Although HIV replication can be suppressed by antiretroviral therapy (ART) and most individuals on ART recover peripheral blood CD4+ T cells and regain immunocompetence to some degree, the expected lifespan of treated individuals is shorter than that of the general population.1 Moreover, among ART-treated, HIV-infected subjects, those with limited CD4+ T-cell recovery (immunologic nonresponders) have lifespans that are even shorter2 and an increased incidence of non–AIDS-related complications (eg, cardiovascular disease and neurocognitive decline)3 compared with those with more complete CD4+ T-cell recovery (immunologic responders).
T-cell activation is associated with HIV disease and is recognized as a more accurate predictor of disease progression than is CD4+ T-cell count or viral load alone.4–6 T-cell activation does not fully normalize after the initiation of ART, and the extent of residual elevation is associated with poor CD4+ T-cell recovery.7,8 In nonresponders, T-cell activation is elevated in association with a lack of peripheral blood CD4+ T-cell recovery.9–11 Such T-cell activation may be induced by inflammatory cytokines such as type 1 interferon (IFN),12,13 tumor necrosis factor (TNF),6 and/or interleukin 6 (IL-6),11,14 which are elevated during untreated and treated HIV infection.10,11,14–20 These inflammatory cytokines are elevated concomitantly with T-cell activation,11 consistent with the hypothesis that the lack of CD4+ T-cell recovery during ART may be caused by persistent inflammation. If so, it would be important to understand which inflammatory pathways are active in which sites during ART to facilitate the design of interventions to dampen inflammation and to thereby decrease the incidence of inflammation-associated non-AIDS complications.
Many of the inflammatory pathways associated with HIV disease are induced on stimulation of innate immune cells by viral or bacterial products. Previous reports have conflicted as to whether residual virus burden is elevated in nonresponders,19,21,22 whereas other reports have demonstrated that markers of microbial translocation, thought to arise as a result of HIV-induced intestinal immune dysfunction, are associated both with limited CD4+ T-cell gains on therapy and with T-cell activation.11,23–25 These previous studies, however, have presented only limited data from the gastrointestinal tract. Understanding what takes place in the intestinal mucosal tissues is essential to understanding persistent inflammation, as this is a site where HIV replicates during untreated infection,26,27 and where HIV RNA and DNA (and possibly virus replication) may persist during otherwise suppressive ART.28,29
To understand whether gastrointestinal inflammation or HIV reservoirs are associated with the lack of CD4+ T-cell repopulation or persistent T-cell activation during ART, we studied plasma, peripheral blood mononuclear cells (PBMC), and rectosigmoid colon biopsies from immunologic responders and nonresponders, where CD4+ T-cell levels and T-cell activation levels are widely different in peripheral blood. In this study, we confirmed previous observations that there are higher levels of T-cell activation, systemic inflammation, and soluble CD14 (sCD14) in nonresponders than in responders,9–11 and analyzed rectosigmoid colon biopsies for evidence of inflammation and virus load. While T-cell activation tended to be higher in the mucosal tissue of nonresponders, we found little evidence for a difference in colon inflammatory gene expression or HIV RNA or DNA levels between nonresponders and responders. Rather, the inflammatory signature differentiating these 2 groups is strongest in the peripheral blood and is associated with plasma sCD14 levels. This observation, due either to nonintestinal determinants of inflammation or to the limited and possibly nonrepresentative sampling afforded by isolated colonic biopsies, may be instructive in the design and implementation of future attempts to intervene against inflammation in nonresponders.
Subjects were recruited from the Study of the Consequences of the Protease Inhibitor Era30 or options cohort at San Francisco General Hospital. All subjects provided informed consent, and the protocol was approved by the UCSF Committee on Human Research. Subjects on ART had at least 2 years of plasma viral load of <40 copies per milliliter and were assigned as immunologic nonresponders or responders based on CD4+ T-cell levels (<350 cells/mm3 or >500 cells/mm3, respectively). Untreated subjects with viremia (>1000 copies/mL) or who were HIV seronegative were recruited as comparator groups. Subjects receiving immunomodulatory or immunosuppressive therapies and/or those with a recent acute illness were excluded.
