Experimental bacterial dysbiosis with consequent immune alterations increase intrarectal SIV acquisition susceptibility.

Variations in the composition of the intestinal bacterial microbiome correlate with acquisition of some sexually transmitted pathogens. To experimentally assess the contribution of intestinal dysbiosis to rectal lentiviral acquisition, we induce dysbiosis in rhesus macaques (RMs) with the antibiotic vancomycin prior to repeated low-dose intrarectal challenge with simian immunodeficiency virus (SIV) SIVmac239X. Vancomycin administration reduces T helper 17 (TH17) and TH22 frequencies, increases expression of host bacterial sensors and antibacterial peptides, and increases numbers of transmitted-founder (T/F) variants detected upon SIV acquisition. We observe that SIV acquisition does not correlate with measures of dysbiosis but rather associates with perturbations in the host antimicrobial program. These findings establish a functional association between the intestinal microbiome and susceptibility to lentiviral acquisition across the rectal epithelial barrier.

Butyrate administration is not sufficient to improve immune reconstitution in antiretroviral-treated SIV-infected macaques.

Defective gastrointestinal barrier function and, in turn, microbial translocation have been identified as significant contributors to persistent inflammation in antiretroviral (ARV)-treated people living with HIV. Metabolic supplementation of short-chain fatty acids (SCFAs), generally produced by the commensal microbiome, may improve these outcomes. Butyrate is a SCFA that is essential for the development and maintenance of intestinal immunity and has a known role in supporting epithelial integrity. Herein we assessed whether supplementation with the dietary supplement sodium butyrate would improve immune reconstitution and reduce inflammation in ARV-treated, simian immunodeficiency virus (SIV)-infected rhesus macaques. We demonstrate that butyrate supplementation does not significantly improve immune reconstitution, with no differences observed in systemic CD4+ T-cell frequencies, T-cell functionality or immune activation, microbial translocation, or transcriptional regulation. Our findings demonstrate that oral administration of sodium butyrate is insufficient to reduce persistent inflammation and microbial translocation in ARV-treated, SIV-infected macaques, suggesting that this therapeutic may not reduce co-morbidities and co-mortalities in treated people living with HIV.

Biomarkers of Cellular Stress Do Not Associate with sCD14 in Progressive HIV and SIV Infections in Vivo.

BACKGROUND: Microbial translocation occurs after damage to the structural and/or immunological barrier of the gastrointestinal (GI) tract into circulation. Microbial components that trans-locate from the lumen of the GI tract directly stimulate the immune system and contribute to inflammation. When microbial translocation becomes chronic, the inflammation has detrimental consequences. Given that microbial translocation is an important phenomenon in many diseases, defining biomarkers that reliably reflect microbial translocation is critical. Measurement of systemic microbial products is difficult since: 1) robust assays to measure microbial antigens simultaneously are lacking; 2) confounding factors influence assays used to detect microbial products; and 3) biological clearance mechanisms limit their detection in circulation. Thus, host proteins produced in response to microbial stimulation are used as surrogates for microbial translocation; however, many of these proteins are also produced in response to host proteins expressed by dying cells. METHODS: We measured plasma levels of biomarkers associated with GI tract damage, immune responses to microbial products, and cell-death in people living with HIV before and after antiretroviral administration, and in macaque nonhuman primates before and after SIV infection. RESULTS: Proteins secreted during cellular stress (receptor for advanced glycation endproducts-RAGE and high motility group box 1-HMGB1), which can induce sCD14 production in vitro and in vivo, do not associate with elevated levels of biomarkers associated with microbial translocation in progressively HIV-infected individuals and SIV-infected NHPs. CONCLUSIONS: Bystander cell death and generalized inflammation do not contribute to elevated levels of sCD14 observed in HIV/SIV-infected individuals.

HIV-1-induced cytokines deplete homeostatic innate lymphoid cells and expand TCF7-dependent memory NK cells.

