Altered CD4+ T cell homing to the gut impairs mucosal immune reconstitution in treated HIV-infected individuals

Abstract

Depletion of CD4+ T cells from the gut occurs rapidly during acute HIV-1 infection. This has been linked to systemic inflammation and disease progression as a result of translocation of microbial products from the gut lumen into the bloodstream. Combined antiretroviral therapy (cART) substantially restores CD4+ T cell numbers in peripheral blood, but the gut compartment remains largely depleted of such cells for poorly understood reasons. Here, we show that a lack of recruitment of CD4+ T cells to the gut could be involved in the incomplete mucosal immune reconstitution of cART-treated HIV-infected individuals. We investigated the trafficking of CD4+ T cells expressing the gut-homing receptors CCR9 and integrin α4β7 and found that many of these T cells remained in the circulation rather than repopulating the mucosa of the small intestine. This is likely because expression of the CCR9 ligand CCL25 was lower in the small intestine of HIV-infected individuals. The defective gut homing of CCR9+β7+ CD4+ T cells - a population that we found included most gut-homing Th17 cells, which have a critical role in mucosal immune defense - correlated with high plasma concentrations of markers of mucosal damage, microbial translocation, and systemic T cell activation. Our results thus describe alterations in CD4+ T cell homing to the gut that could prevent efficient mucosal immune reconstitution in HIV-infected individuals despite effective cART.

Figures

Figure 1. CD4+ T cell depletion and HIV-1 persistence in the gut mucosa of HIV-infected individuals despite sustained effective cART.

(A) Frequencies of CD4+ in CD3+ T cells in the peripheral blood (PB) and jejunum mucosa of HIV-infected individuals (n = 20) and uninfected individuals (n = 9). Percentages of cells were determined by flow cytometry. Horizontal lines indicate median values. Each symbol represents an individual. (B) Absolute numbers of CD4+ T cells per surface unit of jejunum mucosa in HIV-infected individuals (n = 20) and uninfected individuals (n = 9). Horizontal lines indicate median values. Each symbol represents an individual. Immunohistochemical detection of intestinal CD4+ T cells (brown staining) in a representative HIV-infected individual and an uninfected control are shown. Original magnification, ×100. (C) Correlation between the frequency of CD4+ in CD3+ T cells in peripheral blood and jejunum mucosa (n = 20 HIV-infected individuals, and n = 9 uninfected individuals). Percentages of cells were determined by flow cytometry. Each symbol represents an individual. (D) Paired HIV-1 DNA loads in CD4+ T cells of the peripheral blood and jejunum mucosa (n = 20). HIV-1 DNA was quantified by real-time PCR.

Figure 2. Distribution of CCR9+β7hi CD4+ T cells in the peripheral blood and jejunum compartments.

(A) Flow cytometry analysis of CCR9 and β7 on CD4+ T cells in the peripheral blood and jejunum mucosa of a representative HIV-infected individual and an uninfected control. Numbers indicate the percentage of gated cells. (B) Correlation between the frequency of CCR9+β7hi in CD4+ T cells in peripheral blood and jejunum mucosa of HIV-infected individuals (n = 20) and uninfected individuals (n = 9). Percentages of cells were determined by flow cytometry. Each symbol represents an individual. (C) Frequency of CCR9+β7hi in CD4+ T cells in the peripheral blood and jejunum mucosa of HIV-infected individuals (n = 20) and uninfected individuals (n = 9). Percentages of cells were determined by flow cytometry. Horizontal lines indicate median values. Each symbol represents an individual.

Figure 3. Alterations in the CCL25-CCR9 axis that drives the gut homing of CCR9+β7hi CD4+ T cells in HIV-infected individuals.

(A) CCL25 mRNA expression in the jejunum epithelial cells of HIV-infected individuals (n = 18) and uninfected individuals (n = 7). Horizontal lines indicate median values. CCL25 mRNA was quantified by real-time RT-PCR and normalized to GAPDH. (B) CCL25 chemokine expression in the jejunum mucosa of HIV-infected individuals (n = 19) and uninfected individuals (n = 9). Horizontal lines indicate median values. A representative HIV-infected individual and an uninfected control are shown. CCL25 chemokine (brown) was stained by immunohistochemistry and automatically quantified with NIS-element (Nikon). Original magnification, ×400. (C) Correlation between CCL25 mRNA expression and the frequency of CCR9+β7hi in CD4+ T cells in the jejunum mucosa (n = 18 HIV-infected individuals, and n = 7 uninfected individuals). (D) Correlation between CCL25 mRNA expression and the frequency of CCR9+β7hi in CD4+ T cells in the peripheral blood (n = 18 HIV-infected individuals, and n = 7 uninfected individuals). Throughout, each symbol represents an individual.

Figure 4. CCR9+β7hi CD4+ T cells include most gut-homing Th17 cells.

(A) Paired frequencies of CCR6+ cells in CCR9+β7hi and CCR9–β7hi CD4+ T cell subsets in the peripheral blood (n = 20 HIV-infected individuals, and n = 9 uninfected individuals). Percentages of cells were determined using flow cytometry. (B) Paired frequencies of IL-17–producing cells in CCR9+β7hi and CCR9–β7hi CD4+ T cell subsets in the peripheral blood (n = 5 HIV-infected individuals, and n = 5 uninfected individuals). Percentages of cells were determined by flow cytometry.

Figure 5. Correlation between defective homing of CCR9+β7hi CD4+ T cells to the gut and mucosal damage, microbial translocation, and systemic T cell activation.

(A) Concentrations of I-FABP, LPS, and sCD14 in HIV-infected individuals (n = 20 for I-FABP and sCD14 concentrations, and n = 19 for LPS concentrations) and uninfected individuals (n = 9). Plasma I-FABP and sCD14 concentrations were measured by ELISA. Plasma LPS was measured by the Limulus amoebocyte lysate assay. Horizontal lines indicate median values. (B) Correlation between the frequency of CCR9+β7hi in CD4+ T cells in the peripheral blood and the plasma concentrations of LPS (n = 19 HIV-infected individuals, and n = 9 uninfected individuals) and sCD14 (n = 20 HIV-infected individuals, and n = 9 uninfected individuals). (C) Correlation between the frequency of CCR9+β7hi in CD4+ T cells in the jejunum mucosa and the plasma concentrations of LPS (n = 19 HIV-infected individuals, and n = 9 uninfected individuals) and sCD14 (n = 20 HIV-infected individuals, and n = 9 uninfected individuals). (D) Correlation between CCL25 mRNA expression in the jejunum mucosa and the plasma concentrations of LPS (n = 17 HIV-infected individuals, and n = 7 uninfected individuals) and sCD14 (n = 18 HIV-infected individuals, and n = 7 uninfected individuals). (E) Correlation between the frequency of CCR9+β7hi in CD4+ T cells in the peripheral blood and the frequency of Ki67+ in CD4+ T cells in the peripheral blood (n = 20 HIV-infected individuals). (F) Correlation between CCL25 mRNA expression in jejunum epithelial cells and the frequency of Ki67+ in CD4+ T cells in peripheral blood (n = 18 HIV-infected individuals). Throughout, each symbol represents an individual.