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TIMI-1130; No. of Pages 9pathologies including encephalitis and severe arthritis. En-cephalitic alphaviruses such as Venezuelan equine enceph-alitis virus (VEEV), Western equine encephalitis virus(WEEV) and Eastern equine encephalitis virus (EEEV)are neuroinvasive and frequently cause encephalitic man-ifestations [1]. By contrast, arthritogenic alphaviruses suchas Ross River virus (RRV), Barmah Forest virus (BFV),onyong-nyong virus (ONNV), Mayaro virus (MAYV), chi-kungunya virus (CHIKV), Sindbis virus (SINV), and Sem-liki Forest virus (SFV) cause acute febrile illnesses, malaise,maculopapular rashes, myalgia, and severe arthralgia[28]. The potential of these viruses to cause chronic jointsymptoms combined with their ability to cause large-scaleepidemics makes alphavirus-associated disease an illness of

trast to the widespread distribution of CHIKV, RRV isendemic to Australia, Papua New Guinea, and WesternPacific Islands and causes approximately 6000 casesannually [15].

Recent alphavirus outbreaks have provided increasingevidence of painful and persistent arthritic manifestationsfollowing infection. For example, patients with acuteCHIKV infection during the 20052006 La Reunion out-break presented with chronic polyarthritis in the ankles,wrists, knees, and small joints of the extremities [16]. Fif-teen months after CHIKV disease onset, only 43% ofpatients reported full remission, with more than half ofthe patients still experiencing arthritic rheumatic jointsymptoms [17]. Other prospective longitudinal studiesinvolving SINV-related alphaviruses in Northern Europe[18,19] and RRV in Australia [15,20] have reported similararthritic symptoms to CHIKV infection, with over 95% ofthese patients developing polyarthritis during the acutephase of infection. Joint symptoms in these patients canlast from months to years. Thus, arthritogenic alpha-viruses can cause arthralgia that is likely to evolve into

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2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tim.2014.09.005

Corresponding author: Mahalingam, S. (s.mahalingam@griffith.edu.au).Keywords: alphavirus; bone loss; arthritis; inflammation; Ross River virus;chikungunya virus.*These authors contributed equally to this manuscript.insights into arthritpathologyWeiqiang Chen1*, Suan-Sin Foo1*, NatalieNicole C. Walsh2, and Suresh Mahalingam1 Institute for Glycomics, Griffith University, Gold Coast, QLD 42St Vincents Institute of Medical Research and Department of Melbourne, VIC 3065, Australia

Arthritogenic alphaviral infection begins as a febrileillness and often progresses to joint pain and rheumaticsymptoms that are described as polyarthritis. Alphaviralarthritis and classical arthritides share many similarcellular and immune mediators involved in their patho-genesis. Recent in vitro and in vivo evidence suggeststhat bone loss resulting from increased expression ofbone resorption mediators may accompany alphaviralinfection. In addition, several longitudinal studies havereported more severe and delayed recovery of alphaviraldisease in patients with pre-existing arthritic conditions.This review aims to provide insights into alphavirus-induced bone loss and focuses on aspects of diseaseexacerbation in patients with underlying arthritis and onpossible therapeutic targets.

Epidemiological and clinical burden of arthritogenicalphavirus infectionArthropod-borne alphaviruses are globally distributed andcause considerable human morbidity and mortality. Alpha-viruses cause a range of human diseases, with manifesta-tions varying from fever or rash to significant inflammatoryiruses: news and bone

. Sims2, Lara J. Herrero1,

, Australiadicine, St. Vincents Hospital, The University of Melbourne,

major socioeconomic concern [9]. Typical acute clinical diag-nosis of arthritogenic alphaviruses involves virus detectionthrough reverse transcription-polymerase chain reaction(RT-PCR) [10]. Serological testing for alphavirus-specificantibodies may also be performed using ELISA. Specific-IgM and IgG against alphavirus may be detected 37 daysafter disease onset, while specific-IgG could be detected upto 4 months post-onset [11].