Peripheral blood was collected by venipuncture, and mononuclear cells were isolated using ficoll-hypaque (GE Healthcare, Piscataway, NJ). Mucosal biopsies (26–30) from the rectosigmoid colon were obtained during sigmoidoscopy using jumbo biopsy forceps. Biopsies were preserved in RNAlater (Qiagen, Valencia, CA) for mRNA measurements (n = 2–4), snap frozen for virological analysis (n = 6), or placed in RPMI (Life Technologies, Carlsbad, CA) for cell isolation (n = 12–16). Cells were prepared from biopsies in RPMI by collagenase digestion (Sigma, St. Louis, MO) followed by mechanical disruption and filtration without density enrichment.31
Gene and Virus Quantification
RNA was extracted from whole blood using the PaxGene Blood RNA Kit (Qiagen) or from biopsies frozen in RNAlater (Life Technologies). Biopsies were homogenized in Trizol (Life Technologies), and RNA was purified using the RNeasy mini-kit (Qiagen). cDNA was prepared using Omniscript RT Kit (Qiagen). Quantitative polymerase chain reaction for blood-derived cDNA was performed using TaqMan Gene Expression MasterMix (Life Technologies) in a StepOne Plus Real-Time PCR system (Life Technologies). Commercially available primer–probe sets from Applied Biosystems were used: GBP1 Hs00977005_m1, IFI27 HSs00271467_m1, MX1 Hs00895601_m1, and OAS1 Hs00973637_m1. In the case of biopsy specimens, gene expression was measured by Fluidigm (Fluidigm, South San Francisco, CA) with gene-specific preamplification using computationally designed primers (DELTAgene). Relative expression was calculated using the ΔΔCT method, using hypoxanthine guanine phosphoribosyl transferase as housekeeping gene. HIV RNA and DNA levels were measured in Trizol extracts from snap-frozen biopsies, using quantitative polymerase chain reaction for the HIV long terminal repeat.32,33
Freshly isolated cells were stained with an antibody cocktail using standard procedures. Antibodies used were [target (fluorophore, clone, dilution)]: CD3 (QDot655, S4.1, 1:2500); CD4 (QDot605, S3.5, 1:1000); CD8 (QDot 705, 3B5, 1:1000) from Life Technologies, Green Island, NY; CD45RO (ECD, UCHL1, 1:50) from Beckman Coulter, Brea, CA; CD27 (PerCP-Cy5.5, M-T271, 1:50); HLA-DR (FITC, L243, 1:25), and CD38 (PE, HB7, 1:25) from BD Bioscience (San Jose, CA). Dead cells were stained with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies). Stained cells were acquired on an LSRII using FACSDiVa software (BD Biosciences) and analyzed using FlowJo software (Treestar, Ashland, OR). An example gating strategy is shown in Figure S1 (see Supplemental Digital Content,http://links.lww.com/QAI/A463).
Plasma levels of IL-6, TNF-RI, TNF-RII, and sCD14 were measured by enzyme-linked immunosorbent assay using commercially available kits (R&D Systems, Minneapolis MN).
Two sample comparisons were made using the Mann–Whitney U test, whereas multi-sample comparisons were made using the Kruskal–Wallis test and Dunn's posts-tests, both performed in the Prism software package (GraphPad, San Diego, CA). Correlations were measured using the Spearman correlation coefficient in the JMP software package (SAS, Cary, NC).
