Stephen N. Waggoner

Hepatitis C Virus Core Selectively Suppresses Interleukin-12 Synthesis in Human Macrophages by Interfering with AP-1 Activation

Hepatitis C virus (HCV) is remarkably efficient at establishing persistent infection, suggesting that it has evolved one or more strategies aimed at evading the host immune response. T cell responses, including interferon-γ production, are severely suppressed in chronic HCV patients. The HCV core protein has been previously shown to circulate in the bloodstream of HCV-infected patients and inhibit host immunity through an interaction with gC1qR. To determine the role of the HCV core-gC1qR interaction in modulation of inflammatory cytokine production, we examined interleukin (IL)-12 production, which is critical for the induction of interferon-γ synthesis, in lipopolysaccharide-stimulated human monocyte/macrophages. We found that core protein binds the gC1qR displayed on the cell surface of monocyte/macrophages and inhibits the production of IL-12p70 upon lipopolysaccharide stimulation. This inhibition was found to be selective in that HCV core failed to affect the production of IL-6, IL-8, IL-1β, and tumor necrosis factor α. In addition, suppression of IL-12 production by core protein occurred at the transcriptional level by inhibition of IL-12p40 mRNA synthesis. Importantly, core-induced inhibition of IL-12p40 mRNA synthesis resulted from impaired activation of AP-1 rather than enhanced IL-10 production. These results suggest that the HCV core-gC1qR interaction may play a pivotal role in establishing persistent infection by dampening TH1 responses.

Direct Binding of Hepatitis C Virus Core to gC1qR on CD4+ and CD8+ T Cells Leads to Impaired Activation of Lck and Akt

ABSTRACT Complement plays a pivotal role in the regulation of innate and adaptive immunity. It has been shown that the binding of C1q, a natural ligand of gC1qR, on T cells inhibits their proliferation. Here, we demonstrate that direct binding of the hepatitis C virus (HCV) core to gC1qR on T cells leads to impaired Lck/Akt activation and T-cell function. The HCV core associates with the surface of T cells specifically via gC1qR, as this binding is inhibited by the addition of either anti-gC1qR antibody or soluble gC1qR. The binding affinity constant of core protein for gC1qR, as determined by BIAcore analysis, is 3.8 × 10−7 M. The specificity of the HCV core-gC1qR interaction is confirmed by reduced core binding on Molt-4 T cells treated with gC1qR-silencing small interfering RNA and enhanced core binding on GPC-16 guinea pig cells transfected with human gC1qR. Interestingly, gC1qR is expressed at higher levels on CD8+ than on CD4+ T cells, resulting in more severe core-induced suppression of the CD8+-T-cell population. Importantly, T-cell receptor-mediated activation of the Src kinases Lck and ZAP-70 but not Fyn and the phosphorylation of Akt are impaired by the HCV core, suggesting that it inhibits the very early events of T-cell activation.

HCV core protein interaction with gC1q receptor inhibits Th1 differentiation of CD4+ T cells via suppression of dendritic cell IL-12 production

Dendritic cells (DCs) isolated from patients with chronic hepatitis C virus (HCV) infection display an impaired capacity to generate type 1 CD4+ T cell immunity. Several reports have described an immunomodulatory function for the HCV core protein, and circulating core has been shown to associate with the putative gC1q receptor, gC1qR, expressed on host immune cells. However, the molecular mechanism(s) of HCV core‐mediated DC dysfunction has not been defined. Herein, ligation of gC1qR on human monocyte‐derived DCs (MDDCs) with HCV core or anti‐gC1qR agonist antibody was shown to inhibit TLR‐induced IL‐12 production but not the production of other TLR‐stimulated cytokines. Furthermore, engagement of gC1qR on MDDCs resulted in reduced IFN‐γ secretion by allogeneic CD4+ T lymphocytes during mixed lymphocyte culture. Differentiation of CD4+ T cells cocultured with HCV core‐ or anti‐gC1qR antibody‐treated MDDCs was also skewed toward production of Th2 cytokines, including IL‐4. Importantly, that addition of IL‐12 rescued IFN‐γ production and Th1 differentiation by CD4+ T cells. Therefore, engagement of gC1qR on DCs by HCV core limits the induction of Th1 responses and may contribute to viral persistence.

gC1q Receptor Ligation Selectively Down-Regulates Human IL-12 Production through Activation of the Phosphoinositide 3-Kinase Pathway

gC1qR, a complement receptor for C1q, plays a pivotal role in the regulation of inflammatory and antiviral T cell responses. Several pathogens, including hepatitis C virus, exploit gC1qR-dependent regulatory pathways to manipulate host immunity. However, the molecular mechanism(s) of gC1qR signaling involved in regulating inflammatory responses remains unknown. We report the selective inhibition of TLR4-induced IL-12 production after cross-linking of gC1qR on the surface of macrophages and dendritic cells. Suppression of IL-12 did not result from increased IL-10 or TGF-β, but was dependent on PI3K activation. Activation of PI3K and subsequent phosphorylation of Akt define an intracellular pathway mediating gC1qR signaling and cross-talk with TLR4 signaling. This is the first report to identify signaling pathways used by gC1qR-mediated immune suppression, and it establishes a means of complement-mediated immune suppression to inhibit Th1 immunity crucial for clearing pathogenic infection.

