and lung homogenates Theseresults suggest that the improvement in survival rates asso-ciated with CAM premedication could be attributed to an anti-inflammatory effect exerted prior to the inoculation of micewith S pneumoniaeTaken together CAM premedication reduced the susceptibil-
ity of COPDmice infected with influenza virus to subsequent Spneumoniae infection by decreasing the overall inflammatoryresponse and ultimately improved the survival of COPD modelmice infected with both pathogens Our results obtained usingCOPD-model mice are similar to previous reports demonstrat-ing that macrolides offer clinical benefits in reducing acuteexacerbations of COPD (Albert et al 2011 Ni et al 2015)Erythromycin or azithromycin therapy for 6ndash12 months couldeffectively reduce the frequency of exacerbations and improvethe quality of life in patients with COPD (Albert et al 2011 Niet al 2015) Unlike previous studies reporting that macrolidesexert direct antiviral effects on the influenza virus (Miyamotoet al 2008 Yamaya et al 2010) CAM premedication did notinfluence viral titers in our study Rather CAM premedicationappeared to act via a chemokine-mediated mechanism bydisrupting the cycle of infection-inflammation and strengthen-ing lung defenses as reported previously (Spagnolo et al 2013)Interferons play critical and complex roles in host defense
against viral and bacterial infections For example IFN-a andIFN-b (Type-1 IFNs) prevent the progression of local lunginfection with S pneumoniae (Trinchieri 2010 LeMessurieret al 2013) and IFN-g (Type-2 IFN Type-1 T-helper cellcytokine) combats the early stages of viral infection (Mulleret al 1994 Schroder et al 2004) However high levels of IFN-aIFN-b and IFN-g induced by influenza virus infection enhancesusceptibility to secondary pneumococcal infection (Sun andMetzger 2008 Nakamura et al 2011 Li et al 2012 Lee et al2015) via various mechanisms including IFN-g-mediated in-hibition of initial bacterial clearance from the lung by alveolarmacrophages (Sun and Metzger 2008) The trends towardreduced IFN-a and IFN-b and the significantly lower levels ofIFN-g observed after CAM premedication in our study and inprevious studies [ie reduced IFN-g levels were detected aftermacrolide therapy in mice with influenza virusndashinduced lunginjury (Sato et al 1998)]mighthaveenhancedbacterial clearancefrom the lung and reduced the susceptibility of influenza-infectedCOPD mice to secondary pneumococcal pneumoniaInfluenza-infected COPD mice showed a more advanced
pathology characterized by significantly more neutrophilsin the lung tissue and BALF compared with control miceExcessive neutrophil infiltration into the lungs has beenreported during influenza virus infection and disrupts thealveolar-capillary barrier leading to acute lung injury anddeath (Seki et al 2010 Galani and Andreakos 2015 Heroldet al 2015) Conversely reductions in neutrophils or neu-trophil function increase the severity of influenza virusinfection (Tate et al 2009) CAM has been reported to en-hance the nonphlogistic clearance of apoptotic neutrophils byalveolar macrophages (Yamaryo et al 2003) Indeed CAMpremedication in our study reduced neutrophil numbers in thelung tissue and BALF of influenza-infected COPD mice andmight have attenuated the potential for inflammation-inducedlung damage
Fig 9 Anti-inflammatory effect of CAM on IFN-g levels in (A) lunghomogenates and (B) serum samples Data are presented asmeans6 SEn = 5ndash9 mice NS no significant difference P 005 significantdifference
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While vaccination strategies are most efficient with respectto protection against influenza and pneumococcal infection inCOPD patients the global supply of influenza vaccines isinadequate during influenza pandemics and is of particularconcern in low- and middle-income countries (Partridge andKieny 2013) where the incidence of COPD and the demandfor influenza vaccinations are expected to increase Ourresults suggest that macrolide premedication may be aneffective strategy in COPD patients at a high risk of infectionwith influenza virus and S pneumoniae to prevent severe oreven fatal secondary bacterial pneumonia in these high-riskand low-resource settings Additional studies are required toidentify the ideal patient group and the optimal timing andduration of macrolide premedication to avoid injudiciousadministration over extended periods (Ni et al 2015)In conclusion administration of CAM initiated 4 days prior
to influenza virus infection in COPD mice reduced IFN-gproduction and enhanced the immune response against sec-ondary bacterial pneumonia to reduce bacterial counts andprolong survival rates Future studies should evaluate the useof macrolide premedication in COPD patients at a high risk ofinfection with influenza virus and S pneumoniae to preventsevere secondary bacterial pneumonia
Acknowledgments
The authors thankMr A Yokoyama for excellent technical supportEditorial support in the form of medical writing based on the
authorsrsquo detailed instructions collating author comments copy-editing fact checking and referencing was provided by CactusCommunications
Authorship Contributions
Participated in research design Harada Ishimatsu Hara MoritaConducted experiments Harada Hara Morita Nakashima
Kakugawa Sakamoto KosaiContributed new reagents or analytic tools Kosai Izumikawa
Yanagihara KohnoPerformed data analysis Harada Nakashima IshimatsuWrote or contributed to the writing of the manuscript Harada
Ishimatsu Kakugawa Sakamoto Mukae
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Dai MY Qiao JP Xu YH and Fei GH (2015) Respiratory infectious phenotypes inacute exacerbation of COPD an aid to length of stay and COPD Assessment TestInt J Chron Obstruct Pulmon Dis 102257ndash2263
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Fukuda Y Yanagihara K Higashiyama Y Miyazaki Y Hirakata Y Mukae H TomonoK Mizuta Y Tsukamoto K and Kohno S (2006) Effects of macrolides on pneumolysinof macrolide-resistant Streptococcus pneumoniae Eur Respir J 271020ndash1025
Galani IE and Andreakos E (2015) Neutrophils in viral infections current conceptsand caveats J Leukoc Biol 98557ndash564
Herold S Becker C Ridge KM and Budinger GR (2015) Influenza virus-induced lunginjury pathogenesis and implications for treatment Eur Respir J 451463ndash1478
Kanoh S and Rubin BK (2010) Mechanisms of action and clinical application ofmacrolides as immunomodulatory medications Clin Microbiol Rev 23590ndash615
Kikuchi E Yamazaki K Kikuchi J Hasegawa N Hashimoto S Ishizaka Aand Nishimura M (2008) Pharmacokinetics of clarithromycin in bronchial epithe-lial lining fluid Respirology 13221ndash226
Kurai D Saraya T Ishii H and Takizawa H (2013) Virus-induced exacerbations inasthma and COPD Front Microbiol 4293
Lee B Robinson KM McHugh KJ Scheller EV Mandalapu S Chen C Di YP ClayME Enelow RI and Dubin PJ et al (2015) Influenza-induced type I interferonenhances susceptibility to gram-negative and gram-positive bacterial pneumoniain mice Am J Physiol Lung Cell Mol Physiol 309L158ndashL167
LeMessurier KS Haumlcker H Chi L Tuomanen E and Redecke V (2013) Type I in-terferon protects against pneumococcal invasive disease by inhibiting bacterialtransmigration across the lung PLoS Pathog 9e1003727
Li W Moltedo B and Moran TM (2012) Type I interferon induction during influenzavirus infection increases susceptibility to secondary Streptococcus pneumoniae in-fection by negative regulation of gd T cells J Virol 8612304ndash12312
Mathers CD and Loncar D (2006) Projections of global mortality and burden of dis-ease from 2002 to 2030 PLoS Med 3e442
McCarty JM (2000) Clarithromycin in the management of community-acquiredpneumonia Clin Ther 22281ndash294 discussion 265
Miyamoto D Hasegawa S Sriwilaijaroen N Yingsakmongkon S Hiramatsu HTakahashi T Hidari K Guo CT Sakano Y and Suzuki T et al (2008) Clari-thromycin inhibits progeny virus production from human influenza virus-infectedhost cells Biol Pharm Bull 31217ndash222
Morens DM Taubenberger JK and Fauci AS (2008) Predominant role of bacterialpneumonia as a cause of death in pandemic influenza implications for pandemicinfluenza preparedness J Infect Dis 198962ndash970
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Murata Y Walsh EE and Falsey AR (2007) Pulmonary complications of in-terpandemic influenza A in hospitalized adults J Infect Dis 1951029ndash1037
Nakamura S Davis KM and Weiser JN (2011) Synergistic stimulation of type Iinterferons during influenza virus coinfection promotes Streptococcus pneumoniaecolonization in mice J Clin Invest 1213657ndash3665
Ni W Shao X Cai X Wei C Cui J Wang R and Liu Y (2015) Prophylactic use ofmacrolide antibiotics for the prevention of chronic obstructive pulmonary diseaseexacerbation a meta-analysis PLoS One 10e0121257
Otsu Y Yanagihara K Fukuda Y Miyazaki Y Tsukamoto K Hirakata Y Tomono KKadota J Tashiro T and Murata I et al (2003) In vivo efficacy of a new quinoloneDQ-113 against Streptococcus pneumoniae in a mouse model Antimicrob AgentsChemother 473699ndash3703
Partridge J and Kieny MP (2013) Global production capacity of seasonal influenzavaccine in 2011 Vaccine 31728ndash731
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Sapey E and Stockley RA (2006) COPD exacerbations 2 aetiology Thorax 61250ndash258
Sato K Suga M Akaike T Fujii S Muranaka H Doi T Maeda H and Ando M (1998)Therapeutic effect of erythromycin on influenza virus-induced lung injury in miceAm J Respir Crit Care Med 157853ndash857
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Seki M Kohno S Newstead MW Zeng X Bhan U Lukacs NW Kunkel SLand Standiford TJ (2010) Critical role of IL-1 receptor-associated kinase-M inregulating chemokine-dependent deleterious inflammation in murine influenzapneumonia J Immunol 1841410ndash1418
Sethi S Evans N Grant BJ and Murphy TF (2002) New strains of bacteria and ex-acerbations of chronic obstructive pulmonary disease N Engl J Med 347465ndash471
Spagnolo P Fabbri LM and Bush A (2013) Long-term macrolide treatment forchronic respiratory disease Eur Respir J 42239ndash251
Sun K and Metzger DW (2008) Inhibition of pulmonary antibacterial defense byinterferon-gamma during recovery from influenza infection Nat Med 14558ndash564
Tate MD Deng YM Jones JE Anderson GP Brooks AG and Reading PC (2009)Neutrophils ameliorate lung injury and the development of severe disease duringinfluenza infection J Immunol 1837441ndash7450
Trinchieri G (2010) Type I interferon friend or foe J Exp Med 2072053ndash2063van der Sluijs KF van der Poll T Lutter R Juffermans NP and Schultz MJ (2010)Bench-to-bedside review bacterial pneumonia with influenzamdashpathogenesis andclinical implications Crit Care 14219
Yamaryo T Oishi K Yoshimine H Tsuchihashi Y Matsushima K and Nagatake T(2003) Fourteen-member macrolides promote the phosphatidylserine receptor-dependent phagocytosis of apoptotic neutrophils by alveolar macrophages Anti-microb Agents Chemother 4748ndash53
Yamaya M Azuma A Takizawa H Kadota J Tamaoki J and Kudoh S (2012)Macrolide effects on the prevention of COPD exacerbations Eur Respir J 40485ndash494
Yamaya M Shinya K Hatachi Y Kubo H Asada M Yasuda H Nishimura Hand Nagatomi R (2010) Clarithromycin inhibits type a seasonal influenza virusinfection in human airway epithelial cells J Pharmacol Exp Ther 33381ndash90
Zarogoulidis P Papanas N Kioumis I Chatzaki E Maltezos E and Zarogoulidis K(2012) Macrolides from in vitro anti-inflammatory and immunomodulatory prop-erties to clinical practice in respiratory diseases Eur J Clin Pharmacol 68479ndash503
Address correspondence to Dr Yuji Ishimatsu Department of Cardiopul-monary Rehabilitation Sciences Nagasaki University Graduate School ofBiomedical Sciences 1-7-1 Sakamoto Nagasaki 852-8520 Japan E-mail yuji-inagasaki-uacjp
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studies (Albert et al 2011 Yamaya et al 2012 Donath et al2013 Spagnolo et al 2013) A variety of pharmacologicalactivities including direct antibacterial antiviral (eg againstinfluenza virus and rhinovirus) anti-inflammatory andimmunomodulatory activity as well as inhibitory effectson mucus secretion bacterial virulence and biofilm forma-tion (Kanoh and Rubin 2010 Zarogoulidis et al 2012Spagnolo et al 2013) are thought to contribute to thisprotective effect but the precise mechanisms of action andthe effects of long-termmacrolide therapy on influenza virusand S pneumoniae coinfection in COPD patients have notbeen clarifiedIn the present study we investigated the effects and mech-
anism of action of clarithromycin (CAM) in a mouse model ofCOPD with secondary infection by influenza virus and subse-quent coinfection by CAM-resistant S pneumoniae
Materials and MethodsMicroorganisms Influenza virus (strain APR834 H1N1 type)
was purchased from the American Type Culture Collection (ATCCManassas VA) The virus was cultured inMadin-Darby canine kidneycells for 3 days Cells were then centrifuged at 2000g for 15 minutesand the supernatant was stored at ndash80degC as a primary virus stockA mouse-adapted influenza virus was prepared Briefly primaryvirus stock solutions were inoculated intranasally to anesthetizedC57BL6J mouse After 3 days lung homogenates were collected andcentrifuged at 2000g for 15 minutes The supernatant was termed thefirst-generation mouse-adapted influenza virus a third-generationmouse-adapted influenza virus was prepared by repeating the abovemethods and storing the stock solutions at ndash80degC Viral stocks werethawed and diluted with phosphate-buffered saline (PBS) to thedesired concentration prior to use
The macrolide-susceptible S pneumoniae strain ATCC49619and a clinical isolate of macrolide-resistant S pneumoniae strainNU4471 were used ATCC49619 (minimum inhibitory concentra-tion of clarithromycin 0125 mgml serotype 19F) was purchased fromATCC NU4471 (minimum inhibitory concentration of clarithromycin256 mgml serotype 19) was obtained from the Nagasaki UniversitySchool of Medicine (Nagasaki Japan) The presence of both erm B andmef EA resistance genes in NU4471 was confirmed by polymerasechain reaction as previously reported (Fukuda et al 2006) Bacteriawere stored at ndash80degC until use
Viral Titer and 50 Tissue Culture Infective Dose The virustiter in Madin-Darby canine kidney cells was determined bymeasuring the 50 tissue culture infective dose (TCID50) followingthe methods described by Reed and Muench (1938) Briefly whencells reached 80 confluence in a 96-well plate they were infectedwith 50 ml of 10-fold serial virus dilutions (four replicates)Minimal essential medium supplemented with 05 bovine serumalbumin and 25 mgml trypsin was used for the virus dilutionsAfter 2 hours the solutions were removed and the cells wereincubated at 37degC Seventy-two hours later the cytopathic effectwas monitored using an inverted microscope and TCID50 wasdetermined
Emphysema Mouse Model with Influenza and SecondaryBacterial Pneumonia Themouse model is described in Fig 1A Allexperimental protocols and procedures were carried out in accordancewith theGuide for the Care andUse of Laboratory Animals as adoptedand promulgated by the US National Institutes of Health andperformed in compliance with Nagasaki University guidelines foranimal experiments Specific-pathogenndashfree male C57BL6J mice
Fig 1 Experimental schedule of treatment Mice were treated withclarithromycin (CAM 200 mgkg per day) or vehicle once daily Thepostexposure medication group was treated from Day 1 and the pre-medication group was treated from Day ndash4
Fig 2 Survival curve of secondary bacterial pneumonia mice treatedwith or without CAM (A) Survival curve of the postexposure medicationgroup Open triangle mice treated with vehicle from Day 0 (control) (n =6) solid triangle mice treated with CAM from Day 0 (n = 10) (B) Survivalcurve of the premedication group Open circle mice treated with vehiclefrom Day ndash4 (control) (n = 9) solid circle mice treated with CAM from Dayndash4 (n = 9) NS no significant difference versus control P 005significant difference versus control SP Streptococcus pneumoniae
