Enhanced innate antiviral gene expression, IFN-α, and cytolytic responses are predictive of mucosal immune recovery during simian immunodeficiency virus infection.

The mucosa that lines the respiratory and gastrointestinal (GI) tracts is an important portal of entry for pathogens and provides the first line of innate immune defense against infections. Although an abundance of memory CD4(+) T cells at mucosal sites render them highly susceptible to HIV infection, the gut and not the lung experiences severe and sustained CD4(+) T cell depletion and tissue disruption. We hypothesized that distinct immune responses in the lung and gut during the primary and chronic stages of viral infection contribute to these differences. Using the SIV model of AIDS, we performed a comparative analysis of the molecular and cellular characteristics of host responses in the gut and lung. Our findings showed that both mucosal compartments harbor similar percentages of memory CD4(+) T cells and displayed comparable cytokine (IL-2, IFN-γ, and TNF-α) responses to mitogenic stimulations prior to infection. However, despite similar viral replication and CD4(+) T cell depletion during primary SIV infection, CD4(+) T cell restoration kinetics in the lung and gut diverged during acute viral infection. The CD4(+) T cells rebounded or were preserved in the lung mucosa during chronic viral infection, which correlated with heightened induction of type I IFN signaling molecules and innate viral restriction factors. In contrast, the lack of CD4(+) T cell restoration in the gut was associated with dampened immune responses and diminished expression of viral restriction factors. Thus, unique immune mechanisms contribute to the differential response and protection of pulmonary versus GI mucosa and can be leveraged to enhance mucosal recovery.

In vivo CD8+ T-cell suppression of siv viremia is not mediated by CTL clearance of productively infected cells.

The CD8+ T-cell is a key mediator of antiviral immunity, potentially contributing to control of pathogenic lentiviral infection through both innate and adaptive mechanisms. We studied viral dynamics during antiretroviral treatment of simian immunodeficiency virus (SIV) infected rhesus macaques following CD8+ T-cell depletion to test the importance of adaptive cytotoxic effects in clearance of cells productively infected with SIV. As previously described, plasma viral load (VL) increased following CD8+ T-cell depletion and was proportional to the magnitude of CD8+ T-cell depletion in the GALT, confirming a direct relationship between CD8+ T-cell loss and viral replication. Surprisingly, first phase plasma virus decay following administration of antiretroviral drugs was not slower in CD8+ T-cell depleted animals compared with controls indicating that the short lifespan of the average productively infected cell is not a reflection of cytotoxic T-lymphocyte (CTL) killing. Our findings support a dominant role for non-cytotoxic effects of CD8+ T-cells on control of pathogenic lentiviral infection and suggest that cytotoxic effects, if present, are limited to early, pre-productive stages of the viral life cycle. These observations have important implications for future strategies to augment immune control of HIV.

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.

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.

Novel p53/p130 axis in bladder tumors.

OBJECTIVES: To identify the relationships between key components of the proliferative and apoptotic pathways in bladder tumors. METHODS: A tissue array of 88 bladder tumors was assembled. Immunohistochemical analyses were used to investigate the relationship between nine different parameters: stage, proliferation (Ki67), apoptosis (in situ DNA nick end labeling), the anti-apoptotic protein Bcl-2, tumor suppressors p53 and retinoblastoma protein (Rb), the Rb-related protein p130, cyclin E, and the cyclin-dependent kinase inhibitor p27. The protein expression in each tumor was reported as the percentage of positively staining cells. RESULTS: The analysis focused on Stage 1 to 3 tumors. Analysis found that p53 expression increased progressively with stage, and Rb and p27 decreased with increasing stage. Overall, the cyclin E levels correlated with the proliferative index. Cyclin E levels were low in Stage 1 tumors and elevated in Stage 2 tumors, but were decreased in Stage 3 tumors. Multivariate analysis uncovered a correlation between cyclin E and proliferation (Ki67) and a weak correlation between p53 and Bcl-2 and between p27 and Rb. A strong correlation was found between the expression of p53 and p130, which was apparent in Stages 1 and 3, but not in Stage 2. Furthermore, high levels of p130 protein were detected primarily in the cytoplasm. CONCLUSIONS: These results suggest a novel p53/p130 axis in bladder tumors.

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.

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.

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.

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.

Apoptosis of gastric lymphocytes in Helicobacter pylori-infected rhesus macaques.

In vitro studies suggest that H. pylori induces apoptosis in gastric epithelial cells and perhaps in gastric lymphocytes as well. However, the early effects of H. pylori infection on lymphocyte apoptosis have not been examined in experimental animal models, nor have studies been performed using markers specific for T cells and T-cell subsets. Gastric T-cell apoptosis and Fas ligand expression were examined by flow cytometry after experimental infection of rhesus macaques with H. pylori. Infection induced transient apoptosis of gastric CD4+ and CD8+ T-cells, which began as soon as three days after inoculation and declined to baseline within eight weeks. Fas ligand expression showed a similar transient induction, suggesting that it mediates gastric T-cell apoptosis. We propose that transient, Fas-mediated apoptosis in gastric lymphocytes is a compensatory response to the initial T-cell inflammatory response after acute H. pylori infection.

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.