Interleukin-17 contributes to Ross River virus-induced arthritis and myositis.

Arthritogenic alphaviruses are mosquito-borne viruses that are a major cause of infectious arthropathies worldwide, and recent outbreaks of chikungunya virus and Ross River virus (RRV) infections highlight the need for robust intervention strategies. Alphaviral arthritis can persist for months after the initial acute disease, and is mediated by cellular immune responses. A common strategy to limit inflammation and pathology is to dampen the overwhelming inflammatory responses by modulating proinflammatory cytokine pathways. Here, we investigate the contribution of interleukin-17 (IL-17), a cytokine involved in arthropathies such as rheumatoid arthritis, in the development RRV-induced arthritis and myositis. IL-17 was quantified in serum from RRV-infected patients, and mice were infected with RRV and joints and muscle tissues collected to analyse cellular infiltrates, tissue mRNA, cytokine expression, and joint and muscle histopathology. IL-17 expression was increased in musculoskeletal tissues and serum of RRV-infected mice and humans, respectively. IL-17-producing T cells and neutrophils contributed to the cellular infiltrate in the joint and muscle tissue during acute RRV disease in mice. Blockade of IL-17A/F using a monoclonal antibody (mAb) reduced disease severity in RRV-infected mice and led to decreased proinflammatory proteins, cellular infiltration in synovial tissues and cartilage damage, without affecting viral titers in inflamed tissues. IL-17A/F blockade triggered a shift in transcriptional profile of both leukocyte infiltrates and musculoskeletal stromal cells by downregulating proinflammatory genes. This study highlights a previously uncharacterized role for an effector cytokine in alphaviral pathology and points towards potential therapeutic benefit in targeting IL-17 to treat patients presenting with RRV-induced arthropathy.

N-Linked Glycans Shape Skin Immune Responses during Arthritis and Myositis after Intradermal Infection with Ross River Virus.

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.

Anti-inflammatory actions of Pentosan polysulfate sodium in a mouse model of influenza virus A/PR8/34-induced pulmonary inflammation.

Introduction: There is an unmet medical need for effective anti-inflammatory agents for the treatment of acute and post-acute lung inflammation caused by respiratory viruses. The semi-synthetic polysaccharide, Pentosan polysulfate sodium (PPS), an inhibitor of NF-kB activation, was investigated for its systemic and local anti-inflammatory effects in a mouse model of influenza virus A/PR8/1934 (PR8 strain) mediated infection. Methods: Immunocompetent C57BL/6J mice were infected intranasally with a sublethal dose of PR8 and treated subcutaneously with 3 or 6 mg/kg PPS or vehicle. Disease was monitored and tissues were collected at the acute (8 days post-infection; dpi) or post-acute (21 dpi) phase of disease to assess the effect of PPS on PR8-induced pathology. Results: In the acute phase of PR8 infection, PPS treatment was associated with a reduction in weight loss and improvement in oxygen saturation when compared to vehicle-treated mice. Associated with these clinical improvements, PPS treatment showed a significant retention in the numbers of protective SiglecF+ resident alveolar macrophages, despite uneventful changes in pulmonary leukocyte infiltrates assessed by flow cytometry. PPS treatment in PR8- infected mice showed significant reductions systemically but not locally of the inflammatory molecules, IL-6, IFN-g, TNF-a, IL-12p70 and CCL2. In the post-acute phase of infection, PPS demonstrated a reduction in the pulmonary fibrotic biomarkers, sICAM-1 and complement factor C5b9. Discussion: The systemic and local anti-inflammatory actions of PPS may regulate acute and post-acute pulmonary inflammation and tissue remodeling mediated by PR8 infection, which warrants further investigation.

A booster regime of liposome-delivered live-attenuated CHIKV vaccine RNA genome protects against chikungunya virus disease in mice.