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(8ndash9 weeks old Charles River Laboratories Japan Inc YokohamaJapan) were anesthetized by intraperitoneal injection of 125 (wv)pentobarbital Pulmonary emphysema was induced by injecting3 units of porcine pancreatic elastase (Wako Pure Chemical IndustriesLtd Osaka Japan) dissolved in 50 ml of sterile PBS into the trachea(Day ndash21) Pulmonary emphysema was verified by microscopic exam-ination 14 days later in a preliminary evaluation Twenty-one daysafter the porcine pancreatic elastase injection (Day 0) mice wereinoculated intranasally with 1 104 TCID50mouse influenza virusFour days later (Day 4) a subgroup of mice were also inoculated with1 106 colony-forming units of S pneumoniae NU4471 to inducesecondary bacterial pneumonia Eight to ten mice were used in eachexperimental group
Clarithromycin Medication and Survival Analysis CAMwas obtained from Taisho Toyama Pharmaceutical (Tokyo Japan)
CAM powder was dissolved in 5 gum arabic solution and diluted toa concentration of 25 mgml This solution was stored at 4degC for nomore than 7 days CAM was administered orally once daily(200 mgkg per day) either from Day ndash4 (premedication group) orfrom Day 1 (postexposure medication group) until Day 14 In thepremedication and postexposure medication groups sets of micewere administered 5 gum arabic solution (200 ml) orally once dailyas the vehicle control group A survival analysis was performedusing the log-rank test and the Kaplan-Meier method was used toestimate short-term (14 days) survival for comparisons between theCAM premedication and postexposure medication groups and therespective vehicle controls
Pathologic and Bacteriological Examination of LungTissues On Day 6 (ie 6 days after inoculation with influenzavirus and 2 days after coinfection with S pneumoniae NU4471) asubset of mice from the CAM premedication group were sacri-ficed and lung tissue was taken for pathologic (n 5 3 mice) and
Fig 3 Representative hematoxylin and eosinndashstained tissue sections of the vehicle controlgroup (A B) and CAM premedication group (CD) showing pathologic inflammatory changes andbacterial proliferation on Day 6 Magnification40 (A C) and 200 (B D)
Fig 4 Bacterial counts in lung homogenates (n = 7 micegroup) Data arepresented as means6 SE P 005 significant difference versus control
Fig 5 Local antibacterial activity of CAM in the lung Agar platesinoculated with (A) NU4471 (macrolide-resistant S pneumoniae) and (B)ATCC49619 (macrolide-susceptible S pneumoniae) Four paper discswere arranged as follows upper left lung homogenates of vehicle-treatedmice lower left lung homogenates of CAM-treated mice upper rightgarenoxacin and lower right CAM
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bacteriological (n 5 7 mice) examinations For the pathologic exam-ination the tissue sample was fixed in 10 buffered formalin andstained with hematoxylin and eosin For the bacteriological examina-tion the number of bacteria in the lungs was determined as describedpreviously (Otsu et al 2003) Briefly the lungs were dissected underaseptic conditions and suspended in 1 ml of sterile saline at Day 6Organs were homogenized quantitatively inoculated onto blood agarplates by serial dilution and incubated at 37degC for 24 hours Colonynumbers were counted and the number of bacteria was calculated ascolony-forming units per milliliter
Local Antibacterial Activity and Viral Titers in LungTissue On Day 4 (ie 4 days after inoculation with influenzavirus and immediately prior to coinfection with S pneumoniaeNU4471) a subset of mice from the CAM premedication group weresacrificed Local antibacterial activity in the lung was determinedusing the paper disc agar diffusion method Petri dishes containingblood agar (TSA II 5 Sheep Blood Agar M Japan BectonDickinson and Company Tokyo Japan) were prepared and eachplate was inoculated with cultures of macrolide-susceptible Spneumoniae (ATCC49619) or macrolide-resistant S pneumoniae(NU4471) adjusted to McFarland No 05 by swabbing on the wholeagar surface with cotton wool Lungs were homogenized andcentrifuged at 3000 rpm for 10 minutes and the supernatant wasevenly spread on both sides of filter paper discs (6-mm diameterpaper disc for antibiotic assay Toyo Roshi Kaisha Ltd TokyoJapan) The discs were aseptically placed on the plates CAM discs(15 mg Eiken Chemical Co Ltd Tokyo Japan) and garenoxacindiscs (5 mg Eiken Chemical Co Ltd) were used as positive controlsThe Petri dishes were incubated at 370degC for 24 hours At the endof this period inhibition zones that formed on the media wererecorded
The influenza viral titer in the lung was determined using thesupernatant of the lung homogenates as viral solutions as describedpreviously (50 Tissue Culture Infective Dose TCID50ml)
Bronchoalveolar Lavage Cell Counts and Cytokine Analy-sis On Day 4 (ie 4 days after inoculation with influenza virus andimmediately prior to coinfection with S pneumoniae NU4471) asubset of mice from the CAM premedication group and vehicle controlgroup were sacrificed for a bronchoalveolar lavage fluid (BALF)analysis Additional control mice that were inoculated with PBSinstead of influenza virus onDay 0were also investigated The effect ofCAM premedication on inflammation in the lung was examined
The anesthetized mice were sacrificed by incision of the axillaryartery and vein The lung blood was perfused by injection of 3 ml of
sterile saline into the right ventricle BAL was performed bywashing the lungs and airways with 1 ml of sterile saline threetimes per mouse The number of live cells in the BALF wasdetermined using a LUNA-FL Dual Fluorescence Cell Counter(Logos Biosystems Inc Annandale VA) BALF was centrifuged at1100 rpm for 2 minutes using the Cytospin III (Thermo FisherScientific KK Kanagawa Japan) and differential cell counts wereanalyzed using Diff Quick staining (Sysmex Kobe Japan) TheBALF supernatants were analyzed for cytokines and chemokinesassociated with the immune response to viral infections [interleu-kin (IL)-4 IL-10 IL-12 macrophage inflammatory protein-2 (MIP-2) IFN-a IFN-b and IFN-g] by enzyme-linked immunosorbentassay (ELISA RampD Systems Inc Minneapolis MN) according tothe manufacturerrsquos instructions
Cytokine and Chemokine Analysis in Lung Homogenatesand Serum On the basis of ELISA results from the BALF analysisthe levels of IL-10 and IFN-gwere examined in lung homogenates andserum on Day 4 using ELISA
Statistical Analysis Data are presented as means 6 SE Asurvival analysis was performed using the log-rank test and survivalrates were calculated using the Kaplan-Meier method Comparisonsbetween two groups were performed using Mann-Whitney U testsComparisons among four groups were performed using the Tukey-Kramer method A P-value of 005 was considered statisticallysignificant
Fig 6 Influenza viral titers of lung homogenates at Day 4 Data arepresented as means6 SE n = 4mice NS no significant difference betweentwo groups P 005 significant difference versus the vehicle control group
Fig 7 Anti-inflammatory effect of CAM on BALF on the basis of thenumber of (A) total cells and (B) neutrophils in each group Data arepresented as means 6 SE n = 5ndash9 mice NS no significant differencebetween two groups P 005 significant difference between two groups
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ResultsPremedication with CAM Improved Survival Rates
Postexposure treatment with CAM did not improve the sur-vival rate compared with that of the vehicle control Howeverpremedication with CAM resulted in a significantly highersurvival rate than that of the vehicle control (P 005 Fig 2A and B)CAM Premedication Suppressed Lung Inflammation
and Bacterial Counts Figure 3 shows representative im-ages of hematoxylin and eosinndashstained lung sections from thepremedication group at Day 6 Vehicle control mice exhibitedbronchopneumonia characterized by abundant neutrophilsparticularly in peribronchiolar lesions as well as in airwaysalveolar spaces and the interstitium (Fig 3 A and B) Incontrast the premedication group showed significantly lessneutrophilic inflammation than that of the control group (Fig3 C and D) on the basis of a quantitative analysis of histologicimages (control group 102 6 6 neutrophilsmm2 premedica-tion group 656 4 neutrophilsmm2P 00001) The bacterialcount was significantly lower in the lungs of the premedicationgroup than those of the control group (P 005 Fig 4)CAM Did Not Exhibit Antibacterial Activity against
NU4471 in the Lungs The discs with lung homogenates(Fig 5A lower left) and thosewith 15mg of CAM (Fig 5A lowerright) did not form inhibition zones on the macrolide-resistant
NU4471 plate but both discs formed inhibition zones on themacrolide-susceptible ATCC49619 plate (Fig 5B)CAM Did Not Affect Viral Titers at the Time of
Inoculation with NU4471 There was no significant differ-ence between the premedication and control groups in theviral titers of the lung homogenates on Day 4 (Fig 6)CAM Reduced the Number of Neutrophils in BALF
The total number of cells and neutrophils in BALF from theCAM-negativeinfluenza virusndashpositive group was significantlyhigher than that observed in the BALF from the CAM-negativeinfluenza virusndashnegative group Although no significant dif-ferences in cell number were observed after CAM medicationthere was a significant reduction in the number of neutrophilscompared with the control group (Fig 7) No significant differ-ences between groups were observed for other cell subsets(lymphocytes basophils and eosinophils data not shown)Effects of CAM on Cytokines and Chemokines The
levels of IFN-a IFN-b and INF-g were significantly elevatedin BALF at Day 4 of influenza infection compared with thelevels observed in mice without influenza (Fig 8) There wereno significant differences in the levels of IL-4 IL-12 andmacrophage inflammatory protein-2 (data not shown) CAMmedication significantly reduced IFN-g levels in BALFwhereas there were no significant changes in IFN-a IFN-band IL-10 levels after CAM medication (Fig 8) Further
Fig 8 Anti-inflammatory effect of CAM on cytokine levels in BALF (A) IFN-a (B) IFN-b (C) IFN-g and (D) IL-10 Data are presented asmeans6 SEn = 5ndash9 mice NS no significant difference between two groups P 005 significant difference between two groups
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investigation of IFN-g levels in the serumand lung revealed thatINF-g levels were significantly higher in the lung and serumduring influenza infection than in the controls (Fig 9) CAMmedication significantly reduced IFN-g levels in lung homoge-nates but did not significant influence its serum levels (Fig 9)
DiscussionOn the basis of our results premedication with CAM
initiated 4 days before influenza virus inoculation in COPD-model mice but not postexposure medication improved thesurvival of mice against subsequent coinfection with theinfluenza virus and S pneumoniae Mice with improvedsurvival also had a less severe inflammatory response in lungtissues than those of mice in the control group Although viraltiters were not affected by CAM premedication viable bacte-rial counts decreased in lung tissues even though S pneumo-niae NU4471 is macrolide-resistant CAM accumulates tohigher concentrations in the bronchial epithelial fluid than inthe serum (McCarty 2000 Kikuchi et al 2008) accordinglywe examined the local antimicrobial activity of CAM in lungtissues (Fig 5) and confirmed that the lung homogenatesdid not possess antibacterial activity against S pneumoniae
NU4471 Further investigation revealed that CAM premed-ication significantly reduced the number of total inflammatorycells including neutrophils in BALF and significantly atten-uated IFN-g levels in BALF and lung homogenates Theseresults suggest that the improvement in survival rates asso-ciated with CAM premedication could be attributed to an anti-inflammatory effect exerted prior to the inoculation of micewith S pneumoniaeTaken together CAM premedication reduced the susceptibil-
ity of COPDmice infected with influenza virus to subsequent Spneumoniae infection by decreasing the overall inflammatoryresponse and ultimately improved the survival of COPD modelmice infected with both pathogens Our results obtained usingCOPD-model mice are similar to previous reports demonstrat-ing that macrolides offer clinical benefits in reducing acuteexacerbations of COPD (Albert et al 2011 Ni et al 2015)Erythromycin or azithromycin therapy for 6ndash12 months couldeffectively reduce the frequency of exacerbations and improvethe quality of life in patients with COPD (Albert et al 2011 Niet al 2015) Unlike previous studies reporting that macrolidesexert direct antiviral effects on the influenza virus (Miyamotoet al 2008 Yamaya et al 2010) CAM premedication did notinfluence viral titers in our study Rather CAM premedicationappeared to act via a chemokine-mediated mechanism bydisrupting the cycle of infection-inflammation and strengthen-ing lung defenses as reported previously (Spagnolo et al 2013)Interferons play critical and complex roles in host defense
against viral and bacterial infections For example IFN-a andIFN-b (Type-1 IFNs) prevent the progression of local lunginfection with S pneumoniae (Trinchieri 2010 LeMessurieret al 2013) and IFN-g (Type-2 IFN Type-1 T-helper cellcytokine) combats the early stages of viral infection (Mulleret al 1994 Schroder et al 2004) However high levels of IFN-aIFN-b and IFN-g induced by influenza virus infection enhancesusceptibility to secondary pneumococcal infection (Sun andMetzger 2008 Nakamura et al 2011 Li et al 2012 Lee et al2015) via various mechanisms including IFN-g-mediated in-hibition of initial bacterial clearance from the lung by alveolarmacrophages (Sun and Metzger 2008) The trends towardreduced IFN-a and IFN-b and the significantly lower levels ofIFN-g observed after CAM premedication in our study and inprevious studies [ie reduced IFN-g levels were detected aftermacrolide therapy in mice with influenza virusndashinduced lunginjury (Sato et al 1998)]mighthaveenhancedbacterial clearancefrom the lung and reduced the susceptibility of influenza-infectedCOPD mice to secondary pneumococcal pneumoniaInfluenza-infected COPD mice showed a more advanced
pathology characterized by significantly more neutrophilsin the lung tissue and BALF compared with control miceExcessive neutrophil infiltration into the lungs has beenreported during influenza virus infection and disrupts thealveolar-capillary barrier leading to acute lung injury anddeath (Seki et al 2010 Galani and Andreakos 2015 Heroldet al 2015) Conversely reductions in neutrophils or neu-trophil function increase the severity of influenza virusinfection (Tate et al 2009) CAM has been reported to en-hance the nonphlogistic clearance of apoptotic neutrophils byalveolar macrophages (Yamaryo et al 2003) Indeed CAMpremedication in our study reduced neutrophil numbers in thelung tissue and BALF of influenza-infected COPD mice andmight have attenuated the potential for inflammation-inducedlung damage
Fig 9 Anti-inflammatory effect of CAM on IFN-g levels in (A) lunghomogenates and (B) serum samples Data are presented asmeans6 SEn = 5ndash9 mice NS no significant difference P 005 significantdifference
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While vaccination strategies are most efficient with respectto protection against influenza and pneumococcal infection inCOPD patients the global supply of influenza vaccines isinadequate during influenza pandemics and is of particularconcern in low- and middle-income countries (Partridge andKieny 2013) where the incidence of COPD and the demandfor influenza vaccinations are expected to increase Ourresults suggest that macrolide premedication may be aneffective strategy in COPD patients at a high risk of infectionwith influenza virus and S pneumoniae to prevent severe oreven fatal secondary bacterial pneumonia in these high-riskand low-resource settings Additional studies are required toidentify the ideal patient group and the optimal timing andduration of macrolide premedication to avoid injudiciousadministration over extended periods (Ni et al 2015)In conclusion administration of CAM initiated 4 days prior
to influenza virus infection in COPD mice reduced IFN-gproduction and enhanced the immune response against sec-ondary bacterial pneumonia to reduce bacterial counts andprolong survival rates Future studies should evaluate the useof macrolide premedication in COPD patients at a high risk ofinfection with influenza virus and S pneumoniae to preventsevere secondary bacterial pneumonia
Acknowledgments
The authors thankMr A Yokoyama for excellent technical supportEditorial support in the form of medical writing based on the
authorsrsquo detailed instructions collating author comments copy-editing fact checking and referencing was provided by CactusCommunications
