People living with HIV demonstrate increased mortality and comorbidity, especially when antiretroviral therapy (ART) is delayed [1,2]. Persistent systemic inflammation despite ART may contribute to these adverse health outcomes [3,4]; immune alterations include blood CD8+ T-cell immune activation  and elevated soluble markers of coagulation and inflammation [4,6,7]. Therefore, clinical interventions to reduce persistent inflammation may improve the health of ART-treated individuals.
One major driver of systemic inflammation is the translocation of gut luminal microbes into the bloodstream , resulting from a combination of mucosal immunodeficiency , epithelial barrier dysfunction , and dysbiosis of the gut microbiota . Although standard ART generally reconstitutes peripheral blood immune cells, gut mucosal restoration is often delayed and incomplete . Mucosal CD4+ T-cell subsets, including regulatory T cells (Tregs), Th17, Th1, and Th22 cells are important in host mucosal defense but are rapidly altered following HIV infection [12–15]. Th22 cells normally maintain and repair the gut by increasing enterocyte survival , whereas Th17 cells play a role in tissue repair and defense against extracellular bacteria and fungi , but both are reduced in number and/or function during untreated and early treated HIV infection [12,13,17].
Rapid initiation of ART reduces AIDS and non-AIDS-related morbidity  and levels of inflammatory cytokines [4,19]. Although less is known about how the timing of ART initiation impacts gut immunology, treatment initiated in the earliest stages of acute HIV infection preserved gut immune function and reversed immune activation . Moreover, individuals with prolonged continuous viral suppression on ART (>6 years) demonstrated complete restoration of gut CD4+ T cells and of Th17 numbers and function [12,17,21]. However, in clinical practice, it is unusual to diagnose and start ART during acute HIV infection, and the slow gut immune restoration when ART is initiated later is discouraging.
We hypothesized that newer antiviral agents with improved drug penetration into gut tissues, specifically maraviroc [chemokine receptor type 5 (CCR5) inhibitor] and raltegravir (integrase inhibitor), might accelerate gut T-cell homeostasis . We therefore randomized individuals who were starting ART early after HIV acquisition to receive either a standard protease inhibitor-based ART regimen, or a regimen intensified through the addition of raltegravir and maraviroc.
This protocol was a predefined substudy nested within a larger prospective, randomized, double-blinded, placebo-controlled trial (NCT01154763) , in which men recently infected by a CCR5-tropic HIV strain were randomized 1 : 1 to receive either emtricitabine 200 mg/tenofovir disoproxil fumarate 300 mg QD + lopinavir 400 mg/ritonavir 100 mg BID (standard regimen; sART) or to receive the standard regimen and raltegravir 400 mg BID and maraviroc 150 mg BID (intensified regimen; iART). Specifically, a subset of participants from that trial (n = 22) consented to provide paired sigmoid biopsies and peripheral blood at baseline and week 48.
Participants with documented early HIV infection (≤7 months) and HIV-uninfected controls from the same community were enrolled; details regarding sexual practices were not collected (Maple Leaf Medical Clinic, Toronto, Ontario, Canada). A multiassay algorithm on baseline sera was used to estimate timing of HIV infection , as previously described . The research ethics boards at University of Toronto and St Michael's Hospital approved the study, and all participants provided written informed consent.
Immune cell isolation from blood and gut
Peripheral blood mononuclear cells were isolated by Ficoll–Hypaque density centrifugation. Sigmoid biopsies were collected 25–30 cm from the anal verge as previously described  and immediately placed into RPMI solution (RPMI-1640 media containing 100 U/ml penicillin, 100 μg/ml streptomycin, and 1× GlutaMAX-1; Invitrogen, Carlsbad, California, USA). Gut mononuclear cells were extracted by two serial collagenase type II digestions at 0.5 and 1.0 mg/ml for 30 min each on a shaking heated block (Clostridiopeptidase A; Sigma-Aldrich, St Louis, Missouri, USA).