Mosquito-transmitted chikungunya virus (CHIKV) is the causal pathogen of CHIKV disease and is responsible for global epidemics of arthritic disease. CHIKV infection can lead to severe chronic and debilitating arthralgia, significantly impacting patient mobility and quality of life. Our previous studies have shown a live-attenuated CHIKV vaccine candidate, CHIKV-NoLS, to be effective in protecting against CHIKV disease in mice vaccinated with one dose. Further studies have demonstrated the value of a liposome RNA delivery system to deliver the RNA genome of CHIKV-NoLS directly in vivo, promoting de novo production of live-attenuated vaccine particles in vaccinated hosts. This system, designed to bypass live-attenuated vaccine production bottlenecks, uses CAF01 liposomes. However, one dose of CHIKV-NoLS CAF01 failed to provide systemic protection against CHIKV challenge in mice, with low levels of CHIKV-specific antibodies. Here we describe CHIKV-NoLS CAF01 booster vaccination regimes designed to increase vaccine efficacy. C57BL/6 mice were vaccinated with three doses of CHIKV-NoLS CAF01 either intramuscularly or subcutaneously. CHIKV-NoLS CAF01 vaccinated mice developed a systemic immune response against CHIKV that shared similarity to vaccination with CHIKV-NoLS, including high levels of CHIKV-specific neutralising antibodies in subcutaneously inoculated mice. CHIKV-NoLS CAF01 vaccinated mice were protected against disease signs and musculoskeletal inflammation when challenged with CHIKV. Mice given one dose of live-attenuated CHIKV-NoLS developed a long lasting protective immune response for up to 71 days. A clinically relevant CHIKV-NoLS CAF01 booster regime can overcome the challenges faced by our previous one dose strategy and provide systemic protection against CHIKV disease.

Macrophage ACE2 is necessary for SARS-CoV-2 replication and subsequent cytokine responses that restrict continued virion release.

Macrophages are key cellular contributors to the pathogenesis of COVID-19, the disease caused by the virus SARS-CoV-2. The SARS-CoV-2 entry receptor ACE2 is present only on a subset of macrophages at sites of SARS-CoV-2 infection in humans. Here, we investigated whether SARS-CoV-2 can enter macrophages, replicate, and release new viral progeny; whether macrophages need to sense a replicating virus to drive cytokine release; and, if so, whether ACE2 is involved in these mechanisms. We found that SARS-CoV-2 could enter, but did not replicate within, ACE2-deficient human primary macrophages and did not induce proinflammatory cytokine expression. By contrast, ACE2 overexpression in human THP-1-derived macrophages permitted SARS-CoV-2 entry, processing and replication, and virion release. ACE2-overexpressing THP-1 macrophages sensed active viral replication and triggered proinflammatory, antiviral programs mediated by the kinase TBK-1 that limited prolonged viral replication and release. These findings help elucidate the role of ACE2 and its absence in macrophage responses to SARS-CoV-2 infection.

The Delta SARS-CoV-2 Variant of Concern Induces Distinct Pathogenic Patterns of Respiratory Disease in K18-hACE2 Transgenic Mice Compared to the Ancestral Strain from Wuhan.

