A Summary of the First HIV Microbiome Workshop 2015.

The role of microbiota in the pathogenesis of HIV infection has become the subject of intense research in recent years. A rapidly growing amount of data suggest that microbial dysbiosis-in the gut or the genital tract-can influence HIV transmission and/or disease progression; however, a deeper understanding of the mechanisms involved is lacking. To better understand the relationship between the microbiome and HIV infection, investigators from a wide variety of disciplines, including those working in basic and clinical HIV studies, cardiovascular disease, reproductive health, and bioinformatics, gathered at the first International Workshop on Microbiome in HIV Pathogenesis, Prevention and Treatment, at NIH on 7 and 8 April, 2015.

Differential CD4+ cell count increase and CD4+ :  CD8+ ratio normalization with maraviroc compared with tenofovir.

OBJECTIVE: Studies exploring the immunologic effects of maraviroc (MVC) have produced mixed results; hence, it remains unclear whether MVC has unique immunologic effects in comparison with other antiretroviral drugs. We sought to determine whether MVC has differential effects compared with tenofovir disoproxil fumarate (TDF) during initial antiretroviral therapy. DESIGN: Prospective study in AIDS Clinical Trials Group A5303, a double-blind, placebo-controlled trial (N = 262) of MVC vs. TDF, each combined with boosted darunavir and emtricitabine. METHODS: A total of 31 cellular and soluble biomarkers were assayed at weeks 0 and 48. Polychromatic flow cytometry was performed on cryopreserved peripheral blood mononuclear cells. Soluble markers were assayed in plasma using ELISA kits. Analyses were as treated. RESULTS: Analyses included 230 participants (119 in MVC arm and 111 in TDF arm). Over 48 weeks of treatment, no significant differences were detected in declines in markers of inflammation and activation with MVC vs. TDF. A greater CD4 T-cell count increase (median +234 vs. +188 cells/µl, P = 0.036), a smaller CD8 T-cell count decrease (-6 vs. -109 cells/µl, P = 0.008), and a smaller CD4 : CD8 ratio increase (0.26 vs. 0.39, P = 0.003) occurred with MVC. Among participants with a baseline CD4 : CD8 ratio less than 1, a smaller proportion of MVC group normalized to a ratio greater than 1 at week 48 (15 and 36%, P < 0.001). CONCLUSION: MVC resulted in less improvement in the CD4 : CD8 ratio driven by greater increase in CD4 cell count but smaller decline in CD8 cell count. Changes in soluble or cellular biomarkers of inflammation and immune activation were not different between MVC and TDF.

Glycogen Levels in Undiluted Genital Fluid and Their Relationship to Vaginal pH, Estrogen, and Progesterone.

BACKGROUND: Colonization of the female lower genital tract with Lactobacillus provides protection against STIs and adverse pregnancy outcomes. Growth of genital Lactobacillus is postulated to depend on epithelial cell-produced glycogen. However, the amount of cell-free glycogen in genital fluid available for utilization by Lactobacillus is not known. METHODS: Eighty-five genital fluid samples from 7 pre-menopausal women taken over 4-6 weeks were obtained using the Instead SoftCup® (EvoFem, Inc., San Diego, CA, USA) by consented donors. Cell-free glycogen and glucose in genital fluids and estrogen and progesterone in blood were quantified. FINDINGS: Glycogen ranged from 0.1-32 µg/µl. There were significant differences between women in glycogen over the observation period. There was a strong negative correlation between glycogen and vaginal pH (r = -0.542, p<0.0001). In multivariable analysis, free glycogen levels were significantly negatively associated with both vaginal pH and progesterone (p < 0.001 and p = 0.004, respectively). Estrogen, glucose, age, sexual intercourse 24 hours prior to visit, and days after the initial visit were not significantly associated with free glycogen levels. CONCLUSION: Cell-free glycogen concentrations can be very high, up to 3% of genital fluid, and are strongly associated with acidic vaginal pH. However, the fluctuations in glycogen levels in individuals and differences between individuals do not appear to be associated with estrogen.

Does systemic inflammation and immune activation contribute to fracture risk in HIV?