Authorship Contributions
Participated in research design Harada Ishimatsu Hara MoritaConducted experiments Harada Hara Morita Nakashima
Kakugawa Sakamoto KosaiContributed new reagents or analytic tools Kosai Izumikawa
Yanagihara KohnoPerformed data analysis Harada Nakashima IshimatsuWrote or contributed to the writing of the manuscript Harada
Ishimatsu Kakugawa Sakamoto Mukae
References
Albert RK Connett J Bailey WC Casaburi R Cooper JA Jr Criner GJ Curtis JLDransfield MT Han MK and Lazarus SC et al COPD Clinical Research Network(2011) Azithromycin for prevention of exacerbations of COPD N Engl J Med 365689ndash698
Bautista E Chotpitayasunondh T Gao Z Harper SA Shaw M Uyeki TM Zaki SRHayden FG Hui DS and Kettner JD et al Writing Committee of the WHO Consul-tation on Clinical Aspects of Pandemic (H1N1) 2009 Influenza (2010) Clinical aspects ofpandemic 2009 influenza A (H1N1) virus infection N Engl J Med 3621708ndash1719
Brundage JF and Shanks GD (2008) Deaths from bacterial pneumonia during1918ndash49 influenza pandemic Emerg Infect Dis 141193ndash1199
Cilloniz C Pantin-Jackwood MJ Ni C Carter VS Korth MJ Swayne DE TumpeyTM and Katze MG (2012) Molecular signatures associated with Mx1-mediatedresistance to highly pathogenic influenza virus infection mechanisms of survival JVirol 862437ndash2446
Dai MY Qiao JP Xu YH and Fei GH (2015) Respiratory infectious phenotypes inacute exacerbation of COPD an aid to length of stay and COPD Assessment TestInt J Chron Obstruct Pulmon Dis 102257ndash2263
Donath E Chaudhry A Hernandez-Aya LF and Lit L (2013) A meta-analysis on theprophylactic use of macrolide antibiotics for the prevention of disease exacerbations inpatients with chronic obstructive pulmonary disease Respir Med 1071385ndash1392
Fukuda Y Yanagihara K Higashiyama Y Miyazaki Y Hirakata Y Mukae H TomonoK Mizuta Y Tsukamoto K and Kohno S (2006) Effects of macrolides on pneumolysinof macrolide-resistant Streptococcus pneumoniae Eur Respir J 271020ndash1025
Galani IE and Andreakos E (2015) Neutrophils in viral infections current conceptsand caveats J Leukoc Biol 98557ndash564
Herold S Becker C Ridge KM and Budinger GR (2015) Influenza virus-induced lunginjury pathogenesis and implications for treatment Eur Respir J 451463ndash1478
Kanoh S and Rubin BK (2010) Mechanisms of action and clinical application ofmacrolides as immunomodulatory medications Clin Microbiol Rev 23590ndash615
Kikuchi E Yamazaki K Kikuchi J Hasegawa N Hashimoto S Ishizaka Aand Nishimura M (2008) Pharmacokinetics of clarithromycin in bronchial epithe-lial lining fluid Respirology 13221ndash226
Kurai D Saraya T Ishii H and Takizawa H (2013) Virus-induced exacerbations inasthma and COPD Front Microbiol 4293
Lee B Robinson KM McHugh KJ Scheller EV Mandalapu S Chen C Di YP ClayME Enelow RI and Dubin PJ et al (2015) Influenza-induced type I interferonenhances susceptibility to gram-negative and gram-positive bacterial pneumoniain mice Am J Physiol Lung Cell Mol Physiol 309L158ndashL167
LeMessurier KS Haumlcker H Chi L Tuomanen E and Redecke V (2013) Type I in-terferon protects against pneumococcal invasive disease by inhibiting bacterialtransmigration across the lung PLoS Pathog 9e1003727
Li W Moltedo B and Moran TM (2012) Type I interferon induction during influenzavirus infection increases susceptibility to secondary Streptococcus pneumoniae in-fection by negative regulation of gd T cells J Virol 8612304ndash12312
Mathers CD and Loncar D (2006) Projections of global mortality and burden of dis-ease from 2002 to 2030 PLoS Med 3e442
McCarty JM (2000) Clarithromycin in the management of community-acquiredpneumonia Clin Ther 22281ndash294 discussion 265
Miyamoto D Hasegawa S Sriwilaijaroen N Yingsakmongkon S Hiramatsu HTakahashi T Hidari K Guo CT Sakano Y and Suzuki T et al (2008) Clari-thromycin inhibits progeny virus production from human influenza virus-infectedhost cells Biol Pharm Bull 31217ndash222
Morens DM Taubenberger JK and Fauci AS (2008) Predominant role of bacterialpneumonia as a cause of death in pandemic influenza implications for pandemicinfluenza preparedness J Infect Dis 198962ndash970
Muumlller U Steinhoff U Reis LF Hemmi S Pavlovic J Zinkernagel RM and Aguet M(1994) Functional role of type I and type II interferons in antiviral defense Science2641918ndash1921
Murata Y Walsh EE and Falsey AR (2007) Pulmonary complications of in-terpandemic influenza A in hospitalized adults J Infect Dis 1951029ndash1037
Nakamura S Davis KM and Weiser JN (2011) Synergistic stimulation of type Iinterferons during influenza virus coinfection promotes Streptococcus pneumoniaecolonization in mice J Clin Invest 1213657ndash3665
Ni W Shao X Cai X Wei C Cui J Wang R and Liu Y (2015) Prophylactic use ofmacrolide antibiotics for the prevention of chronic obstructive pulmonary diseaseexacerbation a meta-analysis PLoS One 10e0121257
Otsu Y Yanagihara K Fukuda Y Miyazaki Y Tsukamoto K Hirakata Y Tomono KKadota J Tashiro T and Murata I et al (2003) In vivo efficacy of a new quinoloneDQ-113 against Streptococcus pneumoniae in a mouse model Antimicrob AgentsChemother 473699ndash3703
Partridge J and Kieny MP (2013) Global production capacity of seasonal influenzavaccine in 2011 Vaccine 31728ndash731
Reed LJ and Muench H (1938) A simple method of estimating fifty percent endpointsAm J Hyg 27493ndash497
Rohde G Wiethege A Borg I Kauth M Bauer TT Gillissen A Bufe A and Schultze-Werninghaus G (2003) Respiratory viruses in exacerbations of chronic obstructivepulmonary disease requiring hospitalisation a case-control study Thorax 5837ndash42
Sapey E and Stockley RA (2006) COPD exacerbations 2 aetiology Thorax 61250ndash258
Sato K Suga M Akaike T Fujii S Muranaka H Doi T Maeda H and Ando M (1998)Therapeutic effect of erythromycin on influenza virus-induced lung injury in miceAm J Respir Crit Care Med 157853ndash857
Schroder K Hertzog PJ Ravasi T and Hume DA (2004) Interferon-gamma anoverview of signals mechanisms and functions J Leukoc Biol 75163ndash189
Seki M Kohno S Newstead MW Zeng X Bhan U Lukacs NW Kunkel SLand Standiford TJ (2010) Critical role of IL-1 receptor-associated kinase-M inregulating chemokine-dependent deleterious inflammation in murine influenzapneumonia J Immunol 1841410ndash1418
Sethi S Evans N Grant BJ and Murphy TF (2002) New strains of bacteria and ex-acerbations of chronic obstructive pulmonary disease N Engl J Med 347465ndash471
Spagnolo P Fabbri LM and Bush A (2013) Long-term macrolide treatment forchronic respiratory disease Eur Respir J 42239ndash251
Sun K and Metzger DW (2008) Inhibition of pulmonary antibacterial defense byinterferon-gamma during recovery from influenza infection Nat Med 14558ndash564
Tate MD Deng YM Jones JE Anderson GP Brooks AG and Reading PC (2009)Neutrophils ameliorate lung injury and the development of severe disease duringinfluenza infection J Immunol 1837441ndash7450
Trinchieri G (2010) Type I interferon friend or foe J Exp Med 2072053ndash2063van der Sluijs KF van der Poll T Lutter R Juffermans NP and Schultz MJ (2010)Bench-to-bedside review bacterial pneumonia with influenzamdashpathogenesis andclinical implications Crit Care 14219
Yamaryo T Oishi K Yoshimine H Tsuchihashi Y Matsushima K and Nagatake T(2003) Fourteen-member macrolides promote the phosphatidylserine receptor-dependent phagocytosis of apoptotic neutrophils by alveolar macrophages Anti-microb Agents Chemother 4748ndash53
Yamaya M Azuma A Takizawa H Kadota J Tamaoki J and Kudoh S (2012)Macrolide effects on the prevention of COPD exacerbations Eur Respir J 40485ndash494
Yamaya M Shinya K Hatachi Y Kubo H Asada M Yasuda H Nishimura Hand Nagatomi R (2010) Clarithromycin inhibits type a seasonal influenza virusinfection in human airway epithelial cells J Pharmacol Exp Ther 33381ndash90
Zarogoulidis P Papanas N Kioumis I Chatzaki E Maltezos E and Zarogoulidis K(2012) Macrolides from in vitro anti-inflammatory and immunomodulatory prop-erties to clinical practice in respiratory diseases Eur J Clin Pharmacol 68479ndash503
Address correspondence to Dr Yuji Ishimatsu Department of Cardiopul-monary Rehabilitation Sciences Nagasaki University Graduate School ofBiomedical Sciences 1-7-1 Sakamoto Nagasaki 852-8520 Japan E-mail yuji-inagasaki-uacjp
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(8ndash9 weeks old Charles River Laboratories Japan Inc YokohamaJapan) were anesthetized by intraperitoneal injection of 125 (wv)pentobarbital Pulmonary emphysema was induced by injecting3 units of porcine pancreatic elastase (Wako Pure Chemical IndustriesLtd Osaka Japan) dissolved in 50 ml of sterile PBS into the trachea(Day ndash21) Pulmonary emphysema was verified by microscopic exam-ination 14 days later in a preliminary evaluation Twenty-one daysafter the porcine pancreatic elastase injection (Day 0) mice wereinoculated intranasally with 1 104 TCID50mouse influenza virusFour days later (Day 4) a subgroup of mice were also inoculated with1 106 colony-forming units of S pneumoniae NU4471 to inducesecondary bacterial pneumonia Eight to ten mice were used in eachexperimental group
Clarithromycin Medication and Survival Analysis CAMwas obtained from Taisho Toyama Pharmaceutical (Tokyo Japan)
CAM powder was dissolved in 5 gum arabic solution and diluted toa concentration of 25 mgml This solution was stored at 4degC for nomore than 7 days CAM was administered orally once daily(200 mgkg per day) either from Day ndash4 (premedication group) orfrom Day 1 (postexposure medication group) until Day 14 In thepremedication and postexposure medication groups sets of micewere administered 5 gum arabic solution (200 ml) orally once dailyas the vehicle control group A survival analysis was performedusing the log-rank test and the Kaplan-Meier method was used toestimate short-term (14 days) survival for comparisons between theCAM premedication and postexposure medication groups and therespective vehicle controls
Pathologic and Bacteriological Examination of LungTissues On Day 6 (ie 6 days after inoculation with influenzavirus and 2 days after coinfection with S pneumoniae NU4471) asubset of mice from the CAM premedication group were sacri-ficed and lung tissue was taken for pathologic (n 5 3 mice) and
Fig 3 Representative hematoxylin and eosinndashstained tissue sections of the vehicle controlgroup (A B) and CAM premedication group (CD) showing pathologic inflammatory changes andbacterial proliferation on Day 6 Magnification40 (A C) and 200 (B D)
Fig 4 Bacterial counts in lung homogenates (n = 7 micegroup) Data arepresented as means6 SE P 005 significant difference versus control
Fig 5 Local antibacterial activity of CAM in the lung Agar platesinoculated with (A) NU4471 (macrolide-resistant S pneumoniae) and (B)ATCC49619 (macrolide-susceptible S pneumoniae) Four paper discswere arranged as follows upper left lung homogenates of vehicle-treatedmice lower left lung homogenates of CAM-treated mice upper rightgarenoxacin and lower right CAM
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bacteriological (n 5 7 mice) examinations For the pathologic exam-ination the tissue sample was fixed in 10 buffered formalin andstained with hematoxylin and eosin For the bacteriological examina-tion the number of bacteria in the lungs was determined as describedpreviously (Otsu et al 2003) Briefly the lungs were dissected underaseptic conditions and suspended in 1 ml of sterile saline at Day 6Organs were homogenized quantitatively inoculated onto blood agarplates by serial dilution and incubated at 37degC for 24 hours Colonynumbers were counted and the number of bacteria was calculated ascolony-forming units per milliliter
Local Antibacterial Activity and Viral Titers in LungTissue On Day 4 (ie 4 days after inoculation with influenzavirus and immediately prior to coinfection with S pneumoniaeNU4471) a subset of mice from the CAM premedication group weresacrificed Local antibacterial activity in the lung was determinedusing the paper disc agar diffusion method Petri dishes containingblood agar (TSA II 5 Sheep Blood Agar M Japan BectonDickinson and Company Tokyo Japan) were prepared and eachplate was inoculated with cultures of macrolide-susceptible Spneumoniae (ATCC49619) or macrolide-resistant S pneumoniae(NU4471) adjusted to McFarland No 05 by swabbing on the wholeagar surface with cotton wool Lungs were homogenized andcentrifuged at 3000 rpm for 10 minutes and the supernatant wasevenly spread on both sides of filter paper discs (6-mm diameterpaper disc for antibiotic assay Toyo Roshi Kaisha Ltd TokyoJapan) The discs were aseptically placed on the plates CAM discs(15 mg Eiken Chemical Co Ltd Tokyo Japan) and garenoxacindiscs (5 mg Eiken Chemical Co Ltd) were used as positive controlsThe Petri dishes were incubated at 370degC for 24 hours At the endof this period inhibition zones that formed on the media wererecorded
The influenza viral titer in the lung was determined using thesupernatant of the lung homogenates as viral solutions as describedpreviously (50 Tissue Culture Infective Dose TCID50ml)
Bronchoalveolar Lavage Cell Counts and Cytokine Analy-sis On Day 4 (ie 4 days after inoculation with influenza virus andimmediately prior to coinfection with S pneumoniae NU4471) asubset of mice from the CAM premedication group and vehicle controlgroup were sacrificed for a bronchoalveolar lavage fluid (BALF)analysis Additional control mice that were inoculated with PBSinstead of influenza virus onDay 0were also investigated The effect ofCAM premedication on inflammation in the lung was examined
The anesthetized mice were sacrificed by incision of the axillaryartery and vein The lung blood was perfused by injection of 3 ml of
sterile saline into the right ventricle BAL was performed bywashing the lungs and airways with 1 ml of sterile saline threetimes per mouse The number of live cells in the BALF wasdetermined using a LUNA-FL Dual Fluorescence Cell Counter(Logos Biosystems Inc Annandale VA) BALF was centrifuged at1100 rpm for 2 minutes using the Cytospin III (Thermo FisherScientific KK Kanagawa Japan) and differential cell counts wereanalyzed using Diff Quick staining (Sysmex Kobe Japan) TheBALF supernatants were analyzed for cytokines and chemokinesassociated with the immune response to viral infections [interleu-kin (IL)-4 IL-10 IL-12 macrophage inflammatory protein-2 (MIP-2) IFN-a IFN-b and IFN-g] by enzyme-linked immunosorbentassay (ELISA RampD Systems Inc Minneapolis MN) according tothe manufacturerrsquos instructions
Cytokine and Chemokine Analysis in Lung Homogenatesand Serum On the basis of ELISA results from the BALF analysisthe levels of IL-10 and IFN-gwere examined in lung homogenates andserum on Day 4 using ELISA
Statistical Analysis Data are presented as means 6 SE Asurvival analysis was performed using the log-rank test and survivalrates were calculated using the Kaplan-Meier method Comparisonsbetween two groups were performed using Mann-Whitney U testsComparisons among four groups were performed using the Tukey-Kramer method A P-value of 005 was considered statisticallysignificant
Fig 6 Influenza viral titers of lung homogenates at Day 4 Data arepresented as means6 SE n = 4mice NS no significant difference betweentwo groups P 005 significant difference versus the vehicle control group
Fig 7 Anti-inflammatory effect of CAM on BALF on the basis of thenumber of (A) total cells and (B) neutrophils in each group Data arepresented as means 6 SE n = 5ndash9 mice NS no significant differencebetween two groups P 005 significant difference between two groups
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ResultsPremedication with CAM Improved Survival Rates
Postexposure treatment with CAM did not improve the sur-vival rate compared with that of the vehicle control Howeverpremedication with CAM resulted in a significantly highersurvival rate than that of the vehicle control (P 005 Fig 2A and B)CAM Premedication Suppressed Lung Inflammation
and Bacterial Counts Figure 3 shows representative im-ages of hematoxylin and eosinndashstained lung sections from thepremedication group at Day 6 Vehicle control mice exhibitedbronchopneumonia characterized by abundant neutrophilsparticularly in peribronchiolar lesions as well as in airwaysalveolar spaces and the interstitium (Fig 3 A and B) Incontrast the premedication group showed significantly lessneutrophilic inflammation than that of the control group (Fig3 C and D) on the basis of a quantitative analysis of histologicimages (control group 102 6 6 neutrophilsmm2 premedica-tion group 656 4 neutrophilsmm2P 00001) The bacterialcount was significantly lower in the lungs of the premedicationgroup than those of the control group (P 005 Fig 4)CAM Did Not Exhibit Antibacterial Activity against