Human immunodeficiency virus 1 (HIV-1) infection is associated with heightened inflammation and excess risk of cardiovascular disease, cancer and other complications. These pathologies persist despite antiretroviral therapy. In two independent cohorts, we found that innate lymphoid cells (ILCs) were depleted in the blood and gut of people with HIV-1, even with effective antiretroviral therapy. ILC depletion was associated with neutrophil infiltration of the gut lamina propria, type 1 interferon activation, increased microbial translocation and natural killer (NK) cell skewing towards an inflammatory state, with chromatin structure and phenotype typical of WNT transcription factor TCF7-dependent memory T cells. Cytokines that are elevated during acute HIV-1 infection reproduced the ILC and NK cell abnormalities ex vivo. These results show that inflammatory cytokines associated with HIV-1 infection irreversibly disrupt ILCs. This results in loss of gut epithelial integrity, microbial translocation and memory NK cells with heightened inflammatory potential, and explains the chronic inflammation in people with HIV-1.

SIV-specific CD8+ T cells are clonotypically distinct across lymphoid and mucosal tissues.

CD8+ T cell responses are necessary for immune control of simian immunodeficiency virus (SIV). However, the key parameters that dictate antiviral potency remain elusive, conceivably because most studies to date have been restricted to analyses of circulating CD8+ T cells. We conducted a detailed clonotypic, functional, and phenotypic survey of SIV-specific CD8+ T cells across multiple anatomical sites in chronically infected rhesus macaques with high (>10,000 copies/mL plasma) or low burdens of viral RNA (<10,000 copies/mL plasma). No significant differences in response magnitude were identified across anatomical compartments. Rhesus macaques with low viral loads (VLs) harbored higher frequencies of polyfunctional CXCR5+ SIV-specific CD8+ T cells in various lymphoid tissues and higher proportions of unique Gag-specific CD8+ T cell clonotypes in the mesenteric lymph nodes relative to rhesus macaques with high VLs. In addition, public Gag-specific CD8+ T cell clonotypes were more commonly shared across distinct anatomical sites than the corresponding private clonotypes, which tended to form tissue-specific repertoires, especially in the peripheral blood and the gastrointestinal tract. Collectively, these data suggest that functionality and tissue localization are important determinants of CD8+ T cell-mediated efficacy against SIV.

Simian Immunodeficiency Virus Infection of Rhesus Macaques Results in Delayed Zika Virus Clearance.

Flaviviruses are controlled by adaptive immune responses but are exquisitely sensitive to interferon-stimulated genes (ISGs). How coinfections, particularly simian immunodeficiency viruses (SIVs), that induce robust ISG signatures influence flavivirus clearance and pathogenesis is unclear. Here, we studied how Zika virus (ZIKV) infection is modulated in SIV-infected nonhuman primates. We measured ZIKV replication, cellular ZIKV RNA levels, and immune responses in non-SIV-infected and SIV-infected rhesus macaques (RMs), which we infected with ZIKV. Coinfected animals had a 1- to 2-day delay in peak ZIKV viremia, which was 30% of that in non-SIV-infected animals. However, ZIKV viremia was significantly prolonged in SIV-positive (SIV+) RMs. ISG levels at the time of ZIKV infection were predictive for lower ZIKV viremia in the SIV+ RMs, while prolonged ZIKV viremia was associated with muted and delayed adaptive responses in SIV+ RMs.IMPORTANCE Immunocompromised individuals often become symptomatic with infections which are normally fairly asymptomatic in healthy individuals. The particular mechanisms that underlie susceptibility to coinfections in human immunodeficiency virus (HIV)-infected individuals are multifaceted. ZIKV and other flaviviruses are sensitive to neutralizing antibodies, whose production can be limited in HIV-infected individuals but are also sensitive to type I interferons, which are expressed at high levels in HIV-infected individuals. Data in this study highlight how individual components of the innate and adaptive immune responses which become perturbed in HIV-infected individuals influence ZIKV infection.

Simian Immunodeficiency Virus Infects Functionally Polarized Memory CD4 T Cells Equivalently In Vivo.