In this review we focus on arthritogenic CHIKV andRRV, which have caused sporadic outbreaks worldwide.CHIKV was first isolated during an outbreak of dengue-like disease in southern Tanzania in 19521953 [12]. Therecent reemergence of CHIKV in La Reunion in 2005 ledto 300 000 infections with an estimated 250 deaths[13]. CHIKV then spread to India, where it affected over1.3 million people [14] and continued to spread across theAsia-Pacific. Recently, CHIKV entered several Caribbeanislands in the Americas, and to date the Pan AmericanHealth Organization (PAHO) has reported an estimated715 000 suspected and confirmed cases as of September 12,2014 (http://www.paho.org/hq/index.php?option=com_content&view=article&id=9053&Itemid=39843). In con-Trends in Microbiology xx (2014) 19 1

persistent arthritis. Despite a high percentage of patientsdeveloping persistent arthralgia, there is a lack of routineclinical surveillance of bone pathology. Hence, the impacton bone during alphavirus-induced arthritis warrants fur-ther investigation.

Inflammation and bone lossThe maintenance of normal healthy bone structure is atightly regulated process requiring the coordinated actionsof bone-resorbing osteoclasts and bone-forming osteo-blasts. Signals elicited from cells within the local bonemicroenvironment, including signals arising from cellswithin the bone matrix [21] and the bone matrix itself[22], are crucial for regulating the activity of osteoblastsand osteoclasts. It is increasingly recognized that immunecells and their molecular mediators may contribute tonormal bone homeostasis and are key drivers of bone lossin chronic inflammatory conditions including the prototyp-ical inflammatory arthritis, rheumatoid arthritis (RA)[23]. In RA, periarticular (or juxta-articular) bone loss isevident in the inner network of trabecular bone in themetaphyseal region of the bone adjacent to the inflamedjoint; this often precedes focal bone erosion that occurs oncortical bone surfaces at the sites of inflammation(Figure 1). Systemic bone loss remote from the affectedjoints within both the appendicular and axial skeleton isalso commonplace in RA patients and is associated withincreased fracture risk (reviewed in [24,25]). A study in

diagnosis and progressive bone loss in patients with activedisease (defined by serum C-reactive protein (CRP) levels>20 mg/dL) over 12 months [26]. Inflammation in RA leadsto bone loss by both increasing osteoclast-mediated boneresorption [27] and impairing osteoblast-mediated boneformation ([28], reviewed in [24]).

The differentiation of osteoclasts from myeloid precur-sor cells requires the local presence of a pro-osteoclasto-genic cytokine termed RANKL [receptor activator ofnuclear factor (NF)-kB ligand] [29,30] and its interactionwith its receptor, RANK [receptor activator of (NF)-kB][31], which is expressed on the cell surface of osteoclastprecursors. The actions of RANKL can be inhibited byosteoprotegerin (OPG), a natural decoy receptor for RANKLthat prevents its interaction with RANK [32]. The relativeexpression of RANKL to OPG (RANKL:OPG ratio) withinthe local bone microenvironment controls osteoclast differ-entiation [33,34], with a high ratio leading to increasedosteoclastogenesis.

In bone homeostasis, RANKL is supplied by cells of theosteoblast lineage, including early to midstage osteoblasts[35,36] and osteocytes [37]. In inflammatory arthritis suchas RA, other cell types including synovial fibroblasts[38,39] and T cells [38,40] provide additional sources ofRANKL, leading to increased RANKL:OPG in the inflamedsynovial tissue [41] and at the pannusbone interface [42],thus contributing to increased osteoclastogenesis withininflamed joints [27,43]. A study in treatment nave RA

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TIMI-1130; No. of Pages 9treatment-nave RA patients showed reduced bone miner-al density in patients with disease duration >6 months at

Normaljoint

Alphaviral join

Bone

Inamedmuscle

Ligament

Synovialmembrane

Synovialuid

Arcularcarlage

Alphavirus

Figure 1. Comparative joint pathology in rheumatoid arthritis (RA) and alphavi

macrophages, synoviocytes, and bone-forming osteoblasts leading to the produc

mouse model of alphavirus infection, both periarticular and systemic bone loss are local inflammation, with proliferation of the synovial lining cells and infiltration of this tis

inflamed synovial tissue promote destruction of the articular cartilage and bone. Bone lo

bone erosion at the margins of cartilage and bone; systemic bone loss away from the

