A storm is born: immune activation and pathogenesis in arthritogenic alphavirus infections.

Arthritogenic alphaviruses are mosquito-borne arboviruses that include several re-emerging human pathogens, including the chikungunya (CHIKV), Ross River (RRV), Mayaro (MAYV), and o'nyong-nyong (ONNV) virus. Arboviruses are transmitted via a mosquito bite to the skin. Herein, we describe intradermal RRV infection in a mouse model that replicates the arthritis and myositis seen in humans with Ross River virus disease (RRVD). We show that skin infection with RRV results in the recruitment of inflammatory monocytes and neutrophils, which together with dendritic cells migrate to draining lymph nodes (LN) of the skin. Neutrophils and monocytes are productively infected and traffic virus from the skin to LN. We show that viral envelope N-linked glycosylation is a key determinant of skin immune responses and disease severity. RRV grown in mammalian cells elicited robust early antiviral responses in the skin, while RRV grown in mosquito cells stimulated poorer early antiviral responses. We used glycan mass spectrometry to characterize the glycan profile of mosquito and mammalian cell-derived RRV, showing deglycosylation of the RRV E2 glycoprotein is associated with curtailed skin immune responses and reduced disease following intradermal infection. Altogether, our findings demonstrate skin infection with an arthritogenic alphavirus leads to musculoskeletal disease and envelope glycoprotein glycosylation shapes disease outcome. IMPORTANCE Arthritogenic alphaviruses are transmitted via mosquito bites through the skin, potentially causing debilitating diseases. Our understanding of how viral infection starts in the skin and how virus systemically disseminates to cause disease remains limited. Intradermal arbovirus infection described herein results in musculoskeletal pathology, which is dependent on viral envelope N-linked glycosylation. As such, intradermal infection route provides new insights into how arboviruses cause disease and could be extended to future investigations of skin immune responses following infection with other re-emerging arboviruses.

Amongst the arthritis-causing arboviruses, i.e. those spread by insects, the alphavirus group is of special interest. These viruses occasionally cause vast outbreaks, such as O'nyong-nyong in Africa in 1959. In Fennoscandia, Sindbis-related Ockelbo, Pogosta, or Karelian fever viruses have been found to cause significant morbidity. The major symptoms in addition to joint inflammation are fever, fatigue, headache and rash. The joint symptoms may persist for weeks, even months. The diagnosis is based on the clinical picture and serology. The causative viruses are closely related but not identical. It appears that at least in Finland the Pogosta disease is more common than thought, and the symptoms may often be overlooked. Several factors related to the viruses, their hosts, and global environmental changes may affect the spread of these viruses. All over the world arbovirus-caused diseases have increased, because of global changes.

Arthritogenic alphaviruses, such as Ross River virus, chikungunya virus and O’nyong-nyong virus, cause endemic disease globally and are a major public health concern. The hallmarks of arthritogenic alphavirus disease are debilitating pain, and potentially chronic inflammation of the muscles, thus influencing quality of life. The type I IFN response is a major component of the innate immune response against arthritogenic alphaviruses, and is essential in inhibiting viral replication and dissemination. Type I IFNs are induced during early stages of infection and are essential for the activation of the antiviral innate immune response. They also link the innate immune response and the activation of adaptive immunity. This review focuses on the host immune response, particularly that involving type I IFN, in arthritogenic alphavirus disease.

Human mAbs Broadly Protect against Arthritogenic Alphaviruses by Recognizing Conserved Elements of the Mxra8 Receptor-Binding Site

