Zhisheng Her

IL-1β, IL-6, and RANTES as Biomarkers of Chikungunya Severity

Background Little is known about the immunopathogenesis of Chikungunya virus. Circulating levels of immune mediators and growth factors were analyzed from patients infected during the first Singaporean Chikungunya fever outbreak in early 2008 to establish biomarkers associated with infection and/or disease severity. Methods and Findings Adult patients with laboratory-confirmed Chikungunya fever infection, who were referred to the Communicable Disease Centre/Tan Tock Seng Hospital during the period from January to February 2008, were included in this retrospective study. Plasma fractions were analyzed using a multiplex-microbead immunoassay. Among the patients, the most common clinical features were fever (100%), arthralgia (90%), rash (50%) and conjunctivitis (40%). Profiles of 30 cytokines, chemokines, and growth factors were able to discriminate the clinical forms of Chikungunya from healthy controls, with patients classified as non-severe and severe disease. Levels of 8 plasma cytokines and 4 growth factors were significantly elevated. Statistical analysis showed that an increase in IL-1β, IL-6 and a decrease in RANTES were associated with disease severity. Conclusions This is the first comprehensive report on the production of cytokines, chemokines, and growth factors during acute Chikungunya virus infection. Using these biomarkers, we were able to distinguish between mild disease and more severe forms of Chikungunya fever, thus enabling the identification of patients with poor prognosis and monitoring of the disease.

Persistent Arthralgia Induced by Chikungunya Virus Infection is Associated with Interleukin-6 and Granulocyte Macrophage Colony-Stimulating Factor

Background. Chikungunya virus (CHIKV) infection induces arthralgia. The involvement of inflammatory cytokines and chemokines has been suggested, but very little is known about their secretion profile in CHIKV-infected patients. Methods. A case-control longitudinal study was performed that involved 30 adult patients with laboratory-confirmed Chikungunya fever. Their profiles of clinical disease, viral load, and immune mediators were investigated. Results. When patients were segregated into high viral load and low viral load groups during the acute phase, those with high viremia had lymphopenia, lower levels of monocytes, neutrophilia, and signs of inflammation. The high viral load group was also characterized by a higher production of pro-inflammatory cytokines, such as interferon-α and interleukin (IL)–6, during the acute phase. As the disease progressed to the chronic phase, IL-17 became detectable. However, persistent arthralgia was associated with higher levels of IL-6 and granulocyte macrophage colony-stimulating factor, whereas patients who recovered fully had high levels of Eotaxin and hepatocyte growth factor. Conclusions. The level of CHIKV viremia during the acute phase determined specific patterns of pro-inflammatory cytokines, which were associated with disease severity. At the chronic phase, levels of IL-6, and granulocyte macrophage colony-stimulating factor found to be associated with persistent arthralgia provide a possible explanation for the etiology of arthralgia that plagues numerous CHIKV-infected patients.

Active Infection of Human Blood Monocytes by Chikungunya Virus Triggers an Innate Immune Response

Chikungunya virus (CHIKV) is an alphavirus that causes chronic and incapacitating arthralgia in humans. To date, interactions between the immune system and the different stages of the virus life cycle remain poorly defined. We demonstrated for the first time that CHIKV Ags could be detected in vivo in the monocytes of acutely infected patients. Using in vitro experimental systems, whole blood and purified monocytes, we confirmed that monocytes could be infected and virus growth could be sustained. CHIKV interactions with monocytes, and with other blood leukocytes, induced a robust and rapid innate immune response with the production of specific chemokines and cytokines. In particular, high levels of IFN-α were produced rapidly after CHIKV incubation with monocytes. The identification of monocytes during the early phase of CHIKV infection in vivo is significant as infected monocyte/macrophage cells have been detected in the synovial tissues of chronically CHIKV-infected patients, and these cells may behave as the vehicles for virus dissemination. This may explain the persistence of joint symptoms despite the short duration of viremia. Our results provide a better understanding on the basic mechanisms of infection and early antiviral immune responses and will help in the development of future effective control strategies.

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.

Chikungunya: a bending reality.