2patients also showed elevated systemic levels of serumRANKL:OPG at diagnosis [44].

ris Rheumatoid arthrisjoint

Inamedsynovial membrane

Focal boneerosion

Thinningarcular carlage

Periarcular bone loss

Auto-anbody

TRENDS in Microbiology

nduced arthritis. In alphavirus infection the virus infects skeletal muscle cells,

of proinflammatory cytokines and chemokines which promote bone loss. In the

rved. In RA, the joint lining (synovium) of diarthrodial joints is the primary target ofsue by inflammatory cells. Cytokines and chemokines produced by cells within this

ss is evident in RA joints as periarticular bone loss adjacent to the joint, and as focal

affected joint is also evident.

TIMI-1130; No. of Pages 9Emerging evidence for alphavirus-induced arthritisaffecting boneAlphaviral infection shares some features with inflamma-tory arthritic diseases such as RA. Whether these infec-tions affect bone in a similar manner to RA remains to beclearly determined. In the few studies that have includedanalysis of bone structure, the majority of CHIKV-infectedpatients do not show radiographic signs of focal boneerosion [45,46]. However, several case reports suggest thatfocal bone erosion does occur in a small number of CHIKV-infected patients. CHIKV-infected patients with no priorhistory of inflammatory arthritis showed magnetic reso-nance imaging (MRI) evidence of joint inflammation [47]and bone erosion [48] in the hands, and abnormal uptake ofradioactive tracers in the wrists and ankles. After the20052006 CHIKV outbreaks in La Reunion, Bouquillardand colleagues [49] reported 21 cases of CHIKV-infectedpatients diagnosed with RA according to the 1987 Ameri-can College of Rheumatology (ACR) criteria. Of these21 cases, five exhibited focal bone erosion at RA diagnosis(mean time of 10 months post-viral infection) and thisincreased to 17 patients 2 years post RA diagnosis [49]. Ra-diographic focal bone erosion was also reported by Mani-munda et al. [50] in three CHIKV-infected patients withoutpre-existing joint pain from a cohort of 20 patients report-ing persistent joint pain 10 months post-infection. Further-more, in this cohort MRI demonstrated that inflammationof the tendon, joint effusion, and bone marrow edema werecommon in the arthritis-affected joints [50]. Urinary hy-droxyproline in CHIKV-infected patients during activeviral infection has been reported to be higher than inhealthy controls, suggesting that bone resorption may beincreased in these patients [51]. In support of increasedosteoclast activity in alphaviral patients, a higher level ofcirculating serum TRAP5b activity was recently reported,together with greater synovial expression of RANKL:OPGratio in RRV-infected patients [52].

The capacity of alphaviral infection to have a directeffect on skeletal health was highlighted by a recentpreclinical study in a mouse model of RRV infection[52]. Although focal bone erosion was not observed withinthe joints of RRV-infected mice, there was clear evidencefor periarticular bone loss within the tibial epiphysisand systemic bone loss within the vertebrae of these mice[52]. This bone loss was associated with increasedRANKL:OPG ratio and increased osteoclastogenesis[52,53]. Combined with the case reports in human alpha-viral infections, these findings suggest that effects ofalphaviral infection on skeletal health should be consid-ered in these patients, including study not only of thearthritis-affected joint but also of systemic bone health.It is worth noting that persistent low-grade inflammation,diagnosed by serum high-sensitivity CRP levels, is anindependent risk predictor for non-traumatic fractures[54]. This suggests that persistent low-grade inflamma-tion in patients with persistent alphavirus-induced ar-thritis might also place these patients at risk of systemicbone loss and therefore at greater risk of fracture. Future

Reviewstudies investigating the effects of alphaviral infection onpatient bone mineral density over time are needed toconfirm this hypothesis.Recent evidence also suggests that alphaviruses canreplicate in joint tissues and stimulate an immune re-sponse in the bone microenvironment. Synovial biopsiesfrom RRV- [55] and CHIKV-infected [56] patients clearlyindicate the presence of viral antigens within cells of theinfected joints. Phuklia et al. demonstrated direct infectionof primary synoviocytes by CHIKV, and reported thatthese infected cells have the ability to drive monocyte/macrophage migration and osteoclast formation [57]. Re-cent studies using primary human osteoblasts have dem-onstrated that these cells also have high susceptibility toinfection with RRV and CHIKV, and that the infectedosteoblasts can induce the formation of bone-resorbingosteoclasts through RANKL expression [52,53], providinga mechanism by which alphaviruses could mediate boneloss.