NU4471 in the Lungs The discs with lung homogenates(Fig 5A lower left) and thosewith 15mg of CAM (Fig 5A lowerright) did not form inhibition zones on the macrolide-resistant
NU4471 plate but both discs formed inhibition zones on themacrolide-susceptible ATCC49619 plate (Fig 5B)CAM Did Not Affect Viral Titers at the Time of
Inoculation with NU4471 There was no significant differ-ence between the premedication and control groups in theviral titers of the lung homogenates on Day 4 (Fig 6)CAM Reduced the Number of Neutrophils in BALF
The total number of cells and neutrophils in BALF from theCAM-negativeinfluenza virusndashpositive group was significantlyhigher than that observed in the BALF from the CAM-negativeinfluenza virusndashnegative group Although no significant dif-ferences in cell number were observed after CAM medicationthere was a significant reduction in the number of neutrophilscompared with the control group (Fig 7) No significant differ-ences between groups were observed for other cell subsets(lymphocytes basophils and eosinophils data not shown)Effects of CAM on Cytokines and Chemokines The
levels of IFN-a IFN-b and INF-g were significantly elevatedin BALF at Day 4 of influenza infection compared with thelevels observed in mice without influenza (Fig 8) There wereno significant differences in the levels of IL-4 IL-12 andmacrophage inflammatory protein-2 (data not shown) CAMmedication significantly reduced IFN-g levels in BALFwhereas there were no significant changes in IFN-a IFN-band IL-10 levels after CAM medication (Fig 8) Further
Fig 8 Anti-inflammatory effect of CAM on cytokine levels in BALF (A) IFN-a (B) IFN-b (C) IFN-g and (D) IL-10 Data are presented asmeans6 SEn = 5ndash9 mice NS no significant difference between two groups P 005 significant difference between two groups
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investigation of IFN-g levels in the serumand lung revealed thatINF-g levels were significantly higher in the lung and serumduring influenza infection than in the controls (Fig 9) CAMmedication significantly reduced IFN-g levels in lung homoge-nates but did not significant influence its serum levels (Fig 9)
DiscussionOn the basis of our results premedication with CAM
initiated 4 days before influenza virus inoculation in COPD-model mice but not postexposure medication improved thesurvival of mice against subsequent coinfection with theinfluenza virus and S pneumoniae Mice with improvedsurvival also had a less severe inflammatory response in lungtissues than those of mice in the control group Although viraltiters were not affected by CAM premedication viable bacte-rial counts decreased in lung tissues even though S pneumo-niae NU4471 is macrolide-resistant CAM accumulates tohigher concentrations in the bronchial epithelial fluid than inthe serum (McCarty 2000 Kikuchi et al 2008) accordinglywe examined the local antimicrobial activity of CAM in lungtissues (Fig 5) and confirmed that the lung homogenatesdid not possess antibacterial activity against S pneumoniae
NU4471 Further investigation revealed that CAM premed-ication significantly reduced the number of total inflammatorycells including neutrophils in BALF and significantly atten-uated IFN-g levels in BALF and lung homogenates Theseresults suggest that the improvement in survival rates asso-ciated with CAM premedication could be attributed to an anti-inflammatory effect exerted prior to the inoculation of micewith S pneumoniaeTaken together CAM premedication reduced the susceptibil-
ity of COPDmice infected with influenza virus to subsequent Spneumoniae infection by decreasing the overall inflammatoryresponse and ultimately improved the survival of COPD modelmice infected with both pathogens Our results obtained usingCOPD-model mice are similar to previous reports demonstrat-ing that macrolides offer clinical benefits in reducing acuteexacerbations of COPD (Albert et al 2011 Ni et al 2015)Erythromycin or azithromycin therapy for 6ndash12 months couldeffectively reduce the frequency of exacerbations and improvethe quality of life in patients with COPD (Albert et al 2011 Niet al 2015) Unlike previous studies reporting that macrolidesexert direct antiviral effects on the influenza virus (Miyamotoet al 2008 Yamaya et al 2010) CAM premedication did notinfluence viral titers in our study Rather CAM premedicationappeared to act via a chemokine-mediated mechanism bydisrupting the cycle of infection-inflammation and strengthen-ing lung defenses as reported previously (Spagnolo et al 2013)Interferons play critical and complex roles in host defense
against viral and bacterial infections For example IFN-a andIFN-b (Type-1 IFNs) prevent the progression of local lunginfection with S pneumoniae (Trinchieri 2010 LeMessurieret al 2013) and IFN-g (Type-2 IFN Type-1 T-helper cellcytokine) combats the early stages of viral infection (Mulleret al 1994 Schroder et al 2004) However high levels of IFN-aIFN-b and IFN-g induced by influenza virus infection enhancesusceptibility to secondary pneumococcal infection (Sun andMetzger 2008 Nakamura et al 2011 Li et al 2012 Lee et al2015) via various mechanisms including IFN-g-mediated in-hibition of initial bacterial clearance from the lung by alveolarmacrophages (Sun and Metzger 2008) The trends towardreduced IFN-a and IFN-b and the significantly lower levels ofIFN-g observed after CAM premedication in our study and inprevious studies [ie reduced IFN-g levels were detected aftermacrolide therapy in mice with influenza virusndashinduced lunginjury (Sato et al 1998)]mighthaveenhancedbacterial clearancefrom the lung and reduced the susceptibility of influenza-infectedCOPD mice to secondary pneumococcal pneumoniaInfluenza-infected COPD mice showed a more advanced
pathology characterized by significantly more neutrophilsin the lung tissue and BALF compared with control miceExcessive neutrophil infiltration into the lungs has beenreported during influenza virus infection and disrupts thealveolar-capillary barrier leading to acute lung injury anddeath (Seki et al 2010 Galani and Andreakos 2015 Heroldet al 2015) Conversely reductions in neutrophils or neu-trophil function increase the severity of influenza virusinfection (Tate et al 2009) CAM has been reported to en-hance the nonphlogistic clearance of apoptotic neutrophils byalveolar macrophages (Yamaryo et al 2003) Indeed CAMpremedication in our study reduced neutrophil numbers in thelung tissue and BALF of influenza-infected COPD mice andmight have attenuated the potential for inflammation-inducedlung damage
Fig 9 Anti-inflammatory effect of CAM on IFN-g levels in (A) lunghomogenates and (B) serum samples Data are presented asmeans6 SEn = 5ndash9 mice NS no significant difference P 005 significantdifference
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While vaccination strategies are most efficient with respectto protection against influenza and pneumococcal infection inCOPD patients the global supply of influenza vaccines isinadequate during influenza pandemics and is of particularconcern in low- and middle-income countries (Partridge andKieny 2013) where the incidence of COPD and the demandfor influenza vaccinations are expected to increase Ourresults suggest that macrolide premedication may be aneffective strategy in COPD patients at a high risk of infectionwith influenza virus and S pneumoniae to prevent severe oreven fatal secondary bacterial pneumonia in these high-riskand low-resource settings Additional studies are required toidentify the ideal patient group and the optimal timing andduration of macrolide premedication to avoid injudiciousadministration over extended periods (Ni et al 2015)In conclusion administration of CAM initiated 4 days prior
to influenza virus infection in COPD mice reduced IFN-gproduction and enhanced the immune response against sec-ondary bacterial pneumonia to reduce bacterial counts andprolong survival rates Future studies should evaluate the useof macrolide premedication in COPD patients at a high risk ofinfection with influenza virus and S pneumoniae to preventsevere secondary bacterial pneumonia
Acknowledgments
The authors thankMr A Yokoyama for excellent technical supportEditorial support in the form of medical writing based on the
authorsrsquo detailed instructions collating author comments copy-editing fact checking and referencing was provided by CactusCommunications
Authorship Contributions
Participated in research design Harada Ishimatsu Hara MoritaConducted experiments Harada Hara Morita Nakashima
Kakugawa Sakamoto KosaiContributed new reagents or analytic tools Kosai Izumikawa
Yanagihara KohnoPerformed data analysis Harada Nakashima IshimatsuWrote or contributed to the writing of the manuscript Harada
Ishimatsu Kakugawa Sakamoto Mukae
References
Albert RK Connett J Bailey WC Casaburi R Cooper JA Jr Criner GJ Curtis JLDransfield MT Han MK and Lazarus SC et al COPD Clinical Research Network(2011) Azithromycin for prevention of exacerbations of COPD N Engl J Med 365689ndash698
Bautista E Chotpitayasunondh T Gao Z Harper SA Shaw M Uyeki TM Zaki SRHayden FG Hui DS and Kettner JD et al Writing Committee of the WHO Consul-tation on Clinical Aspects of Pandemic (H1N1) 2009 Influenza (2010) Clinical aspects ofpandemic 2009 influenza A (H1N1) virus infection N Engl J Med 3621708ndash1719
Brundage JF and Shanks GD (2008) Deaths from bacterial pneumonia during1918ndash49 influenza pandemic Emerg Infect Dis 141193ndash1199
Cilloniz C Pantin-Jackwood MJ Ni C Carter VS Korth MJ Swayne DE TumpeyTM and Katze MG (2012) Molecular signatures associated with Mx1-mediatedresistance to highly pathogenic influenza virus infection mechanisms of survival JVirol 862437ndash2446
Dai MY Qiao JP Xu YH and Fei GH (2015) Respiratory infectious phenotypes inacute exacerbation of COPD an aid to length of stay and COPD Assessment TestInt J Chron Obstruct Pulmon Dis 102257ndash2263
Donath E Chaudhry A Hernandez-Aya LF and Lit L (2013) A meta-analysis on theprophylactic use of macrolide antibiotics for the prevention of disease exacerbations inpatients with chronic obstructive pulmonary disease Respir Med 1071385ndash1392
Fukuda Y Yanagihara K Higashiyama Y Miyazaki Y Hirakata Y Mukae H TomonoK Mizuta Y Tsukamoto K and Kohno S (2006) Effects of macrolides on pneumolysinof macrolide-resistant Streptococcus pneumoniae Eur Respir J 271020ndash1025
Galani IE and Andreakos E (2015) Neutrophils in viral infections current conceptsand caveats J Leukoc Biol 98557ndash564
Herold S Becker C Ridge KM and Budinger GR (2015) Influenza virus-induced lunginjury pathogenesis and implications for treatment Eur Respir J 451463ndash1478
Kanoh S and Rubin BK (2010) Mechanisms of action and clinical application ofmacrolides as immunomodulatory medications Clin Microbiol Rev 23590ndash615
Kikuchi E Yamazaki K Kikuchi J Hasegawa N Hashimoto S Ishizaka Aand Nishimura M (2008) Pharmacokinetics of clarithromycin in bronchial epithe-lial lining fluid Respirology 13221ndash226
Kurai D Saraya T Ishii H and Takizawa H (2013) Virus-induced exacerbations inasthma and COPD Front Microbiol 4293
Lee B Robinson KM McHugh KJ Scheller EV Mandalapu S Chen C Di YP ClayME Enelow RI and Dubin PJ et al (2015) Influenza-induced type I interferonenhances susceptibility to gram-negative and gram-positive bacterial pneumoniain mice Am J Physiol Lung Cell Mol Physiol 309L158ndashL167
LeMessurier KS Haumlcker H Chi L Tuomanen E and Redecke V (2013) Type I in-terferon protects against pneumococcal invasive disease by inhibiting bacterialtransmigration across the lung PLoS Pathog 9e1003727
Li W Moltedo B and Moran TM (2012) Type I interferon induction during influenzavirus infection increases susceptibility to secondary Streptococcus pneumoniae in-fection by negative regulation of gd T cells J Virol 8612304ndash12312
Mathers CD and Loncar D (2006) Projections of global mortality and burden of dis-ease from 2002 to 2030 PLoS Med 3e442
McCarty JM (2000) Clarithromycin in the management of community-acquiredpneumonia Clin Ther 22281ndash294 discussion 265
Miyamoto D Hasegawa S Sriwilaijaroen N Yingsakmongkon S Hiramatsu HTakahashi T Hidari K Guo CT Sakano Y and Suzuki T et al (2008) Clari-thromycin inhibits progeny virus production from human influenza virus-infectedhost cells Biol Pharm Bull 31217ndash222
Morens DM Taubenberger JK and Fauci AS (2008) Predominant role of bacterialpneumonia as a cause of death in pandemic influenza implications for pandemicinfluenza preparedness J Infect Dis 198962ndash970
Muumlller U Steinhoff U Reis LF Hemmi S Pavlovic J Zinkernagel RM and Aguet M(1994) Functional role of type I and type II interferons in antiviral defense Science2641918ndash1921
Murata Y Walsh EE and Falsey AR (2007) Pulmonary complications of in-terpandemic influenza A in hospitalized adults J Infect Dis 1951029ndash1037
Nakamura S Davis KM and Weiser JN (2011) Synergistic stimulation of type Iinterferons during influenza virus coinfection promotes Streptococcus pneumoniaecolonization in mice J Clin Invest 1213657ndash3665
Ni W Shao X Cai X Wei C Cui J Wang R and Liu Y (2015) Prophylactic use ofmacrolide antibiotics for the prevention of chronic obstructive pulmonary diseaseexacerbation a meta-analysis PLoS One 10e0121257
Otsu Y Yanagihara K Fukuda Y Miyazaki Y Tsukamoto K Hirakata Y Tomono KKadota J Tashiro T and Murata I et al (2003) In vivo efficacy of a new quinoloneDQ-113 against Streptococcus pneumoniae in a mouse model Antimicrob AgentsChemother 473699ndash3703
Partridge J and Kieny MP (2013) Global production capacity of seasonal influenzavaccine in 2011 Vaccine 31728ndash731
Reed LJ and Muench H (1938) A simple method of estimating fifty percent endpointsAm J Hyg 27493ndash497
Rohde G Wiethege A Borg I Kauth M Bauer TT Gillissen A Bufe A and Schultze-Werninghaus G (2003) Respiratory viruses in exacerbations of chronic obstructivepulmonary disease requiring hospitalisation a case-control study Thorax 5837ndash42
Sapey E and Stockley RA (2006) COPD exacerbations 2 aetiology Thorax 61250ndash258
Sato K Suga M Akaike T Fujii S Muranaka H Doi T Maeda H and Ando M (1998)Therapeutic effect of erythromycin on influenza virus-induced lung injury in miceAm J Respir Crit Care Med 157853ndash857
Schroder K Hertzog PJ Ravasi T and Hume DA (2004) Interferon-gamma anoverview of signals mechanisms and functions J Leukoc Biol 75163ndash189
Seki M Kohno S Newstead MW Zeng X Bhan U Lukacs NW Kunkel SLand Standiford TJ (2010) Critical role of IL-1 receptor-associated kinase-M inregulating chemokine-dependent deleterious inflammation in murine influenzapneumonia J Immunol 1841410ndash1418
Sethi S Evans N Grant BJ and Murphy TF (2002) New strains of bacteria and ex-acerbations of chronic obstructive pulmonary disease N Engl J Med 347465ndash471
Spagnolo P Fabbri LM and Bush A (2013) Long-term macrolide treatment forchronic respiratory disease Eur Respir J 42239ndash251
Sun K and Metzger DW (2008) Inhibition of pulmonary antibacterial defense byinterferon-gamma during recovery from influenza infection Nat Med 14558ndash564
Tate MD Deng YM Jones JE Anderson GP Brooks AG and Reading PC (2009)Neutrophils ameliorate lung injury and the development of severe disease duringinfluenza infection J Immunol 1837441ndash7450
Trinchieri G (2010) Type I interferon friend or foe J Exp Med 2072053ndash2063van der Sluijs KF van der Poll T Lutter R Juffermans NP and Schultz MJ (2010)Bench-to-bedside review bacterial pneumonia with influenzamdashpathogenesis andclinical implications Crit Care 14219
Yamaryo T Oishi K Yoshimine H Tsuchihashi Y Matsushima K and Nagatake T(2003) Fourteen-member macrolides promote the phosphatidylserine receptor-dependent phagocytosis of apoptotic neutrophils by alveolar macrophages Anti-microb Agents Chemother 4748ndash53
Yamaya M Azuma A Takizawa H Kadota J Tamaoki J and Kudoh S (2012)Macrolide effects on the prevention of COPD exacerbations Eur Respir J 40485ndash494
Yamaya M Shinya K Hatachi Y Kubo H Asada M Yasuda H Nishimura Hand Nagatomi R (2010) Clarithromycin inhibits type a seasonal influenza virusinfection in human airway epithelial cells J Pharmacol Exp Ther 33381ndash90
