Specific inhibition of NLRP3 in chikungunya disease reveals a role for inflammasomes in alphavirus-induced inflammation.

Mosquito-borne viruses can cause severe inflammatory diseases and there are limited therapeutic solutions targeted specifically at virus-induced inflammation. Chikungunya virus (CHIKV), a re-emerging alphavirus responsible for several outbreaks worldwide in the past decade, causes debilitating joint inflammation and severe pain. Here, we show that CHIKV infection activates the NLRP3 inflammasome in humans and mice. Peripheral blood mononuclear cells isolated from CHIKV-infected patients showed elevated NLRP3, caspase-1 and interleukin-18 messenger RNA expression and, using a mouse model of CHIKV infection, we found that high NLRP3 expression was associated with peak inflammatory symptoms. Inhibition of NLRP3 activation using the small-molecule inhibitor MCC950 resulted in reduced CHIKV-induced inflammation and abrogated osteoclastogenic bone loss and myositis, but did not affect in vivo viral replication. Mice treated with MCC950 displayed lower expression levels of the cytokines interleukin-6, chemokine ligand 2 and tumour necrosis factor in joint tissue. Interestingly, MCC950 treatment abrogated disease signs in mice infected with a related arthritogenic alphavirus, Ross River virus, but not in mice infected with West Nile virus-a flavivirus. Here, using mouse models of alphavirus-induced musculoskeletal disease, we demonstrate that NLRP3 inhibition in vivo can reduce inflammatory pathology and that further development of therapeutic solutions targeting inflammasome function could help treat arboviral diseases.

Role of pentraxin 3 in shaping arthritogenic alphaviral disease: from enhanced viral replication to immunomodulation.

The rising prevalence of arthritogenic alphavirus infections, including chikungunya virus (CHIKV) and Ross River virus (RRV), and the lack of antiviral treatments highlight the potential threat of a global alphavirus pandemic. The immune responses underlying alphavirus virulence remain enigmatic. We found that pentraxin 3 (PTX3) was highly expressed in CHIKV and RRV patients during acute disease. Overt expression of PTX3 in CHIKV patients was associated with increased viral load and disease severity. PTX3-deficient (PTX3(-/-)) mice acutely infected with RRV exhibited delayed disease progression and rapid recovery through diminished inflammatory responses and viral replication. Furthermore, binding of the N-terminal domain of PTX3 to RRV facilitated viral entry and replication. Thus, our study demonstrates the pivotal role of PTX3 in shaping alphavirus-triggered immunity and disease and provides new insights into alphavirus pathogenesis.

A Systematic Meta-analysis of Immune Signatures in Patients With Acute Chikungunya Virus Infection.

BACKGROUND: Individuals infected with chikungunya virus (CHIKV) normally exhibit a variety of clinical manifestations during the acute phase of infection. However, studies in different patient cohorts have revealed that disease manifestations vary in frequency. METHODS: Disease profiles between patients with acute CHIKV-infection and febrile patients without CHIKV were compared and examined to determine whether any clinical presentations were associated with the clinical outcome of CHIKV infection. Circulatory immune mediators profiles were then characterized and compared with data from 14 independent patient cohort studies. The particular immune mediator signature that defines acute CHIKV infection was determined. RESULTS: Our findings revealed a specific pattern of clinical presentations of joint-specific arthralgia from this CHIKV cohort. More importantly, we identified an immune mediator signature dominated by proinflammatory cytokines, which include interferon α and γ and interleukin 2, 2R, 6, 7, 12, 15, 17, and 18, across different patient cohorts of CHIKV load associated with arthralgia. CONCLUSIONS: To our knowledge, this is the first study that associated levels of CHIKV load with arthralgia as an indicator of acute CHIKV infection. Importantly, our findings also revealed specific immune mediator signatures that can be used to better define CHIKV infection.

Loss of TLR3 aggravates CHIKV replication and pathology due to an altered virus-specific neutralizing antibody response.

RNA-sensing toll-like receptors (TLRs) mediate innate immunity and regulate anti-viral response. We show here that TLR3 regulates host immunity and the loss of TLR3 aggravates pathology in Chikungunya virus (CHIKV) infection. Susceptibility to CHIKV infection is markedly increased in human and mouse fibroblasts with defective TLR3 signaling. Up to 100-fold increase in CHIKV load was observed in Tlr3-/- mice, alongside increased virus dissemination and pro-inflammatory myeloid cells infiltration. Infection in bone marrow chimeric mice showed that TLR3-expressing hematopoietic cells are required for effective CHIKV clearance. CHIKV-specific antibodies from Tlr3-/- mice exhibited significantly lower in vitro neutralization capacity, due to altered virus-neutralizing epitope specificity. Finally, SNP genotyping analysis of CHIKF patients on TLR3 identified SNP rs6552950 to be associated with disease severity and CHIKV-specific neutralizing antibody response. These results demonstrate a key role for TLR3-mediated antibody response to CHIKV infection, virus replication and pathology, providing a basis for future development of immunotherapeutics in vaccine development.

