VIRAL IMMUNITY

THE JOURNAL OF EXPERIMENTAL MEDICINE SELECTED ARTICLES JANUARY 2015 www.jem.org

WelcomeViral Immunity

The Journal of Experimental Medicine now prints topic-specific mini collections to showcase a handful of our recent publications. In this installment, we highlight papers focusing on host-virus interactions and their implications for disease outcome.

Our collection begins with Insights and articles relevant to the pathogenesis of influenza viral infections in mice. Flu infection can cause respiratory as well as gastrointestinal symptoms, even though the virus only exhibits tropism for respiratory tissues. An Insight from Carolina Amezcua, Nicola Gagliani, and Richard Flavell highlights findings from Wang et al., who provide an explanation for gut inflammation in the absence of detectable virus in the gastrointestinal tract. They find that infection in mice recruits lung-derived IFNg secreting CCR9+CD4+ T cells into the small intestine that alter the composition of the gut microbiota. Th17 cells then expand in the small intestine and neutralization with IL-17A or antibiotic treatment reduces intestinal injury.

In another flu study, Heaton et al. demonstrate how club cells in the respiratory tract are infected by influenza, survive acute infection, and establish a proinflammatory environment that contributes to lung pathology. Depletion of club cells reduces lung tissue damage associated with the infection. An accompanying Insight by Thomas Braciale and Taeg Kim discusses this mechanism of flu pathogenesis and poses questions about viral and immune evasion strategies as well as therapeutic potential.

An article by Woodruff et al. relies on imaging techniques and examines aspects of flu infection relevant to vaccination. The authors describe how during immunization, resident lymph node dendritic cells can rapidly relocate to sites of viral influenza antigen, driving early activation of T cells, and contributing to germinal center formation and B cell memory to establish an appropriate immune response.

In a human study of hepatitis B and C, Kurktschiev et al. implicate the transcription factor T-bet in viral clearance. The authors find that acute resolving infections are characterized by high expression of T-bet in CD8+ T cells which is correlated with enhanced IFNg production, while absence of T-bet is more often seen in patients whose infections become chronic. IFN-g induction and T-bet expression are restored in dysfunctional T cells upon IL-2 and IL-12 supplementation.

Rapid and effective adaptive immune responses to viral pathogens rely on immunological memory and are curtailed by lymphocyte exhaustion. An article by Penaloza-Macmaster et al. investigates how regulatory T cells (T reg) can modulate CD8+ T cell exhaustion during chronic lymphocytic choriomeningitis virus infection in mice. Their findings show that depletion of T regs can expand functional virus-specific CD8+ T cells, rescuing exhausted CD8+ T cell subpopulations. T reg depletion also upregulates the programmed-death ligand 1 receptor (PD-L1) on CD8+ T cells, but it is a combination of PD-L1 blockade and T reg depletion that is able to reduce viral load. These findings suggest that T regs have the ability to contribute to the maintenance of exhausted CD8+ T cells during chronic infection.

Collectively, the presented articles identify pathways and processes related to viral pathogenesis and immunity that may contribute to therapeutic approaches to combat viral infection and disease. We hope you enjoy this complimentary copy of our Viral Immunity collection. We invite you to explore additional collections at www.jem.org and to follow JEM on Facebook, Google+, and Twitter.

FLUshing in the bathroomCarolina Amezcua, Nicola Gagliani, and Richard Flavell

Respiratory influenza virus infection induces intestinal immune injury via microbiota-mediated Th17 celldependent inflammationJian Wang, Fengqi Li, Haiming Wei, Zhe-Xiong Lian, Rui Sun, and Zhigang Tian

Influenza pathogenesis: Club cells take the cureThomas Braciale and Taeg Kim

Selected Articles January 2015

Long-term survival of influenza virus infected club cells drives immunopathologyNicholas S. Heaton, Ryan A. Langlois, David Sachs, Jean K. Lim, Peter Palese, and Benjamin R. tenOever

Trans-nodal migration of resident dendritic cells into medullary interfollicular regions initiates immunity to influenza vaccineMatthew C. Woodruff, Balthasar A. Heesters, Caroline N. Herndon, Joanna R. Groom, Paul G. Thomas, Andrew D. Luster, Shannon J. Turley, and Michael C. Carroll

Dysfunctional CD8+ T cells in hepatitis B and C are characterized by a lack of antigen-specific T-bet inductionPeter D. Kurktschiev, Bijan Raziorrouh, Winfried Schraut, Markus Backmund, Martin Wchtler, Clemens-Martin Wendtner, Bertram Bengsch, Robert Thimme, Gerald Denk, Reinhart Zachoval, Andrea Dick, Michael Spannagl, Jrgen Haas, Helmut M. Diepolder, Maria-Christina Jung, and Norbert H. Gruener

Interplay between regulatory T cells and PD-1 in modulating T cell exhaustion and viral control during chronic LCMV infectionPablo Penaloza-MacMaster, Alice O. Kamphorst, Andreas Wieland, Koichi Araki, Smita S. Iyer, Erin E. West, Leigh OMara, Shu Yang, Bogumila T. Konieczny, Arlene H. Sharpe, Gordon J. Freeman, Alexander Y. Rudensky, and Rafi Ahmed

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INSIGHTS

4 INSIGHTS | The Journal of Experimental Medicine

Seasonal influenza represents a contagious family of respiratory viruses that infect 530% of the global population yearly and account for as many as 500,000 deaths annually. Flu infection is characterized by symptoms such as fever, cough, sore throat, runny nose, body aches, and fatigue. Interestingly, some people also develop diarrhea, even though the virus tropism is for respiratory tissue. Despite being a well-studied viral infection, the underlying mechanisms involved in the development of gastro-enteritis-like syndrome with flu infection are poorly understood.

Now, Wang et al. have used a mouse model to show that influenza infection not only causes lung inflammation but also causes intestinal inflammation, even though influenza virus was not detectable in the gastrointestinal tract. They showed that during intranasal flu infection, CCR9+CD4+ T cells migrate from the lung into the intestinal mucosa in a CCL25/CCR9-dependent manner and alter the composition of the gut microbiota by secreting IFN-g. Homeostasis of the intestinal microbiota was altered, and increased numbers of Escherichia coli were detected after flu infection. Antibiotic treatment of intranasal flu-infected mice protected them against infection-induced diarrhea. These data suggest that the dysbiosis generated after flu infection leads to intestinal injury. The changes generated in the gut microbiota induced the production of IL-15 by intestinal epithelial cells. IL-15 induced the expansion of Th17 cells in the small intestine, which then mediated intestinal immune injury.

It is noteworthy that not all flu-infected patients develop gastroenteritis-like symptoms. Why is this? One possibility could be that the migration of pathogenic cells into the intestine depends on the severity of the infection. Consequently only highly infected patients may get diarrhea. Alternatively, the specific microbial landscape in some individuals and the existence of regulatory bacteria, could obstruct the growth of E. coli or promote regulatory T cells which in turn can control intestinal inflammation.

FLUshing in the bathroom

Insight from (left to right) Carolina Amezcua, Nicola Gagliani, and Richard Flavell

The data presented in this paper corroborate the hypothesis that the intestine is a suitable place to defuse an immune response. The abundance of antiinflammatory cytokines, such as IL-10 and TGF-b, regulatory cells and continuous regenera-tion of the tissue predispose the intestine with an ability to control effector cells. Moreover, if effector T cells escape all possible regulatory mechanisms, diverting them to the lumenin essence flushing them awaycould still be less dangerous than allowing them to remain in situ and risking lung tissue damage.

Wang, J., et al. 2014. J. Exp. Med. http://dx.doi.org/10.1084/jem.20140625.

Working model of intestinal injury induced by respiratory influenza virus infection

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Carolina Amezcua, Nicola Gagliani, and richard flavell; Howard Hughes Medical Institute, yale School of Medicine: richard.flavell@yale.edu, caro.amezcua@yale.edu, and nicola.gagliani@yale.edu

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The Rockefeller University Press $30.00J. Exp. Med. 2014 Vol. 211 No. 12 23972410www.jem.org/cgi/doi/10.1084/jem.20140625

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Influenza is an infectious respiratory disease af-fecting many bird and mammal species (Laver and Webster, 1979; Reid et al., 1999). Clinically, the most common symptoms include cough, fever, headache, and weakness (Monto et al., 2000). These symptoms are often accompanied by gastroenteritis-like symptoms in many influ-enza patients, such as abdominal pain, nausea, vomiting, and diarrhea, especially in young chil-dren (Baden et al., 2009; Shinde et al., 2009; Dilantika et al., 2010). However, the immune mechanisms underlying these clinical manifes-tations in the intestine during a lung-tropic viral influenza infection remain unclear.