Flow cytometry immune studies
Mononuclear cells were stained immediately after isolation or stimulated in RPMI solution containing 10% fetal bovine serum (FBS) with phorbol 12-myristate 13-acetate (1 ng/ml) and ionomycin (1 μmol/ml) for 6 h at 37 °C, and with Brefeldin A for the final 5 h (1 μmol/ml). Cells were washed in 1% FBS-1× PBS, permeabilized, stained for 30 min with fluorochrome-labeled mAbs and a viability dye and fixed. Antibodies included anti-CD3, CD4+, CD8+, human leukocyte antigen – antigen D related (HLA-DR), CD38, IL-17a, IL-22, IFNγ, and tumor necrosis factor. Cells were acquired on a FACSCanto II or LSR II (BD Biosciences, La Jolla, California, USA). Data analysis was performed using Flow Jo software v9.8 (Treestar, Ashland, Oregon, USA) and SPICE v5.3 (NIAID/NIH, Rockville, Maryland, USA) for polyfunctionality analysis. Flow cytometric gating was determined using media control and/or all fluorescence minus one controls, and values for functional cell subsets were background-corrected.
Blood biomarker measurement
Assays were performed in duplicate according to the manufacturers’ protocols. Plasma assays used commercially available ELISA kits to quantify soluble CD14 (sCD14) (R&D), D-dimer (Sekisui Diagnostics, Lexington, Massachussets, USA) and LPS (Lonza Cambrex, Charles City, Iowa, USA), and a custom-designed multiplex ELISA for IL-6, IL-10, IL-17, macrophage inflammatory protein (MIP)-1b, and tumor necrosis factor (Meso Scale Discovery, Rockville, Maryland, USA).
Mann–Whitney and Wilcoxon tests were performed using Prism 6 (GraphPad, La Jolla, California, USA) and SPICE software (v5.3). P < 0.05 was considered statistically significant.
Study participant blood characteristics
Participants in the two groups were similar in age (P = 0.557) and infected with HIV, a median of 4 months ago (P = 0.809; Table 1). The sART and iART arms did not differ in blood CD4+ cell count, CD4+ : CD8+ ratio, CD8+ T-cell immune activation (HLA-DR + CD38 + coexpression), or soluble markers of inflammation/microbial translocation at baseline and demonstrated a similar change in all parameters at week 48 (Table 1). Time to HIV viral suppression following ART initiation was also similar (sART, 2.0 vs. iART 1.5 months; P = 0.753).
Overall impact of early antiretroviral therapy on soluble and cellular immune parameters in blood
As ART intensification did not impact blood immune parameters, data from the two groups were pooled to assess the overall impact of early ART initiation. At baseline, blood CD8+ T-cell activation was elevated almost 10-fold compared with HIV-uninfected controls (20.6 vs. 2.9%; P < 0.001) and decreased but remained higher than uninfected controls by 48 weeks (5.95%; P = 0.031; Supplemental Fig. 1a, http://links.lww.com/QAD/B95). Neither the frequency nor function of blood Th17 cells differed between participants with early HIV infection and HIV-uninfected controls, or between sART and iART groups at baseline or week 48 (P > 0.05 for all). Plasma biomarkers of inflammation and indirect marker of microbial translocation (sCD14) were also increased at baseline, but only D-dimer normalized after 48 weeks of ART (P = 0.226); IL-10, IL-17, and MIP-1b decreased on ART but remained significantly elevated, whereas IL-6, IL-17, and sCD14 levels did not change on ART (Supplemental Fig. 1b–g, http://links.lww.com/QAD/B95). Plasma LPS levels did not differ from uninfected controls at baseline or week 48 (P > 0.05; data not shown). Significantly, tumor necrosis factor levels were also similar at baseline to HIV-uninfected controls but significantly increased after ART (Supplemental Fig. 1h, http://links.lww.com/QAD/B95).
No impact of antiretroviral therapy intensification on gut immunology
In the gut, all measured immune variables were comparable at baseline between the two groups (Fig. 1; P > 0.05 for all), and intensification did not alter the change after 48 weeks in CD8+ T-cell immune activation, % CD4+ T cells, CD4+ : CD8+ ratio, %Th1, %Th22, or %Th17 cells (P > 0.05).
Impact of early antiretroviral therapy on gut immunology
As ART intensification did not alter gut immune restoration, data were again pooled from the two arms to characterize the impact of early ART initiation. At baseline, gut CD8+ T-cell immune activation was substantially elevated compared with uninfected controls, with a reduced CD4+ T-cell proportion and a CD4+ : CD8+ ratio that improved but did not normalize on ART (Fig. 1a–c). Frequencies of CD4+ sigmoid Th1 cells (IFNγ+) and regulatory T cells (Foxp3+CD25+) were similar to HIV-uninfected controls, both before and after ART (P > 0.05), whereas Th22 cell frequencies (IL-22+IFNγ−IL17a−) were dramatically reduced at baseline, there was near-complete reconstitution after ART (Fig. 1d and e). Th17 cell frequencies (IL-17a+) were comparable with HIV-uninfected controls at baseline and week 48, but their capacity to produce cytokines was dramatically reduced, both before and after a year of ART (Fig. 1f and g).
