Research ArticleEMERGING INFECTIONS

Fingolimod treatment abrogates chikungunya virus–induced arthralgia

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Science Translational Medicine  01 Feb 2017:
Vol. 9, Issue 375, eaal1333
DOI: 10.1126/scitranslmed.aal1333

Taming T cells to ameliorate chikungunya arthritis

Mosquito-borne chikungunya virus causes fever and joint pain; some patients suffer from arthritis for years with no treatment options. In this issue, two studies investigated targeting pathogenic CD4+ T cells to prevent arthritis symptoms in a mouse model of chikungunya virus infection. Teo et al. demonstrated that fingolimod, a drug that sequesters immune cells to lymphoid organs, was able to relieve arthritis symptoms without affecting viral replication. Miner et al. used a combination of abatacept, which blocks T cell costimulation, and a human chikungunya neutralizing antibody to reduce both viral replication and disease severity. Repurposing these clinically available therapies could provide treatment options for chikungunya patients.

Abstract

Chikungunya virus (CHIKV) is one of the many rheumatic arthropod-borne alphaviruses responsible for debilitating joint inflammation in humans. Despite the severity in many endemic regions, clinically approved intervention targeting the virus remains unavailable. CD4+ T cells have been shown to mediate CHIKV-induced joint inflammation in mice. We demonstrate here that transfer of splenic CD4+ T cells from virus-infected C57BL/6 mice into virus-infected T cell receptor–deficient (TCR−/−) mice recapitulated severe joint pathology including inflammation, vascular leakages, subcutaneous edema, and skeletal muscle necrosis. Proteome-wide screening identified dominant CD4+ T cell epitopes in nsP1 and E2 viral antigens. Transfer of nsP1- or E2-specific primary CD4+ T cell lines into CHIKV-infected TCR−/− recipients led to severe joint inflammation and vascular leakage. This pathogenic role of virus-specific CD4+ T cells in CHIKV infections led to the assessment of clinically approved T cell–suppressive drugs for disease intervention. Although drugs targeting interleukin-2 pathway were ineffective, treatment with fingolimod, an agonist of sphingosine 1-phosphate receptor, successfully abrogated joint pathology in CHIKV-infected animals by blocking the migration of CD4+ T cells into the joints without any effect on viral replication. These results set the stage for further clinical evaluation of fingolimod in the treatment of CHIKV-induced joint pathologies.

INTRODUCTION

Arthritogenic alphaviruses are arthropod-borne infectious agents that include chikungunya virus (CHIKV), O’nyong-nyong virus, Ross River virus, Barmah Forest virus, Mayaro virus, and Sindbis virus (SINV) (1). Infected patients typically endure arthritic manifestation in the joints such as synovitis and necrosis of muscle fibers at sites of inflammation (17). Among these, CHIKV has become a serious public health concern across the globe since mid-2004 (8). Major outbreaks have been reported from India (9, 10), Southeast Asia (1113), and the islands of the Indian Ocean (14, 15). Local transmissions of CHIKV have also expanded to the Americas, resulting in more than 1.2 million cases (16). Despite the threats of CHIKV disease, no U.S. Food and Drug Administration (FDA)–approved drugs are available. Understanding the disease pathogenesis in mammalian hosts would greatly aid in the development of clinically viable host-targeted interventions.

Studies on chikungunya fever (CHIKF) patients have provided evidence that CHIKV-induced joint swelling is immune-mediated, involving the production of proinflammatory cytokines (17) and extensive cellular infiltrates into the inflamed tissues (7, 18). However, limited access to tissue samples from CHIKF patients (18) has resulted in the development of CHIKV mouse models that could recapitulate viremia and joint pathology such as tenosynovitis, myositis, edema, and cellular infiltration (1923). In this model, CD4+ T cells were demonstrated to be the primary mediators of joint inflammation (23), but the precise mechanisms remain unknown.

Here, we sought to define and characterize the pathogenic role of CD4+ T cells during CHIKV infections through systematic screening across the entire virus proteome to identify CD4+ T cell “pathogenic” epitopes. Transfer of primary CD4+ T cell pathogenic epitope-specific cell lines into virus-infected T cell receptor–deficient (TCR−/−) mice recapitulated joint swelling, vascular leakages, edema, and inflammation and necrosis of the muscles. The action of CD4+ T cells as a mediator of joint pathology in CHIKV infection led to the assessment of clinically approved T cell–suppressive drugs as a targeted option for disease intervention. We showed that therapeutic use of fingolimod (FTY720), an agonist of sphingosine 1-phosphate receptor, successfully limited the migration of CD4+ T cells to the joints and effectively suppressed virus-induced joint pathology. This paves the way for therapeutic intervention to reduce severe disease morbidity in CHIKV-infected patients.

RESULTS

CD4+ T cells mediate severe vascular leakages, edema, and muscle necrosis in the mouse paw during CHIKV infection

Inoculation of CHIKV via subcutaneous injection into the footpad of C57BL/6 induces joint pathologies characterized by tenosynovitis, myositis, edema, and cellular infiltration (19, 20, 23). To quantify vascular leakages in this model, a fluorescent tracer was injected intravenously and the degree of plasma leakage was determined by measuring the intensity of fluorescent signal in the joints (24). First, CD4+ T cells were demonstrated to mediate vascular leakage with CHIKV-infected CD4−/− mice displaying significant reduced tracer signals (Fig. 1A). Cellular infiltration of CD45+ leukocytes into the joints of virus-infected CD4−/− mice did not differ from that of wild-type (WT) mice (Fig. 1B).