Compared to the original ancestral strain of SARS-CoV-2, the Delta variant of concern has shown increased transmissibility and resistance toward COVID-19 vaccines and therapies. However, the pathogenesis of the disease associated with Delta is still not clear. In this study, using K18-hACE2 transgenic mice, we assessed the pathogenicity of the Delta variant by characterizing the immune response following infection. We found that Delta induced the same clinical disease manifestations as the ancestral SARS-CoV-2, but with significant dissemination to multiple tissues, such as brain, intestine, and kidney. Histopathological analysis showed that tissue pathology and cell infiltration in the lungs of Delta-infected mice were the same as in mice infected with the ancestral SARS-CoV-2. Delta infection caused perivascular inflammation in the brain and intestinal wall thinning in K18-hACE2 transgenic mice. Increased cell infiltration in the kidney was observed in both ancestral strain- and Delta-infected mice, with no clear visible tissue damage identified in either group. Interestingly, compared with mice infected with the ancestral strain, the numbers of CD45+ cells, T cells, B cells, inflammatory monocytes, and dendritic cells were all significantly lower in the lungs of the Delta-infected mice, although there was no significant difference in the levels of proinflammatory cytokines between the two groups. Our results showed distinct immune response patterns in the lungs of K18-hACE2 mice infected with either the ancestral SARS-CoV-2 or Delta variant of concern, which may help to guide therapeutic interventions for emerging SARS-CoV-2 variants. IMPORTANCE SARS-CoV-2 variants, with the threat of increased transmissibility, infectivity, and immune escape, continue to emerge as the COVID-19 pandemic progresses. Detailing the pathogenesis of disease caused by SARS-CoV-2 variants, such as Delta, is essential to better understand the clinical threat caused by emerging variants and associated disease. This study, using the K18-hACE2 mouse model of severe COVID-19, provides essential observation and analysis on the pathogenicity and immune response of Delta infection. These observations shed light on the changing disease profile associated with emerging SARS-CoV-2 variants and have potential to guide COVID-19 treatment strategies.

Interleukin-17 Contributes to Chikungunya Virus-Induced Disease.

Alphaviral arthritides caused by mosquito-borne arboviruses such as chikungunya virus (CHIKV) can persist for months after the initial acute disease. Here, we investigated the contribution of interleukin-17 (IL-17), a cytokine involved in chronic autoimmune arthropathies such as rheumatoid arthritis, to the development of alphaviral arthropathy. Sera from CHIKV-infected patients who displayed both acute and chronic disease showed high levels of IL-17, IL-6, IL-21, IL-22, and IL-23, especially during the chronic phase of disease. We sought to validate these findings using a mouse model of CHIKV infection and disease using wild-type and IL-17A-deficient mice. Mice were infected with CHIKV, and joint and muscle tissues were harvested at designated time points. Tissue infiltrates were examined by immunohistochemistry, and tissue mRNA and protein expression of cytokines was assessed. Joint and muscle pathology was assessed using histology. CHIKV-infected mice lacking IL-17A showed reduced tissue inflammation and neutrophil infiltration, compared to wild-type mice. These investigations showed a role for IL-17 in the acute phase of CHIKV infection and also during the postacute disease resolution phase. IMPORTANCE CHIKV has been prevalent in Africa, Asia, and the Indian Ocean Islands for decades. There are currently no clinically approved vaccines or specific antiviral drugs targeting CHIKV. The upregulation of IL-17 detected in CHIKV disease patients and the reduced disease seen in IL-17-deficient mice suggest a correlation between IL-17 signaling pathways and CHIKV-induced arthritic inflammation. With an established role in contributing to the pathogenesis of immune-mediated diseases, such as psoriatic arthritis and rheumatoid arthritis, IL-17 signaling plays an important role in alphavirus arthritides.

Liposomal Delivery of the RNA Genome of a Live-Attenuated Chikungunya Virus Vaccine Candidate Provides Local, but Not Systemic Protection After One Dose.