Recent preclinical models have provided clear evidencethat alphavirus infections result in inflammatory arthrop-athy affecting the joint, muscle, tendon, and bone. Existingmouse models of CHIKV arthritis using ventral foot injec-tion of the virus recapitulated the rheumatic symptoms ofhuman CHIKV infection, including localized foot swelling,tenosynovitis, and myositis [58,59]. By contrast, subcuta-neous and intradermal injections of CHIKV in this mousemodel produced viremia without foot swelling [58]. Withthis in mind, it is possible that the virus can still bedisseminated into joints and induces bone activity in theabsence of foot swelling. Earlier work with SINV-relatedalphavirus in adult mice demonstrated that the virusreplicated within the epiphyses of long bones adjacent toarticular joints when inoculated through intravenous,intraperitoneal, or subcutaneous routes [60]. This is sup-ported by recent work with RRV, where high levels ofinfectious virus were recovered from the femur, tibia,patella, and foot of infected mice [52]. In addition, thereis clear evidence of systemic bone loss in the tibial epiphy-sis and vertebrae in subcutaneously RRV-infected mice,suggesting that alphaviral infection of osteoblasts in ani-mals may directly lead to inflammation within the jointthat may share features with other human inflammatoryarthritides.

Impact of the alphavirus-induced immune response onboneThe innate immune system acts as the frontline defenseagainst alphaviral invasion. Upon activation by alpha-virus, a sophisticated network of immune responses iselicited. Growing evidence has pinpointed the cell-mediat-ed immune response as the primary contributor to theimmunopathology and persistence of joint manifestationsduring alphaviral infection.

There is a strong body of evidence that infiltratingmacrophages determine the severity and persistence ofalphaviral infection. Persistent infection of perivascularsynovial macrophages by CHIKV and constitutive infiltra-tion of CD14+ monocytic cells into the synovial cavity werereported in a CHIKV-infected patient 18 months postillness onset [56]. Extensive mononuclear cell infiltration

Trends in Microbiology xxx xxxx, Vol. xxx, No. xand persistent CHIKV infection of macrophages are alsoevident in animal models [58,61], and depletion of macro-phages protects RRV-infected mice from disease.

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TIMI-1130; No. of Pages 9A vast array of cytokines, chemokines, and growth factorswere detected in the plasma of CHIKV-infected patients,including type I interferons (IFNs), C-X-C motif chemokineligand 10 (CXCL10/IP-10), interleukins (IL)-1b, IL-5, IL-6,IL-7, IL-10, IL-15, IL-17, chemokine (C-C motif) ligand(CCL)-3 (also known as macrophage inflammatory pro-tein-1a MIP-1a), CCL-4 (MIP-1b), CCL-2 (monocyte che-moattractant protein-1, MCP-1), granulocyte macrophagecolony-stimulating factor (GM-CSF), CCL-11 (eotaxin), andCCL-5 (also known as RANTES regulated on activation,normal T cell expressed and secreted) [62,63]. In the syno-vial fluid and tissue of CHIKV-infected patients, elevatedlevels of CCL-2, IL-6, and IL-8 have been reported [56]. Asimilar elevation of these immune mediators was also ob-served in RRV infection. Synovial fluid samples obtainedfrom RRV-infected patients exhibited significantly higherlevels of tumor necrosis factor (TNF)-a, IFN-g, IL-6, andreactive nitrogen intermediates (RNI) when compared toosteoarthritis (OA) patients [64]. Consistent with this ob-servation, RRV- and CHIKV-infected primary osteoblastsalso showed a greater increase in RANKL:OPG ratio andhigher expression of IL-6 and CCL-2; all are associated withenhanced osteoclastogenesis in response to alphaviral in-fection [52,53].