PURPOSE OF REVIEW: There is increasing evidence pointing toward an important role of heightened immune activation and inflammation in people living with HIV contributing to the development of non-AIDS comorbidities. This review aims to explore low bone mineral density (BMD) in HIV with a focus on the underlying mechanisms and relationships between the immune and skeletal systems. RECENT FINDINGS: Baseline immune activation and inflammation negatively impact BMD at antiretroviral therapy (ART) initiation. B- and T-cell alterations in HIV lead to an imbalance in the osteoblastic osteoprotegerin (OPG) and osteoclastic receptor activator of NF-κB ligand (RANKL) cytokines which favours osteoclastogenesis and bone resorption. These findings suggest an important role for immune-mediated mechanisms in the pathogenesis of low BMD in HIV. SUMMARY: Bone homeostasis is in part regulated by cells of the immune system through complex interactions with the RANK/RANKL/OPG axis. Disturbances in the normal functioning of T, B cells, and monocytes in HIV and the resulting proinflammatory state may contribute to dysregulation of this finely controlled balance leading to increased bone loss. Pre-ART levels of immune activation and inflammation have a consistently negative effect on BMD and further suggest the immunocentric basis of bone loss in HIV alongside supporting the benefits of earlier ART initiation. Further longitudinal studies will help determine the effect this will have on fracture risk in people living with HIV.

Exploratory comparison of vaginal glycogen and Lactobacillus levels in premenopausal and postmenopausal women.

OBJECTIVE: Previous studies have suggested that glycogen expression in the vaginal epithelium decreases during menopause, resulting in reduced levels of lactobacilli. However, free glycogen in genital fluids and its relationship with Lactobacillus levels have not been compared in premenopausal and postmenopausal women. METHODS: Eighty-two cervicovaginal lavage samples were collected at different phases of the menstrual cycle from 11 premenopausal (4 HIV-uninfected and 7 HIV-infected) and 12 postmenopausal (7 HIV-uninfected and 5 HIV-infected) women during a 1- to 3-month period. Free glycogen was quantified in genital fluids. Lactobacillus levels were quantified by real-time polymerase chain reaction. Estrogen and progesterone levels in blood were determined by enzyme-linked immunosorbent assay. RESULTS: Free glycogen was detected in both premenopausal and postmenopausal women. Across all samples, those from postmenopausal women had significantly lower levels of free glycogen than those from premenopausal women (median, 0.002 vs 0.065 µg/µL, respectively; P = 0.03). Lactobacillus levels correlated positively with free glycogen in both premenopausal (Spearman r = 0.68, P < 0.0001) and postmenopausal (r = 0.60, P < 0.002) women. Samples from premenopausal women had higher Lactobacillus levels and lower vaginal pH (median log, 8.1; median pH, 4) than those from postmenopausal women (median log, 7.1; median pH, 4.6), although these differences were not significant. HIV status had no significant effect on these relationships. CONCLUSIONS: Free glycogen is detected in both premenopausal and postmenopausal women and correlates with Lactobacillus in both groups. These results point to the complexity of the relationship between menopause and vaginal microbiota and indicate that more careful studies of the role of glycogen are warranted.

Free glycogen in vaginal fluids is associated with Lactobacillus colonization and low vaginal pH.

OBJECTIVE: Lactobacillus dominates the lower genital tract microbiota of many women, producing a low vaginal pH, and is important for healthy pregnancy outcomes and protection against several sexually transmitted pathogens. Yet, factors that promote Lactobacillus remain poorly understood. We hypothesized that the amount of free glycogen in the lumen of the lower genital tract is an important determinant of Lactobacillus colonization and a low vaginal pH. METHODS: Free glycogen in lavage samples was quantified. Pyrosequencing of the 16S rRNA gene was used to identify microbiota from 21 African American women collected over 8-11 years. RESULTS: Free glycogen levels varied greatly between women and even in the same woman. Samples with the highest free glycogen had a corresponding median genital pH that was significantly lower (pH 4.4) than those with low glycogen (pH 5.8; p<0.001). The fraction of the microbiota consisting of Lactobacillus was highest in samples with high glycogen versus those with low glycogen (median = 0.97 vs. 0.05, p<0.001). In multivariable analysis, having 1 vs. 0 male sexual partner in the past 6 months was negatively associated, while BMI ≥30 was positively associated with glycogen. High concentrations of glycogen corresponded to higher levels of L. crispatus and L. jensenii, but not L. iners. CONCLUSION: These findings show that free glycogen in genital fluid is associated with a genital microbiota dominated by Lactobacillus, suggesting glycogen is important for maintaining genital health. Treatments aimed at increasing genital free glycogen might impact Lactobacillus colonization.