Fig. 1. CHIKV-specific CD4+ T cells mediate vascular leakage in the joints.

(A) Joint vascular leakages and of WT + CHIKV (n = 5) and CD4−/− + CHIKV (n = 5) on 6 days post-infection (dpi). Representative fluorescence images used to quantify leakage from the tracer assays (left). Data are representative of two independent experiments. (B) Cellular infiltration into the joints of WT + CHIKV (n = 5) and CD4−/− + CHIKV (n = 5) on 6 dpi. Both (A) and (B) were analyzed by Mann-Whitney U two-tailed analysis (**P < 0.01). The inflamed joints are indicated by yellow arrows. (C) Joint swelling and (D) viral load of WT + CHIKV (n = 5) and TCR−/− + CHIKV (n = 4) groups. Data were analyzed by Mann-Whitney U two-tailed analysis (**P < 0.01). Graphs are representative of two independent experiments. (E) Joint swelling and (F) viral load of TCR−/− + naïve donor CD4 + CHIKV (n = 7), TCR−/− + virus-infected (IF) donor CD4 + CHIKV (n = 6), and TCR−/− mock + IF donor CD4 (n = 4) groups. Data comparison between TCR−/− + naïve donor CD4 + CHIKV and TCR−/− + IF donor CD4 + CHIKV groups was done by Mann-Whitney U two-tailed analysis (**P < 0.01). (G) Joint vascular leakages and (I) cellular infiltration into the joints of WT + CHIKV, TCR−/− + CHIKV, and TCR−/− + IF donor CD4 + CHIKV groups on 6 dpi (n ≥ 5). Data were analyzed by one-way analysis of variance (ANOVA) with Tukey post-test (**P < 0.01 and ***P < 0.001). Graphs are representative of two independent experiments. (H) Joint vascular leakages of TCR−/− + naïve donor CD4 + CHIKV (n = 7) and TCR−/− + IF donor CD4 + CHIKV (n = 6) groups. Data were analyzed by Mann-Whitney U two-tailed analysis (**P < 0.01). For all quantification of joint vascular leakages, average tracer reading from WT mock-infected mice (n = 2) was shown as black dashed line in (A), (G), and (H).

To further characterize the role of CD4+ T cells, we performed adoptive transfer of CD4+ T cells into TCR−/− mice. The absence of T cells in CHIKV-infected TCR−/− mice prevented joint swelling with no effects on viremia (Fig. 1, C and D). To show that CHIKV-specific CD4+ T cells mediate CHIKV-induced joint pathology, we transferred an equal number of CD4+ T cells from naïve or virus-infected (6 dpi) WT donors into their respective virus-infected TCR−/− recipients at 1 dpi. Despite no change in viremia, the transfer of CD4+ T cells from virus-infected donors into TCR−/−-infected recipients not only recapitulated peak swelling on 6 dpi like the infected WT mice but also hastened the development of severe joint swelling to start at 3 dpi (Fig. 1, E and F). Transfer of virus-specific CD4+ T cells into CHIKV-infected TCR−/− recipients resulted in severe joint pathology, as demonstrated by the greater degree of vascular leakage and cellular infiltration into the joints during peak swelling (Fig. 1, G to I). The recapitulation of severe joint pathology was complemented by the restoration of CHIKV-specific CD4+ T cells in the spleen, popliteal lymph node (pLN), and joints in the TCR−/− recipient mice (fig. S1). In the absence of CHIKV infection, transfer of CD4+ T cells from infected donor into TCR−/− mock recipients did not induce any joint pathology (Fig. 1E and fig. S2), demonstrating that CHIKV-induced joint swelling is mediated by virus-specific CD4+ T cells recognizing virus-infected cells.

The effects of CD4+ T cells transfer from virus-infected donors into infected TCR−/− recipients on joint pathology during the acute phase (6 dpi) were further assessed by histology (Fig. 2). Infection with virus distended the joint footpad as observed by the presence of a diffused and marked degree of edema. This was also coupled with a combination of various inflammatory cells: lymphocytes, macrophages, few neutrophils, and occasional plasma cells in the subcutaneous tissue (Fig. 2A). Skeletal muscle fibers were degenerated and necrosed because they were irregular in shape (Fig. 2A). Necrosed myofibers had pyknotic nuclei and were infiltrated with mononuclear cells (Fig. 2A). Minimal subperiosteal fibrosis resulted in irregular cortical area in metatarsal bone. This was coupled with minimal synovitis with infiltration of mononuclear cells at the intermetatarsal-phalangeal joint and tendonitis. Although the cartilage at the joint footpad was not affected and vasculitis was rarely noticed (Fig. 2 and fig. S3), the presence of F4/80+ macrophages was high in the edematous region compared to the muscle area (Fig. 2).

Fig. 2. Virus-specific CD4+ T cells mediate CHIKV-induced edema, inflammation, and muscle necrosis in the joint footpad.

