Viral Immunology

In addition to genetic factors, environmental triggers, including viruses and other pathogens, are thought to play a major role in the development of autoimmune disease. Recent findings have shown that viral-induced autoimmunity is likely to be genetically determined. In large-scale genetic analyses, an association of interferon induced with helicase C domain 1 (IFIH1) gene variants encoding a viral RNA-sensing helicase with susceptibility to several autoimmune diseases was found. To date, the precise role of IFIH1 in pathogenic mechanisms of viral-induced autoimmunity has yet to be fully elucidated. However, recent reports suggest that IFIH1 may play a role in the etiology of type 1 diabetes. Rare IFIH1 alleles have been shown to be protective against diabetes, and their carriage correlates with lower production of this helicase and its functional disruption. In contrast, upregulation of IFIH1 expression by viruses is associated with more severe disease, and could exacerbate the autoimmune process in susceptible individuals.

Studies from our laboratory and those of others have implicated lipopolysaccharide (LPS)-induced MAPK signaling as an important pathway in the regulation of cytokine expression. In this article, the regulation of IL-12 expression in two different human myeloid cell populations was evaluated. In primary monocytes, the inhibition of p38 enhanced IL-12 production, whereas it downregulated IL-12 production in THP-1 cells. The role of MAPK signaling in transcription factor binding to the IL-12p40 promoter was subsequently determined. In primary monocytes, ERK and p38 inhibition increased binding of AP-1 and Sp1, respectively, to the IL-12p40 promoter, while JNK inhibition increased NF-κB, AP-1, and Sp1 binding. In THP-1 cells, p38, ERK, and JNK inhibition increased NF-κB and Sp1 binding to the IL-12p40 promoter, while inhibiting AP-1 binding. In monocytes, mutations in the NF-κB, AP-1, Sp1, or Ets-2 binding sites resulted in complete inhibition of LPS-stimulated IL-12p40 promoter activity using a luciferase-based assay. In contrast, promoter activity was abrogated in THP-1 cells only when the Sp1 or Ets-2 binding sites were mutated. Transcription factor binding to the IL-12p40 promoter following in-vitro HIV infection demonstrated several differences between monocytes and THP-1 cells. Infection with HIV produced an increase in NF-κB, AP-1, and Sp1 binding in primary monocytes. In contrast, binding of Ets-2 was dramatically impaired following HIV infection of monocytes, but was unaffected in THP-1 cells. These data clearly show that although LPS induces IL-12p40 expression in primary monocytes and THP-1 cells, the signaling pathways involved and the effect of HIV infection differ and can have disparate effects in these two cell types.

Foot-and-mouth disease virus (FMDV) causes an acute, highly contagious disease of livestock. Though FMDV is very sensitive to interferon-α (IFN-α), IFN-β, and IFN-γ, the virus has evolved mechanisms to evade such innate responses. For instance, during acute infection, FMDV suppresses IFN-α production by skin and myeloid dendritic cells (DCs). We have previously reported that FMDV infection induces a transient lymphopenia and interruption of T-lymphocyte responses to mitogenic stimuli. To further understand the immunopathogenesis of FMD, we have now analyzed the serum IFN-α response in relation to lymphopenia, and the number and function of plasmacytoid DCs (pDCs) following infection of pigs with multiple serotypes of FMDV. Serum IFN-α peaked 2–3 d post-infection (PI), regardless of FMDV serotype. Lymphopenia coincided with peak viremia and the serum IFN-α response. Circulating pDC numbers and in-vitro pDC IFN-α secretion transiently declined by 48 h following infection. Infection of lymphocytes or pDCs was never detected regardless of the FMDV serotype inoculated or the age of the animal infected. These data indicate that, like other DC subsets, there is suppression of interferon production by pDCs, which abrogates this important innate response. Rapid induction of serum IFN-α, albeit short-lived, may contribute to the rapid resolution of FMDV viremia prior to induction of specific immunity.