Hepatitis C virus: immunosuppression by complement regulatory pathway.

Hepatitis C virus (HCV) infection in humans is almost invariably associated with viral persistence and chronic hepatitis. HCV-induced chronic hepatitis is a major risk factor for the development of hepatocellular carcinoma. The high incidence of HCV persistence suggests that this virus has evolved one or more mechanisms to evade and possibly suppress host immune responses. To understand the mechanism(s) involved in the establishment of HCV persistence, we have identified an HCV core protein as an immunomodulatory molecule to suppress host immune response. We have further determined a molecular mechanism of HCV core-mediated immune suppression by searching for a potential host protein(s) capable of associating with the HCV core protein. Interestingly, the Clq complement receptor, gC1qR, can bind to the HCV core. Clq is a ligand of gClqR and is involved in the early defense against viral infection as well as regulation of adaptive immune response. Similar to Clq, the HCV core can inhibit human T-lymphocyte proliferative response through its interaction with the gC1qR. It implicates that HCV core/gClqR-induced immune suppression may play a critical role in the establishment of persistent infection.

SOCS1 and SOCS3 Are Targeted by Hepatitis C Virus Core/gC1qR Ligation To Inhibit T-Cell Function

ABSTRACT T cells play an important role in the control of hepatitis C virus (HCV) infection. We have previously demonstrated that the HCV core inhibits T-cell responses through interaction with gC1qR. We show here that core proteins from chronic and resolved HCV patients differ in sequence, gC1qR-binding ability, and T-cell inhibition. Specifically, chronic core isolates bind to gC1qR more efficiently and inhibit T-cell proliferation as well as gamma interferon (IFN-γ) production more profoundly than resolved core isolates. This inhibition is mediated by the disruption of STAT phosphorylation through the induction of SOCS molecules. Silencing either SOCS1 or SOCS3 by small interfering RNA dramatically augments the production of IFN-γ in T cells, thereby abrogating the inhibitory effect of core. Additionally, the ability of core proteins from patients with chronic infections to induce SOCS proteins and suppress STAT activation greatly exceeds that of core proteins from patients with resolved infections. These results suggest that the HCV core/gC1qR-induced T-cell dysfunction involves the induction of SOCS, a powerful inhibitor of cytokine signaling, which represents a novel mechanism by which a virus usurps the host machinery for persistence.

Roles of natural killer cells in antiviral immunity

Natural killer (NK) cells are important in immune defense against virus infections. This is predominantly considered a function of rapid, innate NK-cell killing of virus-infected cells. However, NK cells also prime other immune cells through the release of interferon gamma (IFN-γ) and other cytokines. Additionally, NK cells share features with long-lived adaptive immune cells and can impact disease pathogenesis through the inhibition of adaptive immune responses by virus-specific T and B cells. The relative contributions of these diverse and conflicting functions of NK cells in humans are poorly defined and likely context-dependent, thereby complicating the development of therapeutic interventions. Here we focus on the contributions of NK cells to disease in diverse virus infections germane to human health.

Fas Ligand Is Responsible for CXCR3 Chemokine Induction in CD4 + T Cell-Dependent Liver Damage

Immune-mediated hepatic damage has been demonstrated in the pathogenesis of hepatitis C virus (HCV) and other hepatotrophic infections. Fas/Fas ligand (FasL) interaction plays a critical role in immune-mediated hepatic damage. To understand the molecular mechanism(s) of FasL-mediated liver inflammation, we examined the effect of CD4+ T cells expressing high levels of FasL on the initiation of hepatic damage through analysis of chemokine and chemokine receptor expression in HCV core × TCR (DO11.10) double-transgenic mice. In vivo antigenic stimulation triggers a marked influx of core-expressing Ag-specific CD4+ T cells into the liver of the immunized core+ TCR mice but not their core− TCR littermates. Strikingly, the inflammatory process in the liver of core+ TCR mice was accompanied by a dramatic increase in IFN-inducible protein 10 and monokine induced by IFN-γ production. The intrahepatic lymphocytes were primarily CXCR3-positive and anti-CXCR3 Ab treatment abrogates migration of CXCR3+ lymphocytes into the liver and hepatic damage. Importantly, the blockade of Fas/FasL interaction reduces the expression of IFN-inducible protein 10 and monokine induced by IFN-γ and cellular infiltration into the liver. These findings suggest that activated CD4+ T cells with elevated FasL expression are involved in promoting liver inflammation and hepatic damage through the induction of chemokines.