Initiation of ART during Early Acute HIV Infection Preserves Mucosal Th17 Function and Reverses HIV-Related Immune Activation

<div><p>Mucosal Th17 cells play an important role in maintaining gut epithelium integrity and thus prevent microbial translocation. Chronic HIV infection is characterized by mucosal Th17 cell depletion, microbial translocation and subsequent immune-activation, which remain elevated despite antiretroviral therapy (ART) correlating with increased mortality. However, when Th17 depletion occurs following HIV infection is unknown. We analyzed mucosal Th17 cells in 42 acute HIV infection (AHI) subjects (Fiebig (F) stage I-V) with a median duration of infection of 16 days and the short-term impact of early initiation of ART. Th17 cells were defined as IL-17+ CD4+ T cells and their function was assessed by the co-expression of IL-22, IL-2 and IFNγ. While intact during FI/II, depletion of mucosal Th17 cell numbers and function was observed during FIII correlating with local and systemic markers of immune-activation. ART initiated at FI/II prevented loss of Th17 cell numbers and function, while initiation at FIII restored Th17 cell numbers but not their polyfunctionality. Furthermore, early initiation of ART in FI/II fully reversed the initially observed mucosal and systemic immune-activation. In contrast, patients treated later during AHI maintained elevated mucosal and systemic CD8+ T-cell activation post initiation of ART. These data support a loss of Th17 cells at early stages of acute HIV infection, and highlight that studies of ART initiation during early AHI should be further explored to assess the underlying mechanism of mucosal Th17 function preservation.</p></div

Clinical, immunological and virological baseline characteristics and demographics of study participants.

A<p>range;</p>B<p>MHAbce: Multi-region hybridization assay distinguishing between subtypes B, C and CRF01_AE. One subject was CRF01_AE/B, 8 were non typable and results for 3 subjects were not typed by the time the manuscript was written;</p>C<p>Western blot positive with p31 band; MSM: Men who have sex with men; Fiebig I - positive HIV RNA, negative p24 antigen, negative 3rd generation EIA; Fiebig II – positive HIV RNA, positive p24 antigen, negative 3rd generation EIA; Fiebig III - positive HIV RNA, positive p24 antigen, positive 3rd generation EIA, negative western blot; Fiebig IV - positive HIV RNA, positive or negative p24 antigen, positive 3rd generation EIA, indeterminate western blot; Fiebig V - positive HIV RNA, positive p24 antigen, positive 3rd generation EIA, positive western blot except p31; NA: Not Applicable; ND: Not Determined</p><p>Clinical, immunological and virological baseline characteristics and demographics of study participants.</p

Proportion and absolute number of CD4+, CD4+CCR5+ and CD8+ T cells in sigmoid colon and peripheral blood at baseline in HIV-, FI/II, FIII, FIV/V and CHI subjects.

<p>All data are median (interquartile rang); All comparisons were made to FI/II; *p≤0.05, **p≤0.01 and ***p≤0.001; CHI: Chronically HIV-infected patients; ND: Not Determined; abs numbers in the sigmoid colon are shown as 10⁶ cells per gram tissue and in the peripheral blood as cells/mm^{3; &}Percentage of CD4+ and CD8+T cells based on population of CD3+T cells.</p><p>Proportion and absolute number of CD4+, CD4+CCR5+ and CD8+ T cells in sigmoid colon and peripheral blood at baseline in HIV-, FI/II, FIII, FIV/V and CHI subjects.</p

Frequency of IL-17 and/or IL-22 expressing mucosal CD4+T cells decreases by progression of Fiebig stage.