Zarogoulidis P Papanas N Kioumis I Chatzaki E Maltezos E and Zarogoulidis K(2012) Macrolides from in vitro anti-inflammatory and immunomodulatory prop-erties to clinical practice in respiratory diseases Eur J Clin Pharmacol 68479ndash503
Address correspondence to Dr Yuji Ishimatsu Department of Cardiopul-monary Rehabilitation Sciences Nagasaki University Graduate School ofBiomedical Sciences 1-7-1 Sakamoto Nagasaki 852-8520 Japan E-mail yuji-inagasaki-uacjp
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bacteriological (n 5 7 mice) examinations For the pathologic exam-ination the tissue sample was fixed in 10 buffered formalin andstained with hematoxylin and eosin For the bacteriological examina-tion the number of bacteria in the lungs was determined as describedpreviously (Otsu et al 2003) Briefly the lungs were dissected underaseptic conditions and suspended in 1 ml of sterile saline at Day 6Organs were homogenized quantitatively inoculated onto blood agarplates by serial dilution and incubated at 37degC for 24 hours Colonynumbers were counted and the number of bacteria was calculated ascolony-forming units per milliliter
Local Antibacterial Activity and Viral Titers in LungTissue On Day 4 (ie 4 days after inoculation with influenzavirus and immediately prior to coinfection with S pneumoniaeNU4471) a subset of mice from the CAM premedication group weresacrificed Local antibacterial activity in the lung was determinedusing the paper disc agar diffusion method Petri dishes containingblood agar (TSA II 5 Sheep Blood Agar M Japan BectonDickinson and Company Tokyo Japan) were prepared and eachplate was inoculated with cultures of macrolide-susceptible Spneumoniae (ATCC49619) or macrolide-resistant S pneumoniae(NU4471) adjusted to McFarland No 05 by swabbing on the wholeagar surface with cotton wool Lungs were homogenized andcentrifuged at 3000 rpm for 10 minutes and the supernatant wasevenly spread on both sides of filter paper discs (6-mm diameterpaper disc for antibiotic assay Toyo Roshi Kaisha Ltd TokyoJapan) The discs were aseptically placed on the plates CAM discs(15 mg Eiken Chemical Co Ltd Tokyo Japan) and garenoxacindiscs (5 mg Eiken Chemical Co Ltd) were used as positive controlsThe Petri dishes were incubated at 370degC for 24 hours At the endof this period inhibition zones that formed on the media wererecorded
The influenza viral titer in the lung was determined using thesupernatant of the lung homogenates as viral solutions as describedpreviously (50 Tissue Culture Infective Dose TCID50ml)
Bronchoalveolar Lavage Cell Counts and Cytokine Analy-sis On Day 4 (ie 4 days after inoculation with influenza virus andimmediately prior to coinfection with S pneumoniae NU4471) asubset of mice from the CAM premedication group and vehicle controlgroup were sacrificed for a bronchoalveolar lavage fluid (BALF)analysis Additional control mice that were inoculated with PBSinstead of influenza virus onDay 0were also investigated The effect ofCAM premedication on inflammation in the lung was examined
The anesthetized mice were sacrificed by incision of the axillaryartery and vein The lung blood was perfused by injection of 3 ml of
sterile saline into the right ventricle BAL was performed bywashing the lungs and airways with 1 ml of sterile saline threetimes per mouse The number of live cells in the BALF wasdetermined using a LUNA-FL Dual Fluorescence Cell Counter(Logos Biosystems Inc Annandale VA) BALF was centrifuged at1100 rpm for 2 minutes using the Cytospin III (Thermo FisherScientific KK Kanagawa Japan) and differential cell counts wereanalyzed using Diff Quick staining (Sysmex Kobe Japan) TheBALF supernatants were analyzed for cytokines and chemokinesassociated with the immune response to viral infections [interleu-kin (IL)-4 IL-10 IL-12 macrophage inflammatory protein-2 (MIP-2) IFN-a IFN-b and IFN-g] by enzyme-linked immunosorbentassay (ELISA RampD Systems Inc Minneapolis MN) according tothe manufacturerrsquos instructions
Cytokine and Chemokine Analysis in Lung Homogenatesand Serum On the basis of ELISA results from the BALF analysisthe levels of IL-10 and IFN-gwere examined in lung homogenates andserum on Day 4 using ELISA
Statistical Analysis Data are presented as means 6 SE Asurvival analysis was performed using the log-rank test and survivalrates were calculated using the Kaplan-Meier method Comparisonsbetween two groups were performed using Mann-Whitney U testsComparisons among four groups were performed using the Tukey-Kramer method A P-value of 005 was considered statisticallysignificant
Fig 6 Influenza viral titers of lung homogenates at Day 4 Data arepresented as means6 SE n = 4mice NS no significant difference betweentwo groups P 005 significant difference versus the vehicle control group
Fig 7 Anti-inflammatory effect of CAM on BALF on the basis of thenumber of (A) total cells and (B) neutrophils in each group Data arepresented as means 6 SE n = 5ndash9 mice NS no significant differencebetween two groups P 005 significant difference between two groups
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ResultsPremedication with CAM Improved Survival Rates
Postexposure treatment with CAM did not improve the sur-vival rate compared with that of the vehicle control Howeverpremedication with CAM resulted in a significantly highersurvival rate than that of the vehicle control (P 005 Fig 2A and B)CAM Premedication Suppressed Lung Inflammation
and Bacterial Counts Figure 3 shows representative im-ages of hematoxylin and eosinndashstained lung sections from thepremedication group at Day 6 Vehicle control mice exhibitedbronchopneumonia characterized by abundant neutrophilsparticularly in peribronchiolar lesions as well as in airwaysalveolar spaces and the interstitium (Fig 3 A and B) Incontrast the premedication group showed significantly lessneutrophilic inflammation than that of the control group (Fig3 C and D) on the basis of a quantitative analysis of histologicimages (control group 102 6 6 neutrophilsmm2 premedica-tion group 656 4 neutrophilsmm2P 00001) The bacterialcount was significantly lower in the lungs of the premedicationgroup than those of the control group (P 005 Fig 4)CAM Did Not Exhibit Antibacterial Activity against
NU4471 in the Lungs The discs with lung homogenates(Fig 5A lower left) and thosewith 15mg of CAM (Fig 5A lowerright) did not form inhibition zones on the macrolide-resistant
NU4471 plate but both discs formed inhibition zones on themacrolide-susceptible ATCC49619 plate (Fig 5B)CAM Did Not Affect Viral Titers at the Time of
Inoculation with NU4471 There was no significant differ-ence between the premedication and control groups in theviral titers of the lung homogenates on Day 4 (Fig 6)CAM Reduced the Number of Neutrophils in BALF
The total number of cells and neutrophils in BALF from theCAM-negativeinfluenza virusndashpositive group was significantlyhigher than that observed in the BALF from the CAM-negativeinfluenza virusndashnegative group Although no significant dif-ferences in cell number were observed after CAM medicationthere was a significant reduction in the number of neutrophilscompared with the control group (Fig 7) No significant differ-ences between groups were observed for other cell subsets(lymphocytes basophils and eosinophils data not shown)Effects of CAM on Cytokines and Chemokines The
levels of IFN-a IFN-b and INF-g were significantly elevatedin BALF at Day 4 of influenza infection compared with thelevels observed in mice without influenza (Fig 8) There wereno significant differences in the levels of IL-4 IL-12 andmacrophage inflammatory protein-2 (data not shown) CAMmedication significantly reduced IFN-g levels in BALFwhereas there were no significant changes in IFN-a IFN-band IL-10 levels after CAM medication (Fig 8) Further
Fig 8 Anti-inflammatory effect of CAM on cytokine levels in BALF (A) IFN-a (B) IFN-b (C) IFN-g and (D) IL-10 Data are presented asmeans6 SEn = 5ndash9 mice NS no significant difference between two groups P 005 significant difference between two groups
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investigation of IFN-g levels in the serumand lung revealed thatINF-g levels were significantly higher in the lung and serumduring influenza infection than in the controls (Fig 9) CAMmedication significantly reduced IFN-g levels in lung homoge-nates but did not significant influence its serum levels (Fig 9)
DiscussionOn the basis of our results premedication with CAM
initiated 4 days before influenza virus inoculation in COPD-model mice but not postexposure medication improved thesurvival of mice against subsequent coinfection with theinfluenza virus and S pneumoniae Mice with improvedsurvival also had a less severe inflammatory response in lungtissues than those of mice in the control group Although viraltiters were not affected by CAM premedication viable bacte-rial counts decreased in lung tissues even though S pneumo-niae NU4471 is macrolide-resistant CAM accumulates tohigher concentrations in the bronchial epithelial fluid than inthe serum (McCarty 2000 Kikuchi et al 2008) accordinglywe examined the local antimicrobial activity of CAM in lungtissues (Fig 5) and confirmed that the lung homogenatesdid not possess antibacterial activity against S pneumoniae
NU4471 Further investigation revealed that CAM premed-ication significantly reduced the number of total inflammatorycells including neutrophils in BALF and significantly atten-uated IFN-g levels in BALF and lung homogenates Theseresults suggest that the improvement in survival rates asso-ciated with CAM premedication could be attributed to an anti-inflammatory effect exerted prior to the inoculation of micewith S pneumoniaeTaken together CAM premedication reduced the susceptibil-
ity of COPDmice infected with influenza virus to subsequent Spneumoniae infection by decreasing the overall inflammatoryresponse and ultimately improved the survival of COPD modelmice infected with both pathogens Our results obtained usingCOPD-model mice are similar to previous reports demonstrat-ing that macrolides offer clinical benefits in reducing acuteexacerbations of COPD (Albert et al 2011 Ni et al 2015)Erythromycin or azithromycin therapy for 6ndash12 months couldeffectively reduce the frequency of exacerbations and improvethe quality of life in patients with COPD (Albert et al 2011 Niet al 2015) Unlike previous studies reporting that macrolidesexert direct antiviral effects on the influenza virus (Miyamotoet al 2008 Yamaya et al 2010) CAM premedication did notinfluence viral titers in our study Rather CAM premedicationappeared to act via a chemokine-mediated mechanism bydisrupting the cycle of infection-inflammation and strengthen-ing lung defenses as reported previously (Spagnolo et al 2013)Interferons play critical and complex roles in host defense
against viral and bacterial infections For example IFN-a andIFN-b (Type-1 IFNs) prevent the progression of local lunginfection with S pneumoniae (Trinchieri 2010 LeMessurieret al 2013) and IFN-g (Type-2 IFN Type-1 T-helper cellcytokine) combats the early stages of viral infection (Mulleret al 1994 Schroder et al 2004) However high levels of IFN-aIFN-b and IFN-g induced by influenza virus infection enhancesusceptibility to secondary pneumococcal infection (Sun andMetzger 2008 Nakamura et al 2011 Li et al 2012 Lee et al2015) via various mechanisms including IFN-g-mediated in-hibition of initial bacterial clearance from the lung by alveolarmacrophages (Sun and Metzger 2008) The trends towardreduced IFN-a and IFN-b and the significantly lower levels ofIFN-g observed after CAM premedication in our study and inprevious studies [ie reduced IFN-g levels were detected aftermacrolide therapy in mice with influenza virusndashinduced lunginjury (Sato et al 1998)]mighthaveenhancedbacterial clearancefrom the lung and reduced the susceptibility of influenza-infectedCOPD mice to secondary pneumococcal pneumoniaInfluenza-infected COPD mice showed a more advanced
pathology characterized by significantly more neutrophilsin the lung tissue and BALF compared with control miceExcessive neutrophil infiltration into the lungs has beenreported during influenza virus infection and disrupts thealveolar-capillary barrier leading to acute lung injury anddeath (Seki et al 2010 Galani and Andreakos 2015 Heroldet al 2015) Conversely reductions in neutrophils or neu-trophil function increase the severity of influenza virusinfection (Tate et al 2009) CAM has been reported to en-hance the nonphlogistic clearance of apoptotic neutrophils byalveolar macrophages (Yamaryo et al 2003) Indeed CAMpremedication in our study reduced neutrophil numbers in thelung tissue and BALF of influenza-infected COPD mice andmight have attenuated the potential for inflammation-inducedlung damage
Fig 9 Anti-inflammatory effect of CAM on IFN-g levels in (A) lunghomogenates and (B) serum samples Data are presented asmeans6 SEn = 5ndash9 mice NS no significant difference P 005 significantdifference
462 Harada et al
at ASPE
T Journals on June 1 2020
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ownloaded from
While vaccination strategies are most efficient with respectto protection against influenza and pneumococcal infection inCOPD patients the global supply of influenza vaccines isinadequate during influenza pandemics and is of particularconcern in low- and middle-income countries (Partridge andKieny 2013) where the incidence of COPD and the demandfor influenza vaccinations are expected to increase Ourresults suggest that macrolide premedication may be aneffective strategy in COPD patients at a high risk of infectionwith influenza virus and S pneumoniae to prevent severe oreven fatal secondary bacterial pneumonia in these high-riskand low-resource settings Additional studies are required toidentify the ideal patient group and the optimal timing andduration of macrolide premedication to avoid injudiciousadministration over extended periods (Ni et al 2015)In conclusion administration of CAM initiated 4 days prior
to influenza virus infection in COPD mice reduced IFN-gproduction and enhanced the immune response against sec-ondary bacterial pneumonia to reduce bacterial counts andprolong survival rates Future studies should evaluate the useof macrolide premedication in COPD patients at a high risk ofinfection with influenza virus and S pneumoniae to preventsevere secondary bacterial pneumonia
Acknowledgments
The authors thankMr A Yokoyama for excellent technical supportEditorial support in the form of medical writing based on the
authorsrsquo detailed instructions collating author comments copy-editing fact checking and referencing was provided by CactusCommunications
Authorship Contributions
Participated in research design Harada Ishimatsu Hara MoritaConducted experiments Harada Hara Morita Nakashima
Kakugawa Sakamoto KosaiContributed new reagents or analytic tools Kosai Izumikawa
Yanagihara KohnoPerformed data analysis Harada Nakashima IshimatsuWrote or contributed to the writing of the manuscript Harada
Ishimatsu Kakugawa Sakamoto Mukae
References
Albert RK Connett J Bailey WC Casaburi R Cooper JA Jr Criner GJ Curtis JLDransfield MT Han MK and Lazarus SC et al COPD Clinical Research Network(2011) Azithromycin for prevention of exacerbations of COPD N Engl J Med 365689ndash698
Bautista E Chotpitayasunondh T Gao Z Harper SA Shaw M Uyeki TM Zaki SRHayden FG Hui DS and Kettner JD et al Writing Committee of the WHO Consul-tation on Clinical Aspects of Pandemic (H1N1) 2009 Influenza (2010) Clinical aspects ofpandemic 2009 influenza A (H1N1) virus infection N Engl J Med 3621708ndash1719
Brundage JF and Shanks GD (2008) Deaths from bacterial pneumonia during1918ndash49 influenza pandemic Emerg Infect Dis 141193ndash1199
Cilloniz C Pantin-Jackwood MJ Ni C Carter VS Korth MJ Swayne DE TumpeyTM and Katze MG (2012) Molecular signatures associated with Mx1-mediatedresistance to highly pathogenic influenza virus infection mechanisms of survival JVirol 862437ndash2446
Dai MY Qiao JP Xu YH and Fei GH (2015) Respiratory infectious phenotypes inacute exacerbation of COPD an aid to length of stay and COPD Assessment TestInt J Chron Obstruct Pulmon Dis 102257ndash2263
Donath E Chaudhry A Hernandez-Aya LF and Lit L (2013) A meta-analysis on theprophylactic use of macrolide antibiotics for the prevention of disease exacerbations inpatients with chronic obstructive pulmonary disease Respir Med 1071385ndash1392