Arthritogenic alphaviruses, including chikungunya virus (CHIKV), Mayaro virus (MAYV), Ross River virus (RRV), and O’nyong nyong virus (ONNV) are emerging and reemerging viruses that cause disease characterized by fever, rash, and incapacitating joint swelling. Alphavirus infection induces robust immune responses in infected hosts, leading to the upregulation of several cytokines and chemokines, including chemokine C ligand 4 (CCL4). CCL4 is a chemoattractant for immune cells such as T cells, natural killer cells, monocytes/macrophages, and dendritic cells, recruiting these cells to the site of infection, stimulating the release of proinflammatory mediators, and inducing T cell differentiation. CCL4 has been found at high levels in both the acute and chronic phases of chikungunya disease; however, the role of CCL4 in arthritogenic alphavirus disease development remains unexplored. Here, we tested the effect of CCL4 on MAYV infection in mice through antibody depletion and treatment with recombinant mouse CCL4. We observed no differences in mice depleted of CCL4 or treated with recombinant CCL4 in terms of disease progression such as weight loss and footpad swelling or the development of viremia. CCL4 uses the G protein-coupled receptor C-C chemokine receptor type 5 (CCR5). To determine whether CCR5 deficiency would alter disease outcomes or virus replication in mice, we inoculated CCR5 knockout (CCR5−/−) mice with MAYV and observed no effect on disease development and immune cell profile of blood and footpads between CCR5−/− and wild type mice. These studies failed to identify a clear role for CCL4 or its receptor CCR5 in MAYV infection.

Arthritogenic alphaviruses, such as Ross River virus (RRV) and chikungunya virus (CHIKV), particularly affect joints of the extremities and can lead to debilitating and potentially chronic polyarthritis/polyarthralgia. The innate immune response of the host plays a crucial role in inducing proinflammatory host factors, leading to tissue destruction and bone loss in the joints. This study was performed to assess how the inhibition of interleukin-1β (IL-1β) signaling using the clinical rheumatoid arthritis drug anakinra influences bone loss in mice with arthritogenic alphavirus infections. Mice (n = 5 per group) were infected with RRV or CHIKV and then treated with anakinra. Weight gain and disease severity were measured, tissue viral titers were determined, and histologic changes in joint tissues were assessed. Anakinra therapy reduced RRV- and CHIKV-induced bone loss in this murine model (P < 0.001 and P < 0.05, respectively). Histologic analysis of the knee joint showed that treatment with anakinra decreased epiphyseal growth plate thinning, loss of epiphyseal bone volume, and osteoclastogenesis in the tibia. Importantly, pharmacologic IL-1 receptor (IL-1R) blockade did not improve other clinical features, including disease score, weight loss, or viremia. The present findings suggest that anakinra therapy may reduce bone loss in experimental murine models of RRV and CHIKV. Further investigations are needed to assess the potential therapeutic benefits of anakinra in patients with arthritogenic alphavirus disease.

Chikungunya virus (CHIKV) and Ross River virus (RRV) cause a debilitating, and often chronic, musculoskeletal inflammatory disease in humans. Macrophages constitute the major inflammatory infiltrates in musculoskeletal tissues during these infections. However, the precise macrophage effector functions that affect the pathogenesis of arthritogenic alphaviruses have not been defined. We hypothesized that the severe damage to musculoskeletal tissues observed in RRV- or CHIKV-infected mice would promote a wound-healing response characterized by M2-like macrophages. Indeed, we found that RRV- and CHIKV-induced musculoskeletal inflammatory lesions, and macrophages present in these lesions, have a unique gene-expression pattern characterized by high expression of arginase 1 and Ym1/Chi3l3 in the absence of FIZZ1/Relmα that is consistent with an M2-like activation phenotype. Strikingly, mice specifically deleted for arginase 1 in neutrophils and macrophages had dramatically reduced viral loads and improved pathology in musculoskeletal tissues at late times post-RRV infection. These findings indicate that arthritogenic alphavirus infection drives a unique myeloid cell activation program in inflamed musculoskeletal tissues that inhibits virus clearance and impedes disease resolution in an arginase 1-dependent manner.