The intestinal tracts in humans and other animals are inhabited by hundreds of diverse species of commensal bacteria, which are essen-tial in shaping intestinal immune responses dur-ing both health and disease (Hooper and Gordon,

2001; Chervonsky, 2009). Distinct components of commensal bacteria were associated with spe-cial status of the immune system. Although most commensal bacteria are beneficial (Ichinohe et al., 2011), a few can be potentially harmful in some conditions; for example, some commensal bac-teria have been suggested to influence suscepti-bility to inflammatory bowel disease (IBD; Garrett et al., 2007; Mazmanian et al., 2008). Thus, when conditions in the host are unfavorable, such as during infection, the resulting changes within the intestinal tract environment may promote growth of the harmful bacteria that induce in-testinal disease.

It is well known that the respiratory and intes-tinal tracts are both mucosal tissues. Over 30 years ago, John Bienenstock hypothesized that the

CORRESPONDENCE Zhigang Tian: tzg@ustc.edu.cn

Abbreviations used: BALF, bronchoalveolar lavage fluid; IBD, inflammatory bowel dis-ease; IEC, intestinal epithelial cell; i.g., intragastrical(ly); i.n., intranasal(ly); SFB, segmented filamentous bacteria.

*J. Wang and F. Li contributed equally to this paper.

Respiratory influenza virus infection induces intestinal immune injury via microbiota-mediated Th17 celldependent inflammation

Jian Wang,1* Fengqi Li,1* Haiming Wei,1,2 Zhe-Xiong Lian,1,2 Rui Sun,1,2 and Zhigang Tian1,2,3

1Institute of Immunology and CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, Anhui 230027, China

2Hefei National Laboratory for Physical Sciences at Microscale, Hefei, Anhui 230027, China3Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, China

Influenza in humans is often accompanied by gastroenteritis-like symptoms such as diar-rhea, but the underlying mechanism is not yet understood. We explored the occurrence of gastroenteritis-like symptoms using a mouse model of respiratory influenza infection. We found that respiratory influenza infection caused intestinal injury when lung injury oc-curred, which was not due to direct intestinal viral infection. Influenza infection altered the intestinal microbiota composition, which was mediated by IFN- produced by lung-derived CCR9+CD4+ T cells recruited into the small intestine. Th17 cells markedly increased in the small intestine after PR8 infection, and neutralizing IL-17A reduced intestinal injury. Moreover, antibiotic depletion of intestinal microbiota reduced IL-17A production and attenuated influenza-caused intestinal injury. Further study showed that the alteration of intestinal microbiota significantly stimulated IL-15 production from intestinal epithelial cells, which subsequently promoted Th17 cell polarization in the small intestine in situ. Thus, our findings provide new insights into an undescribed mechanism by which respiratory influenza infection causes intestinal disease.

2014 Wang et al. This article is distributed under the terms of an Attribution NoncommercialShare AlikeNo Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (AttributionNoncommercial Share Alike 3.0 Unported license, as described at http://creativecommons.org/ licenses/by-nc-sa/3.0/).

2398 Microbiota cause intestine injury during influenza | Wang et al.

RESULTSIntranasal (i.n.), but not intragastric (i.g.), infection with influenza virus causes intestinal immune injuryTo test whether intestinal injury was also a feature in a mouse model of influenza, we infected mice i.n. with the A/PR/8/34 (PR8) influenza virus strain. Indeed, their body weight grad-ually decreased from days 2 to 9 as compared with saline-treated controls, which maintained their body weight over the same period (Fig. 1 A). Furthermore, both the lung and small intes-tine had severe injury after PR8 infection (Fig. 1 B). Colon length was shortened (Fig. 1 C) and mild diarrhea occurred (Fig. 1 D), further indicating intestinal injury (Zaki et al., 2010; Murray and Rubio-Tapia, 2012). In contrast, nonmucosal liver and kidney tissues appeared normal after PR8 infection (Fig. 1 E), which was also supported by ALT and BUN analysis (Fig. 1 F). Together, these data indicate that respiratory influenza infec-tion causes severe immune injury not only in the lung but also in the intestine.

To rule out the possibility that the influenza virus entered the gastrointestinal tract and directly caused immune injury at this site, we tested for the presence of virus within the small intestine after i.n. infection and found that the influenza virus could not be detected at this site (Fig. 2 A). To test this possi-bility in a more rigorous way, we i.g. infected mice with PR8 and found that live virus could be detected in the intestinal contents and intestinal tissues in a short time after infection, and virus was completely cleared from these sites 3 d after infection

immune cells and structures contained in mucosal tissues were universally connected within the whole body. This common mucosal immune system concept speculated that the mucosal immune system was itself an organ in which the mucosal immune cells distributed throughout the body could inter-play between or among different mucosal tissues or organs (McDermott and Bienenstock, 1979; McDermott et al., 1980). Although this term was coined three decades ago, appreciation of its importance is only just beginning. Much was learned from the numerous studies conducted on the mucosal immune system during this time, which mainly focused on understand-ing its individual components (Holmgren and Czerkinsky, 2005; Sato and Kiyono, 2012). Although a few studies have suggested that the mucosal immune system is a system-wide organ (Gallichan et al., 2001; Sobko et al., 2010), some questions still need to be clarified. For example, how do the different com-ponents affect each other, and how is cross talk achieved among the various mucosal sites (Gill et al., 2010)?

In this study, we found that lymphocytes derived from the respiratory mucosa specifically migrated into the intestinal mucosa during respiratory influenza infection by the CCL25CCR9 chemokine axis and destroyed the intestinal microbiota homeostasis in the small intestine, finally leading to intestinal immune injury. Our findings may provide new insights into not only the mechanisms underlying intestinal immune injury in-duced by influenza infection of the lung but also the interplay of immune cells between or among different mucosal sites.

Figure 1. Respiratory influenza virus infection causes lung and intestinal im-mune injury. C57BL/6 mice were i.n. infected with saline or 0.1 HA of PR8. (A) Body weight was monitored after PR8 infection. (B) The pathology of lung and small intestine was assayed after PR8 infection. (C) The length of colon was recorded after PR8 infection. (D) The severity of the diarrhea was scored after PR8 infection (0, normal stool or absent; 1, slightly wet and soft stool; 2, wet and un-formed stool with moderate perianal staining of the coat; and 3, watery stool with severe perianal staining of the coat). (E) The pathol-ogy of liver and kidney was assayed after PR8 infection. (F) Serum ALT and BUN levels were measured after PR8 infection (dashed lines represent damage threshold). All tissue sec-tions were stained with H&E. Bars, 100 m. Data represent three independent experi-ments with at least five mice/group in A, C, and D or three mice/group in B, E, and F. Data are expressed as mean SEM by a Students t test. ***, P < 0.001.

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into healthy WT mice increased the number of Enterobacteriaceae and caused intestinal immune injury in recipient mice even in the absence of viral infection as compared with the intesti-nal microbiota from saline-treated mice (Fig. 3, E and F). Thus, these data suggest that respiratory influenza infection induces intestinal immune injury by altering the composition of in-testinal microbiota.

Escherichia coli is an important component of Enterobacteria-ceae, and pathogenic E. coli infection often causes vomiting and diarrhea in humans (Ochoa and Contreras, 2011). The number of E. coli in the intestinal tract significantly increased after PR8 infection (Fig. 3 G). Treating mice with streptomycinan antibiotic to which E. coli is sensitiveprotected mice against PR8 infection-induced immune injury to the small intestine by inhibiting the increase of Enterobacteriaceae (Fig. 3, H and I). Furthermore, directly infecting mice i.g. with E. coli caused immune injury in the small intestine (Fig. 3 J). Thus, these data suggest that the increase of E. coli may be the primary cause for intestinal immune injury during influenza infection.