Fukuda Y Yanagihara K Higashiyama Y Miyazaki Y Hirakata Y Mukae H TomonoK Mizuta Y Tsukamoto K and Kohno S (2006) Effects of macrolides on pneumolysinof macrolide-resistant Streptococcus pneumoniae Eur Respir J 271020ndash1025
Galani IE and Andreakos E (2015) Neutrophils in viral infections current conceptsand caveats J Leukoc Biol 98557ndash564
Herold S Becker C Ridge KM and Budinger GR (2015) Influenza virus-induced lunginjury pathogenesis and implications for treatment Eur Respir J 451463ndash1478
Kanoh S and Rubin BK (2010) Mechanisms of action and clinical application ofmacrolides as immunomodulatory medications Clin Microbiol Rev 23590ndash615
Kikuchi E Yamazaki K Kikuchi J Hasegawa N Hashimoto S Ishizaka Aand Nishimura M (2008) Pharmacokinetics of clarithromycin in bronchial epithe-lial lining fluid Respirology 13221ndash226
Kurai D Saraya T Ishii H and Takizawa H (2013) Virus-induced exacerbations inasthma and COPD Front Microbiol 4293
Lee B Robinson KM McHugh KJ Scheller EV Mandalapu S Chen C Di YP ClayME Enelow RI and Dubin PJ et al (2015) Influenza-induced type I interferonenhances susceptibility to gram-negative and gram-positive bacterial pneumoniain mice Am J Physiol Lung Cell Mol Physiol 309L158ndashL167
LeMessurier KS Haumlcker H Chi L Tuomanen E and Redecke V (2013) Type I in-terferon protects against pneumococcal invasive disease by inhibiting bacterialtransmigration across the lung PLoS Pathog 9e1003727
Li W Moltedo B and Moran TM (2012) Type I interferon induction during influenzavirus infection increases susceptibility to secondary Streptococcus pneumoniae in-fection by negative regulation of gd T cells J Virol 8612304ndash12312
Mathers CD and Loncar D (2006) Projections of global mortality and burden of dis-ease from 2002 to 2030 PLoS Med 3e442
McCarty JM (2000) Clarithromycin in the management of community-acquiredpneumonia Clin Ther 22281ndash294 discussion 265
Miyamoto D Hasegawa S Sriwilaijaroen N Yingsakmongkon S Hiramatsu HTakahashi T Hidari K Guo CT Sakano Y and Suzuki T et al (2008) Clari-thromycin inhibits progeny virus production from human influenza virus-infectedhost cells Biol Pharm Bull 31217ndash222
Morens DM Taubenberger JK and Fauci AS (2008) Predominant role of bacterialpneumonia as a cause of death in pandemic influenza implications for pandemicinfluenza preparedness J Infect Dis 198962ndash970
Muumlller U Steinhoff U Reis LF Hemmi S Pavlovic J Zinkernagel RM and Aguet M(1994) Functional role of type I and type II interferons in antiviral defense Science2641918ndash1921
Murata Y Walsh EE and Falsey AR (2007) Pulmonary complications of in-terpandemic influenza A in hospitalized adults J Infect Dis 1951029ndash1037
Nakamura S Davis KM and Weiser JN (2011) Synergistic stimulation of type Iinterferons during influenza virus coinfection promotes Streptococcus pneumoniaecolonization in mice J Clin Invest 1213657ndash3665
Ni W Shao X Cai X Wei C Cui J Wang R and Liu Y (2015) Prophylactic use ofmacrolide antibiotics for the prevention of chronic obstructive pulmonary diseaseexacerbation a meta-analysis PLoS One 10e0121257
Otsu Y Yanagihara K Fukuda Y Miyazaki Y Tsukamoto K Hirakata Y Tomono KKadota J Tashiro T and Murata I et al (2003) In vivo efficacy of a new quinoloneDQ-113 against Streptococcus pneumoniae in a mouse model Antimicrob AgentsChemother 473699ndash3703
Partridge J and Kieny MP (2013) Global production capacity of seasonal influenzavaccine in 2011 Vaccine 31728ndash731
Reed LJ and Muench H (1938) A simple method of estimating fifty percent endpointsAm J Hyg 27493ndash497
Rohde G Wiethege A Borg I Kauth M Bauer TT Gillissen A Bufe A and Schultze-Werninghaus G (2003) Respiratory viruses in exacerbations of chronic obstructivepulmonary disease requiring hospitalisation a case-control study Thorax 5837ndash42
Sapey E and Stockley RA (2006) COPD exacerbations 2 aetiology Thorax 61250ndash258
Sato K Suga M Akaike T Fujii S Muranaka H Doi T Maeda H and Ando M (1998)Therapeutic effect of erythromycin on influenza virus-induced lung injury in miceAm J Respir Crit Care Med 157853ndash857
Schroder K Hertzog PJ Ravasi T and Hume DA (2004) Interferon-gamma anoverview of signals mechanisms and functions J Leukoc Biol 75163ndash189
Seki M Kohno S Newstead MW Zeng X Bhan U Lukacs NW Kunkel SLand Standiford TJ (2010) Critical role of IL-1 receptor-associated kinase-M inregulating chemokine-dependent deleterious inflammation in murine influenzapneumonia J Immunol 1841410ndash1418
Sethi S Evans N Grant BJ and Murphy TF (2002) New strains of bacteria and ex-acerbations of chronic obstructive pulmonary disease N Engl J Med 347465ndash471
Spagnolo P Fabbri LM and Bush A (2013) Long-term macrolide treatment forchronic respiratory disease Eur Respir J 42239ndash251
Sun K and Metzger DW (2008) Inhibition of pulmonary antibacterial defense byinterferon-gamma during recovery from influenza infection Nat Med 14558ndash564
Tate MD Deng YM Jones JE Anderson GP Brooks AG and Reading PC (2009)Neutrophils ameliorate lung injury and the development of severe disease duringinfluenza infection J Immunol 1837441ndash7450
Trinchieri G (2010) Type I interferon friend or foe J Exp Med 2072053ndash2063van der Sluijs KF van der Poll T Lutter R Juffermans NP and Schultz MJ (2010)Bench-to-bedside review bacterial pneumonia with influenzamdashpathogenesis andclinical implications Crit Care 14219
Yamaryo T Oishi K Yoshimine H Tsuchihashi Y Matsushima K and Nagatake T(2003) Fourteen-member macrolides promote the phosphatidylserine receptor-dependent phagocytosis of apoptotic neutrophils by alveolar macrophages Anti-microb Agents Chemother 4748ndash53
Yamaya M Azuma A Takizawa H Kadota J Tamaoki J and Kudoh S (2012)Macrolide effects on the prevention of COPD exacerbations Eur Respir J 40485ndash494
Yamaya M Shinya K Hatachi Y Kubo H Asada M Yasuda H Nishimura Hand Nagatomi R (2010) Clarithromycin inhibits type a seasonal influenza virusinfection in human airway epithelial cells J Pharmacol Exp Ther 33381ndash90
Zarogoulidis P Papanas N Kioumis I Chatzaki E Maltezos E and Zarogoulidis K(2012) Macrolides from in vitro anti-inflammatory and immunomodulatory prop-erties to clinical practice in respiratory diseases Eur J Clin Pharmacol 68479ndash503
Address correspondence to Dr Yuji Ishimatsu Department of Cardiopul-monary Rehabilitation Sciences Nagasaki University Graduate School ofBiomedical Sciences 1-7-1 Sakamoto Nagasaki 852-8520 Japan E-mail yuji-inagasaki-uacjp
Effect of CAM on SBP during Influenza in Emphysema Mice 463
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ResultsPremedication with CAM Improved Survival Rates
Postexposure treatment with CAM did not improve the sur-vival rate compared with that of the vehicle control Howeverpremedication with CAM resulted in a significantly highersurvival rate than that of the vehicle control (P 005 Fig 2A and B)CAM Premedication Suppressed Lung Inflammation
and Bacterial Counts Figure 3 shows representative im-ages of hematoxylin and eosinndashstained lung sections from thepremedication group at Day 6 Vehicle control mice exhibitedbronchopneumonia characterized by abundant neutrophilsparticularly in peribronchiolar lesions as well as in airwaysalveolar spaces and the interstitium (Fig 3 A and B) Incontrast the premedication group showed significantly lessneutrophilic inflammation than that of the control group (Fig3 C and D) on the basis of a quantitative analysis of histologicimages (control group 102 6 6 neutrophilsmm2 premedica-tion group 656 4 neutrophilsmm2P 00001) The bacterialcount was significantly lower in the lungs of the premedicationgroup than those of the control group (P 005 Fig 4)CAM Did Not Exhibit Antibacterial Activity against
NU4471 in the Lungs The discs with lung homogenates(Fig 5A lower left) and thosewith 15mg of CAM (Fig 5A lowerright) did not form inhibition zones on the macrolide-resistant
NU4471 plate but both discs formed inhibition zones on themacrolide-susceptible ATCC49619 plate (Fig 5B)CAM Did Not Affect Viral Titers at the Time of
Inoculation with NU4471 There was no significant differ-ence between the premedication and control groups in theviral titers of the lung homogenates on Day 4 (Fig 6)CAM Reduced the Number of Neutrophils in BALF
The total number of cells and neutrophils in BALF from theCAM-negativeinfluenza virusndashpositive group was significantlyhigher than that observed in the BALF from the CAM-negativeinfluenza virusndashnegative group Although no significant dif-ferences in cell number were observed after CAM medicationthere was a significant reduction in the number of neutrophilscompared with the control group (Fig 7) No significant differ-ences between groups were observed for other cell subsets(lymphocytes basophils and eosinophils data not shown)Effects of CAM on Cytokines and Chemokines The
levels of IFN-a IFN-b and INF-g were significantly elevatedin BALF at Day 4 of influenza infection compared with thelevels observed in mice without influenza (Fig 8) There wereno significant differences in the levels of IL-4 IL-12 andmacrophage inflammatory protein-2 (data not shown) CAMmedication significantly reduced IFN-g levels in BALFwhereas there were no significant changes in IFN-a IFN-band IL-10 levels after CAM medication (Fig 8) Further
Fig 8 Anti-inflammatory effect of CAM on cytokine levels in BALF (A) IFN-a (B) IFN-b (C) IFN-g and (D) IL-10 Data are presented asmeans6 SEn = 5ndash9 mice NS no significant difference between two groups P 005 significant difference between two groups
Effect of CAM on SBP during Influenza in Emphysema Mice 461
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investigation of IFN-g levels in the serumand lung revealed thatINF-g levels were significantly higher in the lung and serumduring influenza infection than in the controls (Fig 9) CAMmedication significantly reduced IFN-g levels in lung homoge-nates but did not significant influence its serum levels (Fig 9)
DiscussionOn the basis of our results premedication with CAM
initiated 4 days before influenza virus inoculation in COPD-model mice but not postexposure medication improved thesurvival of mice against subsequent coinfection with theinfluenza virus and S pneumoniae Mice with improvedsurvival also had a less severe inflammatory response in lungtissues than those of mice in the control group Although viraltiters were not affected by CAM premedication viable bacte-rial counts decreased in lung tissues even though S pneumo-niae NU4471 is macrolide-resistant CAM accumulates tohigher concentrations in the bronchial epithelial fluid than inthe serum (McCarty 2000 Kikuchi et al 2008) accordinglywe examined the local antimicrobial activity of CAM in lungtissues (Fig 5) and confirmed that the lung homogenatesdid not possess antibacterial activity against S pneumoniae
NU4471 Further investigation revealed that CAM premed-ication significantly reduced the number of total inflammatorycells including neutrophils in BALF and significantly atten-uated IFN-g levels in BALF and lung homogenates Theseresults suggest that the improvement in survival rates asso-ciated with CAM premedication could be attributed to an anti-inflammatory effect exerted prior to the inoculation of micewith S pneumoniaeTaken together CAM premedication reduced the susceptibil-
ity of COPDmice infected with influenza virus to subsequent Spneumoniae infection by decreasing the overall inflammatoryresponse and ultimately improved the survival of COPD modelmice infected with both pathogens Our results obtained usingCOPD-model mice are similar to previous reports demonstrat-ing that macrolides offer clinical benefits in reducing acuteexacerbations of COPD (Albert et al 2011 Ni et al 2015)Erythromycin or azithromycin therapy for 6ndash12 months couldeffectively reduce the frequency of exacerbations and improvethe quality of life in patients with COPD (Albert et al 2011 Niet al 2015) Unlike previous studies reporting that macrolidesexert direct antiviral effects on the influenza virus (Miyamotoet al 2008 Yamaya et al 2010) CAM premedication did notinfluence viral titers in our study Rather CAM premedicationappeared to act via a chemokine-mediated mechanism bydisrupting the cycle of infection-inflammation and strengthen-ing lung defenses as reported previously (Spagnolo et al 2013)Interferons play critical and complex roles in host defense
against viral and bacterial infections For example IFN-a andIFN-b (Type-1 IFNs) prevent the progression of local lunginfection with S pneumoniae (Trinchieri 2010 LeMessurieret al 2013) and IFN-g (Type-2 IFN Type-1 T-helper cellcytokine) combats the early stages of viral infection (Mulleret al 1994 Schroder et al 2004) However high levels of IFN-aIFN-b and IFN-g induced by influenza virus infection enhancesusceptibility to secondary pneumococcal infection (Sun andMetzger 2008 Nakamura et al 2011 Li et al 2012 Lee et al2015) via various mechanisms including IFN-g-mediated in-hibition of initial bacterial clearance from the lung by alveolarmacrophages (Sun and Metzger 2008) The trends towardreduced IFN-a and IFN-b and the significantly lower levels ofIFN-g observed after CAM premedication in our study and inprevious studies [ie reduced IFN-g levels were detected aftermacrolide therapy in mice with influenza virusndashinduced lunginjury (Sato et al 1998)]mighthaveenhancedbacterial clearancefrom the lung and reduced the susceptibility of influenza-infectedCOPD mice to secondary pneumococcal pneumoniaInfluenza-infected COPD mice showed a more advanced
pathology characterized by significantly more neutrophilsin the lung tissue and BALF compared with control miceExcessive neutrophil infiltration into the lungs has beenreported during influenza virus infection and disrupts thealveolar-capillary barrier leading to acute lung injury anddeath (Seki et al 2010 Galani and Andreakos 2015 Heroldet al 2015) Conversely reductions in neutrophils or neu-trophil function increase the severity of influenza virusinfection (Tate et al 2009) CAM has been reported to en-hance the nonphlogistic clearance of apoptotic neutrophils byalveolar macrophages (Yamaryo et al 2003) Indeed CAMpremedication in our study reduced neutrophil numbers in thelung tissue and BALF of influenza-infected COPD mice andmight have attenuated the potential for inflammation-inducedlung damage
Fig 9 Anti-inflammatory effect of CAM on IFN-g levels in (A) lunghomogenates and (B) serum samples Data are presented asmeans6 SEn = 5ndash9 mice NS no significant difference P 005 significantdifference
462 Harada et al
at ASPE
T Journals on June 1 2020
jpetaspetjournalsorgD
ownloaded from
While vaccination strategies are most efficient with respectto protection against influenza and pneumococcal infection inCOPD patients the global supply of influenza vaccines isinadequate during influenza pandemics and is of particularconcern in low- and middle-income countries (Partridge andKieny 2013) where the incidence of COPD and the demandfor influenza vaccinations are expected to increase Ourresults suggest that macrolide premedication may be aneffective strategy in COPD patients at a high risk of infectionwith influenza virus and S pneumoniae to prevent severe oreven fatal secondary bacterial pneumonia in these high-riskand low-resource settings Additional studies are required toidentify the ideal patient group and the optimal timing andduration of macrolide premedication to avoid injudiciousadministration over extended periods (Ni et al 2015)In conclusion administration of CAM initiated 4 days prior
to influenza virus infection in COPD mice reduced IFN-gproduction and enhanced the immune response against sec-ondary bacterial pneumonia to reduce bacterial counts andprolong survival rates Future studies should evaluate the useof macrolide premedication in COPD patients at a high risk ofinfection with influenza virus and S pneumoniae to preventsevere secondary bacterial pneumonia
Acknowledgments
The authors thankMr A Yokoyama for excellent technical supportEditorial support in the form of medical writing based on the
authorsrsquo detailed instructions collating author comments copy-editing fact checking and referencing was provided by CactusCommunications
Authorship Contributions
Participated in research design Harada Ishimatsu Hara MoritaConducted experiments Harada Hara Morita Nakashima
Kakugawa Sakamoto KosaiContributed new reagents or analytic tools Kosai Izumikawa
