Differences in HIV burden and immune activation within the gut of HIV-positive patients receiving suppressive antiretroviral therapy

Abstract

Background: The gut is a major reservoir for human immunodeficiency virus (HIV) in patients receiving antiretroviral therapy (ART). We hypothesized that distinct immune environments within the gut may support varying levels of HIV.

Methods: In 8 HIV-1-positive adults who were receiving ART and had CD4(+) T cell counts of >200 cells/μL and plasma viral loads of <40 copies/mL, levels of HIV and T cell activation were measured in blood samples and endoscopic biopsy specimens from the duodenum, ileum, ascending colon, and rectum.

Results: HIV DNA and RNA levels per CD4(+) T cell were higher in all 4 gut sites compared with those in the blood. HIV DNA levels increased from the duodenum to the rectum, whereas the median HIV RNA level peaked in the ileum. HIV DNA levels correlated positively with T cell activation markers in peripheral blood mononuclear cells (PBMCs) but negatively with T cell activation markers in the gut. Multiply spliced RNA was infrequently detected in gut, and ratios of unspliced RNA to DNA were lower in the colon and rectum than in PBMCs, which reflects paradoxically low HIV transcription, given the higher level of T cell activation in the gut.

Conclusions: HIV DNA and RNA are both concentrated in the gut, but the inverse relationship between HIV DNA levels and T cell activation in the gut and the paradoxically low levels of HIV expression in the large bowel suggest that different processes drive HIV persistence in the blood and gut.

Trial registration: ClinicalTrials.gov identifier: NCT00884793 (PLUS1).

Figure 1

CD4 contents are indicated as the percent of all cells (1A) or the percent of T-cells (1B) that express CD4, as measured by flow cytometry on peripheral blood mononuclear cells (PBMC) or suspensions of total gut cells. Whiskers represent the maximum and minimum; upper and lower box borders represent the 75^th and 25^th percentile; the horizontal line indicates the median.

Figure 2

HIV DNA copy numbers (2A) were measured in peripheral blood mononuclear cells (PBMC) and total gut cells using real time PCR, normalized to total cell numbers by DNA mass (NanoDrop), and normalized to CD4 cells by flow cytometry. Total unspliced HIV RNA levels (2B) were measured using qRT-PCR, normalized to cell numbers by GAPDH, and normalized to CD4 cells by flow cytometry. Whiskers represent the maximum and minimum; upper and lower box borders represent the 75^th and 25^th percentile; the horizontal line indicates the median.

Figure 3

The mean plasma RNA correlated with HIV DNA content in PBMC (3A) and with the percent of peripheral CD4+T-cells that are CD38+ (3B) or CD38+HLA-DR+ (3C). Plasma RNA values indicate the mean of two measurements (two weeks apart) that were made by concentrating virions from up to 30ml of plasma using high speed centrifugation on a 10% iodixanol density cushion and then assaying the resuspended pellet using the Abbott M2000 assay. CD38 and HLA-DR were measured by flow cytometry.

Figure 4

To approximate the transcriptional activity per infected cell, we calculated the ratio of unspliced HIV RNA (copies/10⁶ cells) to HIV DNA (copies/10⁶ cells) [both normalized by nucleic acid mass] in peripheral blood mononuclear cells (PBMC) and cells from the four gut sites. Whiskers represent the maximum and minimum; upper and lower box borders represent the 75^th and 25^th percentile; the horizontal line indicates the median.

Figure 5

T-cell “activation” is indicated by the percent of CD4+T-cells (4A) or CD8+T cells (4B) that express both CD38 and HLA-DR, as measured by flow cytometry. Whiskers represent the maximum and minimum; upper and lower box borders represent the 75^th and 25^th percentile; the horizontal line indicates the median.