(A) Representative hematoxylin and eosin (H&E) and F4/80 immunohistochemistry (IHC) images of the inflamed joint footpad on 6 dpi. E, region of edema; N, muscle necrosis; T, tendon; B, bone; *, infiltration of mononuclear cells; #, normal skeletal muscle; black arrows, vacuolated muscle fibers; red arrows, F4/80+ cells. Blue box inserts represent regions taken at a higher magnification. F4/80 IHC images were taken at the subcutaneous region. (B) Histopathological scoring of edema, inflammation in different regions of the joint footpad, muscle pathology, and cellular infiltration (F4/80+ cells) of WT + CHIKV (n = 5), TCR−/− + CHIKV (n = 6), TCR−/− + CD4 transfer + CHIKV (n = 5), and CD4−/− + CHIKV (n = 5) groups in different regions of the joint footpad on 6 dpi. Scoring was done on three sections from each joint footpad, and data were expressed as means ± SEM. All data were analyzed by one-way ANOVA with Tukey post-test (*P < 0.05, **P < 0.01, and ***P < 0.001).

The degree of edema, skeletal muscle necrosis, and inflammation and infiltration of F4/80+ macrophages in CHIKV-infected TCR−/− mice was significantly reduced (Fig. 2). Transfer of CD4+ T cells from WT-infected donors into CHIKV-infected TCR−/− recipients successfully recapitulated the pathological changes in the inflamed joint footpad (Fig. 2). Together, these data demonstrated that CHIKV-specific CD4+ T cells mediate the peak of joint pathology on 6 dpi by mediating vascular leakage, edema of the subcutaneous regions, and necrosis of skeletal muscles in the joints.

Dominant CHIKV CD4+ T cell epitopes are identified in nsP1 and E2 viral proteins

To identify the T cell epitopes recognized by these pathogenic T cells, we developed a peptide-based interferon-γ (IFNγ) enzyme-linked immunospot (ELISPOT) assay using isolated splenocytes harvested from CHIKV-infected mice during peak joint inflammation (on 6 dpi). Screening was done using an overlapping CHIKV peptide library spanning the entire CHIKV proteome (22, 25). Screening was first performed using pools of five overlapping peptides (fig. S4, A and B). Positive pools were selected, and the screening was repeated using individual peptides from the respective pools. A total of seven peptides in the nonstructural proteins (nsPs) and five peptides in the structural regions were found to induce IFNγ production (fig. S4, C and D). Because the screening used total splenocytes, these positive peptides could be either CD4+ or CD8+ T cell epitopes.

To further identify the specific CD4+ T cell epitopes within these positive peptides, screening was repeated using CD4+ T cells isolated from the spleen, pLN, and joints of virus-infected WT mice. As a positive control, CD4+ T cells from each organ were also stimulated with live viruses in the ELISPOT assay. Dominant CD4+ T cell epitopes were identified in nsP1–pool 4–peptide 2 (nsP1-P4-2) at amino acid position 145 to 162 and in E2EP3 (25) at amino acid position 2800 to 2818 of the CHIKV proteome (Fig. 3A). Although CD4+ T cells recognizing the whole CHIKV- and epitope-specific CD4+ T cells were generated as early as 4 dpi in the pLN, these specific CD4+ T cells were only detected in the spleen and joints on 6 dpi (Fig. 3, B to D). These observations were supported by an increase of CD4+ T cells expressing the activation marker LFA-1+ (leukocyte function-associated antigen–1–positive) on 4 dpi, whereas expansion of activated CD4+ T cells in the spleens and joints was only detected on 6 dpi (Fig. 3E).

Fig. 3. CHIKV-specific CD4+ T cell epitopes.

(A) Schematic diagram depicting positions of dominant CD4 epitopes during acute CHIKV infection (6 dpi). Levels of epitope-specific IFNγ-producing CD4+ T cells in the (B) pLN, (C) spleen, and (D) joints on 4 dpi (n = 5) and 6 dpi (n = 5). Epitope-specific CD4+ T cells were determined by IFNγ ELISPOT assay with isolated CD4+ T cells. Data were expressed as means ± SD. Data at each time point were compared against the respective no-antigen controls using Mann-Whitney U two-tailed analysis (*P < 0.05). (E) Total LFA-1+ CD4+ T cell population in the pLN, spleen, and joints of naïve (n = 10), 4 dpi (n = 5), and 6 dpi (n = 5) groups. Data within each organ were compared against their respective naïve controls using Kruskal-Wallis test with Dunn’s multiple comparisons (*P < 0.05, **P < 0.01, and ***P < 0.001).

Virus-specific CD4+ T cell epitopes are pathogenic and cause joint inflammation in TCR−/− mice

To allow for functional study, we generated primary CD4+ T cell lines specific for nsP1-P4-2 or E2EP3. A CD4+ T cell line isolated from OTII-Rag mice (26) and specific for a CD4 epitope in the ovalbumin protein was generated in parallel as control. These lines had more than 98% purity in CD4+ T cells with a T helper 1 (TH1) phenotype, producing primarily IFNγ and tumor necrosis factor–α after restimulation with their respective peptides (fig. S5). Only minimal induction of interleukin-4 (IL-4), IL-10, transforming growth factor–β1, and IL-17 was observed upon restimulation in these primary cell lines (fig. S5).