Human α-amylase present in lower-genital-tract mucosal fluid processes glycogen to support vaginal colonization by Lactobacillus.

Lactobacillus colonization of the lower female genital tract provides protection from the acquisition of sexually transmitted diseases, including human immunodeficiency virus, and from adverse pregnancy outcomes. While glycogen in vaginal epithelium is thought to support Lactobacillus colonization in vivo, many Lactobacillus isolates cannot utilize glycogen in vitro. This study investigated how glycogen could be utilized by vaginal lactobacilli in the genital tract. Several Lactobacillus isolates were confirmed to not grow in glycogen, but did grow in glycogen-breakdown products, including maltose, maltotriose, maltopentaose, maltodextrins, and glycogen treated with salivary α-amylase. A temperature-dependent glycogen-degrading activity was detected in genital fluids that correlated with levels of α-amylase. Treatment of glycogen with genital fluids resulted in production of maltose, maltotriose, and maltotetraose, the major products of α-amylase digestion. These studies show that human α-amylase is present in the female lower genital tract and elucidates how epithelial glycogen can support Lactobacillus colonization in the genital tract.

The barrier to HIV transmission provided by genital tract Lactobacillus colonization.

While resistance to HIV transmission is due to multiple mechanisms such as the epithelium, a lower genital tract microbiota dominated by Lactobacillus appears to play an important role. This article reviews selected recent research on genital tract microbiota in women including how microbiota impacts HIV resistance and factors affecting Lactobacillus colonization.

Effects of Succinic Acid and Other Microbial Fermentation Products on HIV Expression in Macrophages.

Bacterial vaginosis (BV), a common condition in women, is associated with increased shedding of HIV in the female genital tract. While the Lactobacillus species that comprise a healthy vaginal microbiota produce lactic acid, the bacteria common in BV produce high concentrations of short chain fatty acids (SCFAs) and succinic acid. Macrophages are abundant in the lower genital tract mucosa and are thought to play an important role in HIV infection. In this study, we investigated whether SCFAs and succinic acid impacted HIV expression in monocyte-derived macrophages. Monocytes differentiated with either granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) were infected with either HIVBal or an HIV-luciferase reporter virus and treated with SCFAs, succinic acid, or lactic acid. Butyric acid suppressed HIV expression while succinic acid significantly increased expression in macrophages differentiated with either GM-CSF or M-CSF. Acetic, propionic, and lactic acids had no effect on HIV expression. Only succinic acid resulted in a significant increase in interleukin-8 production by infected macrophages. Our results suggest that succinic acid present in increased concentrations in the genital tract of women with BV plays a pro-inflammatory role and increases HIV expression. This could be one factor contributing to increased virus shedding seen in women with BV.

IL-22 levels are associated with Trichomonas vaginalis infection in the lower genital tract.

PROBLEM: IL-22 has important functions at mucosal surfaces, including the induction of antimicrobial peptides and maintenance of epithelium. However, IL-22 has not been investigated in the genital tract during TV infection. METHODS OF STUDY: Women who visited an STD clinic and women from a cohort with frequent Trichomoniasis were studied. IL-22, IL-17, and antimicrobial peptides were measured in cervicovaginal lavage by ELISA. RESULTS: In women visiting the STD clinic, those without STDs (n = 10) had a median IL-22 of 0 pg/mL, while women with infections (n = 30) had 27 pg/mL (P = 0.04). In the cohort, women with Trichomoniasis (n = 19) had significantly higher IL-22 than women with no infections (n = 21, 74 versus 0 pg/mL, P = 0.0001). IL-17 was also significantly increased in Trichomoniasis, and there was a correlation between IL-22 and IL-17 (P = 0.001). CONCLUSION: IL-22 is increased in STDs generally and in Trichomoniasis specifically suggesting an antimicrobial response of the mucosa and an epithelial repair process induced by the STDs.