Ross River virus (RRV) and other alphaviruses cause chronic musculoskeletal syndromes that are associated with viral persistence, which suggests deficits in immune clearance mechanisms, including CD8+ T-cell responses. Here, we used a recombinant RRV-gp33 that expresses the immunodominant CD8+ T-cell epitope of lymphocytic choriomeningitis virus (LCMV) to directly compare responses with a virus, LCMV, that strongly induces antiviral CD8+ T cells. After footpad injection, we detected fewer gp33-specific CD8+ T cells in the draining lymph node (DLN) after RRV-gp33 than LCMV infection, despite similar viral RNA levels in the foot. However, less RRV RNA was detected in the DLN compared to LCMV, with RRV localizing principally to the subcapsular region and LCMV to the paracortical T-cell zones. Single-cell RNA-sequencing analysis of adoptively transferred gp33-specific transgenic CD8+ T cells showed rapid differentiation into effector cells after LCMV but not RRV infection. This defect in RRV-specific CD8+ T effector cell maturation was corrected by local blockade of type I interferon (IFN) signaling, which also resulted in increased RRV infection in the DLN. Studies in Wdfy4-/-, CD11c-Cre B2mfl/fl, or Xcr1-Cre Ifnar1fl/fl mice that respectively lack cross-presenting capacity, MHC-I antigen presentation by dendritic cells (DCs), or type I IFN signaling in the DC1 subset show that RRV-specific CD8+ T-cell responses can be improved by enhanced direct antigen presentation by DCs. Overall, our experiments suggest that antiviral type I IFN signaling in DCs limits direct alphavirus infection and antigen presentation, which likely delays CD8+ T-cell responses.IMPORTANCEChronic arthritis and musculoskeletal disease are common outcomes of infections caused by arthritogenic alphaviruses, including Ross River virus (RRV), due to incomplete virus clearance. Unlike other viral infections that are efficiently cleared by cytotoxic CD8+ T cells, RRV infection is surprisingly unaffected by CD8+ T cells as mice lacking or having these cells show similar viral persistence in joint and lymphoid tissues. To elucidate the basis for this deficient response, we measured the RRV-specific CD8+ T-cell population size and activation state relative to another virus known to elicit a strong T-cell response. Our findings reveal that RRV induces fewer CD8+ T cells due to limited infection of immune cells in the draining lymph node. By increasing RRV susceptibility in antigen-presenting cells, we elicited a robust CD8+ T-cell response. These results highlight antigen availability and virus tropism as possible targets for intervention against RRV immune evasion and persistence.

Worldwide, mosquito-borne alphaviruses are a major cause of infectious arthritis-like disease, but this has not yet significantly impacted the Americas (1). The arthritogenic alphaviruses include Ross River virus (RRV), chikungunya virus (CHIKV), Sindbis-like viruses (SINV), Barmah Forest virus (BFV), Mayaro virus (MAYV), and o’nyong-nyong virus (ONNV), which are typically found from Europe to central Africa, southern Asia, and in portions of northern South America (2). Infection with these viruses begins as a febrile illness, which in a high percentage of cases, can progress to rheumatic disease, which has been described as polyyarthralgia and/or polyarthritis and can be chronic, flaring, and potentially debilitating (2). In PNAS, Chen et al. (3) present evidence that joint pain and associated symptoms of arthritogenic alphavirus infection (in this case a mouse model of RRV) are strikingly similar to a classical arthritis manifestation, exhibiting cytokine induction profiles reminiscent of rheumatoid arthritis and disruption of osteoblast/osteoclast homeostasis, leading to bone loss.

Arthritogenic alphaviruses such as Ross River and Chikungunya viruses cause debilitating muscle and joint pain and pose significant challenges in the light of recent outbreaks. How host immune responses are orchestrated after alphaviral infections and lead to musculoskeletal inflammation remains poorly understood. Here, we show that myositis induced by Ross River virus (RRV) infection is driven by CD11bhi Ly6Chi inflammatory monocytes and followed by the establishment of a CD11bhi Ly6Clo CX3CR1+ macrophage population in the muscle upon recovery. Selective modulation of CD11bhi Ly6Chi monocyte migration to infected muscle using immune-modifying microparticles (IMP) reduced disease score, tissue damage, and inflammation and promoted the accumulation of CX3CR1+ macrophages, enhancing recovery and resolution. Here, we detail the role of immune pathology, describing a poorly characterized muscle macrophage subset as part of the dynamics of alphavirus-induced myositis and tissue recovery and identify IMP as an effective immunomodulatory approach. Given the lack of specific treatments available for alphavirus-induced pathologies, this study highlights a therapeutic potential for simple immune modulation by IMP in infected individuals in the event of large alphavirus outbreaks.IMPORTANCE Arthritogenic alphaviruses cause debilitating inflammatory disease, and current therapies are restricted to palliative approaches. Here, we show that following monocyte-driven muscle inflammation, tissue recovery is associated with the accumulation of CX3CR1+ macrophages in the muscle. Modulating inflammatory monocyte infiltration using immune-modifying microparticles (IMP) reduced tissue damage and inflammation and enhanced the formation of tissue repair-associated CX3CR1+ macrophages in the muscle. This shows that modulating key effectors of viral inflammation using microparticles can alter the outcome of disease by facilitating the accumulation of macrophage subsets associated with tissue repair.