CD4+ T-Cell Levels in Peripheral Blood Are Not Strongly Related to CD4+ T-Cell Levels in Colon Biopsies
Patients on ART with undetectable viremia for at least 2 years and who were either nonresponders (<350 CD4+ T cells/mm3, n = 18) or responders (>500 CD4+ T cells/mm3, n = 16) were studied. For comparison, viremic untreated subjects (n = 9) and HIV-uninfected subjects (n = 23) were enrolled. From each of these subjects, peripheral blood and rectosigmoid colon biopsies were obtained, and assays were performed to quantify CD4+ T-cell levels, T-cell activation, inflammation, and HIV burden. Some assays were phased in over the course of the study, such that it was not possible to perform all measurements in all subjects. The number of subjects studied with each assay is indicated in the text and figure legends.
Confirming previous reports,34 nonresponders had lower CD4+ T-cell nadirs than did responders (see Table S1, Supplemental Digital Content,http://links.lww.com/QAI/A463) and had significantly lower blood CD4+ T-cell levels than did responders or uninfected subjects (Fig. 1A). The differences in current CD4+ T-cell count reflect fewer cells gained over time during ART (responders: 71.1 cells/year; nonresponders: 11.7 cells/year; P < 0.01) rather than shorter follow-up (responders: median, 9.3 years; nonresponders: median, 5.2 years; P = ns) (see Table S1, Supplemental Digital Content,http://links.lww.com/QAI/A463). In contrast, and as previously demonstrated,35–38 both responders and nonresponders had lower levels of CD4+ T cells in the colonic mucosa than uninfected subjects, as measured by flow cytometry and related to either total recovered cells (Fig. 1B) or CD3+ T cells (Fig. 1C). Despite differences in blood CD4+ T cell levels, responders and nonresponders had similar rectal CD4+ T-cell frequencies. Responders, compared with nonresponders, had significantly more CD8+ T cells in peripheral blood (Fig. 1D) but significantly lower CD8+ T-cell frequency among all live cells in colon (Fig. 1E). CD8+ T-cell frequency among CD3+ cells in the colon was not different between responders and nonresponders (Fig. 1F). Among ART-suppressed participants, there was no evidence for a relationship between peripheral blood CD4+ T-cell count or frequency and colon CD4+ T-cell frequencies as a fraction of all extracted live cells (Fig. 1G) or of CD3+ T cells (Fig. 1H) (blood CD4+ T-cell frequency data not shown).
Peripheral and Colon T-Cell Activation Are Correlated and Inversely Related to Peripheral CD4+ T-Cell Levels
The expression of CD38 and HLA-DR was measured on memory (excluding CD45RO-CD27+ naive cells) CD4+ and CD8+ T cells from PBMC and from colon in a subset of patients. In PBMC, the fraction of CD38+HLA-DR+ CD4+ and CD8+ T cells from nonresponders (n = 12) was higher than that found in responders (n = 7) (Figs. 2A, B, respectively), confirming previous reports.34 In colon, the fraction of CD38+HLA-DR+ CD4+ T cells, but not of CD8+ T cells, was also elevated in nonresponders compared with responders (Figs. 2C, D). Higher levels of PBMC (Fig. 2E) or colon (Fig. 2G) CD4+ T-cell activation correlated with lower peripheral blood CD4+ T-cell counts. Although higher peripheral blood and rectal CD8+ T-cell activation also tended to be associated with lower peripheral blood CD4+ T-cell counts, these correlations did not achieve statistical significance (Figs. 2F, H, respectively). No relationship was observed between blood and colon T-cell activation levels (Fig. 2I, CD4+ T cells: P = 0.11; Fig. 2J, rho = 0.37, CD8+ T cells: P = 0.25, rho = 0.28).