Prostaglandins (PGs) play an important role in pulmonary physiology and various pathophysiological processes following infection. The initial step in the biosynthesis of PGs is regulated by two distinct cyclooxygenase enzymes, cyclooxygenase-1 (COX-1) and COX-2. The goal of this study was to investigate the pulmonary cellular localization and distribution of COX-1 and COX-2 in a neonatal lamb model following respiratory syncytial virus (RSV) and parainfluenza virus 3 (PI3) infection, organisms that also cause significant respiratory disease in children. No significant differences were seen in pulmonary COX-1 expression at various microanatomical locations following RSV or PI3 infection compared to controls. In contrast, COX-2 was upregulated following RSV and PI3 infection. Strong expression was restricted to bronchial and bronchiolar epithelial cells and macrophages, while minimal expression was present in the same microanatomical locations in the uninfected lungs. Other microanatomical locations in both the controls and the infected lungs lacked expression. This work suggests that during RSV or PI3 infection: (1) COX-1 cellular expression is not altered, (2) COX-2 cellular expression is upregulated in airway bronchiolar and bronchial epithelial cells and macrophages, (3) respiratory epithelium along with macrophages are important microanatomical compartments regulating the host inflammatory response during viral infection, and (4) COX-2 may be a potential target for RSV and PI3 therapy.

We investigated in-vitro lymphoproliferative responses and T-cell subsets in 38 HIV-1-infected patients showing impaired restoration of CD4 T cells despite prolonged viral suppression (discordant), and in 42 HIV-1-infected patients showing positive immunological and virological responses to highly active antiretroviral therapy (HAART) (concordant). In comparison to concordant patients, discordant patients showed poor lymphocyte proliferation, lower secretion of IL-2 and IFN-γ, a lower percentage of perforin and granzyme-B-producing CD8 T cells, and poor differentiation of effector memory CD8 T_EM cells into CD8 T_EMRA cells in in-vitro stimulation assays, especially against HIV-1 Gag p24 and one of its peptide pools. Functional CD8 T-cell responses of discordant patients after stimulation with recall antigens, Candida albicans, and tetanus toxoid, were also inferior to concordant patients, but comparable to normal healthy controls. Examination of the multifunctional roles of T cells is imperative in describing the overall magnitude of immune responses to HIV-1. Our results suggest that prolonged suppression of plasma viremia alone does not warrant good qualitative and quantitative CD8 T-cell responses to HIV-1, implying that CD4 T cells are required for maintenance of protective CD8 T-cell responses.

The roles of regulatory T cells (Tregs) and PD-1 in hepatitis B have not been clearly described. Also, the role of B and T lymphocyte attenuator (BTLA), which serves as a negative regulator of T-cell activation, is still unknown in hepatitis B. In this study, we analyzed the frequency of circulating CD4⁺CD25^high Tregs in patients with chronic hepatitis B (CHB), and subsequently investigated expression of PD-1 and BTLA on CD4⁺ T cells, as well as their relationships with the clinical index of CHB patients. A total of 39 CHB patients and 19 healthy persons as controls were enrolled in the study. We found that the frequency of CD4⁺CD25^high Tregs and PD-1 expression on CD4⁺ T cells was significantly increased in CHB patients compared with normal controls. However, BTLA expression on CD4⁺ T cells showed no significant difference between the two groups. The frequency of Tregs was significantly higher in patients with HBV DNA titers ≥10⁸ than in those with HBV DNA titers <10⁸. Circulating CD4⁺CD25^high Treg frequency and PD-1 expression on CD4⁺ T cells correlated positively with serum HBV DNA load in CHB patients. Our findings suggest that the increased frequency of CD4⁺CD25^high Tregs and PD-1 expression on CD4⁺ T lymphocytes may inhibit the cellular immune response against HBV and affect viral clearance, leading to the persistence of chronic HBV infection.