<p>To quantify the expression of Il-17 and IL-22 in CD4+T cells, mucosal and peripheral mononuclear cells were stimulated for 5 hours with 40 ng/mL PMA and 1 µM Ionomycin. Gating strategy of sigmoid colon Th17 and Th22 CD4+T cells is shown for a FI subject (<b>a</b>) and representative flow cytometry plots including unstimulated controls (unstim) gated on CD4+T cells showing expression of IL-17 and/or IL-22 in FI (<b>b</b>), FIII (<b>c</b>) and FV (<b>d</b>) in the sigmoid colon. A decrease in the frequency of IL-17 (<b>e</b>), IL-22 (<b>f</b>), IL-17/IL-22 (<b>g</b>)-producing cells was seen as well as in the subpopulation of triple-cytokine producing (IL-2, IL-22, IFNγ) Th17 cells (<b>h</b>) from FI (black circle)/FII (red circle) to FIII and FIV (red square)/FV (black square). Frequency of single and double cytokine producing cells is calculated from percentage CD4+T cells, while triple-cytokine producing cells are calculated from percentage of CD4+IL17+T cells. All comparisons were made to FI/II: *p≤0.05, **p≤0.01 and ***p≤0.001.</p

Impact of early ART initiation on CD4+ and CD8+T cells as well as Th17 cells.

<p>Subjects that initiated ART during FI (black circle)/FII (red circle) for 6 months were able to maintain mucosal CD4+T cells (<b>a</b>), CD4+CCR5+ (<b>b</b>), Th17 cells (<b>c</b>), triple cytokine-producing Th17 cells (<b>d</b>), Th22 cells (<b>e</b>), and IL-17 and/or IL-22 (<b>f</b>) producing CD4+T cells with no differences when compared to HIV-. In addition their CD8 activation in the mucosa (<b>g</b>) and periphery (<b>h</b>) normalized after 6 months of ART. Frequency of single and double cytokine producing cells was calculated from percentage CD4+T cells, while triple-cytokine producing cells were calculated from percentage of CD4+IL17+T cells. *p≤0.05, **p≤0.01 and ***p≤0.001; DR: HLA-DR; blue dotted line: median of HIV- individuals; purple dotted line: median of CHI individuals.</p

Percentage area of lamina propria CD4+ staining (% Area LP) decreases with progression of Fiebig stage.

<p>(<b>a</b>) The percent area of the lamina propria that stained for CD4+T cells (representative images shown in e) decreases by Fiebig stage in the sigmoid colon when compared to FI/II (*p≤0.05, **p≤0.01 and ***p≤0.001). The percent area of the lamina propria that stained for CD4+T cells correlated inversely with the colonic (<b>b</b>) and plasma (<b>c</b>) HIV RNA in FI/II, FIII and FIV/V. HIV viral replication during different Fiebig stages is shown by in <i>situ hybridization</i> displaying HIV-1 vRNA+ cells within the sigmoid mucosa, indicated by blue/black stained cells in nuclear fast red counterstained tissue sections (black arrows highlighting examples of HIV vRNA+ cells) of patients in FI, FII and FIII (<b>d</b>). CD4+T cell depletion is shown by immunohistochemical staining of CD4+T cells (brown) and macrophages (red) in sigmoid mucosa of patients in FI, FII and FIII (<b>e</b>). FI (black circle)/FII (red circle) and FIV (red square)/FV (black square).</p

Clinical, immunological and virological baseline characteristics and demographics of study participants.

A<p>range;</p>B<p>MHAbce: Multi-region hybridization assay distinguishing between subtypes B, C and CRF01_AE. One subject was CRF01_AE/B, 8 were non typable and results for 3 subjects were not typed by the time the manuscript was written;</p>C<p>Western blot positive with p31 band; MSM: Men who have sex with men; Fiebig I - positive HIV RNA, negative p24 antigen, negative 3rd generation EIA; Fiebig II – positive HIV RNA, positive p24 antigen, negative 3rd generation EIA; Fiebig III - positive HIV RNA, positive p24 antigen, positive 3rd generation EIA, negative western blot; Fiebig IV - positive HIV RNA, positive or negative p24 antigen, positive 3rd generation EIA, indeterminate western blot; Fiebig V - positive HIV RNA, positive p24 antigen, positive 3rd generation EIA, positive western blot except p31; NA: Not Applicable; ND: Not Determined</p><p>Clinical, immunological and virological baseline characteristics and demographics of study participants.</p