Th17 cells mediate influenza-induced intestinal immune injuryTo explore the mechanism by which intestinal bacteria caused intestinal immune injury during influenza infection, many dif-ferent types of proinflammatory cells involved in intestinal inflammation (Zhou et al., 2007a; Kleinschek et al., 2009; Leppkes et al., 2009) were examined. Depletion of NK1.1+ by specific antibodies or T cell deficiency could not re-duce the PR8 infection-induced intestinal immune injury in our study (Fig. 4, A and B). However, no intestinal injury was observed in IL-17A/ mice after PR8 infection (Fig. 4 C),

(Fig. 2, B and C). However, pathological injury was not found in any of the examined tissues (Fig. 2, D and E). These results collectively suggest that influenza infection does not directly cause immune injury in the small intestine. Thus, we unexpect-edly observed that influenza infection induced severe immune injury within the intestine only when the virus infected the re-spiratory tract and immune injury occurred in the lung.

Intestinal microbiota is required for influenza-induced intestinal immune injuryChanges in intestinal microbiota are often involved in the oc-currence of intestinal inflammation in many mouse models (Lupp et al., 2007; Maslowski et al., 2009). To determine whether intestinal microbiota was involved in influenza induced intestinal immune injury, we first assayed whether viral infection affected the relative composition of several major bacterial groups within the intestinal microbiota. Although the number of total bacteria remained the same after infec-tion as quantified by both real-time PCR and selective cul-ture (Fig. 3 A), the numbers of segmented filamentous bacteria (SFB) and Lactobacillus/Lactococcus decreased after PR8 infec-tion, whereas the number of Enterobacteriaceae increased; more-over, the numbers of mouse intestinal Bacteroides, Eubacterium rectale/Clostridium coccoides, and Bacteroides were unchanged (Fig. 3 B). We next administered combinatorial antibiotics to the mice via their drinking water to deplete intestinal micro-biota (Ichinohe et al., 2011) 4 wk before infecting them with PR8. In antibiotic-treated mice, the lungs still sustained severe immune-mediated injury after PR8 infection, but the small intestine and colon were protected (Fig. 3, C and D). In another way, transferring intestinal microbiota from PR8-infected mice

Figure 2. Influenza virus does not infect the small intestine directly. (A) C57BL/6 mice were i.n. infected with 0.1 HA of PR8. The levels of the influ-enza virusderived matrix protein gene in lung and small intestine were detected by PCR. (BE) C57BL/6 mice were i.g. infected with saline or 0.1 HA of PR8. Viral titer in intestinal contents was determined by 50% tissue culture infective dose (TCID50) assay after PR8 infection (B). The levels of the influenza virusderived matrix protein gene in small intestine were detected by PCR after PR8 infection (C). The pathol-ogy of lung and small intestine was assayed after PR8 infection, and tissue sections were stained with H&E. Bar, 100 m (D). The length of colon was re-corded after PR8 infection (E). Data represent three independent experiments with at least three mice/group in AE. Data are expressed as mean SEM by a Students t test. NS: not significant.

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Figure 3. Antibiotic treatment reduces influenza-induced intestinal immune injury. (A) Bacteria in the small intestine were assayed by real-time PCR and selective culture in blood plate 7 d after PR8 infection. (B) Several major bacterial groups in intestinal microbiota were assayed by real-time PCR 7 d after PR8 infection. (C and D) C57BL/6 mice were subjected to a 4-wk oral treatment of combinatorial antibiotics in drinking water, followed by i.n. infection with saline or 0.1 HA of PR8. The pathology of lung and small intestine was assayed 7 d after PR8 infection (C). The length of colon was recorded 7 d after PR8 infection (D). (E and F) Transfer of intestinal microbiota from saline-treated or PR8-infected mice into healthy WT mice by the i.g. route. Major bacterial groups in the intestinal microbiota (E) and the pathology of small intestine were assayed 6 d later (F). (G) The number of E. coli in stool was detected by E. coli/Coliform Count Plates 6 d after PR8 infection. (H and I) C57BL/6 mice were subjected to a 1-wk oral treatment of streptomycin in their drinking water and then were i.n. infected with 0.1 HA of PR8. The pathology of lung and small intestine (H) and major bacterial groups in intesti-nal microbiota (I) were assayed 6 d after PR8 infection. (J) C57BL/6 mice were i.g. infected with saline or 5 108 E. coli, and the pathology of small intes-tine was assayed 3 d later. All tissue sections were stained with H&E. Bars, 100 m. Data represent two independent experiments with three mice/group in I and J or three independent experiments with at least three mice/group in AH. Data are expressed as mean SEM by a Students t test. *, P < 0.05; **, P < 0.01; NS: not significant.

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suggesting that Th17 cells might be involved in influenza-induced intestinal immune injury.

To rule out the possibility that lung injury might also be reduced in IL-17A/ mice after influenza infection, which subsequently resulted in reducing the small intestinal injury indirectly, we compared the degree of the lung injury after influenza infection between WT and IL-17A/ mice. The re-sults showed that both IL-17F and IL-17A expressions in lung from WT mice were increased after PR8 infection (Fig. 4 D). Compared with WT mice, IL-17A/ mice exhibited re-duced body weight loss during PR8 infection (Fig. 4 E). How-ever, the degree of lung leak and the levels of total protein and lactate dehydrogenase in bronchoalveolar lavage fluid (BALF) were not significantly different between WT and IL-17A/ mice (Fig. 4, FH), suggesting that the lung injury did not re-duce in IL-17A/ mice after influenza infection when com-pared with WT mice. Thus, these data suggest that the decrease of immune injury in the small intestine from IL-17A/ mice after influenza infection is independent of the decrease of lung injury.

To further determine that Th17 cells were responsible for influenza-induced intestinal immune injury, we detected the expression of Th17-specific transcription factor RORt and IL-17A and found that their expressions increased in the small intestine after PR8 infection (Fig. 5 A). The percentage and number of Th17 cells increased in the small intestine and colon after PR8 infection (Fig. 5, B and C), but not in the liver or kid-ney (Fig. 5 D), consistent with previous observations (Esplugues

et al., 2011). Furthermore, treating mice i.p. with a neutralizing antiIL-17A antibody during PR8 infection effectively reduced intestinal injury (Fig. 5 E). Together, these data suggest that influenza infectioninduced intestinal immune injury is depen-dent on Th17 cells.

Because we observed that influenzainduced intestinal im-mune injury is dependent on both intestinal microbiota and Th17 cells, we wondered whether there was an association between intestinal bacteria and Th17 cells. The results showed that the percentage and number of Th17 cells in the small in-testine were unchanged in antibiotic-treated mice after PR8 infection as compared with uninfected control mice (Fig. 5 F); transferring intestinal microbiota from PR8-infected mice into healthy WT mice promoted IL-17A expression in the small intestine of recipient mice (Fig. 5 G); and streptomycin treat-ment inhibited IL-17A expression in the small intestine dur-ing PR8 infection (Fig. 5 H). Collectively, these data suggest that changes in intestinal microbiota induced by influenza in-fection promote Th17 cell production, which subsequently causes intestinal immune injury.

CCL25/CCR9 mediates the recruitment of lung-derived CD4+ T cells into the small intestineBecause respiratory influenza infection influences the compo-sition of intestinal microbiota, which subsequently promotes Th17 cell production and causes intestinal immune injury, we wanted to know how respiratory influenza infection destroyed the microecological homeostasis of the intestinal microbiota.

Figure 4. IL-17A deficiency reduces influenza-induced immune injury in small intestine but not in lung. (A) The pathology of lung and small intestine from control and PK136-treated mice was assayed 6 d after PR8 infection. (B) The pathology of lung and small intestine from WT and Tcrd/ mice was assayed 6 d after PR8 infection. (C) The pathology of lung and small intestine from WT and IL-17A/ mice was assayed 6 d after PR8 infection. (D) IL-17A and IL-17F expres-sions in the lung from WT mice were de-tected by real-time PCR 6 d after PR8 infection. (E) Body weight of WT and IL-17A/ mice was monitored after PR8 infec-tion. (F) Evans blue dye concentration in BALF from WT and IL-17A/ mice was determined by spectrophotometer 6 d after PR8 infec-tion. (G and H) Total protein (G) and lactate dehydrogenase (H) levels in BALF from WT and IL-17A/ mice were determined by ELISA 6 d after PR8 infection. All tissue sec-tions were stained with H&E. Bars, 100 m. Data represent two independent experiments with five mice/group in EH or three mice/group in AD. Data are expressed as mean SEM by a Students t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS: not significant.