Arthritogenic alphaviruses have infected millions of people globally. Following acute infection, symptoms of joint pain, swelling, and stiffness can last for months or even years, likely due to persistence of viral RNA and antigen, which also is seen in mice. Intriguingly, wild-type and CD8−/− mice show a similar lack of clearance of viral RNA. Despite this, a mechanism for this CD8+ T cell evasion has not been identified. We hypothesized that there are defects in the generation, function, and migration of the CD8+ T cells after infection with Ross River virus (RRV), a prototype, arthritogenic alphavirus. To study the antigen-specific response, we used a genetically modified RRV expressing the lymphocytic choriomeningitis virus (LCMV)-derived gp33 peptide. Compared to LCMV infection, in which CD8+ T cells are required for viral clearance, RRV infection generates fewer gp33-specific CD8+ T cells in the spleen and draining lymph node, especially at early times after infection. Antigen-specific CD8+ T cells from the draining lymph node at 5 days after RRV infection express much less granzyme B than in LCMV-infected mice. In vivo cytolysis assays showed a qualitative difference in CD8+ T cell killing of targets especially when variant peptides were used for pulsing. Finally, intravital imaging revealed that while both RRV and LCMV infection recruit CD8+ T cells and retain them in an antigen specific manner near infected cells, CD8+ T cells appear to have less contact with RRV than LCMV-infected cells. Identification of a specific defect in the CD8+ T cell response to arthritogenic alphaviruses would increase our understanding of viral immune evasion and provide a target for therapy of these debilitating infections.

Chikungunya (CHIKV) and Mayaro (MAYV) viruses are arthritogenic alphaviruses that promote an incapacitating and long-lasting inflammatory muscle-articular disease. Despite studies pointing out the importance of skeletal muscle (SkM) in viral pathogenesis, the long-term consequences on its physiology and the mechanism of persistence of symptoms are still poorly understood. Combining molecular, morphological, nuclear magnetic resonance imaging, and histological analysis, we conduct a temporal investigation of CHIKV and MAYV replication in a wild-type mice model, focusing on the impact on SkM composition, structure, and repair in the acute and late phases of infection. We found that viral replication and induced inflammation promote a rapid loss of muscle mass and reduction in fiber cross-sectional area by upregulation of muscle-specific E3 ubiquitin ligases MuRF1 and Atrogin-1 expression, both key regulators of SkM fibers atrophy. Despite a reduction in inflammation and clearance of infectious viral particles, SkM atrophy persists until 30 days post-infection. The genomic CHIKV and MAYV RNAs were still detected in SkM in the late phase, along with the upregulation of chemokines and anti-inflammatory cytokine expression. In agreement with the involvement of inflammatory mediators on induced atrophy, the neutralization of TNF and a reduction in oxidative stress using monomethyl fumarate, an agonist of Nrf2, decreases atrogen expression and atrophic fibers while increasing weight gain in treated mice. These data indicate that arthritogenic alphavirus infection could chronically impact body SkM composition and also harm repair machinery, contributing to a better understanding of mechanisms of arthritogenic alphavirus pathogenesis and with a description of potentially new targets of therapeutic intervention.