A striking similarity in inflammatory responses be-tween the animal models of alphaviral infection and hu-man disease has been observed. Elevated expression ofimmune mediators such as TNF-a, CCL-2, IFN-g, and IL-6was evident in RRV-infected mice [64] and in CHIKV-infected mice [58] and non-human primates [61]. Inaddition, there was an IL-6-dependent increase in theRANKL:OPG ratio in serum from RRV-infected mice dur-ing peak disease [52], indicating that the osteoclastogenicenvironment induced by RRV could be therapeuticallytargeted by IL-6 inhibition. Furthermore, reduced produc-tion of TNF-a, CCL-2, IFN-g, and RNI was evident inRRV-induced arthritis in mice upon macrophage depletion,indicating that these cells may play a significant role inalphaviral joint pathology [64]. In a recent study, Poo et al.[65] reported that mice deficient in CCR2, a receptor forseven ligands including CCL2, CCL8, and CCL7, displayedexacerbated disease following CHIKV infection. This wascharacterized by neutrophil-dominant cellular infiltration,suggesting a role of neutrophils in mediating cartilagedamage during CHIKV infection [65]. However, treatmentof RRV-infected mice at therapeutic dose with bindarit, aninhibitor of CCL-2, CCL-8, and CCL-7, leads to diseaseamelioration [66]. Together, these studies suggest thatsuppressing the overexpression of alphavirus-inducedproinflammatory factors using a specific anti-inflammato-ry drug at therapeutic dose may be a viable option forpreventing joint inflammation.

Pro-osteoclastic cytokines TNF-a, IL-6, and IL-1, whichare also prominent alphavirus-induced proinflammatorycytokines, have been implicated in alphavirus-inducedosteoclastogenesis and bone loss [67,68]. Moreover, chemo-tactic cytokines such as CXCL1, CCL-2, CCL-3, CCL-4,CCL-5, and CCL-11, which have each been implicated in

Reviewthe egress of osteoclast precursors [69] and osteoclastdifferentiation [53,7072] in pathological bone conditions,are also elevated during alphaviral infection.

4Recent studies have identified an indispensable role foradaptive immunity in modulating alphaviral disease patho-genesis. In particular, increased T cell activation was ob-served during acute CHIKV infection in humans[56,73]. Consistent with these clinical studies, egress of Tcells to the inflamed joints and muscle tissues was alsoobserved in acute models of RRV and CHIKV infection[53,59,74,75]. Further characterization identified a patho-genic role for CD4+T helper (Th) cells in disease pathogenesisthrough mediating inflammatory processes [76]. Similarly,T cell infiltration has been identified as a hallmark of RA, inwhich Th17 cells have been characterized as a T cell subsetthat can support osteoclastogenesis by virtue of theirRANKL expression [77]. Hence, the presence of Th17-asso-ciated cytokines IL-17, RANKL, TNF, and IL-6 during alpha-viral infection strongly suggests that Th17 cells may also beinvolved in alphaviral disease, including osteoclastogenesis.

Taken together, the elevated profiles of inflammatorycytokines, pro-osteoclastic factors, chemokines, and Th17-associated cytokines play a crucial role in alphaviral ar-thritis. Most importantly, the increased RANKL:OPG ratioand osteoclastogenesis in alphavirus infection as a result ofinflammation demonstrate a strong association betweenthe immune response following alphaviral infection andbone health (Figure 2).

Underlying inflammatory arthritis: potential risk factorfor exacerbated alphavirus-induced arthritisThere has been considerable interest in the persistent butperiodically flaring arthralgia that lasts for months afteralphavirus infection. Potential risk factors, including age45 years, initial severe joint pain, and pre-existing jointdisorders, were independently associated with persistenceof CHIKV arthralgia [17].

Patients with underlying degenerative joint disordersmay be predisposed to delayed recovery from alphavirus-induced arthralgia [17,50,78]. For example, Borgheriniet al. reported that 44% of CHIKV-infected patients witha prior history of joint symptoms self-reported an extendedperiod of CHIKV arthralgia [79]. In addition, RRV-infectedpatients with pre-dating joint disease such as OA, gout, orpsoriatic arthritis complained of worse arthralgia monthsafter disease onset [15,20]. Similarly, the SINV-relatedalphavirus that causes Pogosta disease has been reportedto cause exacerbation of joint arthralgia in OA patients[19,80]. These observations suggest that underlying ar-thritic conditions may be associated with disease exacer-bation after alphavirus infection.