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in the small intestine (Fig. 6 D). These results suggest that the CCL25CCR9 axis contributes to altering the composition of the intestinal microbiota after influenza infection and the subsequent development of intestinal inflammation via recruit-ing effector lymphocytes into the intestinal mucosa.

Next, we explored which lymphocyte subsets were recruited by CCL25 in our influenza model. Although the total number of T cells increased in the LPL after PR8 infection, the total number of B cells decreased (Fig. 6 E). Within the T cell popula-tion, the CCR9+CD4+ T cell subset increased (Fig. 6 F), whereas the CCR9+CD8+ T cell subset remained unchanged (Fig. 6 G), indicating that CCR9+CD4+ T cells might play a key role in al-tering the intestinal microbiota. Evaluating this subpopulation in other tissues revealed that the number of CCR9+CD4+ T cells was significantly increased in the lung and in the mediastinal LNs after PR8 infection, but not in the mesenteric LNs (Fig. 6 G),

Given that influenza infection specifically caused immune in-jury in the respiratory and intestinal mucosal tissues, but not in the nonmucosal liver or kidney in our study, an interconnected relationship existed between them was intriguing according to the common mucosal immune system theory (McDermott and Bienenstock, 1979; McDermott et al., 1980). The CCL25 chemokine is expressed by intestinal epithelial cells (IECs) and functions to specifically guide CCR9-expressing effector lym-phocytes into the small intestine as a homing mechanism (Campbell and Butcher, 2002). Consistent with previous ob-servations, CCL25 expression in the small intestine tissue was much higher than any other tissues, including liver, kidney, and lung (Fig. 6 A). Treating mice i.v. with a neutralizing anti-CCL25 antibody during PR8 infection reduced intestinal immune injury (Fig. 6 B), inhibited the changes in intestinal microbiota (Fig. 6 C), and reduced the number of Th17 cells

Figure 5. Increased Th17 cells occur in the small intestine during influenza virus infec-tion. (A) RORt and IL-17A expressions in the small intestine were detected by real-time PCR 7 d after PR8 infection. (B) The percentage and number of Th17 cells in intestinal IEL and LPL were detected 7 d after PR8 infection. (C) The percentage and number of Th17 cells in colonic LPL were detected 7 d after PR8 infection. (D) The number of Th17 cells in liver and kidney was de-tected 7 d after PR8 infection. (E) C57BL/6 mice were i.p. treated with a neutralizing antiIL-17A antibody during PR8 infection. The pathology of lung and small intestine was assayed 6 d after PR8 infection, and tissue sections were stained with H&E. Bars, 100 m. (F) The percentage and number of Th17 cells in IEL and LPL were detected 7 d after PR8 infection in antibiotic-treated mice. (G) Transfer of intestinal microbiota from saline-treated or PR8-infected mice into healthy WT mice by the i.g. route. IL-17A expression in the small intestine was detected by real-time PCR 6 d later. (H) IL-17A expression in the small intestine was detected by real-time PCR at day 6 after PR8 infection in streptomycin-treated mice. Data represent two independent experiments with three mice/group in A, D, E, G, and H or three independent experiments with three mice/group in B, C, and F. Data are expressed as mean SEM by a Students t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS: not significant.

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Figure 6. Anti-CCL25 antibody treatment reduces influenzainduced intestinal immune injury. (A) CCL25 expression in various tissues was de-tected by real-time PCR 4 d after PR8 infection. (BD) C57BL/6 mice were i.v. treated with a neutralizing anti-CCL25 antibody during PR8 infection. The pathology of lung and small intestine (B), major bacterial groups in intestinal microbiota (C), and the number of Th17 cells in IEL and LPL were assayed 7 d after PR8 infection (D). (EG) C57BL/6 mice were i.n. infected with saline or 0.1 HA of PR8. The number of T and B cells in LPL (E), the percentage and number of CCR9+CD4+ T cells in small intestine (F), and the number of CCR9+CD8+ T cells in LPL and CCR9+CD4+ T cells in lung, mediastinal LNs, and mes-enteric LNs were assayed 7 d after PR8 infection (G). (H) ALDH1A2 expression in lung was detected by real-time PCR 6 d after PR8 infection. (I) CD4+ T cells from the lungs of saline- or PR8-infected CD45.1+ mice were adoptively transferred into WT CD45.2+ mice, and the percentage of CD45.1+CD4+ T cells in total CD4+ T cells in LPL from recipient CD45.2+ mice was detected by flow cytometry 48 h later. (J) C57BL/6 mice were i.n. infected with saline or 0.1 HA of PR8. CD4+ T cells in the lung and LPL were purified 6 d later by MACS and then co-cultured with antigen-presenting cells and heat-killed PR8 in an IFN- ELISPOT plate. The number of positive spots was counted 20 h later. (K) Parabiotic pairs of WT mice were established first, and the left partner was i.n. infected with PR8 2 wk later. The pathology of small intestine was assayed 6 d after PR8 infection. All tissue sections were stained with H&E. Bars, 100 m. Data represent three independent experiments with three mice/group in AH and K or three wells/treatment in J. Data are expressed as mean SEM by a Students t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS: not significant.

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Figure 7. Lung-derived CD4+ T cells influence microbiota and intestine injury by secreting IFN-. (A) IFN- expression in CD4+ T cells from lung was detected by flow cytometry 6 d after PR8 infection in WT mice. (B) The pathology of small intestine was assayed at day 7 after PR8 infection in WT and IFN-/ mice, and tissue sections were stained with H&E. Bars, 100 m. (C and D) IL-17A expression in the small intestine (C) and major bacterial groups in intestinal microbiota (D) were assayed at day 7 after PR8 infection in IFN-/ mice. (E and F) IL-17A expression in CD4+ T cells from lung (E) and the percentages of CCR9 Th17 cells and CCR9+ Th17 cells in lung and LPL (F) were detected 6 d after PR8 infection in WT mice. (G) IL-17A expression in CD4+ T cells from mesenteric LNs, Peyers patches, and blood was detected by flow cytometry 6 d after PR8 infection in WT mice. (H) IL-17A level in serum was detected by ELISA 6 d after PR8 infection in WT mice. (I) LPL from WT mice at day 6 after PR8 infection was stimulated by heat-killed E. coli in vitro; 24 h later, the expression of IL-17A in CD4+ T cells was detected by flow cytometry. (J) Lung lymphocytes and LPL from WT mice at day 6 after PR8

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suggesting that the lung and mediastinal LNs might be the main sources of CCR9+CD4+ T cells recruited to the small intestine during PR8 infection. Retinoic acid is reported to promote the expression of CCR9 on T cells (Ohoka et al., 2011), and the production of retinoic acid is regulated by the aldehyde dehy-drogenase (ALDH) 1A2 (Yokota et al., 2009). In our study, the expression of ALDH1A2 in lung increased after influenza infec-tion (Fig. 6 H), suggesting that the increase of retinoic acid in lung after influenza infection might be responsible for promot-ing the CCR9 expression on lung CD4+ T cells.

To determine whether influenza infectionactivated lung CD4+ T cells tended to migrate into the small intestine, we adoptively transferred lung CD4+ T cells from saline- or PR8-infected CD45.1+ mice into recipient WT CD45.2+ mice and found that LPL in recipient mice contained a higher frequency of CD45.1+CD4+ T cells from PR8-infected CD45.1+ mice (Fig. 6 I). Moreover, PR8-specific CD4+ T cells were detected not only in lung but also in the small intestine after PR8 in-fection, as assessed by the IFN- ELISPOT plate (Fig. 6 J), and, in a parabiotic mice model, PR8 infection in one partner caused small intestinal injury to occur in a noninfected partner (Fig. 6 K). Thus, these data suggest that the CCL25CCR9 axis mediates the recruitment of lung-derived effector CD4+ T cells into the small intestine as well as the alterations to the intestinal microbiota composition during influenza infection.