Systemic, but Not Colonic, Inflammation Is Elevated in Nonresponders in Correspondence With Peripheral CD4+ T-Cell Levels
To investigate the activity of the major inflammatory pathways that are increased during HIV infection, markers characteristic of the type 1 IFN,12,13 TNF,6 and/or IL-614 activity were examined in blood and rectosigmoid colon biopsy specimens. The type 1 IFN pathway was assessed by reverse transcription and quantitative polymerase chain reaction analysis of the relative expression of a panel of IFN-stimulated genes (ISG), including myxovirus resistance gene-1 (MX1), 2′-5′-oligoadenylate synthetase-1 (OAS1), IFN alpha-inducible protein-27 (IFI27), and guanylate-binding protein-1 (GBP1). Confirming another report using different ISG,10 the expression of each of these transcripts in peripheral blood was elevated in nonresponders as compared with responders (Fig. 3A). The geometric mean of each of the 4 ISG relative expression values for each subject was calculated (ISG Geomean) to consolidate ISG expression into an index with the intent to minimize variability and derive a more consistent metric.39 Blood ISG Geomean was higher in nonresponders and untreated subjects than in either responders or uninfected subjects (Fig. 3A). ISG expression in the colon biopsies was significantly elevated in each HIV-infected group when compared with uninfected subjects, but was surprisingly not different between nonresponders and responders (Fig. 3B). Although ISG levels in colon were not different between the groups, there was a significant, direct correlation between blood and colon ISG Geomean levels (Fig. 3E; P= 0.037, rho = 0.45).
Soluble TNF receptors (ie, sTNF-RI and sTNF-RII, which are shed on TNF binding) were measured in plasma as biomarkers of TNF activity. The difference in sTNF-RI and sTNF-RII levels did not reach statistical significance between responders and nonresponders (Fig. 3C). Levels of sTNF-RII in nonresponders were, however, higher than those found in uninfected individuals and were more comparable with those observed in viremic subjects (Fig. 3C). TNF transcript levels were measured in colon, and no significant differences were observed between responders and nonresponders (Fig. 3C). TNF transcript levels in colon also had no correlation with sTNF-RI or sTNF-RII levels in blood (Fig. 3E, P = 0.79, rho = 0.06; P = 0.63, rho = 0.11; respectively, data not shown).
Finally, levels of plasma IL-6 protein and of IL-6 mRNA in colon were assessed and, although levels in responders were higher and levels in nonresponders trended higher than those found in uninfected subjects, they were not statistically different between responders and nonresponders (Fig. 3D), nor was there a correlation between blood IL-6 levels and colon IL-6 mRNA expression levels (Fig. 3E; P = 0.31, rho = −0.22, data not shown).
Relationships between the blood and colon inflammatory markers and CD4+ T-cell levels or T-cell activation levels in responders and nonresponders were then studied (Fig. 3F). Plasma inflammatory markers were generally found to be inversely related to blood and colon CD4+ T-cell levels, with statistical significance reached in the inverse relationship between PBMC ISG expression and blood or colon CD4+ T-cell levels, and between plasma sTNF-RII and blood CD4+ T-cell levels (Fig. 3F). Conversely, colon IL-6 mRNA levels correlated directly with the percentage of CD4+ T cells among CD3+ T cells in colon. There was also a general trend for plasma inflammatory markers to correlate directly with levels of PBMC and colon CD4+ T-cell activation, with statistical significance reached in the direct relationship between plasma IL-6 and colon CD4+ and CD8+ T-cell activation, and between colon ISG expression and colon CD8+ T-cell activation. Blood ISG expression levels approached significant correlations with PBMC CD4+ and CD8+ T-cell activation (P = 0.057 and P = 0.052, respectively). However, and surprisingly, there was no evidence for a relationship between colon inflammatory cytokine gene expression and blood CD4+ T-cell levels or activation (Fig. 2E).