Viperin restricts chikungunya virus replication and pathology.

Chikungunya virus (CHIKV) is a mosquito-borne arthralgia arbovirus that is reemergent in sub-Saharan Africa and Southeast Asia. CHIKV infection has been shown to be self-limiting, but the molecular mechanisms of the innate immune response that control CHIKV replication remain undefined. Here, longitudinal transcriptional analyses of PBMCs from a cohort of CHIKV-infected patients revealed that type I IFNs controlled CHIKV infection via RSAD2 (which encodes viperin), an enigmatic multifunctional IFN-stimulated gene (ISG). Viperin was highly induced in monocytes, the major target cell of CHIKV in blood. Anti-CHIKV functions of viperin were dependent on its localization in the ER, and the N-terminal amphipathic α-helical domain was crucial for its antiviral activity in controlling CHIKV replication. Furthermore, mice lacking Rsad2 had higher viremia and severe joint inflammation compared with wild-type mice. Our data demonstrate that viperin is a critical antiviral host protein that controls CHIKV infection and provide a preclinical basis for the design of effective control strategies against CHIKV and other reemerging arthrogenic alphaviruses.

Longitudinal analysis of the human antibody response to Chikungunya virus infection: implications for serodiagnosis and vaccine development.

Chikungunya virus (CHIKV) is an alphavirus which causes chronic and incapacitating arthralgia in humans. Although previous studies have shown that antibodies against the virus are produced during and after infection, the fine specificity of the antibody response against CHIKV is not known. Here, using plasma from patients at different times postinfection, we characterized the antibody response against various proteins of the virus. We have shown that the E2 and E3 glycoproteins and the capsid and nsP3 proteins are targets of the anti-CHIKV antibody response. Moreover, we have identified the different regions in these proteins which contain the linear epitopes recognized by the anti-CHIKV antibodies and determined their structural localization. Data also illustrated the effect of a single K(252)Q amino acid change at the E2 glycoprotein that was able to influence antibody binding and interaction between the antibodies and epitope because of the changes of epitope-antibody binding capacity. This study provides important knowledge that will not only aid in the understanding of the immune response to CHIKV infection but also provide new knowledge in the design of modern vaccine development. Furthermore, these pathogen-specific epitopes could be used for future seroepidemiological studies that will unravel the molecular mechanisms of human immunity and protection from CHIKV disease.

Early appearance of neutralizing immunoglobulin G3 antibodies is associated with chikungunya virus clearance and long-term clinical protection.

BACKGROUND: Chikungunya virus (CHIKV) and related arboviruses have been responsible for large epidemic outbreaks with serious economic and social impact. Although infected individuals clear the virus from the blood, some develop debilitating and prolonged arthralgia. METHODS: We investigated specificity and strength of antibody responses in a longitudinal study on CHIKV-infected patients and analyzed their association with viral load, cytokine profile, and severity. RESULTS: We found that CHIKV-specific response is dominated by immunoglobulin G3 (IgG3) antibodies. The antibodies were neutralizing, and patients with high viremia rapidly developed high levels of anti-CHIKV antibodies of this specific isotype. Although these patients endured a more severe disease progression during the acute viremic phase, they cleared the virus faster and did not experience persistent arthralgia. However, significant persistent arthralgia was observed in patients with low viremia who developed IgG3 at a later stage. CONCLUSIONS: Absence of early CHIKV-specific IgG3 may therefore serve as a specific marker of patients with increased risk of disease.

Stat3 promotes directional cell migration by regulating Rac1 activity via its activator betaPIX.

Stat3 is a member of the signal transducer and activator of transcription family, which is important for cytokine signaling as well as for a number of cellular processes including cell proliferation, anti-apoptosis and immune responses. In recent years, evidence has emerged suggesting that Stat3 also participates in cell invasion and motility. However, how Stat3 regulates these processes remains poorly understood. Here, we find that loss of Stat3 expression in mouse embryonic fibroblasts leads to an elevation of Rac1 activity, which promotes a random mode of migration by reducing directional persistence and formation of actin stress fibers. Through rescue experiments, we demonstrate that Stat3 can regulate the activation of Rac1 to mediate persistent directional migration and that this function is not dependent on Stat3 transcriptional activity. We find that Stat3 binds to betaPIX, a Rac1 activator, and that this interaction could represent a mechanism by which cytoplasmic Stat3 regulates Rac1 activity to modulate the organization of actin cytoskeleton and directional migration.