Lung-derived CD4+ T cells destroy microbiota homeostasis and promote resident Th17 cell polarizationAs we found that lung-derived effector CD4+ T cells are recruited into the small intestine and alter the intestinal mi-crobiota during influenza infection, we wondered how they influenced the intestinal microbiota composition and whether Th17 cells in the small intestine originated from the polariza-tion of them. For the first question, IFN- expression was found to be significantly increased in lung CD4+ T cells after PR8 infection (Fig. 7 A). When IFN- was deficient, the mice ex-hibited reduced intestinal immune injury, normal IL-17A ex-pression, and unchanged intestinal microbiota in the small intestine after PR8 infection (Fig. 7, BD). Thus, these data suggest that lung-derived effector CD4+ T cells destroy the homeostasis of intestinal microbiota by secreting IFN-. For the second question, Th17 cells were not found to be increased in lung after PR8 infection (Fig. 7 E) and, although some CCR9+ Th17 cells were present in the small intestine, most Th17 cells (90%) exhibited a CCR9 phenotype (Fig. 7 F). Mean-while, Th17 cells were also not found increased in the mesen-teric LNs, Peyers patches, and blood (Fig. 7 G), and IL-17A levels in blood did not increased after PR8 infection (Fig. 7 H). More convincing evidences showed that E. colispecific Th17 cells could be detected in the small intestine (Fig. 7 I), but PR8-specific Th17 cells could not be detected both in the lung and

small intestine (Fig. 7 J). Thus, these data suggest that Th17 cell polarization, but not recruitment, occurs in the small intestine in situ during influenza infection.

Intestinal microbiotainduced IL-15 production promotes intestinal Th17 cell polarizationBecause Th17 cell polarization occurs in the small intestine in situ during influenza infection, we next explore what kind of factors mediated this process. IL-6 expression in the small intes-tine was increased after PR8 infection, but IL-23 and TGF- expressions were unchanged (Fig. 8 A). However, treating mice i.v. with a neutralizing antiIL-6 antibody during PR8 infection could not reduce intestinal immune injury (Fig. 8 B). Thus, the increase of IL-6 is not the main reason for Th17 cell polarization in our study. IL-15 has been reported to contribute to intestinal inflammation in various mouse models (Zhou et al., 2007b; Schulthess et al., 2012) and, importantly, it has been shown to in-duce IL-17A expression in both mice and human CD4+ T lym-phocytes (Ziolkowska et al., 2000; Ferretti et al., 2003). In our study, IL-15 expression in the small intestine, but not in serum, was up-regulated after PR8 infection (Fig. 8 C). Transferring in-testinal microbiota from PR8-infected mice also increased IL-15 expression in the small intestine of recipient mice (Fig. 8 D). To explore whether IL-15 contributed to Th17 cell polarization in our study, we first assayed the expression of IL-15 receptor and found that intestinal CD4+ T cells expressed the IL-15 receptor after PR8 infection (Fig. 8 E). Next, treating mice with a neu-tralizing antiIL-15 antibody during PR8 infection effectively reduced intestinal immune injury (Fig. 8 F). Thus, IL-15, which was induced by intestinal bacteria, contributes to intestinal im-mune injury during influenza infection. Further experiments showed that IL-15 neutralization inhibited IL-17A and IL-6 ex-pression in the small intestine after PR8 infection (Fig. 8 G) and, consistent with the previous observations (Ziolkowska et al., 2000; Ferretti et al., 2003), exogenous IL-15 promoted IL-17A secretion in purified CD4+ T cells from LPL in vitro (Fig. 8 H), suggesting that intestinal bacteriainduced IL-15 might promote Th17 cell polarization in the small intestine in situ by a direct and/or indirect way. However, IL-15 neutralization did not in-fluence the changes of the intestinal microbiota (Fig. 8 I), sug-gesting that IL-15 functioned upstream of IL-17A production but downstream of the change in microbiota after PR8 infection. Exploring the in vivo cellular sources of IL-15, high IL-15 expression was detected in IECs after PR8 infection (Fig. 8 J), suggesting that IECs might be an important source of IL-15 in the small intestine during influenza infection.

DISCUSSIONMucosal tissues, including the gastrointestinal, respiratory, and urogenital tracts, etc., are the first line of host defense against ex-ternal invaders. Although much has been learned from studying

infection were stimulated by heat-killed PR8 in vitro; 24 h later, the expression of IL-17A in CD4+ T cells was detected by flow cytometry. Data represent three independent experiments with three mice/group in AH or three wells/treatment in I and J. Data are expressed as mean SEM by a Students t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS: not significant.

2406 Microbiota cause intestine injury during influenza | Wang et al.

particularly at the early stage of infection. Although some stud-ies suggest that influenza virus disseminates into extrapulmo-nary tissues or organs during infection (Korteweg and Gu, 2008), others contradict this finding (Mauad et al., 2010), and direct evidence for viral replication in extrapulmonary tissues or organs has not yet been shown (Kuiken and Taubenberger, 2008). It therefore remains a mystery how influenza infection can be associated with immune injury to extrapulmonary tis-sues or organs if these injuries are not induced by direct virus infection of these tissues or organs (Polakos et al., 2006; Mauad et al., 2010). In our mouse model of respiratory influenza in-fection, no influenza virus was detected in the small intestine, and i.g. administration of the influenza virus directly into the intestine did not lead to intestinal immune injury. Thus, the intestinal immune injury observed in our study was not directly caused by influenza infection of the intestine.

each of these components individually, the mucosal immune system has not yet been examined from a holistic point of view as a system-wide organ (Gill et al., 2010), as conceptualized by the common mucosal immune system hypothesis. Unexpect-edly, we observed that respiratory influenza infection in mice caused immune injury not only in the lung but also specifically in the intestine, as it had no influence on the pathology in non-mucosal organs such as the liver or kidney. Because this resem-bles the symptoms exhibited by humans after influenza infection, these influenza virusinfected mice provide a good model in which to study the mechanisms underlying how respiratory in-fluenza infection causes intestinal immune injury; furthermore, these observations provide further evidence to support the ex-istence of a common mucosal immune system.

Pathogens extensively disseminate beyond the limits of the primary infection site in almost all cases of infectious diseases,

Figure 8. Intestinal microbiota induces Th17 cell polarization in situ via trigger-ing IL-15 production. (A) IL-6, IL-23, and TGF- expressions in the small intestine were detected by real-time PCR 6 d after PR8 in-fection. (B) The pathology of lung and small intestine from control and antiIL-6treated mice was assayed 6 d after PR8 infection. (C) IL-15 expression in the small intestine and serum was detected 6 d after PR8 infection. (D) Transfer of intestinal microbiota from saline-treated or PR8-infected mice into healthy WT mice by the i.g. route. IL-15 ex-pression in the small intestine was detected 6 d later. (E) IL-15R expression on CD4+ T cells in LPL from WT mice was detected at day 6 after PR8 infection. (F and G) C57BL/6 mice were i.p. treated with a neutralizing antiIL-15 antibody during PR8 infection. The pathology of lung and small intestine (F) as well as IL-17A and IL-6 expressions in the small in-testine were assayed 6 d after PR8 infection (G). (H) MACS-purified CD4+ T cells from LPL were stimulated by IL-15 in vitro, and IL-17A levels in supernatant were measured at days 2 and 3 by ELISA. (I) Major bacterial groups in the intestinal microbiota from control and antiIL-15treated mice were assayed by real-time PCR 6 d after PR8 infection. (J) IL-15 expression in IECs was detected 6 d after PR8 infection in WT mice. All tissue sections were stained with H&E. Bars, 100 m. Data repre-sent three independent experiments with three mice/group in AG, I, and J or three wells/treatment in H. Data are expressed as mean SEM by a Students t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS: not significant.

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et al., 2010). These findings suggest that potential exists for an undetermined link between mucosal immune components and that each component is efficient at sharing information distally (Gill et al., 2010). In our study, we found that lung-derived virus-specific effector CCR9+CD4+ T cells were recruited into the small intestine and destroyed the homeostasis of intestinal mi-crobiota by secreting IFN- after influenza infection. Thus, we speculated that the effector CCR9+CD4+ T cells might enter into the small intestine by a special way as described above and remained in the active state to secrete IFN- even in the ab-sence of antigen stimulation.