The delayed recovery from arthralgia in alphavirus-infected patients with pre-existing arthritis may be areflection of ongoing inflammation caused by the underly-ing disease. However, it is very challenging to distinguishbetween the symptoms associated with pre-dating arthriticconditions and those associated with alphavirus-inducedarthritis. To date, alphavirus-infected patients with un-derlying arthritis have not been studied in detail. Thestriking similarities between the immunological responsessuch as the cellular and proinflammatory mediators eli-

Trends in Microbiology xxx xxxx, Vol. xxx, No. xcited during alphaviral infection and arthritis such as RAfurther suggests the likelihood of interplay between theseinflammatory arthritides (Figure 3). It will be of interest to

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TIMI-1130; No. of Pages 9identify and dissect the mechanisms by which arthrito-genic alphaviruses can exacerbate underlying arthritis-

OPGRANKLRANK

Osteoblasts

Alphavirus

Boneformaon

Figure 2. Schematic representation of immune and skeletal responses during alph

forming osteoblasts and bone-resorbing osteoclasts. Following alphaviral infectio

infection of osteoblasts gives rise to the secretion of proinflammatory and pro-ost

RANKL:OPG ratio is increased, favoring osteoclast differentiation and contributing

Reviewinduced bone pathology.

Potential therapeutic strategies to prevent alphavirus-induced arthritisBone loss is a newly identified characteristic of alphavirus-induced disease, which has yet to be addressed in currenttherapeutic approaches. The similarities in the immuneand osteoimmunological responses during alphavirus in-fection and inflammatory arthritis such as RA suggest thatcurrent therapeutics for RA may be useful in preventingalphavirus-induced bone loss. Many of the biologic treat-ments targeting proinflammatory cytokines in RA haveproved effective at halting the progression of focal erosion.These therapies may show similar efficacy in alphavirusinfection.

Anti-IL-6 therapyIL-6 is a potent stimulus for RANKL expression in osteo-blast-lineage cells, which then promotes osteoclastogenesisand bone resorption [81]. IL-6 is produced by immune cells,osteoblasts, and synoviocytes [81,82]. In addition to its rolein alphavirus arthritis, overt expression of IL-6 has alsobeen implicated in bone loss in other bone disorders such asRA [83] and bone metastases [84]. Tocilizumab (TCZ), ahumanized anti-human IL-6R monoclonal antibody, iscurrently used to treat systemic juvenile idiopathic arthri-tis and RA [85,86], in which it successfully controls syno-vial inflammation and protects against progressive jointdestruction. In our mouse model of RRV infection, treat-ment with a neutralizing anti-IL-6 antibody effectivelyprevented the increase in the RANKL:OPG ratio and boneloss triggered by RRV [52]. Inhibition of IL-6 may alsoalleviate chronic joint pain associated with alphavirus

Bone loss

Osteoclasts

Recruit

Monocyc precursors

OsteoclastogenesisRANKL:OPG

Proinammatorycytokines

(IL-6, IL-1, CCL-2)

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al infection. In the bone microenvironment the two major cell subsets are bone-

iruses are disseminated to inflamed joints, where osteoblasts are infected. The

astic factors including IL-6, IL-1b, and CCL-2, as well as RANKL. Furthermore, the

one loss.

Trends in Microbiology xxx xxxx, Vol. xxx, No. xinfection because inflammatory mediators such as IL-6have been associated with hyperalgesia [87,88].

