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HIV Reservoir in Fat Tissue: inflammation, lipodystrophies
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"During HIV infection, the inflammatory profile of adipose tissue has been revealed by the occurrence of lipodystrophies (primarily related to ART)...... During HIV infection, the inflammatory profile of adipose tissue has been revealed by the occurrence of lipodystrophies"
"Overall, we were able to demonstrate the persistence of HIV DNA and RNA within stromal vascular cells in adipose tissue recovered from ART-treated HIV-infected patients. These observations were supported by the results of in vitro reactivation experiments, showing that adipose CD4+ T cells contained replication-competent HIV. This dataset strongly supports the hypothesis whereby adipose tissue constitutes an important viral reservoir. Adipose tissue may thus constitute a favorable environment for viral persistence for several reasons: (a) constant inflammation favors viral replication, (b) the presence of elevated fractions of activated and central memory CD4+ T cells, which are HIV's natural targets, (c) the potentially insufficient distribution of some antiretroviral drugs into adipose tissue [44], which may favor viral persistence, and (d) the specific metabolic and immune activity of adipose tissue, which may affect the effectiveness of immune responses [74].
Our observations have major implications in the context of HIV disease. Firstly, they emphasize the crucial requirement for the broad diffusion of antiretroviral drugs within tissues; combination therapy must include drugs that diffuse not only into adipose tissues but also into tissue CD4+ T cells and macrophages. Two main mechanisms may prevent efficient activity of ART on adipose infected cells: (i) low accessibility of ART to fat tissue [44], (ii) sequestration of drugs inside the lipid droplets at the expense of adipose infected immune cells [75]. Secondly, they provide an interesting rationale for the use of drugs with metabolic activity. It might be interesting to reconsider the anti-inflammatory impact of statins [76,77] by focusing on adipose sites and addressing the drugs' potential impact on viral reservoirs. Thirdly, gender differences in adipose tissue distribution, the inflammatory profile and immune cell content [78] may underlie differential susceptibility to the establishment of viral reservoirs. Fourthly, our results open up new therapeutic strategies for limiting the size of viral reservoirs, chronic inflammation and associated comorbidities (via the modulation of adipose tissue related pathways rather than strictly immune pathways)."
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Adipose Tissue Is a Neglected Viral Reservoir and an Inflammatory Site during Chronic HIV and SIV Infection
PLOS Pathogens Sept 24 2015
http://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1005153
Abstract
Two of the crucial aspects of human immunodeficiency virus (HIV) infection are (i) viral persistence in reservoirs (precluding viral eradication) and (ii) chronic inflammation (directly associated with all-cause morbidities in antiretroviral therapy (ART)-controlled HIV-infected patients). The objective of the present study was to assess the potential involvement of adipose tissue in these two aspects. Adipose tissue is composed of adipocytes and the stromal vascular fraction (SVF); the latter comprises immune cells such as CD4+ T cells and macrophages (both of which are important target cells for HIV). The inflammatory potential of adipose tissue has been extensively described in the context of obesity. During HIV infection, the inflammatory profile of adipose tissue has been revealed by the occurrence of lipodystrophies (primarily related to ART). Data on the impact of HIV on the SVF (especially in individuals not receiving ART) are scarce. We first analyzed the impact of simian immunodeficiency virus (SIV) infection on abdominal subcutaneous and visceral adipose tissues in SIVmac251 infected macaques and found that both adipocytes and adipose tissue immune cells were affected. The adipocyte density was elevated, and adipose tissue immune cells presented enhanced immune activation and/or inflammatory profiles. We detected cell-associated SIV DNA and RNA in the SVF and in sorted CD4+ T cells and macrophages from adipose tissue. We demonstrated that SVF cells (including CD4+ T cells) are infected in ART-controlled HIV-infected patients. Importantly, the production of HIV RNA was detected by in situ hybridization, and after the in vitro reactivation of sorted CD4+ T cells from adipose tissue. We thus identified adipose tissue as a crucial cofactor in both viral persistence and chronic immune activation/inflammation during HIV infection. These observations open up new therapeutic strategies for limiting the size of the viral reservoir and decreasing low-grade chronic inflammation via the modulation of adipose tissue-related pathways.