Chikungunya (CHIKV) and Mayaro (MAYV) viruses are arthritogenic alphaviruses that promote an incapacitating and long-lasting inflammatory muscle-articular disease. Despite studies pointing out the importance of skeletal muscle (SkM) in viral pathogenesis, the long-term consequences on its physiology after acute injury and the mechanism of persistence of symptoms are still poorly understood. Combining molecular, morphological, Nuclear Magnetic Resonance Imaging, and histological analysis, we conduct a temporal investigation of CHIKV and MAYV replication in a wild-type mice model, focusing on the impact on SkM composition, structure, and repair in the acute and late phases of infection. We found that viral replication and induced-inflammation promote fast loss of muscle mass and reduction of fibers cross sectional area, by inducing muscle-specific E3 ubiquitin ligases MuRF1 and Atrogin expression, that trigger protein breakdown, resulting in acute SkM fibers atrophy. After temporal reduction of inflammation and clearance of infectious viral particles, SkM atrophy persists until the late phase of infection. The genomic CHIKV and MAYV RNAs were still detected in SkM at the late phase, along with increased levels of chemokines and anti-inflammatory cytokines expression. In agreement with the involvement of inflammatory mediators on induced atrophy, neutralization of TNF and the reduction of oxidative stress using monomethyl fumarate, an agonist of Nrf2, decreases atrogens expression and atrophic fibers, while increasing weight gain in treated mice. Taken together, these data indicate that arthritogenic alphavirus infection could chronically impact body SkM composition and also harm repair machinery, contributing to a better understanding of mechanisms of arthritogenic alphaviruses pathogenesis and with description of potentially new targets of therapeutic intervention.

Arthritogenic alphaviruses cause disease characterized by fever, rash, and incapacitating joint pain. Alphavirus infection stimulates robust inflammatory responses in infected hosts, leading to the upregulation of several cytokines, including granulocyte colony-stimulating factor (G-CSF). G-CSF is secreted by endothelial cells, fibroblasts, macrophages, and monocytes and binds to colony stimulating factor 3 receptor (CSF3R, also known as G-CSFR) on the surface of myeloid cells. G-CSFR signaling initiates proliferation, differentiation, and maturation of myeloid cells, especially neutrophils. Importantly, G-CSF has been found at high levels in both the acute and chronic phases of chikungunya disease; however, the role of G-CSF in arthritogenic alphavirus disease remains unexplored. Here, we sought to test the effect of G-CSF on chikungunya virus (CHIKV) and Mayaro virus (MAYV) infection using G-CSFR-deficient mice (G-CSFR-/-). We observed sustained weight loss in G-CSFR-/- mice following viand MAYV infection compared to wild-type mice. Furthermore, G-CSFR-/- mice had a significantly higher percentage of inflammatory monocytes and reduction in neutrophils throughout infection. The difference in weight loss in G-CSFR-/- mice induced by alphavirus infection was corrected by blocking type I IFN signaling. In summary, these studies show that type I IFN signaling contributes to G-CSFR mediated control of arthritogenic alphavirus disease.

Arthritogenic alphaviruses, including Ross River virus (RRV) and chikungunya virus (CHIKV), are responsible for explosive epidemics involving millions of cases. These mosquito-transmitted viruses cause inflammation and injury in skeletal muscle and joint tissues that results in debilitating pain. We previously showed that arginase 1 (Arg1) was highly expressed in myeloid cells in the infected and inflamed musculoskeletal tissues of RRV- and CHIKV-infected mice, and specific deletion of Arg1 from myeloid cells resulted in enhanced viral control. Here, we show that Arg1, along with other genes associated with suppressive myeloid cells, is induced in PBMCs isolated from CHIKV-infected patients during the acute phase as well as the chronic phase, and that high Arg1 expression levels were associated with high viral loads and disease severity. Depletion of both CD4 and CD8 T cells from RRV-infected Arg1-deficient mice restored viral loads to levels detected in T cell-depleted wild-type mice. Moreover, Arg1-expressing myeloid cells inhibited virus-specific T cells in the inflamed and infected musculoskeletal tissues, but not lymphoid tissues, following RRV infection in mice, including suppression of interferon-γ and CD69 expression. Collectively, these data enhance our understanding of the immune response following arthritogenic alphavirus infection and suggest that immunosuppressive myeloid cells may contribute to the duration or severity of these debilitating infections.