Among the numerous immunological abnormalities observed in chronically human immunodeficiency virus (HIV)-infected individuals, perturbations in memory CD4 T cells are thought to contribute specifically to disease pathogenesis. Among these, functional imbalances in the frequencies of T regulatory cells (Tregs) and interleukin 17 (IL-17)/IL-22-producing Th cells (Th17/Th22) from mucosal sites and T follicular helper (Tfh) cells in lymph nodes are thought to facilitate specific aspects of disease pathogenesis. However, while preferential infection of Tfh cells is widely thought to create an important viral reservoir in an immunologically privileged site in vivo, whether immunological perturbations among memory CD4 T cell populations are attributable to their relative infectivity by the virus in vivo is unclear. Here we studied peripheral blood and lymphoid tissues from antiretroviral (ARV)-treated and ARV-naive Asian macaques and isolated functionally defined populations of memory CD4 T cells. We then assessed the degree to which these populations were infected by simian immunodeficiency virus (SIV) in vivo, to determine whether particular functionally identified populations of memory CD4 T cells were preferentially infected by the virus. We found that SIV did not preferentially infect Th17 cells, compared to Th1 cells, Th2 cells, or Tregs. Moreover, Th17 cells contributed proportionately to the total pool of infected cells. Taken together, our data suggest that, although Tfh cells are more prone to harbor viral DNA, other functionally polarized cells are equally infected by the virus in vivo and Th17 cells are not preferentially infected.IMPORTANCE Functional perturbations of memory CD4 T cells have been suggested to underlie important aspects of HIV disease progression. However, the mechanisms underlying these perturbations remain unclear. Using a nonhuman primate model of HIV, we show that SIV infects functionally defined populations of memory CD4 T cells equally in different anatomic sites. Thus, preferential infection by the virus is unlikely to cause functional perturbations.

Experimental microbial dysbiosis does not promote disease progression in SIV-infected macaques.

Intestinal microbial dysbiosis has been described in individuals with an HIV-1 infection and may underlie persistent inflammation in chronic infection, thereby contributing to disease progression. Herein, we induced an HIV-1-like intestinal dysbiosis in rhesus macaques (Macaca mulatta) with vancomycin treatment and assessed the contribution of dysbiosis to SIV disease progression. Dysbiotic and control animals had similar disease progression, indicating that intestinal microbial dysbiosis similar to that observed in individuals with HIV is not sufficient to accelerate untreated lentiviral disease progression.

Cytotoxic T Cell Functions Accumulate When CD4 Is Downregulated by CD4+ T Cells in African Green Monkeys.

African green monkeys (AGMs) are a natural host of SIV that do not develop simian AIDS. Adult AGMs naturally have low numbers of CD4+ T cells and a large population of MHC class II-restricted CD8αα T cells that are generated through CD4 downregulation in CD4+ T cells. In this article, we study the functional profiles and SIV infection status in vivo of CD4+ T cells, CD8αα T cells, and CD8αß T cells in lymph nodes, peripheral blood, and bronchoalveolar lavage fluid of AGMs and rhesus macaques (in which CD4 downregulation is not observed). We show that, although CD8αα T cells in AGMs maintain functions associated with CD4+ T cells (including Th follicular functionality in lymphoid tissues and Th2 responses in bronchoalveolar lavage fluid), they also accumulate functions normally attributed to canonical CD8+ T cells. These hyperfunctional CD8αα T cells are found to circulate peripherally, as well as reside within the lymphoid tissue. Due to their unique combination of CD4 and CD8 T cell effector functions, these CD4- CD8αα T cells are likely able to serve as an immunophenotype capable of Th1, follicular Th, and CTL functionalities, yet they are unable to be infected by SIV. These data demonstrate the ambiguity of CD4/CD8 expression in dictating the functional capacities of T cells and suggest that accumulation of hyperfunctional CD8αα T cells in AGMs may lead to tissue-specific antiviral immune responses in lymphoid follicles that limit SIV replication in this particular anatomical niche.

Tissue myeloid cells in SIV-infected primates acquire viral DNA through phagocytosis of infected T cells.

The viral accessory protein Vpx, expressed by certain simian and human immunodeficiency viruses (SIVs and HIVs), is thought to improve viral infectivity of myeloid cells. We infected 35 Asian macaques and African green monkeys with viruses that do or do not express Vpx and examined viral targeting of cells in vivo. While lack of Vpx expression affected viral dynamics in vivo, with decreased viral loads and infection of CD4⁺ T cells, Vpx expression had no detectable effect on infectivity of myeloid cells. Moreover, viral DNA was observed only within myeloid cells in tissues not massively depleted of CD4⁺ T cells. Myeloid cells containing viral DNA also showed evidence of T cell phagocytosis in vivo, suggesting that their viral DNA may be attributed to phagocytosis of SIV-infected T cells. These data suggest that myeloid cells are not a major source of SIV in vivo, irrespective of Vpx expression.

Homeostatic cytokines induce CD4 downregulation in African green monkeys independently of antigen exposure to generate simian immunodeficiency virus-resistant CD8αα T cells.