Both nsP1-P4-2 and E2EP3 lines were then transferred into their respective virus-infected TCR−/− mice just before the peak of viremia (at 1 dpi), and recipient mice were assessed for disease phenotypes. Expansion of transferred nsP1-P4-2 line was observed in the spleen and joints on 6 dpi, whereas expansion of E2EP3 line was observed only in the joints on 6 dpi when CD4+ T cells were profiled in the TCR−/− recipient mice (fig. S6). Expansion of these epitope-specific lines during infection led to the aggravation of joint inflammation and vascular leakage in the TCR−/− recipients, with little effect on viremia (Fig. 4, A, B, D, and E, and fig. S7). No difference in cellular infiltration into the joints upon transfer was observed (Fig. 4, C and F).

Fig. 4. Expansion of epitope-specific CD4+ T cells in CHIKV-infected TCR−/− mice exacerbates vascular leakage.

nsP1-P4-2– and E2EP3-specific primary CD4+ T cells were generated and transferred into CHIKV-infected TCR−/− mice. Virus-infected TCR−/− mice receiving OTII-specific CD4+ T cell transfer were used as a control. (A) Joint swelling, (B) joint vascular leakage (6 dpi), and (C) cellular infiltration into the joint (6 dpi) of TCR−/− + OTII + CHIKV (n = 5) and TCR−/− + nsP1-P4-2 + CHIKV (n = 5) groups. Tracer reading of mock-infected mice (n = 2) was shown as black dashed line in (B). (D) Joint swelling, (E) joint vascular leakage (6 dpi), and (F) cellular infiltration into the joint (6 dpi) of TCR−/− + OTII + CHIKV (n = 5) and TCR−/− + E2EP3 + CHIKV (n = 5) groups. Tracer reading of mock-infected mice (n = 5) was shown as black dashed line in (E). All data were analyzed by Mann-Whitney U two-tailed analysis (ns, not significant, *P < 0.05 and **P < 0.01). Purple dashed lines in (A) and (D) represent the peak of joint swelling in TCR−/− + CHIKV controls shown in Fig. 1. For (B) and (E), representative fluorescence images of the tracer assay were provided, with inflamed joints indicated by yellow arrows.

Clinically approved T cell immunosuppressive drugs are used as treatment for CHIKV-induced musculoskeletal inflammation

Because CHIKV-specific CD4+ T cells play a role in the development of joint inflammation, T cell modulatory drugs would be a plausible treatment option. Cyclosporin A (CsA) and rapamycin are clinically approved drugs known to target different components of the IL-2 pathway and limit the generation of effector T cells (27, 28). To assess whether these two T cell activation blockers can effectively suppress joint inflammation, we therapeutically treated mice daily with 200 μg of rapamycin (fig. S8, A and B) or 750 μg of CsA (fig. S8, C and D) upon virus infection. However, not only did both drugs not control joint inflammation, treatment even aggravated viremia and joint swelling (fig. S8), thus dismissing them as treatment options.

Fingolimod (FTY720) was next assessed because it has been reported to block T cell egress from the lymphoid organs by acting as an agonist of sphingosine 1-phosphate receptor (29). Mice were first treated prophylactically with 20 μg of fingolimod from −1 to 6 dpi (Fig. 5). Results showed that daily treatment successfully prevented joint inflammation with no effect on viremia (Fig. 5, A and B). Treatment was then assessed therapeutically with daily administration of fingolimod in CHIKV-infected mice from 2 to 6 dpi. Fingolimod-treated mice similarly displayed a reduced joint inflammation with no effect on viremia (Fig. 5, C and D).

Fig. 5. Fingolimod (FTY720) treatments alleviate joint pathology during CHIKV infection.

(A) Joint swelling and (B) viral load of CHIKV (n = 6) and CHIKV + FTY720 prophylactic treatment (n = 6) groups. (C) Joint swelling and (D) viral load of CHIKV (n = 5) and CHIKV + FTY720 therapeutic treatment (n = 7). Data were analyzed by Mann-Whitney U two-tailed analysis (*P < 0.05 and **P < 0.01). Treatment periods were indicated by shaded region in the graphs. Data are representative of two independent experiments. (E) Joint vascular leakage and (F) cellular infiltration of CD45+ cells into the joints on 6 dpi in CHIKV, CHIKV + FTY720 prophylactic treatment, and CHIKV + FTY720 therapeutic treatment groups (n ≥ 5 per group). Tracer reading of mock-infected mice (n = 2) was shown as black dashed line in (E). Vascular leakages were analyzed by Mann-Whitney U two-tailed analysis (**P < 0.01). Cellular infiltrations were analyzed by Kruskal-Wallis with Dunn’s multiple comparisons.

To further define the changes in joint phenotype of fingolimod-treated CHIKV-infected mice, we performed the tracer assay, CD45+ cell quantification, and histology of the joint tissues during the peak of joint swelling. Similar to depletion of CD4+ T cells, the suppression of joint inflammation upon fingolimod treatment, whether prophylactic or therapeutic, was primarily due to phenotypic changes in the skeletal muscles and subcutaneous area (Figs. 5, E and F, and 6). Treatment reduced the severity of joint vascular leakages, edema in the subcutaneous region, and inflammation and necrosis of the muscles (Figs. 5, E and F, and 6). No significant changes in the pathology of the tendon, synovial membranes, bones, cartilages, and infiltration of F4/80+ cells were observed in the inflamed joint footpad (Fig. 6 and fig. S9).

