Expression levels of 10 candidate genes in lung tissue of vaccinated and TB-infected cynomolgus macaques.

The expression of ten tuberculosis candidate genes in lung and lymph nodes of cynomolgus macaques vaccinated and experimentally infected with Mycobacterium tuberculosis (Mtb) was quantified. The expression of TNFα, IL10, IL1ß, TLR4, IL17, IL6, IL12, and iNOS in the lungs of vaccinated animals was higher than that of non-vaccinated animals.

Heightened cytotoxic responses and impaired biogenesis contribute to early pathogenesis in the oral mucosa of simian immunodeficiency virus-infected rhesus macaques.

Simian immunodeficiency virus (SIV) infection disseminated into the oropharyngeal tissues of rhesus macaques 6 weeks following intravenous inoculation. Severe local CD4(+) T-cell depletion coincided with increases in NK cell and proinflammatory biomarkers and the disruption of growth-associated gene transcription, demonstrating the rapid establishment of pathogenesis in the oral mucosa.

Simian immunodeficiency virus-induced mucosal interleukin-17 deficiency promotes Salmonella dissemination from the gut.

Salmonella typhimurium causes a localized enteric infection in immunocompetent individuals, whereas HIV-infected individuals develop a life-threatening bacteremia. Here we show that simian immunodeficiency virus (SIV) infection results in depletion of T helper type 17 (TH17) cells in the ileal mucosa of rhesus macaques, thereby impairing mucosal barrier functions to S. typhimurium dissemination. In SIV-negative macaques, the gene expression profile induced by S. typhimurium in ligated ileal loops was dominated by TH17 responses, including the expression of interleukin-17 (IL-17) and IL-22. TH17 cells were markedly depleted in SIV-infected rhesus macaques, resulting in blunted TH17 responses to S. typhimurium infection and increased bacterial dissemination. IL-17 receptor-deficient mice showed increased systemic dissemination of S. typhimurium from the gut, suggesting that IL-17 deficiency causes defects in mucosal barrier function. We conclude that SIV infection impairs the IL-17 axis, an arm of the mucosal immune response preventing systemic microbial dissemination from the gastrointestinal tract.

Antiviral therapy during primary simian immunodeficiency virus infection fails to prevent acute loss of CD4+ T cells in gut mucosa but enhances their rapid restoration through central memory T cells.

Gut-associated lymphoid tissue (GALT) is an early target of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) and a site for severe CD4+ T-cell depletion. Although antiretroviral therapy (ART) is effective in suppressing HIV replication and restoring CD4+ T cells in peripheral blood, restoration in GALT is delayed. The role of restored CD4+ T-cell help in GALT during ART and its impact on antiviral CD8+ T-cell responses have not been investigated. Using the SIV model, we investigated gut CD4+ T-cell restoration in infected macaques, initiating ART during either the primary stage (1 week postinfection), prior to acute CD4+ cell loss (PSI), or during the chronic stage at 10 weeks postinfection (CSI). ART led to viral suppression in GALT and peripheral blood mononuclear cells of PSI and CSI animals at comparable levels. CSI animals had incomplete CD4+ T-cell restoration in GALT. In PSI animals, ART did not prevent acute CD4+ T-cell loss by 2 weeks postinfection in GALT but supported rapid and complete CD4+ T-cell restoration thereafter. This correlated with an accumulation of central memory CD4+ T cells and better suppression of inflammation. Restoration of CD4+ T cells in GALT correlated with qualitative changes in SIV gag-specific CD8+ T-cell responses, with a dominance of interleukin-2-producing responses in PSI animals, while both CSI macaques and untreated SIV-infected controls were dominated by gamma interferon responses. Thus, central memory CD4+ T-cell levels and qualitative antiviral CD8+ T-cell responses, independent of viral suppression, were the immune correlates of gut mucosal immune restoration during ART.

Rapid onset of intestinal epithelial barrier dysfunction in primary human immunodeficiency virus infection is driven by an imbalance between immune response and mucosal repair and regeneration.

Gut-associated lymphoid tissue (GALT) is an early target for human immunodeficiency virus type 1 (HIV-1) infection and is a site for severe CD4(+) T-cell depletion. HIV-associated enteropathy is well-documented in chronic HIV-1 infection. However, the initial host responses to HIV infection in GALT and the early molecular correlates of HIV enteropathogenesis have not been characterized during primary HIV infection. In this study, we provide evidence of viral replication in GALT resident CD4(+) T cells and macrophages in primary-stage patients and identify early patterns of host mucosal responses and changes in the molecular microenvironment through gene expression profiling. High levels of viral replication in GALT and marked CD4(+) T-cell depletion correlated with decreased expression levels of genes regulating epithelial barrier maintenance and digestive/metabolic functions. These changes coincided with a marked increase in the transcription of immune activation-, inflammation-, and apoptosis-associated genes. Our findings indicate that HIV-induced pathogenesis in GALT emerges at both the molecular and cellular levels prior to seroconversion in primary HIV infection, potentially setting the stage for disease progression by impairing the ability to control viral replication and repair and regenerate intestinal mucosal tissues.