UNLABELLED: African green monkeys (AGMs; genus Chlorocebus) are a natural host of simian immunodeficiency virus (SIVAGM). As they do not develop simian AIDS, there is great interest in understanding how this species has evolved to avoid immunodeficiency. Adult African green monkeys naturally have low numbers of CD4 T cells and a large population of major histocompatibility complex class II-restricted CD8α(dim) T cells that are generated through CD4 downregulation in CD4(+) T cells. Mechanisms that drive this process of CD4 downregulation are unknown. Here, we show that juvenile AGMs accelerate CD4-to-CD8αα conversion upon SIV infection and avoid progression to AIDS. The CD4 downregulation induced by SIV infection is not limited to SIV-specific T cells, and vaccination of an adult AGM who had a negligible number of CD4 T cells demonstrated that CD4 downregulation can occur without antigenic exposure. Finally, we show that the T cell homeostatic cytokines interleukin-2 (IL-2), IL-7, and IL-15 can induce CD4 downregulation in vitro. These data identify a mechanism that allows AGMs to generate a large, diverse population of T cells that perform CD4 T cell functions but are resistant to SIV infection. A better understanding of this mechanism may allow the development of treatments to induce protective CD4 downregulation in humans. IMPORTANCE: Many African primate species are naturally infected with SIV. African green monkeys, one natural host species, avoid simian AIDS by creating a population of T cells that lack CD4, the human immunodeficiency virus/SIV receptor; therefore, they are resistant to infection. However, these T cells maintain properties of CD4(+) T cells even after receptor downregulation and preserve immune function. Here, we show that juvenile AGMs, who have not undergone extensive CD4 downregulation, accelerate this process upon SIV infection. Furthermore, we show that in vivo, CD4 downregulation does not occur exclusively in antigen-experienced T cells. Finally, we show that the cytokines IL-2, IL-7, and IL-15, which induce homeostatic T cell proliferation, lead to CD4 downregulation in vitro; therefore, they can provide signals that lead to antigen-independent CD4 downregulation. These results suggest that if a similar process of CD4 downregulation could be induced in humans, it could provide a cure for AIDS.

Rate of AIDS progression is associated with gastrointestinal dysfunction in simian immunodeficiency virus-infected pigtail macaques.

During HIV/SIV infection, mucosal immune system dysfunction and systemic immune activation are associated with progression to AIDS; however, it is unclear to what extent pre-existing gastrointestinal damage relates to disease progression postinfection. Pigtail macaques (PTM) are an excellent model in which to assess mucosal dysfunction in relation to HIV/SIV pathogenesis, as the majority of these animals have high levels of gastrointestinal damage, immune activation, and microbial translocation prior to infection, and rapidly progress to AIDS upon SIV infection. In this study, we characterized the mucosal immune environment prior to and throughout SIV infection in 13 uninfected PTM and 9 SIV-infected PTM, of which 3 were slow progressors. This small subset of slow progressors had limited innate immune activation in mucosal tissues in the periphery, which was associated with a more intact colonic epithelial barrier. Furthermore, we found that preinfection levels of microbial translocation, as measured by LPS-binding protein, in PTM correlated with the rate of progression to AIDS. These data suggest that pre-existing levels of microbial translocation and gastrointestinal tract dysfunction may influence the rate of HIV disease progression.

Probiotic/prebiotic supplementation of antiretrovirals improves gastrointestinal immunity in SIV-infected macaques.

HIV infection results in gastrointestinal (GI) tract damage, microbial translocation, and immune activation, which are not completely ameliorated with suppression of viremia by antiretroviral (ARV) therapy. Furthermore, increased morbidity and mortality of ARV-treated HIV-infected individuals is associated with these dysfunctions. Thus, to enhance GI tract physiology, we treated SIV-infected pigtail macaques with ARVs, probiotics, and prebiotics or with ARVs alone. This synbiotic treatment resulted in increased frequency and functionality of GI tract APCs, enhanced reconstitution and functionality of CD4+ T cells, and reduced fibrosis of lymphoid follicles in the colon. Thus, ARV synbiotic supplementation in HIV-infected individuals may improve GI tract immunity and thereby mitigate inflammatory sequelae, ultimately improving prognosis.

HIV infection and antiretroviral therapy have divergent effects on mitochondria in adipose tissue.