The role of bacterial vaginosis and trichomonas in HIV transmission across the female genital tract.

Bacterial vaginosis (BV) and Trichomonas vaginalis (TV) infections are both very common and are associated with increased risk of sexual transmission of HIV. There are several mechanisms by which BV and TV could affect susceptibility including inducing pro-inflammatory cytokines and disrupting mucosal barrier function. This review highlights recent advances in our understanding of how these genital conditions lead to an increased risk of HIV infection in women.

Longitudinal assessment of pigtailed macaque lower genital tract microbiota by pyrosequencing reveals dissimilarity to the genital microbiota of healthy humans.

Vaginal bacterial communities play an important role in human health and have been shown to influence HIV infection. Pigtailed macaques (Macaca nemestrina) are used as an animal model of HIV vaginal infection of women. Since the bacterial microbiota could influence retrovirus infection of pigtailed macaques, the genital microbiota in 10 cycling macaques was determined by pyrosequencing. The microbiota of all macaques was polymicrobial with a median of 13 distinct genera. Strikingly, the genera Sneathia and Fusobacterium, both in the phylum Fusobacteria, accounted for 18.9% and 13.3% of sequences while the next most frequent were Prevotella (5.6%), Porphyromonas (4.1%), Atopobium (3.6%), and Parvimonas (2.6%). Sequences corresponding to Lactobacillus comprised only 2.2% of sequences on average and were essentially all L. amylovorus. Longitudinal sampling of the 10 macaques over an 8-week period, which spanned at least one full ovulatory cycle, showed a generally stable presence of the major types of bacteria with some exceptions. These studies show that the microbiota of the pigtailed macaques is substantially dissimilar to that found in most healthy humans, where the genital microbiota is usually dominated by Lactobacillus sp. The polymicrobial makeup of the macaque bacterial populations, the paucity of lactobacilli, and the specific types of bacteria present suggest that the pigtailed macaque microbiota could influence vaginal retrovirus infection.

A comparison of lower genital tract glycogen and lactic acid levels in women and macaques: implications for HIV and SIV susceptibility.

Understanding factors that affect heterosexual transmission of HIV in women is of great importance. Lactobacilli in the lower genital tract of women utilize glycogen in vaginal epithelial cells as an energy source and produce lactic acid. The resultant vaginal acidity is believed to provide protection against HIV infection. Conversely, bacterial vaginosis (BV) is characterized by less lactic acid and a higher pH, and is associated with increased susceptibility to HIV infection. Because vaginal infection of macaques with simian immunodeficiency virus (SIV) or simian-human immunodeficiency virus (SHIV) is used as a model to study HIV sexual transmission, and because previous studies have shown a paucity of lactobacilli in rhesus macaques' lower genital tract, we compared lactic acid and glycogen levels in the genital fluid of rhesus and pigtail macaques with levels found in humans. The levels of lactic acid were lower in both rhesus (median=1.2 mol lactate/mg protein) and pigtail macaques (median=0.7 mol/mg) compared to women with healthy genital microbiota (median=4.2 mol/mg). Glycogen levels were significantly lower in both rhesus (median=0.004 µg glycogen/µg protein) and pigtail macaques (median=0 µg/µg) than in women (median=0.2 µg/µg). No significant differences in glycogen or lactate levels were observed comparing longitudinally collected samples from cycling pigtail macaques. These data show that the previously reported scarcity of lactobacilli in macaques correlates with low glycogen and lactic acid levels. These findings have important implications for studies of vaginal infection of macaques with SIV or SHIV and further our understanding of how the bacterial microbiota influences HIV infection.

Short-chain fatty acids induce pro-inflammatory cytokine production alone and in combination with toll-like receptor ligands.