The predefined clinical substudy investigated the mucosal immune impact of ART initiated during early stages of HIV infection, and whether intensification of ART at this stage might provide superior blood and gut immune reconstitution. Intensification through the addition of maraviroc and raltegravir to standard ART did not provide any additional benefit in blood immune parameters after 48 weeks, as seen in prior studies in this and later stages of HIV infection [25–27], and nor did intensification improve gut immune parameters. Substantial increases in blood and gut immune activation were apparent in these early stages of untreated HIV and did not normalize after a year of ART; although most soluble blood inflammatory biomarkers decreased, some did not change (sCD14, LPS, IL-17, and IL-10), and tumor necrosis factor levels actually increased. Mucosal CD4+ T-cell proportions and the mucosal CD4+ : CD8+ ratio increased, and gut CD8+ T-cell activation decreased considerably on ART but did not normalize, whereas mucosal Th22 populations were completely restored. Mucosal Th17 cell proportions were not reduced prior to ART or after ART; however, their function was substantially reduced and did not improve with ART. Overall, substantial mucosal and systemic immune dysfunction was apparent during early HIV infection; this dysfunction was only partially corrected by ART, and intensification with maraviroc and raltegravir did not provide any additional benefit.
ART intensification with newer antivirals that demonstrate enhanced gut tissue penetration showed no systemic or mucosal immune benefit in this small substudy, or virologic benefit in our overall protocol . A prior clinical trial showed that maraviroc had better drug distribution in the gut than efavirenz or raltegravir, and that duodenal concentrations were associated with nonsignificant trends to improved CD4+ : CD8+ ratio, CD8+ T-cell immune activation, and CD4+ frequency . Despite a lack of overall benefit, we did not measure maraviroc levels in the gut and therefore cannot rule out that higher drug concentrations in the gut might better restore gut immune subsets and/or accelerate local virologic suppression.
Reversing HIV-associated mucosal immune damage remains a challenge. ART initiated in the earliest phases of HIV infection can prevent some aspects of immune gut damage , but even treatment started during Fiebig stages I-II failed to completely restore mucosal CD4+ T cells after 2 years . In our study, 1 year of ART initiated 4 months after HIV infection did not normalize gut immune activation or overall mucosal CD4+ T-cell numbers. However, gut CD4+ T-cell immune subsets were affected by HIV to different degrees, and those affected were reconstituted at varying rates. In contrast with other groups who found Th17 depletion at Fiebig stage III or later  but in keeping with our own earlier findings ; whether this represents differences in participant demographics or other factors is unknown. Th22 cell number and Th17 function were dramatically reduced, but although the former had normalized completely by a year, Th17 function did not improve. The timing of numerical and functional reconstitution within each subset beyond 48 weeks or the reasons for this subset heterogeneity are unclear but will be interesting topics for future research. Given the limited ability of current screening practices to identify even early HIV-infected individuals, effective clinical strategies to accelerate gut immune reconstitution remain a research priority.
The current work was supported the Canadian Institutes of Health Research (CIHR; TMI-138656 and THA-11900) and an unrestricted research grant from Merck, and study medications were provided by Gilead, Abbvie, Merck, ViiV, Janssen, and Pfizer. We acknowledge salary support from the CIHR Canadian HIV Trials Network (CJK), the Ontario HIV Treatment Network (OHTN; MO), and the University of Toronto – OHTN Endowed Research Chair (RK). We would like to thank Dr. Tae-Wook Chun from the NIH for technical assistance and the study participants for their time, contribution, and effort.
Conceived and designed the trial: C.J.K., C.K., M.O., and R.K.
Performed experiments and acquired data: C.J.K., R.R., S.H., E.B., K.S., and G.K.
Analyzed and interpreted the data: C.J.K. and R.K.
Drafted the article: C.J.K. and R.K.
All authors read and approved the final draft of the article and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work will be appropriately investigated and resolved.
Conflicts of interest
There are no conflicts of interest.
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