TNF antagonistsTNF is a multifunctional cytokine that modulates inflam-matory and bone remodeling processes. It is produced atelevated levels by activated macrophages, synoviocytes,and T cells in RA synovial tissue [89,90]. Several TNFantagonists such as adalimumab, infliximab, and etaner-cept have been established as potent drugs for treatment ofinflammatory joint diseases including RA [91], psoriaticarthritis [92], and juvenile idiopathic arthritis [93]. Despitethe reported efficacy of etanercept in treating inflammato-ry diseases, detrimental effects of etanercept administra-tion have been shown in an acute RRV disease mousemodel: etanercept treatment at disease onset led to pro-nounced myositis and increased viral titers as a result ofdiminished alphavirus-triggered type I IFN responses[94,95]. These undesirable effects on acute infection indi-cate that caution should be taken during any trials ofetanercept for viral arthritis. These side-effects might beavoided if the drug is administered after the acute phase ofdisease, when the persistent joint manifestations develop.The potential of adalimumab, infliximab, and etanercept totreat persistent alphavirus-induced bone loss warrantsfurther investigation.

Direct targeting of Th17 cells and related cytokinesActivation of T cells plays a pivotal role in the interplaybetween the immune and skeletal systems. Th17 cells arecapable of supporting osteoclast differentiation and there

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(TNF, IL-6, IL-1)

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Reviewis a growing interest in therapeutic targeting of Th17-related cytokines for pathological bone conditions. In par-ticular, IL-17 has been the primary target for therapeuticintervention because it is capable of inducing RANKLexpression on synovial fibroblasts [91]. Anti-IL-17A anti-body treatment is efficacious in alleviating joint pathologyand bone erosions and in lowering RANKL expression inseveral arthritic mouse models including adjuvant-in-duced arthritis (AIA) [96], collagen-induced arthritis(CIA) [97], and antigen-induced experimental arthritis[98]. A Phase II clinical trial is currently underway usingan anti-IL-17 antibody (AIN457) in RA patients [99].

Anti-RANKL therapyRANKL is a pivotal cytokine that stimulates bone resorp-tion. RANKL has been targeted during pathological boneconditions using a neutralizing anti-RANKL antibody,denosumab. Denosumab is efficacious in treating osteopo-rosis [100102] and has been approved by several healthauthorities including Health Canada and the US FDA forthe treatment of postmenopausal women predisposed toosteoporotic fractures [101,103]. In RA patients, despitehaving no effect on disease activity, denosumab givenevery 6 months in combination with standard methotrex-ate treatment resulted in reduced radiographic scores forfocal bone erosion, suppressed serum markers of boneresorption [serum C-telopeptide of type I collagen (CTX-I) and serum N-propeptide of type I collagen (PINP)] and

Figure 3. Comparative diagram of immune responses regulated during alphavirus-ind

alphaviral infection and in RA can affect several immune parameters. During inflamma

which contribute to the clinical outcomes. Following activation, macrophages present an

Nave CD8+ T cells can also be activated by antigen-presenting cells such as macrophag

contribute to radiologic bone erosion. Nave CD4 T helper cells can differentiate into type

express proinflammatory factors (IL-6, IL-1b, and TNF-a) which have the ability to

osteoclastogenesis is pivotal in the induction of bone pathologies.

6RRV-induced arthris

Rheumatoid arthris

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Trends in Microbiology xxx xxxx, Vol. xxx, No. ximproved bone mineral density compared to patients re-ceiving placebo [104]. These clinical observations clearlysuggest that denosumab is effective in amelioratingRANKL-induced bone erosion in RA and may be a potentialtherapeutic option for alphavirus-induced bone loss.

Concluding remarksDebilitating persistent polyarthralgias induced by arthri-togenic alphavirus infection is a clinical enigma. Emergingclinical evidence of bone pathologies in alphavirus-infectedpatients has identified this as a possible risk factor for theaggravation of joint inflammation. In light of the ominousthreat of a global chikungunya epidemic, and the lack oftherapeutic alternatives, intensive research into the exactmechanisms underlying the development of persistentpolyarthralgias is warranted. Preclinical studies suggestthat there is a clear link between alphavirus-inducedinflammation and periarticular and systemic bone loss.The potential for alphavirus infection to precipitate boneloss and increase fracture risk in humans is an importantclinical question (Box 1). Furthermore, the impact of alpha-viral infection on bone health in patients with pre-existingarthritis also remains to be determined. In the prototypicalinflammatory arthritis, RA, local and systemic inflamma-tion leads to focal bone erosion within the cortical bone,bone loss in the periarticular trabecular bone adjacent tothe affected joint, and systemic bone loss. By contrast,inflammation in the mouse models of alphavirus infection

uced and RA bone loss. The expression of host proinflammatory factors during

tion, macrophages take central stage to facilitate several immunological pathways

tigen to CD4 T helper cells, which in turn activate cytotoxic CD8 T cells and B cells.