Fig. 6. Fingolimod (FTY720) treatments reduce CHIKV-induced joint pathology.

(A) Representative H&E and F4/80 IHC images of inflamed joint footpad on 6 dpi. Blue box inserts represent regions taken at higher magnification. Images of F4/80 IHC were taken at the subcutaneous region. (B) Histopathological scoring of edema, inflammation in different regions of the joint footpad, muscle pathology, and cellular infiltration (F4/80+ cells) of CHIKV (n = 5), CHIKV + FTY720 prophylactic treatment (n = 5), and CHIKV + FTY720 therapeutic treatment (n = 5) groups on 6 dpi. Scoring was done on three sections from each joint footpad, and data were expressed as means ± SEM. All data were analyzed by one-way ANOVA with Tukey post-test (*P < 0.05, **P < 0.01, and ***P < 0.001).

Fingolimod alleviates severe joint inflammation in arthritogenic CHIKV infections by limiting cellular infiltration of pathogenic CD4+ T cells

To assess the immunomodulatory effects of fingolimod on CHIKV-specific CD4+ T cell migration, we quantified activated (LFA-1+) and CHIKV-specific CD4+ T cells in the joints and secondary lymphoid organs during the peak of joint swelling. Both treatment regimens successfully abrogated infiltration of activated and CHIKV-specific CD4+ T cells into the virus-infected joints (Fig. 7, A and B) to reduce joint swelling. Both prophylactic and therapeutic treatments also prevented the increase of activated and CHIKV-specific CD4+ T cells in the spleen and pLN (Fig. 7, C to F). However, LFA-1+ CD4+ T cells were significantly increased in the pLN during therapeutic treatment, suggesting the retention of CD4+ T cells in this organ (Fig. 7E), whereas levels of CHIKV-specific CD4+ T cells remain unchanged (Fig. 7F).

Fig. 7. Fingolimod (FTY720) treatments reduce the levels of activated and CHIKV-specific CD4+ T cells in the joint during CHIKV infection.

Activated (LFA-1+) CD4+ T cells in the (A) joints, (C) spleen, and (E) pLN on 6 dpi in CHIKV, CHIKV + FTY720 prophylactic treatment, and CHIKV + FTY720 therapeutic treatment groups (n ≥ 5 per group). IFNγ-producing CHIKV-specific CD4+ T cells in the (B) joints, (D) spleen, and (F) pLN on 6 dpi in CHIKV, CHIKV + FTY720 prophylactic treatment, and CHIKV + FTY720 therapeutic treatment groups (n ≥ 5 per group). CHIKV-specific CD4 T cells were determined by IFNγ ELISPOT assay using isolated CD4+ T cells and live virus restimulation. Values obtained from cells with no virus antigen stimulation were subtracted from values obtained from cells stimulated with their respective virus antigens. All data were analyzed by one-way ANOVA with Tukey post-test (**P < 0.01 and ***P < 0.001). All prophylactic treatments were given from 1 day preinfection to 6 dpi, whereas therapeutic treatments were given from 2 to 6 dpi.

DISCUSSION

The pathogenic role of CD4+ T cells has been demonstrated in several virus-induced infections such as those caused by respiratory syncytial virus (30), Theiler’s virus (31), and mouse hepatitis virus (32, 33). CD4+ T cells were also shown to be a pathogenic mediator in alphavirus infections such as CHIKV (23) and SINV (34, 35). Here, we further demonstrated how functional virus-specific CD4+ T cells act as the pathogenic mediator of vascular leakage, subcutaneous edema, and skeletal muscle necrosis during CHIKV infection.

The involvement of CD4+ T cells as mediator in CHIKV pathogenesis was first indicated with CD4+ T cell activation and the up-regulation of TH1 cytokines in patients (18, 3639). In our study, the recapitulation of joint pathology with the transfer of TH1-skewed virus or epitope-specific CD4+ T cells into TCR−/− mice suggests that these functional CD4+ T cells mediate pathology. Defining the various interacting partners and the precise cytokines used by these CD4+ T cells will provide novel and alternative control strategies in the future.

The identification of specific pathogenic CHIKV CD4+ T cell epitopes in this study has strong implications for vaccine design against CHIKV and other alphaviruses. Currently, most CHIKV vaccine studies have profiled only the kinetics of antibody production (25, 40, 41), whereas the profiling of CHIKV-specific memory CD4+ T cells remains largely neglected. Helper CD4+ T cells are important for antibody protection, but expansion of virus-specific memory CD4+ T cells with pathogenic potential may counterbalance antibody-mediated protection during a recall response. Therefore, it will be important to elucidate and characterize the pathogenic CD4+ T cell epitopes in patients who have recovered from CHIKV infection to formulate vaccine candidates that do not contain these pathogenic epitopes but yet are able to elicit neutralizing antibodies (40, 41). Alternatively, intervention based on tolerization of pathogenic CD4+ T cell epitopes could be envisaged.