BACKGROUND: Although human immunodeficiency virus (HIV) infection and antiretroviral therapy (ART) affect mitochondrial DNA (mtDNA) content and function, comprehensive evaluations of their effects on mitochondria in muscle, adipose tissue, and blood cells are limited. METHODS: Mitochondrial DNA quantification, mitochondrial genome sequencing, and gene expression analysis were performed on muscle, adipose tissue, and peripheral blood mononuclear cell (PBMC) samples from untreated HIV-positive patients, HIV-positive patients receiving nucleoside reverse transcriptase inhibitor (NRTI)-based ART, and HIV-negative controls. RESULTS: The adipose tissue mtDNA/nuclear DNA (nDNA) ratio was increased in untreated HIV-infected patients (ratio, 353) and decreased in those receiving ART (ratio, 162) compared with controls (ratio, 255; P < .05 for both comparisons); the difference between the 2 HIV-infected groups was also significant (P = .002). In HIV-infected participants, mtDNA/nDNA in adipose tissue correlated with the level of activation (CD38+ /HLA-DR+) for CD4+ and CD8+ lymphocytes. No significant differences in mtDNA content were noted in muscle or PMBCs among groups. Exploratory DNA microarray analysis identified differential gene expression between patient groups, including a subset of adipose tissue genes. CONCLUSIONS: HIV infection and ART have opposing effects on mtDNA content in adipose tissue; immune activation may mediate the effects of HIV, whereas NRTIs likely mediate the effects of ART.

Dynamics of simian immunodeficiency virus SIVmac239 infection in pigtail macaques.

Pigtail macaques (PTM) are an excellent model for HIV research; however, the dynamics of simian immunodeficiency virus (SIV) SIVmac239 infection in PTM have not been fully evaluated. We studied nine PTM prior to infection, during acute and chronic SIVmac239 infections, until progression to AIDS. We found PTM manifest clinical AIDS more rapidly than rhesus macaques (RM), as AIDS-defining events occurred at an average of 42.17 weeks after infection in PTM compared to 69.56 weeks in RM (P = 0.0018). However, increased SIV progression was not associated with increased viremia, as both peak and set-point plasma viremias were similar between PTM and RM (P = 0.7953 and P = 0.1006, respectively). Moreover, this increased disease progression was not associated with rapid CD4(+) T cell depletion, as CD4(+) T cell decline resembled other SIV/human immunodeficiency virus (HIV) models. Since immune activation is the best predictor of disease progression during HIV infection, we analyzed immune activation by turnover of T cells by BrdU decay and Ki67 expression. We found increased levels of turnover prior to SIV infection of PTM compared to that observed with RM, which may contribute to their increased disease progression rate. These data evaluate the kinetics of SIVmac239-induced disease progression and highlight PTM as a model for HIV infection and the importance of immune activation in SIV disease progression.

SIV infection of rhesus macaques results in dysfunctional T- and B-cell responses to neo and recall Leishmania major vaccination.

HIV infection is characterized by immune system dysregulation, including depletion of CD4+ T cells, immune activation, and abnormal B- and T-cell responses. However, the immunologic mechanisms underlying lymphocytic dysfunctionality and whether it is restricted to immune responses against neo antigens, recall antigens, or both is unclear. Here, we immunized SIV-infected and uninfected rhesus macaques to induce immune responses against neo and recall antigens using a Leishmania major polyprotein (MML) vaccine given with poly-ICLC adjuvant. We found that vaccinated SIVuninfected animals induced high frequencies of polyfunctional MML-specific CD4+ T cells. However, in SIV-infected animals, CD4+ T-cell functionality decreased after both neo (P = .0025) and recall (P = .0080) MML vaccination. Furthermore, after SIV infection, the frequency of MML-specific antibody-secreting classic memory B cells was decreased compared with vaccinated, SIV-uninfected animals. Specifically, antibody-secreting classic memory B cells that produced IgA in response to either neo (P = .0221) or recall (P = .0356) MML vaccinations were decreased. Furthermore, we found that T-follicular helper cells, which are essential for priming B cells, are preferentially infected with SIV. These data indicate that SIV infection results in dysfunctional T-cell responses to neo and recall vaccinations, and direct SIV infection of T-follicular helper cells, both of which probably contribute to deficient B-cell responses and, presumably, susceptibility to certain opportunistic infections.