Loss of Function of Intestinal IL-17 and IL-22 Producing Cells Contributes to Inflammation and Viral Persistence in SIV-Infected Rhesus Macaques

Abstract

In HIV/SIV-infected humans and rhesus macaques (RMs), a severe depletion of intestinal CD4(+) T-cells producing interleukin IL-17 and IL-22 associates with loss of mucosal integrity and chronic immune activation. However, little is known about the function of IL-17 and IL-22 producing cells during lentiviral infections. Here, we longitudinally determined the levels and functions of IL-17, IL-22 and IL-17/IL-22 producing CD4(+) T-cells in blood, lymph node and colorectum of SIV-infected RMs, as well as how they recover during effective ART and are affected by ART interruption. Intestinal IL-17 and IL-22 producing CD4(+) T-cells are polyfunctional in SIV-uninfected RMs, with the large majority of cells producing four or five cytokines. SIV infection induced a severe dysfunction of colorectal IL-17, IL-22 and IL-17/IL-22 producing CD4(+) T-cells, the extent of which associated with the levels of immune activation (HLA-DR(+)CD38(+)), proliferation (Ki-67+) and CD4(+) T-cell counts before and during ART. Additionally, Th17 cell function during ART negatively correlated with residual plasma viremia and levels of sCD163, a soluble marker of inflammation and disease progression. Furthermore, IL-17 and IL-22 producing cell frequency and function at various pre, on, and off-ART experimental points associated with and predicted total SIV-DNA content in the colorectum and blood. While ART restored Th22 cell function to levels similar to pre-infection, it did not fully restore Th17 cell function, and all cell types were rapidly and severely affected--both quantitatively and qualitatively--after ART interruption. In conclusion, intestinal IL-17 producing cell function is severely impaired by SIV infection, not fully normalized despite effective ART, and strongly associates with inflammation as well as SIV persistence off and on ART. As such, strategies able to preserve and/or regenerate the functions of these CD4(+) T-cells central for mucosal immunity are critically needed in future HIV cure research.

Fig 1. Study timeline and representative cytokine panel staining.

(A) Complete study timeline of longitudinal tissue collections, SIVmac239 i.v. infection (d.0 p.i.), ART treatment (d. 58 to d.256 p.i.), and off-ART time period. (B) Representative staining for IL-17, IL-22, IFNγ, TNFα, and IL-2 within intestinal CD4⁺ T-cells in a representative uninfected RM.

Fig 2. IL-17 producing cell function is higher in colorectum than blood in uninfected RMs.

(A) Comparison between cytokine profiles of Th17, Th22, and Th17/Th22 cells in PBMC and RB in uninfected RMs (d. -20 p.i.). Cytokine profiles were generated for each cell population by SPICE program v. 5.33, and were calculated by Flowjo Boolean gating. (B) Functional scores were compared for all three subsets between PBMC and RB. Functional scores represent average number of cytokines produced per individual cell (see Methods). Averaged data are presented as means ± SEM.

Fig 3. Levels of Th17, Th22, and Th17/Th22 are more drastically depleted by SIV infection in RB than PBMC.

Comparison of frequencies (measured as percentages of total CD4⁺ T-cell populations) of circulating (A) and colorectal (B) Th17 (red), Th22 (blue), and Th17/Th22 (green) cells before (d.-20 p.i.) and after (d. 58 p.i.) SIV infection. Averaged data are presented as means ± SEM.

Fig 4. SIV infection severely ablates intestinal IL-17 and IL-22 producing cell function and levels.

(A) Comparison between PBMC and RB Th17, Th22, and Th17/Th22 cell cytokine profiles at pre-infection (d.-20 p.i.) vs SIV infection (d. 58 p.i.). While all RB cytokine profiles significantly changed after SIV infection, the blood cytokine profiles remained unchanged. (B) No changes in functional score in PBMC after SIV infection. (C) In all three subsets, colorectal functional scores drastically decreased after SIV infection. Th17 cells marked as red, Th22 cells marked as blue, and Th17/Th22 cells marked as green. Averaged data are presented as means ± SEM.

Fig 5. ART treatment is insufficient for full restoration of cell subset levels and function.

(A-C) Longitudinal functional scores of Th17, Th22, and Th17/Th22 cells in RB at pre-infection (d. -20 p.i.), pre-ART (d. 58 p.i.), and throughout ART (d. 84 p.i. through d. 256 p.i.). Data are presented as box and whisker plots, with the median functional score plotted in between the 25% and 75% quartiles. Dotted line marks time of SIV infection and shaded gray box represents time of ART treatment. ART significantly increased all three subsets’ functional scores, but did not bring Th17 cell function back to pre-infection level. (D) Levels of subsets (as percentages of total CD4⁺ T-cell populations) in RB during ART remain significantly lower than pre-infection. (E) Longitudinal cumulative subset scores, calculated by multiplying cell frequencies and functional scores, showed the inability of ART to fully restore the levels and function of Th17, Th22, and Th17/Th22 cells. Th17 cells marked as red, Th22 cells marked as blue, and Th17/Th22 cells marked as green. Averaged data are presented as means ± SEM.

Fig 6. ART interruption severely decreases IL-17 and IL-22 producing cell function and levels.

(A) Levels of RB Th17 (red), Th22 (blue), and Th17/Th22 (green) cells, measured as percentages of total CD4⁺ T-cell population, significantly decreased following ART interruption (180 days off-ART). Averaged data are presented as means ± SEM. (B-D) Longitudinal functional scores of Th17, Th22, and Th17/Th22 cells in RB at pre-infection (d. -20 p.i.), pre-ART (d. 58 p.i.), on-ART treatment (d. 256 p.i.), and after ART interruption (180 days off-ART). ART interruption significantly decreases Th17 and Th17/Th22 cell functional scores from late-ART levels. Dotted line marks time of SIV infection and shaded gray box represents time of ART treatment. Averaged data are presented as box and whisker plots, with the median functional score in between the 25% and 75% quartiles.

Fig 7. Loss of Th17, Th22, and Th17/Th22 cell function correlates with colorectal immune activation, soluble markers of inflammation, and levels of CD4⁺ T-cells.

Correlations between Th17, Th22, and Th17/Th22 cell function and plasma viral loads (copies viral RNA per mL of plasma) (A,B), absolute number of CD4⁺ T-cells (C), levels of activated colorectal (HLA-DR⁺CD38⁺) CD4⁺ T-cells (D,E), the level of proliferating (Ki-67⁺) RB CD4⁺ T-cells (F), as well as soluble markers of inflammation sCD14 and sCD163 (G, H). Pearson product-moment correlation coefficients were measured for all plots except for 7A, which required Spearman’s rank correlation coefficient.

Fig 8. Loss of Th17, Th22, and Th17/Th22 cell function and levels are correlates and predictors of SIV persistence.

Correlations between colorectal SIV-DNA content at late-ART (d. 256 p.i.) and IL-17 and IL-22 producing cell function and levels at both ART (A-C) and pre-ART infection (d. 58 p.i). (D,E). Levels and function of the three subsets at pre-ART and after ART interruption additionally correlated with levels of blood DNA levels after ART interruption (d. 440 p.i.) (F-I). Pearson product-moment correlation coefficients were measured for all plots except for 8E, which required Spearman’s rank correlation coefficient.