The intestinal microbiota is extensively accepted in the field as a virtual metabolic organ in and of itself (OHara and Shanahan, 2006). Beyond this role in metabolism, the intestinal microbiota has a conspicuous effect on host immune functions, as indicated by comparing immune responses between germ-free and conventional animals. A previous study showed that commensal SFBs induce IL-6 and IL-23 production to stimu-late Th17 cell polarization (Ivanov et al., 2009). However, in our mouse model of respiratory influenza infection, the num-ber of SFB decreased while the number of E. coli increased in the intestinal tract after influenza infection; E. coli promoted IL-15 expression in IECs, and this IL-15 then promoted Th17 cell polarization. Moreover, overgrowth of existing strain and/or acquisition of new pathogenic strain are involved in E. colicaused gastrointestinal symptoms (Nguyen et al., 2006; Ochoa and Contreras, 2011). In our study, considering that the mice live in an SPF environment and that intestinal inflamma-tion occurs in different kinds of mice, we think that the over-growth of existing E. coli in the gut may be the primary cause for intestinal immune injury during influenza infection.

The function of IL-15 in regulation of Th17 responses has been studied extensively, but there are still some controversies. Some studies found that IL-15 induces IL-17A expression in both mice and human CD4+ T lymphocytes directly (Ziolkowska et al., 2000; Ferretti et al., 2003), which was also demonstrated in our study. However, another study showed that IL-15 inhib-its Th17 cell polarization in a mouse model of EAE (Pandiyan et al., 2012). We thought that there were two main reasons to explain why IL-15 played the opposite effect in different mouse model: (1) the immuno-microenvironment in different mouse model is different; (2) IL-15 is reported to activate both STAT3 and STAT5 (Johnston et al., 1995), which is inferred either to inhibit or to promote Th17 cells.

MATERIALS AND METHODSMice, virus, and bacteria. Male C57BL/6 mice were purchased from the Shanghai Laboratory Animal Center, Chinese Academy of Sciences. IFN-/, Tcrd/, and IL-17A/ mice were purchased from The Jackson Laboratory. All mice were housed under specific pathogen-free conditions at the School of Life Sciences, University of Science and Technology of China (USTC), and were used at 610 wk of age. Animal care and experimental procedures were followed in accordance with the experimental animal guide-lines at USTC. Mouse-adapted influenza A/PR/8/34 strain (H1N1) was a gift from H. Meng (Institute of Basic Medicine, Shandong Academy of Med-ical Sciences, Shandong, China). For influenza infection studies, mice were anesthetized and infected i.n. with 0.1 HA of PR8 in 50 l sterile saline.

An explosive increase in neutrophils is responsible for influenza-induced acute lung injury and death. IL-17 is a po-tent regulator for the neutrophils recruitment. Previous studies have shown that respiratory influenza infection increases the IL-17A and IL-17F expressions in lung, and IL-17RA/ mice exhibit reduced lung injury and higher survival rates after influenza infection (Crowe et al., 2009; Li et al., 2012). In our mouse model of respiratory influenza infection, IL-17A and IL-17F expressions in lung also increased after infection, but IL-17A deficiency could not reduce influenza-induced lung injury. Thus, based on above results, we speculated that IL-17A and IL-17F played the same function during influ-enza infection, and IL-17F alone might be enough to function to activate IL-17RA and recruit neutrophils when IL-17A was deficiency.

Recruitment and infiltration of inflammatory cells into the gastrointestinal mucosa critically regulates the development as well as progression of IBD (Wurbel et al., 2011). Differential expression of chemokine receptors and adhesion molecules on lymphocytes not only determine their migration into dif-ferent tissues but also their localization within these tissues. CCL25 is constitutively expressed by the epithelium of the small intestine (Papadakis et al., 2000), and the CCL25CCR9 che-mokine axis is considered to be one of the few non-promiscuous chemokine/receptor pairs involved in gut-specific migration of lymphocytes (Stenstad et al., 2006). In our study, the per-centage and number of CCR9+CD4+ T cells in both lung and small intestine increased after influenza infection, and neutral-izing CCL25 with antibody treatment reduced CCR9+CD4+ T cell recruitment and intestinal immune injury. Thus, these data might explain why influenza infection specifically caused immune injury in the intestine, but not the liver or kidney, in our study.

IBD is a common disease characterized by severe inflam-mation of the intestine (Hooper and Macpherson, 2010). How-ever, the exact causes of this disease remain unclear. Some studies suggest that IBD arises from dysregulated control of host microorganism interactions. For example, patients with this disease have an increased number of epithelial cell surface associated bacteria (Swidsinski et al., 2005), suggesting the fail-ure of a mechanism designed to limit direct contact between the epithelium and the microbiota. Similarly, in our study, we also found that CCR9+CD4+ T cell recruitment correlated to intestinal inflammation by the following mechanism: the CCR9+CD4+ T cells destroyed the homeostasis of the intestinal microbiota, the altered microbiota then promoted Th17 cell po-larization in the small intestine in situ, and the resulting IL-17A secretion finally mediated the intestinal immune injury.

This concept of the common mucosal immune system pro-posed by John Bienenstock suggests that the mucosal immune system may be considered as one large interconnected network (McDermott and Bienenstock, 1979; McDermott et al., 1980), which is supported by some recent researches. For example, i.n. immunization results in vaginal protection against genital HSV-2 infection (Neutra and Kozlowski, 2006), and antibiot-ics used in neonates increases the risk to develop asthma (Sobko

2408 Microbiota cause intestine injury during influenza | Wang et al.

or isotype control Abs. Samples were collected by a flow cytometer (LSR II; BD) and analyzed by FlowJo and WinMDI 2.9 software.

Real-time PCR. Total RNA was extracted from tissues using TRIzol (Invi-trogen), and cDNA was then synthesized. Real-time PCR was performed according to the manufacturers instructions using a SYBR Premix Ex Taq (Takara Bio Inc.). For analysis, target gene expression was normalized to the housekeeping gene -actin. Gene expression values were then calculated using the mean from the control samples as a calibrator. Real-time PCR primers were synthesized by Sangon Biotech (Table S1).

Neutralizing antibodies and antibiotic treatment. For in vivo neutral-ization, the following neutralizing antibodies were administered: 100 g/mouse antiIL-17A (TC11-18H10.1), 100 g/mouse anti-CCL25 (89818), 100 g/mouse antiIL-6 (MP5-20F3), or 100 g/mouse antiIL-15 (AIO.3) were administered into mice at days 0, 2, and 4 after PR8 infection. For in vivo cell depletion, 200 g/mouse anti-NK1.1 was administered i.v. into mice 2 d before PR8 infection. For intestinal microbiota depletion, mice were treated with a mixture of antibiotics (1 mg/ml ampicillin, 0.5 mg/ml vancomycin, 1 mg/ml neomycin sulfate, and 1 mg/ml metronidazole [San-gon Biotech]) added to their drinking water beginning 4 wk before PR8 in-fection and continuing until sacrifice, as previously described (Ichinohe et al., 2011). For intestinal E. coli depletion, mice were treated with 1 mg/ml strep-tomycin (Sangon Biotech) added to their drinking water beginning 1 wk before PR8 infection and continuing until sacrifice. Antibiotic-containing water was changed twice a week.

Microbiota transplantation. Cecal contents from saline- or PR8-infected mice were suspended in 1 ml saline and were administered (0.5 ml per mouse) immediately to WT mice by the i.g. route. Transplanted mice were maintained in sterile cages and detected intestinal immune injury 7 d later.

Transfer of T cells and PR8-specific CD4+ T cells assay. For T cells transfer, 5 105 CD4+ T cells from the lungs of saline- or PR8-infected CD45.1+ mice were adoptively transferred i.v. into WT CD45.2+ mice, and the percentage of CD45.1+CD4+ T cells in total CD4+ T cells in LPL from recipient CD45.2+ mice was detected 48 h later by flow cytometry. For PR8-specfic CD4+ T cells assay, CD4+ T cells in the lung and LPL from saline-treated and PR8-infected mice were purified 6 d later by MACS and then co-cultured with antigen-presenting cells and heat-killed PR8 in an IFN- ELISPOT plate. The number of positive spots was counted 20 h later according to the manufacturers instructions.