Author Summary
Chronic immune activation/inflammation and viral persistence in reservoirs are important features of chronic HIV infection-even in patients receiving ART. We sought to evaluate the involvement of adipose tissue in chronic HIV/SIV infections. Adipose tissue accounts for 15 to 20% of the body weight, contains both adipocytes and (within the stromal vascular fraction) immune cells, and exerts crucial metabolic and immune activities. We postulated that adipose tissue might provide an ideal environment for HIV persistence and immune inflammation. We first showed that viremic SIV-infected macaques had elevated levels of immune activation and inflammation in adipose tissue, and that both resident CD4+ T cells and macrophages were infected. In similar experiments in ART-controlled HIV-infected patients, HIV DNA was detected in the stromal vascular fraction of adipose tissue (more specifically, in adipose tissue CD4+ T cells). Replication-competent HIV was detected in ex vivo- activated, sorted adipose tissue CD4+ T cells from six aviremic, ART-treated patients. Thus, adipose tissue may constitute a viral reservoir and be involved in long-term immune activation and inflammation-even in ART-suppressed patients. Given that adipose tissue is strongly regulated by both metabolic and immune pathways, modulating adipose tissue may constitute a valuable means of limiting both viral persistence and chronic inflammation in HIV-infected patients even ART-controlled.
Introduction
Human immunodeficiency virus (HIV) infection is characterized by massive CD4+ T cell depletion in the intestinal mucosa (progressively affecting blood and lymphoid CD4+ T cells) and sustained systemic immune activation and inflammation. The advent of antiretroviral therapy (ART) has drastically changed the outcomes of HIV infection by enabling a reduction in the viral load and the restoration (at least in part) of CD4+ T cell counts. In people receiving ART, chronic HIV infection is characterized by the persistence of viral reservoirs (preventing the eradication of HIV) and chronic immune activation and inflammation (which are associated with all-cause, non-AIDS-related morbidity, such as cardiovascular disease and non-insulin dependent diabetes, and mortality [1-3]. Similar observations (i.e. viral persistence and low level immune activation and inflammation) apply-albeit to a lesser extent-to "HIV-controllers", i.e. patients who are able to spontaneously control viral load [4,5]. The eradication or reduction of viral reservoirs remains a crucial therapeutic objective in the fight against HIV [6], and both cellular and anatomical reservoirs require further investigation [7,8]. A second therapeutic objective is to circumscribe the sustained immune activation. It has been suggested that microbial translocation is a potent factor in the maintenance of chronic immune activation/inflammation [9], along with viral persistence, CD4+ T cell lymphopenia, Th17 loss, a change in the regulatory T cell balance, disruption of the lymph node architecture, viral co-infection, accelerated ageing, the side effects of some antiretroviral drugs, and individual susceptibility [2,10,11]). HIV-infected patients on ART are not always able to reestablish gut mucosa integrity and/or normal CD4+ T cell counts, and chronic, low-levels immune activation appears to persist [12]. Taken as a whole, these data suggest that (i) immune activation and chronic inflammation are driven by multiple factors and (ii) targeting several inflammatory mechanisms may achieve better immune restoration.
We hypothesized that adipose tissue has an important role in both chronic immune activation/inflammation and viral persistence. In fact, adipose tissue is not merely a metabolic and endocrine organ for lipid storage; it also exhibits strong immune activity: (a) adipose tissue is an important site of production of both pro-inflammatory molecules (such as leptin, IL-6, MCP-1 (CCL2), RANTES (CCL5) and TNF-α) and anti-inflammatory adipokines (such as adiponectin) [13-21]; (b) the stromal vascular fraction (SVF) contains immune cells (CD4+ T cells and macrophages) that are potentially important target cells for HIV; and (c) a growing body of evidence demonstrates the close relationship between the immune response and metabolic alterations [22,23], such as the recruitment of activated CD8+ T cells and inflammatory macrophages and their participation in inflammation processes (and then the modifications of the adipose tissue) in obesity and non-insulin dependent diabetes [24-31]. In the context of obesity, the interplay between adipocytes and immune cells is being actively investigated. The two cell types are clearly "partners in inflammation" as their coordinated action leads to adipose inflammation [32]. Studies of the adipose tissue during HIV infection have essentially addressed the toxicity of certain antiretroviral drugs and their induction of metabolic alterations-even though a direct impact of infection per se was clearly documented by early studies [33,34]. Metabolic alterations [34-38] and elevated levels of pro-inflammatory cytokines have been described in both plasma and adipose tissue [39-43]. Previous analyses of adipocytes failed to demonstrate that adipocytes could be infected by HIV in vivo [44]-in contrast to the results of in vitro studies [45-47]. However, it has been shown that the HIV viral proteins Vpr and Nef are present in adipose tissue and have a negative impact on adipose homeostasis [48-50]. These observations provide a strong rationale for reconsidering the impact of HIV infection on adipose tissue by focusing on immune cells rather than adipocytes. We hypothesize that adipose tissue may constitute a neglected partner that drives viral persistence and chronic immune activation via two nonexclusive mechanisms: local infection and the abnormal local activation of immune cells. To assess the putative infection of adipose immune cells more precisely, we first analyzed tissues from chronically SIV-infected macaques. We chose to analyze both subcutaneous adipose tissue (SCAT) and visceral adipose tissue (VAT) because they differ in terms of metabolic activity and immune cell content [14,15,17,51]. Secondly, we extended these analyses to ART-treated HIV-infected patients.