es. In RA, B cells are a source of rheumatoid factors (RF) and autoantibodies, which

17 T helper (Th17) subsets which express IL-17 and RANKL. Macrophages can also

enhance osteoclastogenesis. In both RA and alphaviral arthritis, the enhanced

TIMI-1130; No. of Pages 9leads primarily to periarticular and systemic trabecularbone loss with no evidence of focal bone erosion. Despitedistinct differences in bone pathologies resulting fromthese two forms of arthritides, some similarities have alsobeen observed, such as inflammatory mediators and cellu-lar responses leading to increased osteoclastogenesis. Thesimilarities between RA-induced inflammation and boneloss and that observed in alphavirus infection suggeststherapeutic approaches used in the treatment of RA, suchas cytokine and RANKL inhibition, may also be appropri-ate for the treatment of persistent alphavirus-inducedbone disease.

AcknowledgmentsS.M. is the recipient of an Australian National Health and MedicalResearch Council (NHMRC) Senior Research Fellowship (APP1059167).

References1 Zacks, M.A. and Paessler, S. (2010) Encephalitic alphaviruses. Vet.Microbiol. 140, 281286

2 Rulli, N. et al. (2007) The molecular and cellular aspects of arthritisdue to alphavirus infections: lesson learned from Ross River virus.Ann. N. Y. Acad. Sci. 1102, 96108

3 Suhrbier, A. and La Linn, M. (2004) Clinical and pathologic aspects ofarthritis due to Ross River virus and other alphaviruses. Curr. Opin.Rheumatol. 16, 374379

4 Kiwanuka, N. et al. (1999) Onyong-nyong fever in south-centralUganda, 1996-1997: clinical features and validation of a clinicalcase definition for surveillance purposes. Clin. Infect. Dis. 29,12431250

5 Laine, M. et al. (2004) Sindbis viruses and other alphaviruses as causeof human arthritic disease. J. Intern. Med. 256, 457471

6 Munoz, M. and Navarro, J.C. (2012) Mayaro: a re-emergingarbovirus in Venezuela and Latin America. Biomedica 32, 286302(in Spanish)

7 Phillips, D.A. et al. (1990) Clinical and subclinical Barmah Forestvirus infection in Queensland. Med. J. Aust. 152, 463466

8 Paquet, C. et al. (2006) Chikungunya outbreak in Reunion:epidemiology and surveillance, 2005 to early January 2006. EuroSurveill. 11, E060202E060203

9 Suhrbier, A. and Mahalingam, S. (2009) The immunobiology of viralarthritides. Pharmacol. Ther. 124, 301308

10 Parida, M.M. (2008) Rapid and real-time detection technologies foremerging viruses of biomedical importance. J. Biosci. 33, 617628

Box 1. Outstanding questions

Do underlying arthritic conditions exacerbate alphavirus-inducedbone loss (or vice versa)?

What is the effect of alphaviral infection on bone health? Are thesepatients at risk of fracture as a result of infection?

Does alphavirus infection of bone persist during disease progres-sion? Is alphavirus infection of bone responsible for periodicflares of disease?

Do immune cells contribute to alphavirus-induced bone loss?

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TIMI-1130; No. of Pages 99

Arthritogenic alphaviruses: new insights into arthritis and bone pathologyEpidemiological and clinical burden of arthritogenic alphavirus infectionInflammation and bone lossEmerging evidence for alphavirus-induced arthritis affecting boneImpact of the alphavirus-induced immune response on boneUnderlying inflammatory arthritis: potential risk factor for exacerbated alphavirus-induced arthritisPotential therapeutic strategies to prevent alphavirus-induced arthritisAnti-IL-6 therapyTNF antagonistsDirect targeting of Th17 cells and related cytokinesAnti-RANKL therapy

Concluding remarksAcknowledgmentsReferences