Statistical analysis. A two-tailed Students t test was used for statistical analyses. Data were expressed as the mean SEM, and the data were con-sidered statistically significant when differences achieved values of P < 0.05.

Online supplemental material. Table S1 shows primers used for real-time PCR. Online supplemental material is available at http://www.jem .org/cgi/content/full/jem.20140625/DC1.

We thank H. Meng (Shandong Academy of Medical Sciences) for the influenza A/PR/8/34 strain.

This work was supported by Ministry of Science & Technology of China (#2010CB911901 and #2013CB530506), Natural Science Foundation of China (#31300753 and #31400783), Fundamental Research Funds for the Central Universities (#WK2070000039), and China Postdoctoral Science Foundation (#2013M531532 and #2014T70599).

Author contributions: J. Wang and F. Li performed experiments. J. Wang, F. Li, H. Wei, Z.-X. Lian, R. Sun, and Z. Tian designed the research. J. Wang, F. Li, and Z. Tian wrote the manuscript. Z. Tian supervised the project. J. Wang and F. Li contributed equally to this paper.

Submitted: 3 April 2014Accepted: 14 October 2014

E. coli strain was isolated from stool of PR8-infected mice by the 3M Petri-film E. coli/Coliform Count Plate and was cultured in broth medium for amplification. For E. coli infection, mice were anesthetized and infected i.g. with 5 108 E.coli in 500 l sterile saline.

Histopathology. Lung, small intestine, liver, and kidney tissues were re-moved and fixed immediately in 10% neutral-buffered formalin in PBS for >24 h, embedded in paraffin, and cut into 57-m sections. The sections were deparaffinized and stained with hematoxylin and eosin (H&E) to deter-mine histological changes.

Analysis of lung injury. Lung leakage: 1 h before sacrificing mice, 20 mg/kg Evans blue dye was administered i.v. The lung was instilled with 1 ml of saline, and the BALF was collected. After centrifugation, Evans blue dye con-centration in supernatant was determined by spectrophotometer at 620 nm. Total protein and lactic dehydrogenase in BALF: The lung was instilled with 1 ml saline, and the BALF was collected. After centrifugation, the level of total protein in supernatant was assayed by the BCA protein assay kit, and the level of lactic dehydrogenase in supernatant was assayed by ELISA kit (Cloud-Clone Corp).

Analysis of liver and kidney function. Serum from infected mice or control mice were collected and stored at 80C until analysis. Liver func-tion was determined by measuring serum ALT (alanine aminotransferase) using a commercially available kit (Rong Sheng). Kidney function was assessed by measuring serum BUN (blood urea nitrogen) using a commercially available kit (Jiancheng Bioengineering Institute).

Determination of virus and bacteria. Influenza virus in the lung and small intestine were detected by PCR. The primer sequences to detect the gene encoding the matrix protein within the influenza virus were as follows: 5-GGACTGCAGCGTAGACGCTT-3 (forward) and 5-CATCCTGTT-GTATATGAGGCCCAT-3 (reverse). Intestinal bacterial genomic DNA was extracted from the stool using a stool kit (QIAGEN) according to the manufacturers instruction (the optional high-temperature step was per-formed). The abundance of total and specific intestinal bacterial groups was measured by real-time PCR with corresponding 16S rDNA gene primers (Sangon Biotech; Table S1). The number of E. coli in stool was detected by the 3M Petrifilm E. coli/Coliform Count Plate according to the manufac-turers instructions.

Isolation of IEC, IEL, and LPL. IECs were isolated as described in a pre-vious study (Zhou et al., 2007a). IELs and LPLs were isolated as previously described with minor modifications (Das et al., 2003; Kamanaka et al., 2006; Esplugues et al., 2011). In brief, small intestines were harvested and washed with PBS, and mesentery and Peyers patches were carefully dissected out. In-testines were opened longitudinally and then cut into 1-cm pieces. Intestinal pieces were incubated in 10 ml of extraction buffer (5% FCS, 1 mM DTT, and 5 mM EDTA in PBS) at 37C for 30 min. The released cells were loaded onto a Percoll gradient and centrifuged. The cells at the interface of a 40/70% Percoll solution were collected and used as IELs. The remaining segments were incubated twice in extraction buffer to remove IELs and isolate LPLs. The tissue was digested with prewarmed complete RPMI1640 containing 2 mg/ml collagenase IV at 37C for 60 min, loaded onto a Percoll gradient, and centrifuged. The cells at the interface of a 40/70% Percoll solution were collected and used as LPLs.

Flow cytometry. After blocking the Fc receptor with anti-CD16/CD32, single-cell suspensions were incubated with the fluorescently labeled mAbs at 4C for 30 min in PBS and then washed twice. For intracellular cytokine staining, cells were first stimulated for 4 h at 37C with 50 ng/ml PMA, 1 g/ml ionomycin, and 10 g/ml monensin (all from Sigma-Aldrich); cells were then stained for extracellular markers, fixed, permeabilized, and stained with the fluorescently labeled mAbs against the indicated intracellular cytokines

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Thomas Braciale and Taeg Kim, University of Virginia: tjb2r@virginia.edu

The illness that follows influenza A virus (IAV) infection results from both direct effects of IAV replication in the respiratory tract (RT) and from sequelae of the host immune re-sponse. The virus directly induces apoptosis or necrosis of infected RT cells and NK cells, CD8 T cells lyse infected cells, and damaging inflammatory mediators are produced by vari-ous infiltrating immune cells. Accordingly, the degree of RT pathology is believed to reflect both the extent of virus replication and the magnitude of the host immune response. In this issue, however, Heaton et al. provide evidence for a novel mechanism of IAV pathogenesis. They find that a small fraction of a particular RT cell type, the club cell, can cure itself of IAV but continue to produce inflammatory mediators, which may contribute to sustained RT inflammation and injury following IAV clearance.

Heaton et al. adapted a strategy to indelibly mark IAV-infected cells where any cell infected by the virus will express RFP. Contrary to expectation, they detected RFP+ cells in the RT up to day 21 post-infection (p.i.), long after virus-infected cells are cleared. The authors identify these residual RFP+ cells as club cellsbronchiolar (small airway) exo-crine cells (formerly known as Clara cells), which secrete products that protect the small airways.

Remarkably, by day 10 p.i. the residual RFP+ club cells were cured, that is, they no longer expressed detectable viral RNA. But they retained the type I interferon stimu-lated gene expression signature of infected cells and pro-duction of inflammatory chemokines, suggesting that these cells may contribute to RT pathology. Supporting this idea, selective depletion of the residual RFP+ cells diminished epithelial cell damage in the RT. However, severe RT injury with lethal outcome is associated with infection of alveolar epithelial cells, which are not cured in this model, so the contribution of club cells in IAV pathogenesis remains to be determined.

These provocative findings raise many questions, notably, how do infected club cells escape destruction by IAV and recognition by NK cells or CD8 CTL? Also, what sustains the inflammatory signature of club cells after viral RNA elimination? Nevertheless, these results provide a potential explanation for persistent RT inflammation following virus clearance and may presage the development of new strategies to treat the sequelae of IAV infection.

Heaton, N.S., et al. 2014. J. Exp. Med. http://dx.doi.org/10.1084/jem.20140488.

IAV replicates primarily in the respiratory tract epithelium, resulting in cell death via direct- or immune-mediated lysis (A). A small fraction of club cells can cure themselves of IAV but continue to produce interferon-stimulated gene (ISG) products (B).

Influenza pathogenesis: Club cells take the cure

Insight from Thomas Braciale (left) and Taeg Kim

INSIGHTS

The Rockefeller University Press $30.00J. Exp. Med. 2014 Vol. 211 No. 9 1707-1714www.jem.org/cgi/doi/10.1084/jem.20140488

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Brief Definit ive Report

Influenza A virus (IAV) is a seasonal pathogen with the capacity to cause devastating pandem-ics. IAV infects a variety of cells within the re-spiratory tract, including ciliated epithelial cells, type I and II alveolar cells, and immune cells (Matrosovich et al., 2004; Manicassamy et al., 2010; Shieh et al., 2010; Langlois et al., 2012; Smed-Srensen et al., 2012). Classically, IAV-infected cells are tracked through detection of virus-derived products or reporters (e.g., virus RNA or protein), all of which have short half-lives and are therefore incapable of defining infected cell types in the long-term. Ultimately, acute IAV infections are resolved within 2 wk post-infection (Carrat et al., 2008).