Clinically, there are currently no FDA-approved drugs to treat arthritogenic CHIKV infections. Infected patients are often prescribed with generic nonsteroidal anti-inflammatory drugs to control inflammation (42). However, these drugs do not specifically target the primary pathogenic pathways in virus-induced inflammation, thus limiting their efficacy in disease management. The administration of T cell activation blockers (CsA and rapamycin) or T cell migration blockers (fingolimod) in this study targeted the host’s pathogenic response. Only fingolimod alleviated virus-induced joint pathology, whereas both CsA and rapamycin aggravated joint swelling and delayed recovery. This adverse effect could be due to the suppression of B cells by CsA and rapamycin that led to decreased levels of neutralizing antibodies needed for disease resolution that were elicited very early on 6 dpi after virus infection (22, 43)

Fingolimod has been used in the conventional treatment of multiple sclerosis by limiting the migration of autoreactive lymphocytes into the brain (44). More recently, treatment with fingolimod was shown to reduce the severity of neuroinflammation in a virus-induced encephalitis model (45). Our study provides evidence that these immunomodulatory effects exerted by fingolimod could be further extended to treat virus-induced joint inflammation.

Mechanistically, we have demonstrated that treatment with fingolimod alleviated joint swelling by limiting egress and expansion of functional virus-specific CD4+ T cells. The suppression of total virus-specific CD4+ T cells in the pLN was not seen during therapeutic regimen. This is likely due to the earlier induction of virus-specific CD4+ T cells in the pLN. As such, expansion of CD4+ T cells may have started when drug treatment was initiated at 2 dpi in the therapeutic regimen. Nonetheless, these expanded CD4+ T cells were still sufficiently retained in the pLN to limit the total number of CHIKV-specific CD4+ T cells reaching the joints.

Although the use of fingolimod to alleviate acute CHIKV joint pathology was effective in the joint-footpad CHIKV mouse model, the effectiveness of this drug in the management of CHIKV-induced chronic joint pain remains to be ascertained. Nonetheless, the regulation of functional CD4+ T cells with fingolimod suggests a viable intervention for virus-induced inflammation. An adjacent study using cytotoxic T-lymphocyte associated protein 4–immunoglobulin (CTLA4-Ig) to regulate CD4+ T cells during CHIKV infection in conjunction with a neutralizing antibody demonstrated effectiveness in alleviating severe joint pathology in mice (46). This highlights the possibility in extending the use of fingolimod and other FDA-approved T cell suppressors to treat virus-induced inflammation in other arboviruses that are mediated by virus-specific CD4+ T cells.

MATERIALS AND METHODS

Study design

The role of CD4+ T cells in CHIKV infection was investigated in a murine CHIKV model. The reduction of joint pathology in CD4-deficient (CD4−/−) mice or aggravation of pathology during adoptive transfer of CD4+ T cells isolated from CHIKV-infected donor into CHIKV-infected TCRβ-TCRδ–deficient (TCR−/−) mice was used to identify CD4+ T cells as pathogenic mediator of CHIKV infection. Joint pathology was quantified by the measurement of paw size changes and histological assessments. All histological assessments were performed in a blinded fashion by the histopathologists. To identify dominant CD4+ T cell epitopes within CHIKV, screening using IFNγ ELISPOT with a CHIKV peptide library consisting of overlapping 18-mer peptides that span the entire virus proteome was done. To prove the pathogenicity of these dominant CD4+ T cell epitopes, we generated and adoptively transferred epitope-specific primary CD4+ T cell lines back to CHIKV-infected TCR−/− recipient to recapitulate joint swelling. Last, the viability of treating CHIKV-induced joint pathology with T cell activation blockers (CsA and rapamycin) and T cell migration blocker (fingolimod) was tested in WT-infected mice. All mouse experiments were conducted with at least n ≥ 4 per group to fulfill the minimum requirement for nonparametric statistical analysis. Replication of experiments was indicated in the figure legends.

Mice

Three- or 6-week-old gender-matched WT, OTII-Rag, CD4−/−, and TCR−/− mice in C57BL/6J background were used. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC 140968) of the Agency for Science, Technology and Research, Singapore, in accordance with the guidelines of the Agri-Food and Veterinary Authority and the National Advisory Committee for Laboratory Animal Research of Singapore.

Virus stocks

CHIKV isolate used in this study was isolated from an outbreak in Singapore in 2008 at the National University Hospital (47). Virus was propagated in C6/36 cultures as described in (23).

Virus infection and disease monitoring in mice

Mice were inoculated subcutaneously in the ventral side of the right hind footpad toward the ankle, with 106 plaque-forming units of CHIKV isolate in 30 μl of phosphate-buffered saline (PBS). Viral load in the blood was monitored daily from 1 to 8 dpi and subsequently on every alternate day until 14 dpi. Joint swelling of the footpad was scored daily from 0 to 14 dpi, as previously described in (23, 48).

Viral RNA extraction and quantification

Ten microliters of blood collected from the tail vein was diluted in 120 μl of PBS and 10 μl of citrate-phosphate-dextrose solution (Sigma-Aldrich). Viral RNA was extracted with QIAmp Viral RNA Mini kit (Qiagen) in accordance to the manufacturer’s protocol. CHIKV viral genome copies were quantified using negative-sense nsP1 primers with quantitative reverse transcription polymerase chain reaction as described in (23, 25).

