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

Abstract

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.

FIG. 1.

Suppression of HIV replication and restoration of CD4⁺ T cells in peripheral blood of HIV-infected patients during HAART. Plasma viral loads and CD4⁺ T-cell numbers in peripheral blood during HAART were longitudinally monitored in patients initiating HAART during PHI (A) or CHI (B). The detection limit for the viral load was 50 copies/ml. (C) The rate of CD4⁺ T-cell restoration in PHI and CHI patients, measured as average change in percentage points per month.

FIG. 2.

Suppression of HIV replication and restoration of CD4⁺ T cells in GALT of HIV-infected patients receiving HAART. (A) Viral loads in GALT from PHI and CHI patients were determined prior to and post-HAART by real-time PCR. Relative changes were calculated by comparison of HIV RNA copies to the lowest HIV RNA levels measured in GALT of HIV patients on HAART. Flow cytometric analysis was performed in longitudinal GALT biopsy samples from PHI (B) and CHI (C) patients prior to and following HAART.

FIG. 3.

Localization of CD4⁺ T cells in GALT of HIV-infected patients during therapy by fluorescent immunohistochemistry. Immunohistochemical analysis was performed in GALT biopsy samples using confocal microscopy. Anti-CD4 and -CD3 antibodies were used, followed by secondary anti-mouse Cy3 (red) and anti-rabbit Alexa 488 (green), respectively. CD3⁺ CD4⁺ T cells (yellow), CD3⁺ cells (green), and CD4⁺ T cells (red) were detected.

FIG. 4.

Immunophenotypic analysis demonstrated that memory CD4⁺ T cells repopulate GALT following HAART. (A) Expression of naive/memory T-cell markers CD45RA and CD45RO in repopulating CD4⁺ T cells from peripheral blood and jejunal biopsy samples from HIV-infected patients following HAART. (B) Immunophenotypic analysis showed expression of HIV-1 coreceptors CCR5 and CXCR4, LFA-1 and α_Eβ₇ (trafficking markers) in repopulating CD4⁺ T cells in GALT of HIV-1-infected patients during HAART.

FIG. 5.

HIV-specific CD8⁺ T-cell responses in GALT and peripheral blood of HIV-infected patients during HAART. HIV-specific CD8⁺ T-cell responses were detected in both GALT and peripheral blood of HIV-infected patients during HAART using intracellular cytokine flow cytometry.

FIG. 6.

Alterations in gut mucosal gene expression in HIV-infected patients during therapy. Genes differentially expressed (≥1.5-fold above or below mean baseline in healthy, uninfected controls; P value ≤ 0.05) in GALT were hierarchically clustered to identify patterns of up (red) and down (green) regulation in response to HIV-1 infection and antiretroviral therapy. Up and down regulated genes were then statistically analyzed to determine which biological processes or molecular functions were enriched or overrepresented. EtOH, ethanol.

FIG. 7.

Mucosal gene expression profiles in patients with divergent CD4⁺ T-cell restoration during HAART. Gene expression profiles were determined in GALT biopsy samples pre-HAART (Pre) and at 3 months post-HAART (3 mo) by DNA microarray analysis and genes associated with inflammation and immune activation (A) and lipid metabolism and nutrient digestive and absorption (B) were identified.

FIG. 8.

Suppression of viral replication and increased gene expression of mucosal repair and regeneration during CD4⁺ T-cell repopulation in GALT. Data are presented at 6 months post-HAART for patient 116 (efficient CD4⁺ T-cell restoration) and patient 123 (poor CD4⁺ T-cell restoration).