No Relationship Was Observed Between Colon Virus Levels and CD4+ T-Cell Levels or Inflammation
To understand whether HIV reservoirs or residual virus production in the intestine may contribute to systemic inflammation and the lack of CD4+ T-cell recovery on ART, HIV RNA and DNA were assessed in colon biopsies. HIV RNA and DNA copies were measured in whole biopsies (2 biopsies per subject) and normalized to cell equivalents by nucleic acid input. As expected, untreated subjects (n = 9) had higher levels of HIV RNA than did either responders (n = 7) or nonresponders (n = 16); however, no evidence for a difference in HIV RNA levels was observed between responders and nonresponders (Fig. 4A). Likewise, no difference was observed between responders and nonresponders in HIV DNA levels (Fig. 4B) or the ratio of HIV RNA copies to DNA copies (Fig. 4C). No associations were observed between HIV levels and CD4+ T-cell levels or T-cell activation in colon or PBMC, plasma inflammatory markers, or inflammatory gene expression in PBMC or in the colon (Fig. 4D).
Plasma sCD14 Is Elevated in Nonresponders in Association With Inflammation and Colon T-Cell Activation
Because microbial translocation has been linked to systemic inflammation during chronic HIV infection,24 plasma sCD14 (shed by monocytes and macrophages in response to lipopolysaccharide40) was measured as an indirect marker of microbial translocation. Plasma sCD14 levels from nonresponders were significantly higher than levels in uninfected individuals, but the difference between responders and nonresponders did not reach statistical significance (Fig. 5A). However, sCD14 levels did correlate inversely with peripheral CD4+ T-cell levels (Fig. 5B), a relationship that was found to be stronger when nonresponders were considered separately (nonresponders: P = 0.0014; responders: P = 0.75). Plasma sCD14 levels correlated directly with markers of colon T-cell activation and systemic inflammation, but a relationship was not observed with colon inflammatory gene expression (Fig. 5B).
Despite long-term suppression of HIV replication, nonresponders are more susceptible to non-AIDS complications and have a shorter lifespan than uninfected individuals.2 This clinical state is associated with higher levels of immune activation and inflammation,3 potentially because of a fundamental immunologic defect acquired before the initiation of therapy.41 A proposed cause of this systemic inflammation is microbial translocation across a persistently damaged intestinal barrier,24 thought to be related to inflammation in the intestine. To understand whether blood immune activation and inflammation are associated with inflammation or residual HIV in the intestinal mucosa in the context of treated HIV disease, blood and rectosigmoid colon biopsy specimens were obtained from HIV-infected subjects on suppressive ART who met criteria as nonresponders and responders, as well as from viremic, untreated HIV-infected subjects and uninfected subjects. T-cell activation and peripheral inflammatory markers were confirmed to be elevated in nonresponders, and PBMC ISG expression was found to be one of the strongest signatures differentiating nonresponders from responders. Colonic HIV RNA and DNA levels or expression of inflammatory cytokines, however, did not seem to differ between nonresponders and responders in this small study. Importantly, colon levels of HIV or inflammation also do not seem to correlate with levels of CD4+ T cells, T-cell activation in the peripheral blood, or with plasma inflammatory mediators. Despite the lack of colonic inflammatory or virological differences measured here, monocyte activation (as measured by plasma sCD14, potentially indicative of microbial translocation) was inversely correlated with blood CD4+ T-cell levels and directly related to colon T-cell activation and plasma inflammatory cytokine levels,11,25 markers that were elevated in nonresponders.
This study has several limitations that will be important to address in future studies. First, its design did not control for CD4 nadir, a parameter that was found to be different between responders and nonresponders at baseline (see Table S1, Supplemental Digital Content,http://links.lww.com/QAI/A463), leaving open the possibility that the immune system was more damaged in nonresponders before the initiation of ART and that such damage alone limited CD4+ T-cell recovery. In addition, the study was performed over the course of 3 years, during which time assays and concepts evolved, and some measurements were made only in subgroups of patients. The colon immunologic and virological parameters were also measured in small pinch biopsies, which may not be representative of the tissue as a whole, introducing potentially significant sampling error, particularly because lymphoid aggregates likely have very different cell phenotypes and concentrations of virus than the surrounding lamina propria. Similarly, biopsies were taken only from the distal colon, which is likely not representative of the entire intestine.28 Finally, this study was relatively small and the findings herein, particularly those negative findings related to colon inflammation and HIV levels, should be considered with caution because of limited statistical power.