Chikungunya fever is an acute illness caused by the arbovirus Chikungunya virus. The virus is transmitted primarily in a sylvatic cycle involving the Aedes mosquitoes. Since 2005, a Chikungunya fever outbreak of unprecedented magnitude occurred on several Indian Ocean islands. Since then, the disease has spread to many parts of the world due to imported cases among travellers returning from epidemic areas. Chikungunya virus causes a wide spectrum of illness including fever, a characteristic rash, disabling joint symptoms which can sometimes become severe that lasts months. This review summarises on this history of Chikungunya fever, host specificity, the characteristics of Chikungunya virus, clinical features of disease and current control measures. It focuses on how the re-emergence of an old changed the outlook of managing arboviral diseases in the present social and public health context.

Caribbean and La Réunion Chikungunya Virus Isolates Differ in Their Capacity To Induce Proinflammatory Th1 and NK Cell Responses and Acute Joint Pathology

ABSTRACT Chikungunya virus (CHIKV) is a mosquito-borne arthralgic alphavirus that has garnered international attention as an important emerging pathogen since 2005. More recently, it invaded the Caribbean islands and the Western Hemisphere. Intriguingly, the current CHIKV outbreak in the Caribbean is caused by the Asian CHIKV genotype, which differs from the La Réunion LR2006 OPY1 isolate belonging to the Indian Ocean lineage. Here, we adopted a systematic and comparative approach against LR2006 OPY1 to characterize the pathogenicity of the Caribbean CNR20235 isolate and consequential host immune responses in mice. Ex vivo infection using primary mouse tail fibroblasts revealed a weaker replication efficiency by CNR20235 isolate. In the CHIKV mouse model, CNR20235 infection induced an enervated joint pathology characterized by moderate edema and swelling, independent of mononuclear cell infiltration. Based on systemic cytokine analysis, localized immunophenotyping, and gene expression profiles in the popliteal lymph node and inflamed joints, two pathogenic phases were defined for CHIKV infection: early acute (2 to 3 days postinfection [dpi]) and late acute (6 to 8 dpi). Reduced joint pathology during early acute phase of CNR20235 infection was associated with a weaker proinflammatory Th1 response and natural killer (NK) cell activity. The pathological role of NK cells was further demonstrated as depletion of NK cells reduced joint pathology in LR2006 OPY1. Taken together, this study provides evidence that the Caribbean CNR20235 isolate has an enfeebled replication and induces a less pathogenic response in the mammalian host. IMPORTANCE The introduction of CHIKV in the Americas has heightened the risk of large-scale outbreaks due to the close proximity between the United States and the Caribbean. The immunopathogenicity of the circulating Caribbean CHIKV isolate was explored, where it was demonstrated to exhibit reduced infectivity resulting in a weakened joint pathology. Analysis of serum cytokine levels, localized immunophenotyping, and gene expression profiles in the organs revealed that a limited Th1 response and reduced NK cells activity could underlie the reduced pathology in the host. Interestingly, higher asymptomatic infections were observed in the Caribbean compared to the La Réunion outbreaks in 2005 and 2006. This is the first study that showed an association between key proinflammatory factors and pathology-mediating leukocytes with a less severe pathological outcome in Caribbean CHIKV infection. Given the limited information regarding the sequela of Caribbean CHIKV infection, our study is timely and will aid the understanding of this increasingly important disease.

Interaction between tumour-infiltrating B cells and T cells controls the progression of hepatocellular carcinoma

Objective The nature of the tumour-infiltrating leucocytes (TILs) is known to impact clinical outcome in carcinomas, including hepatocellular carcinoma (HCC). However, the role of tumour-infiltrating B cells (TIBs) remains controversial. Here, we investigate the impact of TIBs and their interaction with T cells on HCC patient prognosis. Design Tissue samples were obtained from 112 patients with HCC from Singapore, Hong Kong and Zurich and analysed using immunohistochemistry and immunofluorescence. RNA expression of CD19, CD8A, IFNG was analysed using quantitative PCR. The phenotype of freshly isolated TILs was analysed using flow cytometry. A mouse model depleted of mature B cells was used for functional study. Results Tumour-infiltrating T cells and B cells were observed in close contact with each other and their densities are correlated with superior survival in patients with HCC. Furthermore, the density of TIBs was correlated with an enhanced expression of granzyme B and IFN-γ, as well as with reduced tumour viability defined by low expression of Ki-67, and an enhanced expression of activated caspase-3 on tumour cells. CD27 and CD40 costimulatory molecules and TILs expressing activation marker CD38 in the tumour were also correlated with patient survival. Mice depleted of mature B cells and transplanted with murine hepatoma cells showed reduced tumour control and decreased local T cell activation, further indicating the important role of B cells. Conclusions The close proximity of tumour-infiltrating T cells and B cells indicates a functional interaction between them that is linked to an enhanced local immune activation and contributes to better prognosis for patients with HCC.