Infected cells are eliminated through two major mechanisms, apoptosis/necrosis driven by virus replication (Sanders et al., 2011; Yatim and Albert, 2011) or clearance mediated through the innate and adaptive arms of the immune system (Zinkernagel and Doherty, 1979; Eichelberger et al., 1991; Julkunen et al., 2001; Takeuchi and Akira, 2009). Clearance of IAV infections can come at the cost of aberrant immune-mediated disease (Damjanovic et al., 2012). Therefore, a balance between virus clearance and immune-mediated

tissue damage is important for recovery from IAV infections.

In this study, we define the long-term fate of virus-infected cells within the lung through an IAV expressing Cre recombinase and transgenic reporter mice (Nagy, 2000). This experimental model system allows for the indelible labeling of virus-infected cells, even at time points well after replication has ceased and virus has been cleared. Surprisingly, despite a potent viral lytic phase and generation of antiviral immune re-sponses, we demonstrate that a small population of cells that were infected by IAV persist after virus clearance. Furthermore, using a combina-tion of next-generation mRNA sequencing and flow cytometry, we determine that in-fected long-term surviving cells were comprised mainly of a single cell lineage, club cells (formerly termed Clara cells; Winkelmann and Noack, 2010), and that these cells have heightened in-terferon stimulated gene (ISG) levels. Specific depletion of surviving cells results in increased pulmonary pathology, suggesting a proinflam-matory role in recovery. This study provides evidence of cellular survival from acute virus infection and details new cellular mechanisms of immunopathology.

CORRESPONDENCE Peter Palese: peter.palese@mssm.edu OR Benjamin tenOever: benjamin.tenOever@mssm.edu

Abbreviations used: IAV, influenza A virus; ISG, interferon-stimulated gene.

N.S. Heaton and R.A. Langlois contributed equally to this paper.R.A. Langloiss present address is Department of Microbiol-ogy, University of Minnesota, Minneapolis, MN 55455.

Long-term survival of influenza virus infected club cells drives immunopathology

Nicholas S. Heaton,1 Ryan A. Langlois,1,2 David Sachs,3 Jean K. Lim,1 Peter Palese,1 and Benjamin R. tenOever1,2

1Department of Microbiology, 2Global Health and Emerging Pathogens Institute, and 3Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029

Respiratory infection of influenza A virus (IAV) is frequently characterized by extensive immunopathology and proinflammatory signaling that can persist after virus clearance. In this report, we identify cells that become infected, but survive, acute influenza virus infection. We demonstrate that these cells, known as club cells, elicit a robust transcrip-tional response to virus infection, show increased interferon stimulation, and induce high levels of proinflammatory cytokines after successful viral clearance. Specific depletion of these surviving cells leads to a reduction in lung tissue damage associated with IAV infection. We propose a model in which infected, surviving club cells establish a proinflamma-tory environment aimed at controlling virus levels, but at the same time contribute to lung pathology.

2014 Heaton et al. This article is distributed under the terms of an Attribution NoncommercialShare AlikeNo Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (AttributionNoncommercial Share Alike 3.0 Unported license, as described at http://creativecommons .org/licenses/by-nc-sa/3.0/).

1708 Surviving flu infected club cells drive pathology | Heaton et al.

To characterize the system, we performed ex vivo experi-ments on mouse lung fibroblasts isolated from the transgenic tdTomato reporter animals. Wild-type IAV or mock-infected fibroblasts failed to express tdTomato; however, upon infec-tion with IAV-Cre, we observe red fluorescence (Fig. 1 C). To demonstrate that viral replication is required to activate the reporter, we pretreated cells with IFN-/ and infected with IAV-Cre. Under these conditions, we observed no red signal, indicating that viral RNA replication and protein expression are required (Fig. 1 C). Finally, to determine if phagocytosis of infected cellular extract was sufficient for tdTomato expres-sion, we applied lysed cell debris from IAV-Cre infections in the presence of a neutralizing antibody but found no evidence for fluorescence (Fig. 1 C). Collectively, these data

RESULTS AND DISCUSSIONTo identify and characterize cells that are productively in-fected by IAV but go on to survive infection, we generated an H1N1 strain (A/Puerto Rico/8/1934) expressing the bacte-riophage protein Cre recombinase after a PTV-1 self-cleavage site with a glycine-serine linker (Kim et al., 2011) on the viral PB2 protein (Fig. 1 A). By infecting mice harboring the ap-propriate transgenic fluorescent reporter cassette, the expres-sion of Cre leads to the excision of a stop cassette (Madisen et al., 2010). After the stop element is removed, the cells will constitutively express the red fluorescent protein tdTomato (Fig. 1 B). Because the host cell harbors the tdTomato expres-sion cassette, the cells continue to express the reporter protein even if viral replication is stalled or eliminated.

Figure 1. Generation of influenza A virus expressing Cre recombinase. (A) Schematic showing insertion of Cre recombinase (Cre) downstream of a PTV-1 2A site at the 3 end of PB2 segment. (B) Model depicting Cre mediated excision of tdTomato reporter stop cassette. (C) Lung fibroblast generated from ROSA26 tdTomato lox-stop mice were mock infected or infected with WT IAV or IAV-Cre at an MOI of 5 (top three panels). Reporter fibroblasts were treated with 100 U of IFN-/ for 6 h and infected with IAV-Cre at MOI of 5 (fourth panel). MDCK cells were infected with IAV Cre at an MOI of 5 for 24 h. Cell debris were treated with anti-HA antibodies for 30 min and placed on reporter fibroblast (bottom). All images were taken 36 hpi. Bar, 50 m. Data are representative of two independent experiments. (D) WT C57BL/6 mice were infected with WT IAV (left) or IAV-Cre (right) at the indicated doses and moni-tored for morbidity and mortality. Calculated LD50 values are 50 PFU for WT IAV and 240PFU for IAV-Cre. n = 5 mice per group. The experiment was performed once.

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Br ief Definit ive Repor t

assessed the presence of reporter-positive cells using flow cytometry at various times after infection (Fig. 2 A). While no tdTomato+ cells were identified in uninfected mice or re-porter mice infected with WT PR8 virus (Fig. 2, A and B), we observed a population of reporter-positive cells during active viral replication (5 d post-infection) as expected. Surprisingly, we also observed a population of tdTomato+ cells at 10 and 21 d post-infection (Fig. 2 A), time points that exceed the physiological course of IAV replication ( Eichelberger et al., 1991; Carrat et al., 2008).

To formally demonstrate that the observed tdTomato+ cells were not a reflection of prolonged replication of IAV-Cre, we determined the amount of recoverable virus from lungs over the same time course as the FACS experiments. While high titers were recovered at 2 and 5 post-infection, no virus was detected at day 10 post-infection (Fig. 2 C). To identify where in the lung architecture these surviving cells exist, we infected reporter animals and collected lungs for histological analysis at 10 d post-infection. Upon examination of the lung sections, tdTomato+ cells were only found in the epithelial layer of larger airway spaces (bronchi), never in the alveoli (Fig. 2 D).

suggest that activation of the tdTomato cellular reporter re-quires active viral replication.

We next characterized the virulence of IAV-Cre in vivo to ensure that the pathogenesis of this recombinant virus was maintained. To this end, C57BL/6 mice were infected with either wild-type IAV or IAV-Cre at a range of infectious doses, and body weight and survival were monitored over time (Fig. 1 D). Excitingly, despite the insertion of Cre recombi-nase, intranasal inoculation of IAV-Cre was sufficient to in-duce morbidity in a manner comparable to the parental IAV stain (Fig. 1 D). Furthermore, mortality upon infection with IAV-Cre was only mildly attenuated when compared with the parental strain, with the median lethal dose (LD50) shifting from 50 to 240 PFU (Fig. 1 D). These data suggest that IAV-Cre retains the pathogenic properties associated with IAV disease and justifies its utilization as a tool to probe for cells that survive replicating virus in vivo.

Although IAV infection and replication generally result in the induction of cell death, here we chose to determine whether any cell types could successful