PROBLEM: Short-chain fatty acids (SCFAs), produced at relatively high levels by anaerobic bacteria in bacterial vaginosis (BV), are believed to be anti-inflammatory. BV, a common alteration in the genital microbiota associated with increased susceptibility to HIV infection, is characterized by increased levels of both pro-inflammatory cytokines and SCFAs. We investigated how SCFAs alone or together with Toll-like receptor (TLR) ligands affected pro-inflammatory cytokine secretion. METHOD OF STUDY: Cytokines were measured by ELISA. Flow was used for phenotyping and reactive oxygen species (ROS) measurement. RESULTS: Short-chain fatty acids, at 20 mM, induced interleukin (IL)-8, IL-6, and IL-1ß release, while lower levels (0.02-2 mM) did not induce cytokine secretion. Levels >20 mM were toxic to cells. Interestingly, lower levels of SCFAs significantly enhanced TLR2 ligand- and TLR7 ligand-induced production of IL-8 and TNFα in a time- and dose-dependent manner, but had little effect on lipopolysaccharide-induced cytokine release. SCFAs mediated their effects on pro-inflammatory cytokine production at least in part by inducing the generation of ROS. CONCLUSION: Our data suggest that SCFAs, especially when combined with specific TLR ligands, contribute to a pro-inflammatory milieu in the lower genital tract and help further our understanding of how BV affects susceptibility to microbial infections.

The effects of commensal bacteria on innate immune responses in the female genital tract.

The innate and adaptive immune systems are important mechanisms for resistance to pathogens in the female lower genital tract. Lactobacilli at this site help maintain a healthy vagina by producing several factors including lactic acid. Indeed, bacterial vaginosis, a condition in which the genital microbiota is altered, is strongly associated with increased rates of a number of infections including HIV. However, the precise factors that contribute to increased rates of microbial and viral infections in bacterial vaginosis remain to be elucidated. We have studied the effects of bacterial microbiota in the lower genital tract on innate immunity and have found that Toll-like receptor ligands and short chain fatty acids, produced by bacterial microbiota, have dramatic effects on immune function. In this review, we will discuss these results, in addition to some recent articles that we believe will enhance our understanding of how microbes might interact with the immune system.

IgE signaling suppresses FcepsilonRIbeta expression.

Activation of the high-affinity receptor for IgE, FcepsilonRI, is known to elicit its rapid down-regulation through internalization and degradation. In keeping with this, expression of all three FcepsilonRI subunits is decreased at the protein level after cross-linkage of IgE with antigen. However, we find that the FcepsilonRI beta-subunit is also selectively suppressed at the mRNA level, through a pathway primarily involving Fyn, Syk, PI3K, and NF-kappaB. IgG or calcium ionophore, stimuli known to mimic portions of the IgE signaling cascade, similarly suppressed beta-subunit expression. LPS, a NF-kappaB-activating TLR ligand, did not alter beta-subunit expression. As IgE increases FcepsilonRI expression, we examined the coordinated regulation of FcepsilonRI subunits during culture with IgE, followed by cross-linkage with antigen. IgE increased the expression of all three FcepsilonRI subunits and strikingly induced expression of the antagonistic beta(T). The ratio of beta:beta(T) protein expression decreased significantly during culture with IgE and was reset to starting levels by antigen cross-linkage. These changes in protein levels were matched by similar fluctuations in beta and beta(T) mRNAs. FcepsilonRIbeta is a key regulator of IgER expression and function, a gene in which polymorphisms correlate with allergic disease prevalence. The ability of IgE and FcepsilonRI signaling to coordinate expression of the beta and beta(T) subunits may comprise a homeostatic feedback loop-one that could promote chronic inflammation and allergic disease if dysregulated.

Escape from suppression: tumor-specific effector cells outcompete regulatory T cells following stem-cell transplantation.