In the present report, we demonstrate that SIV infection is associated with changes in the composition of adipose tissue, such as elevated densities of both adipocytes and stromal vascular cells. Importantly, adipose tissue macrophages and CD4+ and CD8+ T cells exhibited a more intense activation profile (relative to non-infected animals). Furthermore, SIV DNA and RNA was detected in total SVF and in sorted adipose tissue macrophages and CD4+ T cells. We observed similar results in ART-controlled, HIV-infected patients having undergone elective visceral surgery: their SVF samples were positive for HIV DNA. The presence of infected/virus-producing cells within adipose tissue was confirmed by the detection of HIV RNA in tissue sections via in situ hybridization. Lastly, we performed an in vitro reactivation assay on samples from six patients and found that adipose tissue CD4+ T cells were capable of producing replication-competent virus. Taken as a whole, our data show that adipose tissue as a viral reservoir with inflammatory potential.
In conclusion, we performed three levels of detection: (i) HIV DNA using PCRs, (ii) HIV RNA using in situ hybridization and (iii) HIV RNA in supernatants in an in vitro reactivation assay based on ultrasensitive RT-PCR. We demonstrated that latently HIV-infected cells were present in the SVF from both SCAT and VAT in 6 ART-treated patients. HIV DNA was consistently detected in adipose CD4+ T cells-strongly suggesting that adipose tissue is an HIV reservoir in aviremic patients on long-term ART.
Discussion
Residual chronic inflammation and viral persistence are two key features of ART-treated, HIV-infected patients. Although several mechanisms have been described, the establishment and persistence of low-grade inflammation are not fully understood and remain to be characterized. Given that adipose tissue is a major site of inflammation in a context of obesity, we hypothesized that the adipose tissue changes associated with HIV infection may drive low-grade inflammation. Indeed, the literature data reveal a strongly pro-inflammatory profile of adipose tissue during HIV infection-although most of these studies were performed on ART-treated patients [36,41,60]. In order to rule out the potential confounding role of ART and to collect sufficient amounts of tissue, we studied the SIV/macaque infection model. Chronic SIV infection in vivo was associated with a markedly higher adipocyte density (i.e. cell numbers per field) and SVF cell count (expressed per gram of adipose tissue). The elevated adipocyte density has been linked to an abnormally low adipocyte size, which in turn may reflect drastic alterations of adipogenesis [61,62] and/or lipolysis [48,60]. The massive increase in SVF cells in adipose tissue in chronically infected animals essentially reflected the accumulation of CD45- cells. The CD45- cells recovered in the SVF of infected animals need to be identified more accurately. Those in the SVF included pre-adipocytes, the accumulation of which may be related to changes in adipocyte differentiation. These striking changes in CD45- proportion need to be further investigated and could relate to multiple defects affecting adipose tissue during SIV infection such as lipodystrophy, inflammation or viral persistence. The SVF also contains mesenchymal stem cells [63-65], and it remains to be established whether these cells can also be infected. When focusing on immune cells, there was no major change in total leukocyte counts. However, the percentage of CD4+ T cells was severely lower in adipose tissue from SIV-infected animals than in non-infected animals, whereas the CD8+ T cell percentage was markedly higher. We demonstrated that the change in the CD4/CD8 ratio reflected the accumulation of CD8+ T cells, in accordance with previous reports of massive CD8 accumulation during adipose tissue inflammation [27]. Intense recruitment of CD8+ T cells was confirmed by the perivascular location of CD8+ T cells. However, a direct impact of SIV in adipose tissue cannot be ruled out [53,54].