Adoptive transfer of CD4+ T cells into TCR−/− mice

CD4+ T cells isolated from the spleen of naïve or CHIKV-infected WT mice at 6 dpi or primary CD4+ T cell lines were adoptively transferred into TCR−/− recipients on 1 dpi by intravenous injection. All donor CD4+ T cells were isolated using negative selection CD4+ T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s manual. Each TCR−/− recipient received CD4+ T cell numbers equivalent to the CD4+ T cell count in one donor spleen (~1 × 107 to 1.2 × 107 CD4+ T cells). For primary CD4+ T cell lines, cells were subjected to CD4+ T cell negative isolation (Miltenyi Biotec) before transfer, and each TCR−/− recipient received 2 × 106 cells.

Quantification of joint vascular leakage by tracer assay

Tracer 653 Assay (Molecular Targeting Technologies Inc.) was performed as described in (24). Briefly, 100 μl of tracer solution was injected (intravenously) into each mouse, and tracer solution that entered the joints was quantified by in vivo imaging system (IVIS) (PerkinElmer). Regions of interest were drawn using the software Living Image 3.0, and average radiance [log10(photons s−1 cm−2 sr−1)/(μW/cm2)] was determined.

Quantification of cellular infiltration into joints

Isolated joint-footpad cells were counted and stained with LIVE/DEAD determination dye (Invitrogen) for 30 min. Cells were subsequently incubated with a blocking buffer [1% (v/v) rat and mouse serum] for 20 min before surface staining. Total cellular infiltrates into the joints were determined as total CD45+ cells recovered from each joint by flow cytometry.

Determination of CHIKV-specific CD4+ T cells in organs

CHIKV-specific CD4+ T cells in spleen, pLN, and joints were determined by IFNγ ELISPOT assay, as described in (23). Briefly, CD4+ T cells were isolated from 3 × 105 splenocytes, 1 × 105 pLN, or 1 × 105 joint-footpad cells by CD4+ T Cell Isolation Kit (Miltenyi Biotec). Isolated cells were stimulated with 1.5 × 106 CHIKV virions and 2 × 105 naïve splenocytes in IL-2 (60 U/ml) for 15 hours. Data obtained were back-calculated using the cell count from each organ to obtain the numbers of IFNγ+ CD4+ T cells per organ.

Histology of joint footpad and bone tissues

Mice were euthanized on 6 dpi, and harvested joint-footpad tissues were fixed in 10% neutral buffered formalin at room temperature for 24 hours. They were then decalcified in 5% formic acid, trimmed to three parts at 5-mm interval, and processed routinely for histologic evaluation. The sections were cut at 5 μm thickness and stained with H&E. Tissues were viewed under an Olympus BX53 upright microscope (Olympus Life Science), and pictures were taken with an Olympus DP71 digital camera using an Olympus DP controller and DP manager software.

Changes in pathology were evaluated independently by histopathologists using a scoring method in each animal based on the presence of edema, inflammation, muscle necrosis, tendonitis, and synovitis. Severity grades were assigned to the following scale as previously described (20): 1, minimal; 2, mild; 3, moderate; 4, marked; 5, severe; 0, no finding. All histological assessments were performed in a blinded fashion by the histopathologists.

Immunohistochemistry and image analysis

Immunohistochemistry was performed in Bond-Max Automated staining system (Leica Microsystems GmbH). Antigen retrieval was performed after deparaffinization of joint-footpad tissues using IHC Select Proteinase K (20 μg/ml) (Merck Millipore) and incubated for 15 min at room temperature. After washing, endogenous peroxidase was blocked for about 30 min in 3% H2O2. Tissues were then treated with 10% goat serum for 30 min to block any nonspecific reaction. Tissues were incubated overnight with primary antibody, anti-F4/80 antibody (clone A3-1; Abcam), at a dilution of 1:50 at 4°C. Slides were treated with secondary antibody, goat anti-rat IgG H&L horseradish peroxidase (Abcam), at a dilution of 1:100 for 60 min. The slides were then rinsed with Bond Wash Solution and treated with DAB-Chromogen detection reagent (Bond Refine Detection Kit; Leica) for 7 min. Last, the slides were counterstained with hematoxylin for 5 min, rehydrated, and mounted in synthetic mounting medium. As a negative control, Bond Antibody Diluent was used instead of the primary antibody.

The IHC F4/80-stained slides were scanned under Leica SCN400 slide scanner (Leica Microsystems GmbH) at ×20 magnification and exported to SlidePath Digital Image system (Leica Microsystems GmbH). Detection of F4/80 was analyzed using cytoplasmic/membranous algorithms of Leica SlidePath Tissue Image Analysis software. The results among the different groups were computed and analyzed for statistical significance.

Identification of CHIKV CD4 epitopes

Screening was first performed with splenocytes isolated at 6 dpi with IFNγ ELISPOT assay and pooled peptides from CHIKV peptide library (Mimotopes). CHIKV peptide library consists of overlapping synthetic 18-mer peptides that span the entire CHIKV proteome, as described in (22, 25). Each peptide pool consists of five consecutive peptides. Each peptide pool (30 μg/ml) was used to stimulate 3 × 105 splenocytes in IL-2 (60 U/ml) for 15 hours. Peptide pools giving significantly higher IFNγ+-producing cells than no-antigen controls were selected, and ELISPOT screening was repeated using individual peptides with the same condition. Reported peptide 1 (RP1) and RP2 (49) were used as positive controls.