Immune reconstitution of autologous hematopoietic stem-cell transplant recipients with the progeny of mature T cells in the graft leads to profound changes in the emerging functional T-cell repertoire. In the steady state, the host is frequently tolerant to tumor antigens, reflecting dominant suppression of naive and effector T cells by regulatory T cells (T(regs)). We examined the relative frequency and function of these 3 components within the tumor-specific T-cell compartment during immune reconstitution. Grafts from tumor-bearing donors exerted a significant antitumor effect in irradiated, syngeneic tumor-bearing recipients. This was associated with dramatic clonal expansion and interferon-gamma (IFNgamma) production by previously tolerant tumor-specific T cells. While donor-derived T(regs) expanded in recipients, they did not inhibit the antigen-driven expansion of effector T cells in the early posttransplantation period. Indeed, the repopulation of tumor-specific effector T cells significantly exceeded that of T(regs), the expansion of which was limited by IL-2 availability. Although the intrinsic suppressive capacity of T(regs) remained intact, their diminished frequency was insufficient to suppress effector cell function. These findings provide an explanation for the reversal of tolerance leading to tumor rejection in transplant recipients and likely contribute to the efficacy of adoptive T-cell therapies in lymphopenic hosts.

IL-10 inhibits Fc epsilon RI expression in mouse mast cells.

FcepsilonRI expression and function is a central aspect of allergic disease. Using bone marrow-derived mouse mast cell populations, we have previously shown that the Th2 cytokine IL-4 inhibits FcepsilonRI expression and function. In the current study we show that the Th2 cytokine IL-10 has similar regulatory properties, and that it augments the inhibitory effects of IL-4. FcepsilonRI down-regulation was functionally significant, as it diminished inflammatory cytokine production and IgE-mediated FcepsilonRI up-regulation. IL-10 and IL-4 reduced FcepsilonRI beta protein expression without altering the alpha or gamma subunits. The ability of IL-4 and IL-10 to alter FcepsilonRI expression by targeting the beta-chain, a critical receptor subunit known to modulate receptor expression and signaling, suggests the presence of a Th2 cytokine-mediated homeostatic network that could serve to both initiate and limit mast cell effector function.

Interleukin-4 elicits apoptosis of developing mast cells via a Stat6-dependent mitochondrial pathway.

OBJECTIVE: The aim of this study was to assess the effects of interleukin-4 and signal transducer and activator of transcription (Stat)-6 on IL-3+SCF-induced mast cell development. PATIENTS AND METHODS: Unseparated mouse bone marrow cells were cultured in IL-3+SCF, giving rise to mast cells and monocytes/macrophages. The addition of IL-4, the use of Stat6-deficient bone marrow cells, and expression of a constitutively active Stat6 mutant were employed to assess the effects of IL-4 and Stat6 on cell viability, proliferation, and differentiation. Bax-deficient and bcl-2 transgenic bone marrow cells were used to assess the importance of the mitochondria in IL-4-mediated effects. RESULTS: IL-4 elicited apoptosis and limited the cell cycle progression of developing bone marrow cells, without affecting cell differentiation. Apoptosis required that IL-4 be present during the first 8 days of the 21-day culture period. Cell death correlated with loss of mitochondrial membrane potential. Accordingly, IL-4-mediated apoptosis was inhibited by Bax deletion or bcl-2 overexpression. Lastly, Stat6 activation was both necessary and sufficient to inhibit cell survival. CONCLUSION: IL-4 exerts potent apoptotic effects on developing mast cells and monocyte/macrophages through mitochondrial damage and Stat6 activation.

Stat5 expression is critical for mast cell development and survival.

Interleukin-3 (IL-3) and stem cell factor (SCF) are important mast cell growth and differentiation factors. Since both cytokines activate the transcription factor signal transducer and activator of transcription 5 (Stat5), a known regulator of proliferation and survival, we investigated the effects of Stat5 deficiency on mast cell development and survival. Bone marrow-derived mast cell (BMMC) populations cultured from Stat5A/B-deficient mice survived in IL-3 + SCF, but not in either cytokine alone. These cells demonstrated reduced expression of Bcl-2, Bcl-x(L), cyclin A2, and cyclin B1, with increased apoptosis and delayed cell cycle progression during IL-3 or SCF culture. Finally, the absence of Stat5 resulted in loss of in vivo mast cell development, as judged by assessments of Stat5-deficient mice and transplantation of Stat5-deficient bone marrow cells to mast cell-deficient recipient mice. These results indicate that Stat5A and Stat5B are critical regulators of in vitro and in vivo mast cell development and survival.