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.

Macrophage Migration Inhibitory Factor Receptor CD74 Mediates Alphavirus-Induced Arthritis and Myositis in Murine Models of Alphavirus Infection

OBJECTIVE Arthrogenic alphaviruses such as Ross River virus (RRV) and chikungunya virus (CHIKV) circulate worldwide. This virus class causes debilitating illnesses that are characterized by arthritis, arthralgia, and myalgia. In previous studies, we identified macrophage migration inhibitory factor (MIF) as a critical inflammatory factor in the pathogenesis of alphaviral diseases. The present study was undertaken to characterize the role of CD74, a cell surface receptor of MIF, in both RRV- and CHIKV-induced alphavirus arthritides. METHODS Mouse models of RRV and CHIKV infection were used to investigate the immunopathogenesis of arthritic alphavirus infection. The role of CD74 was assessed using histologic analysis, real-time polymerase chain reaction, flow cytometry, and plaque assay. RESULTS In comparison to wild-type mice, CD74-/- mice developed only mild clinical features and had low levels of tissue damage. Leukocyte infiltration, characterized predominantly by inflammatory monocytes and natural killer cells, was substantially reduced in the infected tissue of CD74-/- mice, but production of proinflammatory cytokines and chemokines was not decreased. CD74 deficiency was associated with increased monocyte apoptosis, but had no effect on monocyte migratory capacity. Consistent with these findings, alphaviral infection resulted in a dose-dependent up-regulation of CD74 expression in human peripheral blood mononuclear cells, and serum MIF levels were significantly elevated in patients with RRV or CHIKV infection. CONCLUSION CD74 appears to regulate immune responses to alphaviral infection through its effects on cellular recruitment and survival. These findings suggest that both MIF and CD74 play a critical role in mediating alphaviral disease, and blocking these factors with novel therapeutic agents could substantially ameliorate the pathologic manifestations.

Expanding regulatory T cells alleviates chikungunya virus-induced pathology in mice.

ABSTRACT Chikungunya virus (CHIKV) infection is a reemerging pandemic human arboviral disease. CD4+ T cells were previously shown to contribute to joint inflammation in the course of CHIKV infection in mice. The JES6-1 anti-IL-2 antibody selectively expands mouse regulatory T cells (Tregs) by forming a complex with IL-2. In this study, we show that the IL-2 JES6-1-mediated expansion of Tregs ameliorates CHIKV-induced joint pathology. It does so by inhibiting the infiltration of CD4+ T cells due to the induction of anergy in CHIKV-specific CD4+ effector T cells. These findings suggest that activation of Tregs could also become an alternative approach to control CHIKV-mediated disease. IMPORTANCE Chikungunya virus (CHIKV) has reemerged as a pathogen of global significance. Patients infected with CHIKV suffer from incapacitating joint pain that severely affects their daily functioning. Despite the best efforts, treatment is still inadequate. While T cell-mediated immunopathology in CHIKV infections has been reported, the role of regulatory T cells (Tregs) has not been explored. The JES6-1 anti-interleukin 2 (IL-2) antibody has been demonstrated to selectively expand mouse Tregs by forming a complex with IL-2. We reveal here that IL-2 JES6-1-mediated expansion of Tregs ameliorates CHIKV-induced joint pathology in mice by neutralizing virus-specific CD4+ effector T (Teff) cells. We show that this treatment abrogates the infiltration of pathogenic CD4+ T cells through induction of anergy in CHIKV-specific CD4+ Teff cells. This is the first evidence where the role of Tregs is demonstrated in CHIKV pathogenesis, and its expansion could control virus-mediated immunopathology.