Chikungunya virus (CHIKV) is the causative pathogen of chikungunya fever, a mosquito-borne viral disease causing highly debilitating arthralgia that can persist for months and progress to chronic arthritis. Our previous studies have identified the CHIKV live-attenuated vaccine candidate CHIKV-NoLS. Like most live-attenuated vaccines, attenuated replication of CHIKV-NoLS has the potential to limit scalable production. To overcome production limits, as well as other drawbacks of live-attenuated vaccines, we developed an in vivo liposome RNA delivery system to deliver the self-replicating RNA genome of CHIKV-NoLS directly into mice, allowing the recipients' body to produce the live-attenuated vaccine particles. CAF01 liposomes were able to deliver replication-competent CHIKV-NoLS RNA in vitro. Immunodeficient AG129 mice inoculated with liposome-delivered CHIKV-NoLS RNA developed viremia and disease signs representative of this lethal model of CHIKV infection, demonstrating de novo vaccine particle production in vivo. In immunocompetent C57BL/6 mice, liposome-delivered CHIKV-NoLS RNA inoculation was associated with reduced IgM and IgG levels with low antibody CHIKV-neutralizing capacity, compared to vaccination with the original live-attenuated vaccine CHIKV-NoLS. One dose of liposome-delivered CHIKV-NoLS RNA did not provide systemic protection from CHIKV wild-type (WT) challenge but was found to promote an early onset of severe CHIKV-induced footpad swelling. Liposome-delivered CHIKV-NoLS RNA inoculation did, however, provide local protection from CHIKV-WT challenge in the ipsilateral foot after one dose. Results suggest that in the presence of low CHIKV-specific neutralizing antibody levels, local inflammatory responses, likely brought on by liposome adjuvants, have a role in the protection of CHIKV-induced footpad swelling in the ipsilateral foot of mice inoculated with liposome-delivered CHIKV-NoLS RNA. Low IgG and CHIKV-specific neutralizing antibody levels may be responsible for early onset of severe swelling in the feet of CHIKV-WT-challenged mice. These results support previous studies that suggest CHIKV is vulnerable to antibody-mediated enhancement of disease. Further studies using booster regimes aim to demonstrate the potential for liposomes to deliver the self-replicating RNA genome of live-attenuated vaccines and offer a novel immunization strategy.

Modulation of Monocyte-Driven Myositis in Alphavirus Infection Reveals a Role for CX3CR1+ Macrophages in Tissue Repair.

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 Alphavirus-Induced Immunopathology and Targeting Host Inflammation as A Therapeutic Strategy for Alphaviral Disease.

Arthritogenic alphaviruses are a group of medically important arboviruses that cause inflammatory musculoskeletal disease in humans with debilitating symptoms, such as arthralgia, arthritis, and myalgia. The arthritogenic, or Old World, alphaviruses are capable of causing explosive outbreaks, with some viruses of major global concern. At present, there are no specific therapeutics or commercially available vaccines available to prevent alphaviral disease. Infected patients are typically treated with analgesics and non-steroidal anti-inflammatory drugs to provide often inadequate symptomatic relief. Studies to determine the mechanisms of arthritogenic alphaviral disease have highlighted the role of the host immune system in disease pathogenesis. This review discusses the current knowledge of the innate immune response to acute alphavirus infection and alphavirus-induced immunopathology. Therapeutic strategies to treat arthritogenic alphavirus disease by targeting the host immune response are also examined.

Chikungunya Arthritis: Implications of Acute and Chronic Inflammation Mechanisms on Disease Management.

In the past decade, arboviruses-arthropod-borne viruses-have been the focus of public health institutions worldwide following a spate of devastating outbreaks. Chikungunya virus, an arbovirus that belongs to the alphavirus genus, is a reemerging arthritogenic virus that has caused explosive outbreaks since 2006, notably on Réunion Island, and more recently in the Caribbean, South America, India, and Southeast Asia. The severity of arthritic disease caused by chikungunya virus has prompted public health authorities in affected countries to develop specific guidelines to tackle this pathogen. Chikungunya virus disease manifests first as an acute stage of severe joint inflammation and febrile illness, which later progresses to a chronic stage, during which patients may experience debilitating and persisting articular pain for extended periods. This review aims to provide a broad perspective on current knowledge of chikungunya virus pathogenesis by identifying key clinical and experimental studies that have contributed to our understanding of chikungunya virus to date. In addition, the review explores the practical aspects of treatment and management of both acute and chronic chikungunya virus based on clinical experience during chikungunya virus outbreaks. Finally, recent findings on potential therapeutic solutions-from antiviral agents to immunomodulators-are reviewed to provide both viral immunologists and clinical rheumatologists with a balanced perspective on the nature of a reemerging arboviral disease of significant public health concern, and insight into future therapeutic approaches to better address the treatment and management of chikungunya virus.