IL-21 and probiotic therapy improve Th17 frequencies, microbial translocation, and microbiome in ARV-treated, SIV-infected macaques

Abstract

Increased mortality in antiretroviral (ARV)-treated, HIV-infected individuals has been attributed to persistent immune dysfunction, in part due to abnormalities at the gastrointestinal barrier. In particular, the poor reconstitution of gastrointestinal Th17 cells correlates with residual translocation of dysbiotic, immunostimulatory microflora across a compromised intestinal epithelial barrier. We have previously demonstrated that oral probiotics promote increased intestinal CD4(+) T-cell reconstitution during ARV treatment in a non-human primate model of HIV infection; however, essential mucosal T-cell subsets, such as Th17 cells, had limited recovery. Here, we sought to promote Th17 cell recovery by administering interleukin (IL)-21 to a limited number of ARV-treated, probiotic-supplemented, Simian Immunodeficiency Virus (SIV)-infected pigtailed macaques. We demonstrate that probiotic and IL-21 supplementation of ARVs are associated with enhanced polyfunctional Th17 expansion and reduced markers of microbial translocation and dysbiosis as compared with infected controls receiving ARVs alone. Importantly, treatment resulted in fewer morbidities compared with controls, and was independent of increased immune activation or loss of viral suppression. We propose that combining ARVs with therapeutics aimed at restoring intestinal stasis may significantly improve disease prognosis of ARV-treated, HIV-infected individuals.

Figure 1. Experimental Design

Eleven PTM were infected with SIVmac₂₃₉ at day 0 (dotted red line) and daily ARV therapy (grey shaded region) was initiated at day 98 p.i. Of these animals, 6 PTM were additionally treated with daily probiotic therapy (blue shaded region) as well as two rounds of IL-21 therapy, administered once weekly for five weeks (dotted black lines). One ARV-only PTM was euthanized at day 126 p.i. per protocol specified clinical endpoint. Blood, stool and tissues (BAL, Jej, LN, and RB) were collected at pre-determined intervals, as indicated (green and purple bars).

Figure 2. IL-21 and Probiotic Therapy does not Alter Viral Load or Cellular Distribution in ARV-Treated PTMs

a. Longitudinal, log copies/mL of plasma vRNA from animals treated with ARVs only (open squares) or with ARVs, probiotic VSL#3, and IL-21 (closed circles). Grey shading indicates treatment initiation and duration. Dotted lines denote IL-21 administration timepoints. Symbols and shading are consistent throughout the manuscript unless otherwise noted. b. Copies of vDNA per 100 colonic CD4+ TM at the time of necropsy. Triangles indicate samples normalized to the limit of detection

Figure 3. IL-21 and Probiotic Therapy Improves Clinical Status in ARV-Treated PTMs

Percent of animals remaining free from clinical complications over time. Initial complications per animal are noted alongside changes in the incidence-free curve, with ARV-only animals represented by the dashed line and supplemented animals by the solid line. Significance in the time to the first reported clinical complication between groups was assessed by the Mantel-Cox Log-Rank test.

Figure 4. IL-21 and Probiotic Therapy Promotes the Absolute Expansion of Polyfunctional T_H17 Cells in ARV-Treated PTMs

a. Longitudinal mean (±SEM) percent IL-17+ of CD4+ TM (T_H17) from the Jej (left) and RB (right). b. Absolute percent T_H17 (i.e., the percent of IL-17+ CD4+ TM found within CD3+ T cells) from the Jej and RB at the indicated days p.i. c. Absolute percent polyfunctional T_H17 from the Jej and RB at the indicated days p.i. Line through samples represents mean; lines above samples span timepoints of significance. Significance within groups was assessed by Wilcoxon Signed-Rank test. Significance across groups was assessed by Mann-Whitney U test.

Figure 5. IL-21 and Probiotic Therapy Improve Commensal Colonization and Reduce Circulating Kyn

a–b. Relative frequency of Bifidobacter (a) and Lactobacillus (b) 16S DNA of total 16S DNA in rectal tissues at the indicated timepoints. c. Concentration of plasma Kyn (μM) at the indicated days p.i. d. Ratio of plasma Kyn to Trp concentrations at the indicated days p.i. Line through samples represents mean; lines above samples span timepoints of significance. Significance within groups was assessed by Wilcoxon Signed-Rank test. Significance across groups was assessed by Mann-Whitney U test.