Quantification of epitope-specific CD4+ T cells

To determine whether selected individual peptides present in the pooled preparations are CD4 epitopes, we repeated IFNγ ELISPOT assay using isolated CD4+ T cells from spleen, pLN, and joint footpad on 4 and 6 dpi as with the same ELISPOT conditions.

Generation of OTII-, nsP1-P4-2–, and E2EP3-specific primary CD4+ T cell lines

Primary CD4+ T cell lines against OTII, nsP1-P4-2, and E2EP3 were generated using OTII-Rag and WT mice, respectively. Briefly, on days 0 and 14, mice were primed with 30 μg of peptides emulsified in Incomplete Freund’s adjuvant (Sigma-Aldrich) via subcutaneous injection at the right flank. On day 21, right inguinal lymph nodes were removed, and CD4+ T cells were isolated using CD4+ T Cell Isolation Kit (Miltenyi Biotec). Isolated CD4+ T cells were stimulated with irradiated naïve splenocytes that were prepulsed with respective peptides and incubated at 37°C (5% CO2). Fresh peptide-pulsed irradiated naïve splenocytes were added weekly to ensure consistent CD4+ T cell stimulation.

Rapamycin and CsA treatments

Rapamycin stock (LC Laboratories) was reconstituted in dimethyl sulfoxide (DMSO) and diluted to working concentration with PBS. Rapamycin (200 μg) was given daily (intraperitoneally) from 2 to 9 dpi. CsA powder (Sigma-Aldrich) was reconstituted in DMSO and diluted to working concentration with olive oil. CsA (750 μg) was given daily (subcutaneously) from 2 to 6 dpi.

FTY720 treatments

FTY720 (Cayman Chemical) was reconstituted in DMSO and diluted to working concentration with PBS. Prophylactic and therapeutic FTY720 treatment regimens were given to virus-infected mice. In prophylactic regimen, mice were given 20 μg of the drug daily from 1 day preinfection to 6 dpi intraperitoneally. In therapeutic regimen, mice were given 20 μg of the drug daily from 2 to 6 dpi.

Statistical analysis

All statistical analyses were performed according to the appropriate test depending on the parametric or nonparametric distribution of the data using Prism 6 (GraphPad Software). Test of normality was done by D’Agostino-Pearson omnibus normality test. All data with normal distribution were analyzed by unpaired t test or one-way ANOVA with Tukey post-test. All data that do not meet the normality requirements were analyzed with Mann-Whitney two-tailed analysis or Kruskal-Wallis with Dunn’s multiple comparisons. P values less than 0.05 were considered statistically significant. All data sets used for statistical analysis in the main and supplementary figures are shown in tables S1 and S2.

SUPPLEMENTARY MATERIALS

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Methods

Fig. S1. Restoration of total and CHIKV-specific CD4+ T cells in TCR−/− adoptive transfer model.

Fig. S2. Joint footpad with an absence of musculoskeletal pathology in WT mock and TCR−/− mock + CD4+ T cell transfer mice.

Fig. S3. Histopathological evaluation of bone, cartilages, and synovial membrane in CHIKV-infected mice.

Fig. S4. CHIKV epitopes induce IFNγ in splenocytes of acute CHIKV infection ex vivo.

Fig. S5. Primary CD4+ T cell line against dominant CHIKV CD4+ T cell epitopes respond to antigen stimulation in a TH1-biased manner.

Fig. S6. Expansion of primary nsP1-P4-2 and E2EP3 lines in TCR−/− recipient mice after CHIKV infection.

Fig. S7. Epitope-specific CD4+ T cell line transfer into virus-infected TCR−/− mice recapitulates inflammation with no effect on viremia.

Fig. S8. Rapamycin and CsA treatments aggravate joint pathology during CHIKV infection.

Fig. S9. Histopathological evaluation of bone, cartilages, and synovial membrane of fingolimod-treated CHIKV-infected mice.

Table S1. Data sets of all main figures.

Table S2. Data sets of all supplementary figures.

REFERENCES AND NOTES

  1. Acknowledgments: We thank R. Chua and V. Wirayadi for assistance in the animal studies. We also thank A. Larbi and the Singapore Immunology Network (SIgN) Flow Cytometry core for assistance with cytometry analyses and the SIgN mouse core for support in animal breeding. We also thank the Advanced Molecular Pathology Laboratory, A*STAR for providing assistance in histological sectioning and staining. Funding: This work was supported by the Agency for Science, Technology and Research (A*STAR) core grant (grant number F00018). Y.-H.C. is supported by the A*STAR postgraduate scholarship. Author contributions: L.F.P.N. and L.R. conceived and supervised the study. T.-H.T., Y.-H.C., W.W.L.L., F.-M.L., S.N.A., Z.H., and R.R. conducted the experiments and performed data analysis. A.M. and O.R. provided materials and intellectual inputs. T.-H.T., Y.-H.C., R.R., A.M., L.R., and L.F.P.N. wrote the manuscript. Competing interests: The authors declare that they have no competing interests.
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