Connective tissue growth factor (CTGF) plays a crucial role in the formation and development of hepatic fibrosis. The aim of this study was to establish a method for CTGF determination in order to investigate the level of CTGF in the sera of patients with hepatitis B virus, and to assess the correlation between CTGF concentration and stage of hepatic fibrosis. A CTGF C-terminal region gene was obtained by RT-PCR of human mesangial kidney cells and inserted into pET-32a⁽⁺⁾ vector. Recombinant protein was obtained by expression and purification of the fused protein. Polyclonal and monoclonal antibodies were prepared to establish a sandwich ELISA method. CTGF levels in 18 healthy serum samples and 83 serum samples from patients with hepatitis B virus were assessed. A simple, sensitive, and noninvasive method of determining CTGF levels was successfully established. CTGF levels in the sera of patients with hepatitis B were significantly increased compared controls (p < 0.001). There was a strong correlation between CTGF concentration and fibrotic stage (r = 0.8906, p < 0.005). No significant association was found between CTGF level and the grade of hepatic inflammation (p > 0.05). The area under the receiver operating characteristic (ROC) curves of CTGF was 0.681 for identification of significant fibrosis, and 0.759 for the diagnosis of middle- and late-stage fibrosis. Accuracy of CTGF assessment was independent of age, renal function, liver function, platelet count, or other biochemical markers of liver fibrosis (all p > 0.05). No significant correlation was found between CTGF and several humoral factors associated with liver fibrosis (all p > 0.05). The levels of CTGF in the sera of patients with hepatitis B were strongly associated with the stages of hepatic fibrosis, and CTGF may become a useful diagnostic aid in assessing hepatic fibrosis.

Coxsackievirus B3 (CVB3) induces cardiac inflammation (myocarditis) in male but not female C57BL/6 mice. Protection of females correlates with reduced expression of TNF-α and IL-1β at both the mRNA and protein levels in the heart. Treatment of females with 300 ng/mouse of recombinant TNF-α on days +1 and +3 relative to infection restores myocarditis susceptibility to levels approximating those of infected male mice, showing that TNF-α deficiency is central to disease resistance. Female mice express little CD1d on spleen lymphocytes or cardiac myocytes, while females treated with TNF-α show increased CD1d expression in both cell populations. TNF-α treatment of male or female CD1d knockout (CD1dKO) mice failed to restore myocarditis susceptibility, demonstrating that of the multiple potential TNF-α activities, its ability to upregulate this non-classical major histocompatibility complex antigen is its dominant function in myocarditis susceptibility. Bone marrow chimeric mice were produced between female C57BL/6 and C57BL/6 CD1dKO mice so that either hematopoietic or non-hematopoietic cells were CD1d deficient. TNF-α treatment of chimeric mice having wild-type (CD1d+) hematopoietic cells restored myocarditis susceptibility, while TNF-α treatment of chimeric mice having CD1dKO hematopoietic cells, but CD1d+ myocytes, failed to develop myocarditis, showing that CD1d expression in lymphoid cells controls disease susceptibility.

After initiation of highly-active antiretroviral therapy (HAART), long-term HIV-infected hemophilia patients have been shown to lose autoantibodies against CD4⁺ peripheral blood leukocytes (PBL), suggesting that HAART induces autoimmunity-blocking mechanisms. We compared cytokine levels and subpopulations of lymphocytes and dendritic cells (DC) in the blood of 40 long-term HIV⁺ patients with those of 13 long-term HIV^– hemophilia patients; 23 HIV⁺ patients had a detectable retroviral load. Cell subsets were determined using flow cytometry and cytokine levels were measured using ELISA. HIV⁺ patients showed higher proportions of DC subpopulations with immunostimulatory phenotypes (p < 0.01), CD8⁺ PBL (p < 0.001), and IL-2 (p < 0.001) and sIL-2R plasma levels (p = 0.002) than HIV^– patients. They also exhibited increased proportions of T PBL with immunosuppressive phenotypes such as CD3⁺CD4⁺CD25⁺Foxp3⁺ (p = 0.001), and CD3⁺CD8⁺CD28^–Foxp3⁺ PBL (p < 0.001), and a decreased IL-7R expression on CD3⁺CD8⁺ PBL (p = 0.001) compared to HIV^– patients. Frequencies of CD3⁺CD4⁺CD25⁺ PBL producing IL-2, IL-4, IL-10, IL-12, and/or IFN-γ, and of CD3⁺CD4⁺CD28^– PBL secreting IL-2 and/or IL-4 were lower in HIV⁺ than in HIV^– patients (p ≤ 0.02). Proportions of CD4⁺ PBL coated with IgG, IgM, and C3d were similar in HIV⁺ and HIV^– patients (p = n.s.). However, the proportion of CD4⁺gp120⁺ PBL was higher in HIV⁺ patients (p = 0.002), and associated with low CD3⁺CD4⁺CD25⁺Foxp3⁺ PBL (p = 0.012). We conclude that long-term HIV-infected hemophilia patients on HAART show an adaptive immune response, presumably against HIV, in the presence of upregulated immunosuppressive T PBL, downregulated cytokine-producing CD4⁺ PBL, and downregulated IL-7R expression on CD8⁺ PBL. Increased immunoregulatory T PBL might decrease autoimmunity, thereby contributing to immunological reconstitution and stabilization of long-term HIV-infected hemophilia patients on HAART.