Yanagihara KohnoPerformed data analysis Harada Nakashima IshimatsuWrote or contributed to the writing of the manuscript Harada
Ishimatsu Kakugawa Sakamoto Mukae
References
Albert RK Connett J Bailey WC Casaburi R Cooper JA Jr Criner GJ Curtis JLDransfield MT Han MK and Lazarus SC et al COPD Clinical Research Network(2011) Azithromycin for prevention of exacerbations of COPD N Engl J Med 365689ndash698
Bautista E Chotpitayasunondh T Gao Z Harper SA Shaw M Uyeki TM Zaki SRHayden FG Hui DS and Kettner JD et al Writing Committee of the WHO Consul-tation on Clinical Aspects of Pandemic (H1N1) 2009 Influenza (2010) Clinical aspects ofpandemic 2009 influenza A (H1N1) virus infection N Engl J Med 3621708ndash1719
Brundage JF and Shanks GD (2008) Deaths from bacterial pneumonia during1918ndash49 influenza pandemic Emerg Infect Dis 141193ndash1199
Cilloniz C Pantin-Jackwood MJ Ni C Carter VS Korth MJ Swayne DE TumpeyTM and Katze MG (2012) Molecular signatures associated with Mx1-mediatedresistance to highly pathogenic influenza virus infection mechanisms of survival JVirol 862437ndash2446
Dai MY Qiao JP Xu YH and Fei GH (2015) Respiratory infectious phenotypes inacute exacerbation of COPD an aid to length of stay and COPD Assessment TestInt J Chron Obstruct Pulmon Dis 102257ndash2263
Donath E Chaudhry A Hernandez-Aya LF and Lit L (2013) A meta-analysis on theprophylactic use of macrolide antibiotics for the prevention of disease exacerbations inpatients with chronic obstructive pulmonary disease Respir Med 1071385ndash1392
Fukuda Y Yanagihara K Higashiyama Y Miyazaki Y Hirakata Y Mukae H TomonoK Mizuta Y Tsukamoto K and Kohno S (2006) Effects of macrolides on pneumolysinof macrolide-resistant Streptococcus pneumoniae Eur Respir J 271020ndash1025
Galani IE and Andreakos E (2015) Neutrophils in viral infections current conceptsand caveats J Leukoc Biol 98557ndash564
Herold S Becker C Ridge KM and Budinger GR (2015) Influenza virus-induced lunginjury pathogenesis and implications for treatment Eur Respir J 451463ndash1478
Kanoh S and Rubin BK (2010) Mechanisms of action and clinical application ofmacrolides as immunomodulatory medications Clin Microbiol Rev 23590ndash615
Kikuchi E Yamazaki K Kikuchi J Hasegawa N Hashimoto S Ishizaka Aand Nishimura M (2008) Pharmacokinetics of clarithromycin in bronchial epithe-lial lining fluid Respirology 13221ndash226
Kurai D Saraya T Ishii H and Takizawa H (2013) Virus-induced exacerbations inasthma and COPD Front Microbiol 4293
Lee B Robinson KM McHugh KJ Scheller EV Mandalapu S Chen C Di YP ClayME Enelow RI and Dubin PJ et al (2015) Influenza-induced type I interferonenhances susceptibility to gram-negative and gram-positive bacterial pneumoniain mice Am J Physiol Lung Cell Mol Physiol 309L158ndashL167
LeMessurier KS Haumlcker H Chi L Tuomanen E and Redecke V (2013) Type I in-terferon protects against pneumococcal invasive disease by inhibiting bacterialtransmigration across the lung PLoS Pathog 9e1003727
Li W Moltedo B and Moran TM (2012) Type I interferon induction during influenzavirus infection increases susceptibility to secondary Streptococcus pneumoniae in-fection by negative regulation of gd T cells J Virol 8612304ndash12312
Mathers CD and Loncar D (2006) Projections of global mortality and burden of dis-ease from 2002 to 2030 PLoS Med 3e442
McCarty JM (2000) Clarithromycin in the management of community-acquiredpneumonia Clin Ther 22281ndash294 discussion 265
Miyamoto D Hasegawa S Sriwilaijaroen N Yingsakmongkon S Hiramatsu HTakahashi T Hidari K Guo CT Sakano Y and Suzuki T et al (2008) Clari-thromycin inhibits progeny virus production from human influenza virus-infectedhost cells Biol Pharm Bull 31217ndash222
Morens DM Taubenberger JK and Fauci AS (2008) Predominant role of bacterialpneumonia as a cause of death in pandemic influenza implications for pandemicinfluenza preparedness J Infect Dis 198962ndash970
Muumlller U Steinhoff U Reis LF Hemmi S Pavlovic J Zinkernagel RM and Aguet M(1994) Functional role of type I and type II interferons in antiviral defense Science2641918ndash1921
Murata Y Walsh EE and Falsey AR (2007) Pulmonary complications of in-terpandemic influenza A in hospitalized adults J Infect Dis 1951029ndash1037
Nakamura S Davis KM and Weiser JN (2011) Synergistic stimulation of type Iinterferons during influenza virus coinfection promotes Streptococcus pneumoniaecolonization in mice J Clin Invest 1213657ndash3665
Ni W Shao X Cai X Wei C Cui J Wang R and Liu Y (2015) Prophylactic use ofmacrolide antibiotics for the prevention of chronic obstructive pulmonary diseaseexacerbation a meta-analysis PLoS One 10e0121257
Otsu Y Yanagihara K Fukuda Y Miyazaki Y Tsukamoto K Hirakata Y Tomono KKadota J Tashiro T and Murata I et al (2003) In vivo efficacy of a new quinoloneDQ-113 against Streptococcus pneumoniae in a mouse model Antimicrob AgentsChemother 473699ndash3703
Partridge J and Kieny MP (2013) Global production capacity of seasonal influenzavaccine in 2011 Vaccine 31728ndash731
Reed LJ and Muench H (1938) A simple method of estimating fifty percent endpointsAm J Hyg 27493ndash497
Rohde G Wiethege A Borg I Kauth M Bauer TT Gillissen A Bufe A and Schultze-Werninghaus G (2003) Respiratory viruses in exacerbations of chronic obstructivepulmonary disease requiring hospitalisation a case-control study Thorax 5837ndash42
Sapey E and Stockley RA (2006) COPD exacerbations 2 aetiology Thorax 61250ndash258
Sato K Suga M Akaike T Fujii S Muranaka H Doi T Maeda H and Ando M (1998)Therapeutic effect of erythromycin on influenza virus-induced lung injury in miceAm J Respir Crit Care Med 157853ndash857
Schroder K Hertzog PJ Ravasi T and Hume DA (2004) Interferon-gamma anoverview of signals mechanisms and functions J Leukoc Biol 75163ndash189
Seki M Kohno S Newstead MW Zeng X Bhan U Lukacs NW Kunkel SLand Standiford TJ (2010) Critical role of IL-1 receptor-associated kinase-M inregulating chemokine-dependent deleterious inflammation in murine influenzapneumonia J Immunol 1841410ndash1418
Sethi S Evans N Grant BJ and Murphy TF (2002) New strains of bacteria and ex-acerbations of chronic obstructive pulmonary disease N Engl J Med 347465ndash471
Spagnolo P Fabbri LM and Bush A (2013) Long-term macrolide treatment forchronic respiratory disease Eur Respir J 42239ndash251
Sun K and Metzger DW (2008) Inhibition of pulmonary antibacterial defense byinterferon-gamma during recovery from influenza infection Nat Med 14558ndash564
Tate MD Deng YM Jones JE Anderson GP Brooks AG and Reading PC (2009)Neutrophils ameliorate lung injury and the development of severe disease duringinfluenza infection J Immunol 1837441ndash7450
Trinchieri G (2010) Type I interferon friend or foe J Exp Med 2072053ndash2063van der Sluijs KF van der Poll T Lutter R Juffermans NP and Schultz MJ (2010)Bench-to-bedside review bacterial pneumonia with influenzamdashpathogenesis andclinical implications Crit Care 14219
Yamaryo T Oishi K Yoshimine H Tsuchihashi Y Matsushima K and Nagatake T(2003) Fourteen-member macrolides promote the phosphatidylserine receptor-dependent phagocytosis of apoptotic neutrophils by alveolar macrophages Anti-microb Agents Chemother 4748ndash53
Yamaya M Azuma A Takizawa H Kadota J Tamaoki J and Kudoh S (2012)Macrolide effects on the prevention of COPD exacerbations Eur Respir J 40485ndash494
Yamaya M Shinya K Hatachi Y Kubo H Asada M Yasuda H Nishimura Hand Nagatomi R (2010) Clarithromycin inhibits type a seasonal influenza virusinfection in human airway epithelial cells J Pharmacol Exp Ther 33381ndash90
Zarogoulidis P Papanas N Kioumis I Chatzaki E Maltezos E and Zarogoulidis K(2012) Macrolides from in vitro anti-inflammatory and immunomodulatory prop-erties to clinical practice in respiratory diseases Eur J Clin Pharmacol 68479ndash503
Address correspondence to Dr Yuji Ishimatsu Department of Cardiopul-monary Rehabilitation Sciences Nagasaki University Graduate School ofBiomedical Sciences 1-7-1 Sakamoto Nagasaki 852-8520 Japan E-mail yuji-inagasaki-uacjp
Effect of CAM on SBP during Influenza in Emphysema Mice 463
at ASPE
T Journals on June 1 2020
jpetaspetjournalsorgD
ownloaded from
investigation of IFN-g levels in the serumand lung revealed thatINF-g levels were significantly higher in the lung and serumduring influenza infection than in the controls (Fig 9) CAMmedication significantly reduced IFN-g levels in lung homoge-nates but did not significant influence its serum levels (Fig 9)
DiscussionOn the basis of our results premedication with CAM
initiated 4 days before influenza virus inoculation in COPD-model mice but not postexposure medication improved thesurvival of mice against subsequent coinfection with theinfluenza virus and S pneumoniae Mice with improvedsurvival also had a less severe inflammatory response in lungtissues than those of mice in the control group Although viraltiters were not affected by CAM premedication viable bacte-rial counts decreased in lung tissues even though S pneumo-niae NU4471 is macrolide-resistant CAM accumulates tohigher concentrations in the bronchial epithelial fluid than inthe serum (McCarty 2000 Kikuchi et al 2008) accordinglywe examined the local antimicrobial activity of CAM in lungtissues (Fig 5) and confirmed that the lung homogenatesdid not possess antibacterial activity against S pneumoniae
NU4471 Further investigation revealed that CAM premed-ication significantly reduced the number of total inflammatorycells including neutrophils in BALF and significantly atten-uated IFN-g levels in BALF and lung homogenates Theseresults suggest that the improvement in survival rates asso-ciated with CAM premedication could be attributed to an anti-inflammatory effect exerted prior to the inoculation of micewith S pneumoniaeTaken together CAM premedication reduced the susceptibil-
ity of COPDmice infected with influenza virus to subsequent Spneumoniae infection by decreasing the overall inflammatoryresponse and ultimately improved the survival of COPD modelmice infected with both pathogens Our results obtained usingCOPD-model mice are similar to previous reports demonstrat-ing that macrolides offer clinical benefits in reducing acuteexacerbations of COPD (Albert et al 2011 Ni et al 2015)Erythromycin or azithromycin therapy for 6ndash12 months couldeffectively reduce the frequency of exacerbations and improvethe quality of life in patients with COPD (Albert et al 2011 Niet al 2015) Unlike previous studies reporting that macrolidesexert direct antiviral effects on the influenza virus (Miyamotoet al 2008 Yamaya et al 2010) CAM premedication did notinfluence viral titers in our study Rather CAM premedicationappeared to act via a chemokine-mediated mechanism bydisrupting the cycle of infection-inflammation and strengthen-ing lung defenses as reported previously (Spagnolo et al 2013)Interferons play critical and complex roles in host defense
against viral and bacterial infections For example IFN-a andIFN-b (Type-1 IFNs) prevent the progression of local lunginfection with S pneumoniae (Trinchieri 2010 LeMessurieret al 2013) and IFN-g (Type-2 IFN Type-1 T-helper cellcytokine) combats the early stages of viral infection (Mulleret al 1994 Schroder et al 2004) However high levels of IFN-aIFN-b and IFN-g induced by influenza virus infection enhancesusceptibility to secondary pneumococcal infection (Sun andMetzger 2008 Nakamura et al 2011 Li et al 2012 Lee et al2015) via various mechanisms including IFN-g-mediated in-hibition of initial bacterial clearance from the lung by alveolarmacrophages (Sun and Metzger 2008) The trends towardreduced IFN-a and IFN-b and the significantly lower levels ofIFN-g observed after CAM premedication in our study and inprevious studies [ie reduced IFN-g levels were detected aftermacrolide therapy in mice with influenza virusndashinduced lunginjury (Sato et al 1998)]mighthaveenhancedbacterial clearancefrom the lung and reduced the susceptibility of influenza-infectedCOPD mice to secondary pneumococcal pneumoniaInfluenza-infected COPD mice showed a more advanced
pathology characterized by significantly more neutrophilsin the lung tissue and BALF compared with control miceExcessive neutrophil infiltration into the lungs has beenreported during influenza virus infection and disrupts thealveolar-capillary barrier leading to acute lung injury anddeath (Seki et al 2010 Galani and Andreakos 2015 Heroldet al 2015) Conversely reductions in neutrophils or neu-trophil function increase the severity of influenza virusinfection (Tate et al 2009) CAM has been reported to en-hance the nonphlogistic clearance of apoptotic neutrophils byalveolar macrophages (Yamaryo et al 2003) Indeed CAMpremedication in our study reduced neutrophil numbers in thelung tissue and BALF of influenza-infected COPD mice andmight have attenuated the potential for inflammation-inducedlung damage
Fig 9 Anti-inflammatory effect of CAM on IFN-g levels in (A) lunghomogenates and (B) serum samples Data are presented asmeans6 SEn = 5ndash9 mice NS no significant difference P 005 significantdifference
462 Harada et al
at ASPE
T Journals on June 1 2020
jpetaspetjournalsorgD
ownloaded from
While vaccination strategies are most efficient with respectto protection against influenza and pneumococcal infection inCOPD patients the global supply of influenza vaccines isinadequate during influenza pandemics and is of particularconcern in low- and middle-income countries (Partridge andKieny 2013) where the incidence of COPD and the demandfor influenza vaccinations are expected to increase Ourresults suggest that macrolide premedication may be aneffective strategy in COPD patients at a high risk of infectionwith influenza virus and S pneumoniae to prevent severe oreven fatal secondary bacterial pneumonia in these high-riskand low-resource settings Additional studies are required toidentify the ideal patient group and the optimal timing andduration of macrolide premedication to avoid injudiciousadministration over extended periods (Ni et al 2015)In conclusion administration of CAM initiated 4 days prior
to influenza virus infection in COPD mice reduced IFN-gproduction and enhanced the immune response against sec-ondary bacterial pneumonia to reduce bacterial counts andprolong survival rates Future studies should evaluate the useof macrolide premedication in COPD patients at a high risk ofinfection with influenza virus and S pneumoniae to preventsevere secondary bacterial pneumonia
Acknowledgments
The authors thankMr A Yokoyama for excellent technical supportEditorial support in the form of medical writing based on the
authorsrsquo detailed instructions collating author comments copy-editing fact checking and referencing was provided by CactusCommunications
Authorship Contributions
Participated in research design Harada Ishimatsu Hara MoritaConducted experiments Harada Hara Morita Nakashima
Kakugawa Sakamoto KosaiContributed new reagents or analytic tools Kosai Izumikawa
Yanagihara KohnoPerformed data analysis Harada Nakashima IshimatsuWrote or contributed to the writing of the manuscript Harada
Ishimatsu Kakugawa Sakamoto Mukae
References
Albert RK Connett J Bailey WC Casaburi R Cooper JA Jr Criner GJ Curtis JLDransfield MT Han MK and Lazarus SC et al COPD Clinical Research Network(2011) Azithromycin for prevention of exacerbations of COPD N Engl J Med 365689ndash698
Bautista E Chotpitayasunondh T Gao Z Harper SA Shaw M Uyeki TM Zaki SRHayden FG Hui DS and Kettner JD et al Writing Committee of the WHO Consul-tation on Clinical Aspects of Pandemic (H1N1) 2009 Influenza (2010) Clinical aspects ofpandemic 2009 influenza A (H1N1) virus infection N Engl J Med 3621708ndash1719
Brundage JF and Shanks GD (2008) Deaths from bacterial pneumonia during1918ndash49 influenza pandemic Emerg Infect Dis 141193ndash1199
Cilloniz C Pantin-Jackwood MJ Ni C Carter VS Korth MJ Swayne DE TumpeyTM and Katze MG (2012) Molecular signatures associated with Mx1-mediatedresistance to highly pathogenic influenza virus infection mechanisms of survival JVirol 862437ndash2446
Dai MY Qiao JP Xu YH and Fei GH (2015) Respiratory infectious phenotypes inacute exacerbation of COPD an aid to length of stay and COPD Assessment TestInt J Chron Obstruct Pulmon Dis 102257ndash2263
Donath E Chaudhry A Hernandez-Aya LF and Lit L (2013) A meta-analysis on theprophylactic use of macrolide antibiotics for the prevention of disease exacerbations inpatients with chronic obstructive pulmonary disease Respir Med 1071385ndash1392