The fact that CD4+ T cell counts were generally unaffected was not expected in the context of SIV infection-especially given the high percentage of central memory CD4+ T cells (a major HIV/SIV target) in adipose tissue. Organ-dependent differences in the direction and amplitude of cell depletion have been described [66,67]: CD4+ T cell depletion is rapid and massive in the intestinal mucosa, whereas the follicular helper subset (also a major HIV target) even expands in secondary lymphoid organs [68]. Importantly, CD4+ T cells were mainly found recovered in the vicinity of adipocytes and far from capillaries. This remote site may favor viral persistence, as was recently described in follicular CD4+ T cells [69]. It thus appears that adipose tissue may constitute an additional site for the accumulation of CD4+ T cells. Phenotypic analysis revealed that the Tcm CD4+ T cells fraction was predominant in both SCAT and VAT, thus confirming the high potential for viral persistence of adipose tissue. The predominance of central memory CD4+ T cells, which usually identifies inductive lymphoid site, also raises the question of the lymphoid definition of the adipose tissue: Does adipose tissue constitute a lymphoid site and if so is it an inductive or effector lymphoid site?
We also found that phenotypic changes can be detected in both lymphocytes and macrophages during chronic SIV infection of adipose tissue. Expression levels of the activation marker HLA-DR on adipose tissue T lymphocytes were higher in SIV-infected animals. Macrophages displayed a moderate change in phenotype. SIV infection was associated with an increase in the proportion of CD206-CD163- adipose tissue macrophages and a concomitant decrease in the CD206+CD163- fraction-suggesting a shift from an anti-inflammatory profile towards a more activated profile or the recruitment of migrating blood monocytes. Overall, we observed features commonly associated with obesity-related inflammation: elevated SVF numbers, the specific recruitment of CD8+T cells, a higher proportion of macrophages and greater expression of activation markers. These observations strongly suggest that adipose tissue is involved in inflammation during SIV infection.
We next investigated the mechanisms that might underlie the increase in adipose tissue inflammation in chronically SIV-infected animals. There is increasing evidence to show that HIV infection per se interferes with adipose tissue homeostasis, although the mechanisms remains to be defined [34]. Adipose inflammation may be a consequence of systemic inflammation, with in turn is worsened by adipose inflammation in a vicious circle. Adipose inflammation may also be related to the spread of viral proteins [48-50], CD4+ T cell lymphopenia [70] and/or microbial translocation [71,72], all of which are known to alter adipocyte homeostasis. Furthermore, SIV may directly infect adipose-resident immune cells. Importantly, previous reports essentially failed to demonstrate consistent infection of adipocytes and did not provide any information on adipose immune cells. In the present study, SVF fractions were positive for SIV DNA and SIV RNA in all animals tested. SIV DNA was also detected in all sorted adipose CD4+ T lymphocytes. SIV DNA was detected less consistently in sorted CD14-expressing cells (3 out of 5 in both SCAT and VAT). The results for cell-associated SIV RNA essentially corroborated those for SIV DNA. We demonstrated that in SVF fractions, SIV was consistently present in CD4+ T lymphocytes and less frequently present in macrophages. Our detection of viral DNA and RNA in stromal vascular cells collected from adipose tissue of chronically viremic animals thus confirms that adipose tissue is a site of infection.