Notwithstanding these shortcomings, our findings raise several points. First, we found that elevated levels of inflammation were more evident in the peripheral blood than in the rectosigmoid colon biopsies of nonresponders compared with responders, despite the finding that colon ISG and IL-6 expression levels were higher in both groups relative to uninfected subjects. This observation is—at least on the surface—inconsistent with the hypothesis that local inflammation in the intestinal mucosa is proximally related to (and potentially causative of) microbial translocation and systemic inflammation. The finding, however, has a number of potential nonmutually exclusive interpretations and implications: (1) the rectosigmoid colon may not be a prominent location of breaches permitting microbial translocation across the intestinal epithelium as compared with other regions of the intestine28; (2) breaches may be present in the distal colon, but are highly focal and difficult to sample by a limited set of biopsies; (3) translocated microbial products are not immunostimulatory in the intestine itself, but only when they reach more systemic sites such as the mesenteric lymph node, liver, or systemic lymphoid tissues; and/or (4) the discordance between blood and colon inflammation may indicate that microbial translocation into the lamina propria is ongoing in both responders and nonresponders but is contained, for example, by a “firewall” of mesenteric lymph nodes,42 in responders, preventing the systemic spread of microbial products and inflammation to systemic sites that is seen in nonresponders. Detailed investigation of the dynamics of virus production, microbial products, and inflammatory mediators in various tissue compartments would pinpoint the source and cause of systemic inflammation during treated and untreated lentiviral infection.
Second, systemic levels of activation (eg, elevated CD38 and HLA-DR expression on circulating CD4+ and CD8+ T cells) are more readily correlated with circulating sCD14 levels than with the levels of HIV RNA or DNA found in rectosigmoid colon biopsies, suggesting that systemic T-cell activation may be driven more by microbial translocation than by HIV expression in the rectosigmoid colon. This observation could, however, be attributed to the likely production of HIV in other regions of the intestine28 or in nonintestinal sites such as lymph nodes, spleen, or blood, and that such virus may be more related to systemic inflammation than virus in the distal colon; alternatively, it may simply reflect the nonrepresentative nature of sampling that attends collection of biopsies from circumscribed areas of intestine that have variable cellular composition. It also remains possible that responders and nonresponders are fundamentally different in the relationship between virus and inflammation, and that different results may be obtained when considering a larger group of nonresponders alone. This study was particularly underpowered to detect relationships within the subgroups. Future studies that measure intestinal viral reservoir on extracted and sort-purified HIV target cells—and in a larger number of subjects with longitudinal design—will be important to confirm the present findings.
In sum, our findings support the hypothesis that systemic inflammation and monocyte activation are linked to limited CD4+ T-cell recovery during ART. This hypothesis indicates that interventions that diminish inflammation may improve CD4+ T-cell recovery during ART, limit the incidence of non-AIDS disease, and improve the expected lifespan among treated patients. Not least, the ability to measure such markers in the circulation provides a convenient sampling point to use when testing the efficacy of interventions to block inflammation in vivo. Finally, because differential inflammation or virus levels in rectosigmoid colon biopsies were not observed between responders and nonresponders, these results also demonstrate that larger studies with systematic investigation of multiple tissues to identify the source of inflammation and studies with a longitudinal design may lead to enhanced understanding of the mechanisms responsible for AIDS immunopathogenesis and to novel strategies for clinical management.
The authors would like to acknowledge Dr Timothy Schacker for critical review, Janet Robinson, Brian Clagett, Sohani Sirdeshmukh, and Dominic Dorazio for assistance in plasma marker analyses, and Terence Ho and Alex Carvidi for flow cytometry assistance.
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