Distinct Roles of Interferon Alpha and Beta in Controlling Chikungunya Virus Replication and Modulating Neutrophil-Mediated Inflammation.

Type I interferons (IFNs) are key mediators of the innate immune response. Although members of this family of cytokines signal through a single shared receptor, biochemical and functional variation exists in response to different IFN subtypes. While previous work has demonstrated that type I IFNs are essential to control infection by chikungunya virus (CHIKV), a globally emerging alphavirus, the contributions of individual IFN subtypes remain undefined. To address this question, we evaluated CHIKV pathogenesis in mice lacking IFN-ß (IFN-ß knockout [IFN-ß-KO] mice or mice treated with an IFN-ß-blocking antibody) or IFN-α (IFN regulatory factor 7 knockout [IRF7-KO] mice or mice treated with a pan-IFN-α-blocking antibody). Mice lacking either IFN-α or IFN-ß developed severe clinical disease following infection with CHIKV, with a marked increase in foot swelling compared to wild-type mice. Virological analysis revealed that mice lacking IFN-α sustained elevated infection in the infected ankle and in distant tissues. In contrast, IFN-ß-KO mice displayed minimal differences in viral burdens within the ankle or at distal sites and instead had an altered cellular immune response. Mice lacking IFN-ß had increased neutrophil infiltration into musculoskeletal tissues, and depletion of neutrophils in IFN-ß-KO but not IRF7-KO mice mitigated musculoskeletal disease caused by CHIKV. Our findings suggest disparate roles for the IFN subtypes during CHIKV infection, with IFN-α limiting early viral replication and dissemination and IFN-ß modulating neutrophil-mediated inflammation.IMPORTANCE Type I interferons (IFNs) possess a range of biological activity and protect against a number of viruses, including alphaviruses. Despite signaling through a shared receptor, there are established biochemical and functional differences among the IFN subtypes. The significance of our research is in demonstrating that IFN-α and IFN-ß both have protective roles during acute chikungunya virus (CHIKV) infection but do so by distinct mechanisms. IFN-α limits CHIKV replication and dissemination, whereas IFN-ß protects from CHIKV pathogenesis by limiting inflammation mediated by neutrophils. Our findings support the premise that the IFN subtypes have distinct biological activities in the antiviral response.

Dermal and muscle fibroblasts and skeletal myofibers survive chikungunya virus infection and harbor persistent RNA.

Chikungunya virus (CHIKV) is an arthritogenic alphavirus that acutely causes fever as well as severe joint and muscle pain. Chronic musculoskeletal pain persists in a substantial fraction of patients for months to years after the initial infection, yet we still have a poor understanding of what promotes chronic disease. While replicating virus has not been detected in joint-associated tissues of patients with persistent arthritis nor in various animal models at convalescent time points, viral RNA is detected months after acute infection. To identify the cells that might contribute to pathogenesis during this chronic phase, we developed a recombinant CHIKV that expresses Cre recombinase (CHIKV-3'-Cre). CHIKV-3'-Cre replicated in myoblasts and fibroblasts, and it induced arthritis during the acute phase in mice. Importantly, it also induced chronic disease, including persistent viral RNA and chronic myositis and synovitis similar to wild-type virus. CHIKV-3'-Cre infection of tdTomato reporter mice resulted in a population of tdTomato+ cells that persisted for at least 112 days. Immunofluorescence and flow cytometric profiling revealed that these tdTomato+ cells predominantly were myofibers and dermal and muscle fibroblasts. Treatment with an antibody against Mxra8, a recently defined host receptor for CHIKV, reduced the number of tdTomato+ cells in the chronic phase and diminished the levels of chronic viral RNA, implicating these tdTomato+ cells as the reservoir of chronic viral RNA. Finally, isolation and flow cytometry-based sorting of the tdTomato+ fibroblasts from the skin and ankle and analysis for viral RNA revealed that the tdTomato+ cells harbor most of the persistent CHIKV RNA at chronic time points. Therefore, this CHIKV-3'-Cre and tdTomato reporter mouse system identifies the cells that survive CHIKV infection in vivo and are enriched for persistent CHIKV RNA. This model represents a useful tool for studying CHIKV pathogenesis in the acute and chronic stages of disease.

Therapy with CTLA4-Ig and an antiviral monoclonal antibody controls chikungunya virus arthritis.

In 2013, chikungunya virus (CHIKV) transmission was documented in the Western Hemisphere, and the virus has since spread throughout the Americas with more than 1.8 million people infected in more than 40 countries. CHIKV targets the joints, resulting in symmetric polyarthritis that clinically mimics rheumatoid arthritis and can endure for months to years. At present, no approved treatment is effective in preventing or controlling CHIKV infection or disease. We treated mice with eight different disease-modifying antirheumatic drugs and identified CLTA4-Ig (abatacept) and tofacitinib as candidate therapies based on their ability to decrease acute joint swelling. CTLA4-Ig reduced T cell accumulation in the joints of infected animals without affecting viral infection. Whereas monotherapy with CTLA4-Ig or a neutralizing anti-CHIKV human monoclonal antibody provided partial clinical improvement, therapy with both abolished swelling and markedly reduced levels of chemokines, proinflammatory cytokines, and infiltrating leukocytes. Thus, combination CTLA4-Ig and antiviral antibody therapy controls acute CHIKV infection and arthritis and may be a candidate for testing in humans.

Bhlhe40 controls cytokine production by T cells and is essential for pathogenicity in autoimmune neuroinflammation.

TH1 and TH17 cells mediate neuroinflammation in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Pathogenic TH cells in EAE must produce the pro-inflammatory cytokine granulocyte-macrophage colony stimulating factor (GM-CSF). TH cell pathogenicity in EAE is also regulated by cell-intrinsic production of the immunosuppressive cytokine interleukin 10 (IL-10). Here we demonstrate that mice deficient for the basic helix-loop-helix (bHLH) transcription factor Bhlhe40 (Bhlhe40(-/-)) are resistant to the induction of EAE. Bhlhe40 is required in vivo in a T cell-intrinsic manner, where it positively regulates the production of GM-CSF and negatively regulates the production of IL-10. In vitro, GM-CSF secretion is selectively abrogated in polarized Bhlhe40(-/-) TH1 and TH17 cells, and these cells show increased production of IL-10. Blockade of IL-10 receptor in Bhlhe40(-/-) mice renders them susceptible to EAE. These findings identify Bhlhe40 as a critical regulator of autoreactive T-cell pathogenicity.