Correlation of immune activation with HIV-1 RNA levels assayed by real-time RT-PCR in HIV-1 subtype C infected patients in Northern India.

BACKGROUND: Assays with specificity and cost effectiveness are needed for the measurement of HIV-1 burden to monitor disease progression or response to anti-retroviral therapy (ART) in HIV-1 subtype C infected patients. OBJECTIVES: The objective of this study was to develop and validate an affordable one step real-time RT-PCR assay with high specificity and sensitivity to measure plasma HIV-1 loads in HIV-1 subtype C infected patients. RESULTS: We developed an RT-PCR assay to detect and quantitate plasma HIV-1 levels in HIV-1 subtype C infected patients. An inverse correlation between plasma viral loads (PVL) and CD4+ T-cell numbers was detected at all CDC stages. Significant correlations were found between CD8+ T-cell activation and PVL, as well as with the clinical and immunological status of the patients. CONCLUSIONS: This RT-PCR assay provides a sensitive method to measure PVL in HIV-1 subtype C infected patients. Viral loads correlated with immune activation and can be used to monitor HIV care in India.

Hepatitis C virus core protein induces expression of genes regulating immune evasion and anti-apoptosis in hepatocytes.

Hepatitis C virus (HCV) Core protein is implicated in the development of hepatocellular carcinoma (HCC). We utilized a HepG2 human hepatocyte cell line with inducible expression of HCV Core protein (HCV-1b) to investigate the early effects of Core protein on hepatocyte gene expression and to identify molecular processes modulated by the Core protein. A significant change was observed in the expression of 407 genes, which included genes regulating apoptosis, immune response, and cell cycle. Some of these genes were previously known to be tumor markers. The decreased expression of chemo-attractants such as TNFSF10, CCL20, and osteopontin was observed, which suggested that HCV Core expression could lead to suppression of inflammatory response as well as trafficking of macrophages and neutrophils to the site of HCV infection. An increased expression of anti-apoptosis factors including PAK2, API5, BH1, Tax1BP1, DAXX, and TNFAIP3/A20 was observed. Some of these genes were also linked to the regulation of NFKB activation and that the alteration of their expression levels, by HCV Core, might lead to the suppression NFKB activation of inflammatory responses. Our data suggested that Core expression may contribute to the viral persistence by protecting infected hepatocytes from cell death by the suppressing apoptosis and inflammatory reaction to HCV viral infection.

Viral suppression and immune restoration in the gastrointestinal mucosa of human immunodeficiency virus type 1-infected patients initiating therapy during primary or chronic infection.

Although the gut-associated lymphoid tissue (GALT) is an important early site for human immunodeficiency virus (HIV) replication and severe CD4+ T-cell depletion, our understanding is limited about the restoration of the gut mucosal immune system during highly active antiretroviral therapy (HAART). We evaluated the kinetics of viral suppression, CD4+ T-cell restoration, gene expression, and HIV-specific CD8+ T-cell responses in longitudinal gastrointestinal biopsy and peripheral blood samples from patients initiating HAART during primary HIV infection (PHI) or chronic HIV infection (CHI) using flow cytometry, real-time PCR, and DNA microarray analysis. Viral suppression was more effective in GALT of PHI patients than CHI patients during HAART. Mucosal CD4+ T-cell restoration was delayed compared to peripheral blood and independent of the time of HAART initiation. Immunophenotypic analysis showed that repopulating mucosal CD4+ T cells were predominantly of a memory phenotype and expressed CD11 alpha, alpha(E)beta 7, CCR5, and CXCR4. Incomplete suppression of viral replication in GALT during HAART correlated with increased HIV-specific CD8+ T-cell responses. DNA microarray analysis revealed that genes involved in inflammation and cell activation were up regulated in patients who did not replenish mucosal CD4+ T cells efficiently, while expression of genes involved in growth and repair was increased in patients with efficient mucosal CD4+ T-cell restoration. Our findings suggest that the discordance in CD4+ T-cell restoration between GALT and peripheral blood during therapy can be attributed to the incomplete viral suppression and increased immune activation and inflammation that may prevent restoration of CD4+ T cells and the gut microenvironment.

Gut mucosal T cell responses and gene expression correlate with protection against disease in long-term HIV-1-infected nonprogressors.

Limited information is available on the molecular mechanisms by which long-term HIV-1-infected nonprogressors suppress HIV-1 infection and maintain immune functions. The intestinal mucosal immune system is an early target for HIV-1 infection and severe CD4+ T cell depletion. We evaluated mucosal T lymphocyte subsets, virus-specific cellular responses, gene expression profiles, and viral loads in intestinal mucosal biopsies of long-term nonprogressor (LTNP) patients as compared to chronically HIV-1-infected patients with high viral loads (HVLs) and CD4+ T cell loss, as well as HIV-seronegative healthy individuals. This study aims to identify the mucosal correlates of HIV disease progression and to determine the molecular changes associated with immune and intestinal dysfunction. LTNP patients had undetectable viral loads, normal CD4+ T cell levels, and virus-specific cellular responses in peripheral blood and mucosal compartments. Microarray analysis revealed a significant increase in gene expression regulating immune activation, cell trafficking, and inflammatory response in intestinal mucosa of HVL patients as compared to LTNP patients. Genes associated with cell cycle regulation, lipid metabolism, and epithelial cell barrier and digestive functions were down-regulated in both HVL and LTNP patients. This may adversely influence nutrient adsorption and digestive functions, with the potential to impact the efficacy of antiretroviral therapy. We demonstrate that the maintenance of mucosal T cells, virus-specific responses, and distinct gene expression profiles correlate with clinical outcome in LTNP patients. However, the intestinal mucosal immune system remains an important target of HIV-1 infection in LTNP, and these effects may ultimately contribute toward disease progression.