We next sought to confirm the presence of viral infection in human adipose tissue and, more importantly, to characterize viral persistence in ART-suppressed HIV-infected patients. Three different assays were performed: (i) detection of viral DNA in SVF (in 11 patients) and in sorted CD4+ T cell fractions (from 3 patients), (ii) in situ RNA hybridization on fixed sections of adipose tissue (in 3 patients), and (iii) in vitro viral reactivation (in 6 patients). HIV DNA was detected in all SVF samples tested; this finding is in line with Couturier et al.'s report of HIV DNA in the SVF of 5 ART-treated, HIV-infected patients [73]. Importantly, we were able to detect HIV DNA in sorted adipose CD4+ T cell fractions but not in CD206+ CD14-expressing cells. It is noteworthy that sorted CD4+ T cells recovered from adipose tissue had much the same levels of HIV DNA as PBMCs. These results suggest that the proportion of infected cells is similar in adipose tissue CD4+ T cells and in peripheral blood CD4+ T cells. In contrast to the situation in chronically viremic macaques (in which SIV DNA was detected in a CD14+ fraction recovered from the SVF), we did not detect HIV DNA in CD14+CD206+ cells recovered from the SVF in aviremic patients. We checked whether this discrepancy was related to the difference in macrophage selection (i.e. CD14+ cells vs. CD14+CD206+ cells). CD14+CD206+ cells sorted from viremic macaques were still positive for SIV DNA, suggesting that viral DNA may be present in macrophage subsets in viremic stages but might not persist during long-term ART. One can reasonably presume that the half-life of macrophages is shorter than that of Tcm CD4+ T cells. Alternatively, the small number of macrophages collected may have prevented us from detecting a small proportion of infected cells. This aspect will be investigated further. Lastly, six samples were reserved for a viral replication assay. Although only small numbers of sorted adipose cells were available, we were able to monitor in vitro viral replication by detecting HIV RNA in supernatants after the incubation of adipose tissue CD4+ T cells with allogeneic pre-activated CD4+ T cells. HIV RNA was consistently detected in cultures of CD4+ T cells sorted from the SVF, suggesting that adipose CD4+ T cells are infected by replication-competent HIV. In four patients, ex vivo HIV replication was induced to the same extent in sorted CD4+ T cells from the SVF and from PBMCs. These results suggest that the proportion of infected cells is much the same among adipose tissue CD4+ T cells and peripheral blood CD4+ T cells; this hypothesis is supported by the fact that sorted CD4+ T cells from adipose tissue and PBMCs had similar HIV DNA contents. In contrast, replication-competent HIV was detected in the SVF only in two patients. The high frequency of memory CD4+ T cells in adipose tissue (relative to PBMCs) may explain the high proportion of latently infected cells. Otherwise the number of latently infected CD4+ T cells in adipose tissue might be higher than in PBMCs in some patients on ART. These findings emphasize the need to sample several tissues when studying HIV reservoirs in patients on ART. Further investigation (using limiting dilution assays) is necessary but would be technically challenging (given the low numbers of resident tissue CD4+ T cells in patients).
Overall, we were able to demonstrate the persistence of HIV DNA and RNA within stromal vascular cells in adipose tissue recovered from ART-treated HIV-infected patients. These observations were supported by the results of in vitro reactivation experiments, showing that adipose CD4+ T cells contained replication-competent HIV. This dataset strongly supports the hypothesis whereby adipose tissue constitutes an important viral reservoir. Adipose tissue may thus constitute a favorable environment for viral persistence for several reasons: (a) constant inflammation favors viral replication, (b) the presence of elevated fractions of activated and central memory CD4+ T cells, which are HIV's natural targets, (c) the potentially insufficient distribution of some antiretroviral drugs into adipose tissue [44], which may favor viral persistence, and (d) the specific metabolic and immune activity of adipose tissue, which may affect the effectiveness of immune responses [74].
In the present work, our analyses of SCAT and VAT from SIV-infected macaques and ART-suppressed patients, showed that (i) SIV infection induced immune activation and a pro-inflammatory profile in adipose tissue immune cells and (ii) these immune cells were indeed infected by SIV/HIV. These results indicate that adipose tissue constitutes a new, relatively large reservoir for the virus that could be involved in chronic immune activation and low-grade inflammation. Our observations have major implications in the context of HIV disease. Firstly, they emphasize the crucial requirement for the broad diffusion of antiretroviral drugs within tissues; combination therapy must include drugs that diffuse not only into adipose tissues but also into tissue CD4+ T cells and macrophages. Two main mechanisms may prevent efficient activity of ART on adipose infected cells: (i) low accessibility of ART to fat tissue [44], (ii) sequestration of drugs inside the lipid droplets at the expense of adipose infected immune cells [75]. Secondly, they provide an interesting rationale for the use of drugs with metabolic activity. It might be interesting to reconsider the anti-inflammatory impact of statins [76,77] by focusing on adipose sites and addressing the drugs' potential impact on viral reservoirs. Thirdly, gender differences in adipose tissue distribution, the inflammatory profile and immune cell content [78] may underlie differential susceptibility to the establishment of viral reservoirs. Fourthly, our results open up new therapeutic strategies for limiting the size of viral reservoirs, chronic inflammation and associated comorbidities (via the modulation of adipose tissue related pathways rather than strictly immune pathways).
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