Previous studies have shown that enteroviral RNA can be detected in blood at the onset of type 1 diabetes (T1D). The infection may play a role in triggering T1D and genetic host factors may contribute to this process. We investigated (1) whether enterovirus is present at the onset of T1D in peripheral blood mononuclear cells (PBMC), plasma, throat, or stool, and (2) whether enteroviral presence is linked with HLA-DR type and/or polymorphisms in melanoma differentiation-associated gene 5 (MDA5) and 2′-5′ oligoadenylate synthetase 1 (OAS1), factors of antiviral immunity. To this end, PBMC, plasma, throat, and stool samples from 10 T1D patients and 20 unrelated controls were tested for the presence of enteroviruses (RT-PCR), for HLA-DR type, and polymorphisms in MDA5 and OAS1. Enterovirus RNA was detected in PBMC of 4/10 T1D patients, but none of 20 controls. Plasma was positive in 2/10 T1D patients and none of 20 controls, suggesting that enteroviruses found at the onset of T1D are mainly present in PBMC. All throat samples from positive T1D patients were virus-negative and only 1 fecal sample was positive. The negative results for all throat and most stool samples argues against acute infection. Enterovirus presence was linked with HLA-DR4, but not with polymorphisms in MDA5 or OAS1.

The occurrence of swine H1N1 pandemic was unexpected because our previous focus was concentrated on highly pathogenic avian H5N1 outbreaks. The H1N1 pandemic means that cross-species infection and cross-subtype mutation is not as rare as we had previously thought, and the barriers between species and between subtypes are not strong for influenza A virus. In this study, we use ANOVA to determine if there are barriers between species and between subtypes in the matrix protein 1 family from influenza A virus. The results show that the inter-species/subtype variations are generally much smaller than the intra-species/subtype ones, indicating that the barriers between species and between subtypes are not strong for influenza A viruses, which provides statistical evidence for cross-species infections and cross-subtype mutations.

Chikungunya virus infection has recently emerged in several countries. The inflammatory response is suggested to be involved in the pathology observed in this infectious disease. Interleukin-18 (IL-18) is an inducer of interferon-γ (IFN-γ) production and has been shown to play a role in several inflammatory diseases. IL-18 binding protein (IL-18BP) is a natural regulator of IL-18. In this study, we determined the levels of IL-18 and IL-18BP in patients with Chikungunya virus infection. Acute and convalescent sera were collected from each patient. The levels of both IL-18 and IL-18BP were measured by ELISA assays. IL-18 and IL-18BP levels were higher in patients than in controls. In addition, the level of IL-18 was higher in convalescent than in acute sera. However, the level of IL-18BP was lower in convalescent than in acute sera. These data suggest that production of both IL-18 and IL-18BP was induced following Chikungunya virus infection. IL-18BP was increased to regulate the activity of IL-18. The ratio of IL-18 to IL-18BP was higher in convalescent than in acute sera. The lower level of IL-18BP in convalescent sera was probably due to loss following IL-18 neutralization. Our data suggest that Chikungunya virus infection promotes the T helper-1 (Th-1) response by inducing IL-18 production. Manipulation of IL-18 and IL-18BP levels could be a promising therapeutic approach to alleviate symptoms in patients with Chikungunya virus infection.