Early antiretroviral therapy for simian immunodeficiency virus infection leads to mucosal CD4+ T-cell restoration and enhanced gene expression regulating mucosal repair and regeneration.

Simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) infections lead to rapid depletion of CD4(+) T cells from gut-associated lymphoid tissue (GALT). Although the administration of antiretroviral therapy (ART) has been shown to increase CD4(+) T-cell levels in the peripheral blood in both SIV and HIV infections, its efficacy in restoring intestinal mucosal CD4(+) T cells has not been well investigated. To gain insights into the molecular mechanisms of virally induced disruptions in the mucosal immune system, we have evaluated longitudinal changes in viral burden, T-cell subsets, and mucosal gene expression profiles in SIV-infected rhesus macaques in the absence or presence of ART. Our results demonstrate a dramatic suppression of mucosal viral loads and rapid reconstitution of CD4(+) T cells in GALT in animals receiving ART that were not observed in untreated SIV-infected animals. DNA microarray-based gene expression profiling indicated that CD4(+) T-cell restoration in GALT was associated with up regulation of growth factors and genes involved in repair and regeneration of the mucosal epithelium. In contrast, untreated SIV-infected animals increased expression of lymphocyte activation and inflammatory response-associated genes and did not up regulate mucosal growth and repair associated transcription. In conclusion, these data indicate that initiating ART in primary SIV infection may lead to the restoration of the mucosal immune system through reduction of inflammation and promotion of epithelial repair in the intestinal mucosa.

Severe CD4+ T-cell depletion in gut lymphoid tissue during primary human immunodeficiency virus type 1 infection and substantial delay in restoration following highly active antiretroviral therapy.

Gut-associated lymphoid tissue (GALT) harbors the majority of T lymphocytes in the body and is an important target for human immunodeficiency virus type 1 (HIV-1). We analyzed longitudinal jejunal biopsy samples from HIV-1-infected patients, during both primary and chronic stages of HIV-1 infection, prior to and following the initiation of highly active antiretroviral therapy (HAART) to determine the onset of CD4(+) T-cell depletion and the effect of HAART on the restoration of CD4(+) T cells in GALT. Severe depletion of intestinal CD4(+) T cells occurred during primary HIV-1 infection. Our results showed that the restoration of intestinal CD4(+) T cells following HAART in chronically HIV-1-infected patients was substantially delayed and incomplete. In contrast, initiation of HAART during early stages of infection resulted in near-complete restoration of intestinal CD4(+) T cells, despite the delay in comparison to peripheral blood CD4(+) T-cell recovery. DNA microarray analysis of gene expression profiles and flow-cytometric analysis of lymphocyte homing and cell proliferation markers demonstrated that cell trafficking to GALT and not local proliferation contributed to CD4(+) T-cell restoration. Evaluation of jejunal biopsy samples from long-term HIV-1-infected nonprogressors showed maintenance of normal CD4(+) T-cell levels in both GALT and peripheral blood. Our results demonstrate that near-complete restoration of mucosal immune system can be achieved by initiating HAART early in HIV-1 infection. Monitoring of the restoration and/or maintenance of CD4(+) T cells in GALT provides a more accurate assessment of the efficacy of antiviral host immune responses as well as HAART.

High-throughput gene expression profiling indicates dysregulation of intestinal cell cycle mediators and growth factors during primary simian immunodeficiency virus infection.

During primary simian immunodeficiency virus (SIV) infection, CD4+ T cells are severely depleted in gut-associated lymphoid tissue (GALT), while CD8+ T-cell numbers dramatically increase. To gain an understanding of the molecular basis of this disruption in T-cell homeostasis, host gene expression was monitored in longitudinal jejunum tissue biopsies from SIV-infected rhesus macaques by DNA microarray analysis. Transcription of cyclin E1, CDC2, retinoblastoma, transforming growth factor (TGF), fibroblast growth factor (FGF), and interleukin-2 was repressed while cyclins B1 and D2 and transcription factor E2F were upregulated, indicating a complex dysregulation of growth and proliferation within the intestinal mucosa. Innate, cell-mediated, and humoral immune responses were markedly upregulated in animals that significantly reduced their viral loads and retained more intestinal CD4+ T cells. We conclude that the alterations in intestinal gene expression during primary SIV infection were characteristic of a broad-range immune response, and reflective of the efficacy of viral suppression.