Fukuda Y Yanagihara K Higashiyama Y Miyazaki Y Hirakata Y Mukae H TomonoK Mizuta Y Tsukamoto K and Kohno S (2006) Effects of macrolides on pneumolysinof macrolide-resistant Streptococcus pneumoniae Eur Respir J 271020ndash1025
Galani IE and Andreakos E (2015) Neutrophils in viral infections current conceptsand caveats J Leukoc Biol 98557ndash564
Herold S Becker C Ridge KM and Budinger GR (2015) Influenza virus-induced lunginjury pathogenesis and implications for treatment Eur Respir J 451463ndash1478
Kanoh S and Rubin BK (2010) Mechanisms of action and clinical application ofmacrolides as immunomodulatory medications Clin Microbiol Rev 23590ndash615
Kikuchi E Yamazaki K Kikuchi J Hasegawa N Hashimoto S Ishizaka Aand Nishimura M (2008) Pharmacokinetics of clarithromycin in bronchial epithe-lial lining fluid Respirology 13221ndash226
Kurai D Saraya T Ishii H and Takizawa H (2013) Virus-induced exacerbations inasthma and COPD Front Microbiol 4293
Lee B Robinson KM McHugh KJ Scheller EV Mandalapu S Chen C Di YP ClayME Enelow RI and Dubin PJ et al (2015) Influenza-induced type I interferonenhances susceptibility to gram-negative and gram-positive bacterial pneumoniain mice Am J Physiol Lung Cell Mol Physiol 309L158ndashL167
LeMessurier KS Haumlcker H Chi L Tuomanen E and Redecke V (2013) Type I in-terferon protects against pneumococcal invasive disease by inhibiting bacterialtransmigration across the lung PLoS Pathog 9e1003727
Li W Moltedo B and Moran TM (2012) Type I interferon induction during influenzavirus infection increases susceptibility to secondary Streptococcus pneumoniae in-fection by negative regulation of gd T cells J Virol 8612304ndash12312
Mathers CD and Loncar D (2006) Projections of global mortality and burden of dis-ease from 2002 to 2030 PLoS Med 3e442
McCarty JM (2000) Clarithromycin in the management of community-acquiredpneumonia Clin Ther 22281ndash294 discussion 265
Miyamoto D Hasegawa S Sriwilaijaroen N Yingsakmongkon S Hiramatsu HTakahashi T Hidari K Guo CT Sakano Y and Suzuki T et al (2008) Clari-thromycin inhibits progeny virus production from human influenza virus-infectedhost cells Biol Pharm Bull 31217ndash222
Morens DM Taubenberger JK and Fauci AS (2008) Predominant role of bacterialpneumonia as a cause of death in pandemic influenza implications for pandemicinfluenza preparedness J Infect Dis 198962ndash970
Muumlller U Steinhoff U Reis LF Hemmi S Pavlovic J Zinkernagel RM and Aguet M(1994) Functional role of type I and type II interferons in antiviral defense Science2641918ndash1921
Murata Y Walsh EE and Falsey AR (2007) Pulmonary complications of in-terpandemic influenza A in hospitalized adults J Infect Dis 1951029ndash1037
Nakamura S Davis KM and Weiser JN (2011) Synergistic stimulation of type Iinterferons during influenza virus coinfection promotes Streptococcus pneumoniaecolonization in mice J Clin Invest 1213657ndash3665
Ni W Shao X Cai X Wei C Cui J Wang R and Liu Y (2015) Prophylactic use ofmacrolide antibiotics for the prevention of chronic obstructive pulmonary diseaseexacerbation a meta-analysis PLoS One 10e0121257
Otsu Y Yanagihara K Fukuda Y Miyazaki Y Tsukamoto K Hirakata Y Tomono KKadota J Tashiro T and Murata I et al (2003) In vivo efficacy of a new quinoloneDQ-113 against Streptococcus pneumoniae in a mouse model Antimicrob AgentsChemother 473699ndash3703
Partridge J and Kieny MP (2013) Global production capacity of seasonal influenzavaccine in 2011 Vaccine 31728ndash731
Reed LJ and Muench H (1938) A simple method of estimating fifty percent endpointsAm J Hyg 27493ndash497
Rohde G Wiethege A Borg I Kauth M Bauer TT Gillissen A Bufe A and Schultze-Werninghaus G (2003) Respiratory viruses in exacerbations of chronic obstructivepulmonary disease requiring hospitalisation a case-control study Thorax 5837ndash42
Sapey E and Stockley RA (2006) COPD exacerbations 2 aetiology Thorax 61250ndash258
Sato K Suga M Akaike T Fujii S Muranaka H Doi T Maeda H and Ando M (1998)Therapeutic effect of erythromycin on influenza virus-induced lung injury in miceAm J Respir Crit Care Med 157853ndash857
Schroder K Hertzog PJ Ravasi T and Hume DA (2004) Interferon-gamma anoverview of signals mechanisms and functions J Leukoc Biol 75163ndash189
Seki M Kohno S Newstead MW Zeng X Bhan U Lukacs NW Kunkel SLand Standiford TJ (2010) Critical role of IL-1 receptor-associated kinase-M inregulating chemokine-dependent deleterious inflammation in murine influenzapneumonia J Immunol 1841410ndash1418
Sethi S Evans N Grant BJ and Murphy TF (2002) New strains of bacteria and ex-acerbations of chronic obstructive pulmonary disease N Engl J Med 347465ndash471
Spagnolo P Fabbri LM and Bush A (2013) Long-term macrolide treatment forchronic respiratory disease Eur Respir J 42239ndash251
Sun K and Metzger DW (2008) Inhibition of pulmonary antibacterial defense byinterferon-gamma during recovery from influenza infection Nat Med 14558ndash564
Tate MD Deng YM Jones JE Anderson GP Brooks AG and Reading PC (2009)Neutrophils ameliorate lung injury and the development of severe disease duringinfluenza infection J Immunol 1837441ndash7450
Trinchieri G (2010) Type I interferon friend or foe J Exp Med 2072053ndash2063van der Sluijs KF van der Poll T Lutter R Juffermans NP and Schultz MJ (2010)Bench-to-bedside review bacterial pneumonia with influenzamdashpathogenesis andclinical implications Crit Care 14219
Yamaryo T Oishi K Yoshimine H Tsuchihashi Y Matsushima K and Nagatake T(2003) Fourteen-member macrolides promote the phosphatidylserine receptor-dependent phagocytosis of apoptotic neutrophils by alveolar macrophages Anti-microb Agents Chemother 4748ndash53
Yamaya M Azuma A Takizawa H Kadota J Tamaoki J and Kudoh S (2012)Macrolide effects on the prevention of COPD exacerbations Eur Respir J 40485ndash494
Yamaya M Shinya K Hatachi Y Kubo H Asada M Yasuda H Nishimura Hand Nagatomi R (2010) Clarithromycin inhibits type a seasonal influenza virusinfection in human airway epithelial cells J Pharmacol Exp Ther 33381ndash90
Zarogoulidis P Papanas N Kioumis I Chatzaki E Maltezos E and Zarogoulidis K(2012) Macrolides from in vitro anti-inflammatory and immunomodulatory prop-erties to clinical practice in respiratory diseases Eur J Clin Pharmacol 68479ndash503
Address correspondence to Dr Yuji Ishimatsu Department of Cardiopul-monary Rehabilitation Sciences Nagasaki University Graduate School ofBiomedical Sciences 1-7-1 Sakamoto Nagasaki 852-8520 Japan E-mail yuji-inagasaki-uacjp
Effect of CAM on SBP during Influenza in Emphysema Mice 463
at ASPE
T Journals on June 1 2020
jpetaspetjournalsorgD
ownloaded from
While vaccination strategies are most efficient with respectto protection against influenza and pneumococcal infection inCOPD patients the global supply of influenza vaccines isinadequate during influenza pandemics and is of particularconcern in low- and middle-income countries (Partridge andKieny 2013) where the incidence of COPD and the demandfor influenza vaccinations are expected to increase Ourresults suggest that macrolide premedication may be aneffective strategy in COPD patients at a high risk of infectionwith influenza virus and S pneumoniae to prevent severe oreven fatal secondary bacterial pneumonia in these high-riskand low-resource settings Additional studies are required toidentify the ideal patient group and the optimal timing andduration of macrolide premedication to avoid injudiciousadministration over extended periods (Ni et al 2015)In conclusion administration of CAM initiated 4 days prior
to influenza virus infection in COPD mice reduced IFN-gproduction and enhanced the immune response against sec-ondary bacterial pneumonia to reduce bacterial counts andprolong survival rates Future studies should evaluate the useof macrolide premedication in COPD patients at a high risk ofinfection with influenza virus and S pneumoniae to preventsevere secondary bacterial pneumonia
Acknowledgments
The authors thankMr A Yokoyama for excellent technical supportEditorial support in the form of medical writing based on the
authorsrsquo detailed instructions collating author comments copy-editing fact checking and referencing was provided by CactusCommunications
Authorship Contributions
Participated in research design Harada Ishimatsu Hara MoritaConducted experiments Harada Hara Morita Nakashima
Kakugawa Sakamoto KosaiContributed new reagents or analytic tools Kosai Izumikawa
Yanagihara KohnoPerformed data analysis Harada Nakashima IshimatsuWrote or contributed to the writing of the manuscript Harada
Ishimatsu Kakugawa Sakamoto Mukae
References
Albert RK Connett J Bailey WC Casaburi R Cooper JA Jr Criner GJ Curtis JLDransfield MT Han MK and Lazarus SC et al COPD Clinical Research Network(2011) Azithromycin for prevention of exacerbations of COPD N Engl J Med 365689ndash698
Bautista E Chotpitayasunondh T Gao Z Harper SA Shaw M Uyeki TM Zaki SRHayden FG Hui DS and Kettner JD et al Writing Committee of the WHO Consul-tation on Clinical Aspects of Pandemic (H1N1) 2009 Influenza (2010) Clinical aspects ofpandemic 2009 influenza A (H1N1) virus infection N Engl J Med 3621708ndash1719
Brundage JF and Shanks GD (2008) Deaths from bacterial pneumonia during1918ndash49 influenza pandemic Emerg Infect Dis 141193ndash1199
Cilloniz C Pantin-Jackwood MJ Ni C Carter VS Korth MJ Swayne DE TumpeyTM and Katze MG (2012) Molecular signatures associated with Mx1-mediatedresistance to highly pathogenic influenza virus infection mechanisms of survival JVirol 862437ndash2446
Dai MY Qiao JP Xu YH and Fei GH (2015) Respiratory infectious phenotypes inacute exacerbation of COPD an aid to length of stay and COPD Assessment TestInt J Chron Obstruct Pulmon Dis 102257ndash2263
Donath E Chaudhry A Hernandez-Aya LF and Lit L (2013) A meta-analysis on theprophylactic use of macrolide antibiotics for the prevention of disease exacerbations inpatients with chronic obstructive pulmonary disease Respir Med 1071385ndash1392
Fukuda Y Yanagihara K Higashiyama Y Miyazaki Y Hirakata Y Mukae H TomonoK Mizuta Y Tsukamoto K and Kohno S (2006) Effects of macrolides on pneumolysinof macrolide-resistant Streptococcus pneumoniae Eur Respir J 271020ndash1025
Galani IE and Andreakos E (2015) Neutrophils in viral infections current conceptsand caveats J Leukoc Biol 98557ndash564
Herold S Becker C Ridge KM and Budinger GR (2015) Influenza virus-induced lunginjury pathogenesis and implications for treatment Eur Respir J 451463ndash1478
Kanoh S and Rubin BK (2010) Mechanisms of action and clinical application ofmacrolides as immunomodulatory medications Clin Microbiol Rev 23590ndash615
Kikuchi E Yamazaki K Kikuchi J Hasegawa N Hashimoto S Ishizaka Aand Nishimura M (2008) Pharmacokinetics of clarithromycin in bronchial epithe-lial lining fluid Respirology 13221ndash226
Kurai D Saraya T Ishii H and Takizawa H (2013) Virus-induced exacerbations inasthma and COPD Front Microbiol 4293
Lee B Robinson KM McHugh KJ Scheller EV Mandalapu S Chen C Di YP ClayME Enelow RI and Dubin PJ et al (2015) Influenza-induced type I interferonenhances susceptibility to gram-negative and gram-positive bacterial pneumoniain mice Am J Physiol Lung Cell Mol Physiol 309L158ndashL167
LeMessurier KS Haumlcker H Chi L Tuomanen E and Redecke V (2013) Type I in-terferon protects against pneumococcal invasive disease by inhibiting bacterialtransmigration across the lung PLoS Pathog 9e1003727
Li W Moltedo B and Moran TM (2012) Type I interferon induction during influenzavirus infection increases susceptibility to secondary Streptococcus pneumoniae in-fection by negative regulation of gd T cells J Virol 8612304ndash12312
Mathers CD and Loncar D (2006) Projections of global mortality and burden of dis-ease from 2002 to 2030 PLoS Med 3e442
McCarty JM (2000) Clarithromycin in the management of community-acquiredpneumonia Clin Ther 22281ndash294 discussion 265
Miyamoto D Hasegawa S Sriwilaijaroen N Yingsakmongkon S Hiramatsu HTakahashi T Hidari K Guo CT Sakano Y and Suzuki T et al (2008) Clari-thromycin inhibits progeny virus production from human influenza virus-infectedhost cells Biol Pharm Bull 31217ndash222
Morens DM Taubenberger JK and Fauci AS (2008) Predominant role of bacterialpneumonia as a cause of death in pandemic influenza implications for pandemicinfluenza preparedness J Infect Dis 198962ndash970
Muumlller U Steinhoff U Reis LF Hemmi S Pavlovic J Zinkernagel RM and Aguet M(1994) Functional role of type I and type II interferons in antiviral defense Science2641918ndash1921
Murata Y Walsh EE and Falsey AR (2007) Pulmonary complications of in-terpandemic influenza A in hospitalized adults J Infect Dis 1951029ndash1037
Nakamura S Davis KM and Weiser JN (2011) Synergistic stimulation of type Iinterferons during influenza virus coinfection promotes Streptococcus pneumoniaecolonization in mice J Clin Invest 1213657ndash3665
Ni W Shao X Cai X Wei C Cui J Wang R and Liu Y (2015) Prophylactic use ofmacrolide antibiotics for the prevention of chronic obstructive pulmonary diseaseexacerbation a meta-analysis PLoS One 10e0121257
Otsu Y Yanagihara K Fukuda Y Miyazaki Y Tsukamoto K Hirakata Y Tomono KKadota J Tashiro T and Murata I et al (2003) In vivo efficacy of a new quinoloneDQ-113 against Streptococcus pneumoniae in a mouse model Antimicrob AgentsChemother 473699ndash3703
Partridge J and Kieny MP (2013) Global production capacity of seasonal influenzavaccine in 2011 Vaccine 31728ndash731
Reed LJ and Muench H (1938) A simple method of estimating fifty percent endpointsAm J Hyg 27493ndash497
Rohde G Wiethege A Borg I Kauth M Bauer TT Gillissen A Bufe A and Schultze-Werninghaus G (2003) Respiratory viruses in exacerbations of chronic obstructivepulmonary disease requiring hospitalisation a case-control study Thorax 5837ndash42
Sapey E and Stockley RA (2006) COPD exacerbations 2 aetiology Thorax 61250ndash258
Sato K Suga M Akaike T Fujii S Muranaka H Doi T Maeda H and Ando M (1998)Therapeutic effect of erythromycin on influenza virus-induced lung injury in miceAm J Respir Crit Care Med 157853ndash857
Schroder K Hertzog PJ Ravasi T and Hume DA (2004) Interferon-gamma anoverview of signals mechanisms and functions J Leukoc Biol 75163ndash189
Seki M Kohno S Newstead MW Zeng X Bhan U Lukacs NW Kunkel SLand Standiford TJ (2010) Critical role of IL-1 receptor-associated kinase-M inregulating chemokine-dependent deleterious inflammation in murine influenzapneumonia J Immunol 1841410ndash1418
Sethi S Evans N Grant BJ and Murphy TF (2002) New strains of bacteria and ex-acerbations of chronic obstructive pulmonary disease N Engl J Med 347465ndash471
Spagnolo P Fabbri LM and Bush A (2013) Long-term macrolide treatment forchronic respiratory disease Eur Respir J 42239ndash251
Sun K and Metzger DW (2008) Inhibition of pulmonary antibacterial defense byinterferon-gamma during recovery from influenza infection Nat Med 14558ndash564
Tate MD Deng YM Jones JE Anderson GP Brooks AG and Reading PC (2009)Neutrophils ameliorate lung injury and the development of severe disease duringinfluenza infection J Immunol 1837441ndash7450
Trinchieri G (2010) Type I interferon friend or foe J Exp Med 2072053ndash2063van der Sluijs KF van der Poll T Lutter R Juffermans NP and Schultz MJ (2010)Bench-to-bedside review bacterial pneumonia with influenzamdashpathogenesis andclinical implications Crit Care 14219
Yamaryo T Oishi K Yoshimine H Tsuchihashi Y Matsushima K and Nagatake T(2003) Fourteen-member macrolides promote the phosphatidylserine receptor-dependent phagocytosis of apoptotic neutrophils by alveolar macrophages Anti-microb Agents Chemother 4748ndash53
Yamaya M Azuma A Takizawa H Kadota J Tamaoki J and Kudoh S (2012)Macrolide effects on the prevention of COPD exacerbations Eur Respir J 40485ndash494
Yamaya M Shinya K Hatachi Y Kubo H Asada M Yasuda H Nishimura Hand Nagatomi R (2010) Clarithromycin inhibits type a seasonal influenza virusinfection in human airway epithelial cells J Pharmacol Exp Ther 33381ndash90
Zarogoulidis P Papanas N Kioumis I Chatzaki E Maltezos E and Zarogoulidis K(2012) Macrolides from in vitro anti-inflammatory and immunomodulatory prop-erties to clinical practice in respiratory diseases Eur J Clin Pharmacol 68479ndash503
Address correspondence to Dr Yuji Ishimatsu Department of Cardiopul-monary Rehabilitation Sciences Nagasaki University Graduate School ofBiomedical Sciences 1-7-1 Sakamoto Nagasaki 852-8520 Japan E-mail yuji-inagasaki-uacjp
Effect of CAM on SBP during Influenza in Emphysema Mice 463
at ASPE
T Journals on June 1 2020
jpetaspetjournalsorgD
ownloaded from