Increased genital mucosal cytokines in Canadian women associate with higher antigen-presenting cells, inflammatory metabolites, epithelial barrier disruption, and the depletion of L. crispatus.

BACKGROUND: Cervicovaginal inflammation has been linked to negative reproductive health outcomes including the acquisition of HIV, other sexually transmitted infections, and cervical carcinogenesis. While changes to the vaginal microbiome have been linked to genital inflammation, the molecular relationships between the functional components of the microbiome with cervical immunology in the reproductive tract are understudied, limiting our understanding of mucosal biology that may be important for reproductive health. RESULTS: In this study, we used a multi'-omics approach to profile cervicovaginal samples collected from 43 Canadian women to characterize host, immune, functional microbiome, and metabolome features of cervicovaginal inflammation. We demonstrate that inflammation is associated with lower amounts of L. crispatus and higher levels of cervical antigen-presenting cells (APCs). Proteomic analysis showed an upregulation of pathways related to neutrophil degranulation, complement, and leukocyte migration, with lower levels of cornified envelope and cell-cell adherens junctions. Functional microbiome analysis showed reductions in carbohydrate metabolism and lactic acid, with increases in xanthine and other metabolites. Bayesian network analysis linked L. crispatus with glycolytic and nucleotide metabolism, succinate and xanthine, and epithelial proteins SCEL and IVL as major molecular features associated with pro-inflammatory cytokines and increased APCs. CONCLUSIONS: This study identified key molecular and immunological relationships with cervicovaginal inflammation, including higher APCs, bacterial metabolism, and proteome alterations that underlie inflammation. As APCs are involved in HIV transmission, parturition, and cervical cancer progression, further studies are needed to explore the interactions between these cells, bacterial metabolism, mucosal immunity, and their relationship to reproductive health. Video Abstract.

Vaginal epithelial dysfunction is mediated by the microbiome, metabolome, and mTOR signaling.

Bacterial vaginosis (BV) is characterized by depletion of Lactobacillus and overgrowth of anaerobic and facultative bacteria, leading to increased mucosal inflammation, epithelial disruption, and poor reproductive health outcomes. However, the molecular mediators contributing to vaginal epithelial dysfunction are poorly understood. Here we utilize proteomic, transcriptomic, and metabolomic analyses to characterize biological features underlying BV in 405 African women and explore functional mechanisms in vitro. We identify five major vaginal microbiome groups: L. crispatus (21%), L. iners (18%), Lactobacillus (9%), Gardnerella (30%), and polymicrobial (22%). Using multi-omics we show that BV-associated epithelial disruption and mucosal inflammation link to the mammalian target of rapamycin (mTOR) pathway and associate with Gardnerella, M. mulieris, and specific metabolites including imidazole propionate. Experiments in vitro confirm that type strain G. vaginalis and M. mulieris supernatants and imidazole propionate directly affect epithelial barrier function and activation of mTOR pathways. These results find that the microbiome-mTOR axis is a central feature of epithelial dysfunction in BV.

Nonoptimal bacteria species induce neutrophil-driven inflammation and barrier disruption in the female genital tract.

Neutrophil recruitment and activation within the female genital tract are often associated with tissue inflammation, loss of vaginal epithelial barrier integrity, and increased risk for sexually transmitted infections, such as HIV-1. However, the direct role of neutrophils on vaginal epithelial barrier function during genital inflammation in vivo remains unclear. Using complementary proteome and immunological analyses, we show high neutrophil influx into the lower female genital tract in response to physiological surges in progesterone, stimulating distinct stromal, immunological, and metabolic signaling pathways. However, despite the release of extracellular matrix-modifying proteases and inflammatory mediators, neutrophils contributed little to physiological mucosal remodeling events such as epithelial shedding or re-epithelialization during transition from diestrus to estrus phase. In contrast, the presence of bacterial vaginosis-associated bacteria resulted in a rapid and sustained neutrophil recruitment, resulting in vaginal epithelial barrier leakage and decreased cell-cell junction protein expression in vivo. Thus, neutrophils are important mucosal sentinels that rapidly respond to various biological cues within the female genital tract, dictating the magnitude and duration of the ensuing inflammatory response at steady state and during disease processes.

The Female Reproductive Tract Microbiome-Implications for Gynecologic Cancers and Personalized Medicine.

The microbial colonization of the lower female reproductive tract has been extensively studied over the past few decades. In contrast, the upper female reproductive tract including the uterine cavity and peritoneum where the ovaries and fallopian tubes reside were traditionally assumed to be sterile under non-pathologic conditions. However, recent studies applying next-generation sequencing of the bacterial 16S ribosomal RNA gene have provided convincing evidence for the existence of an upper female reproductive tract microbiome. While the vaginal microbiome and its importance for reproductive health outcomes has been extensively studied, the microbiome of the upper female reproductive tract and its relevance for gynecologic cancers has been less studied and will be the focus of this article. This targeted review summarizes the pertinent literature on the female reproductive tract microbiome in gynecologic malignancies and its anticipated role in future research and clinical applications in personalized medicine.

Simian Immunodeficiency Virus Susceptibility, Immunology, and Microbiome in the Female Genital Tract of Adolescent Versus Adult Pigtail Macaques.

In Sub-Saharan Africa, young women 15-24 years of age account for nearly 30% of all new HIV infections, however, biological and epidemiological factors underlying this disproportionate infection rate are unclear. In this study, we assessed biological contributors of SIV/HIV susceptibility in the female genital tract (FGT) using adolescent (n = 9) and adult (n = 10) pigtail macaques (PTMs) with weekly low-dose intravaginal challenges of SIV. Immunological variables were captured in vaginal tissue of PTMs by flow cytometry and cytokine assays. Vaginal biopsies were profiled by proteomic analysis. The vaginal microbiome was assessed by 16S rRNA sequencing. We were powered to detect a 2.2-fold increase in infection rates between age groups, however, we identified no significant differences in susceptibility. This model cannot capture epidemiological factors or may not best represent biological differences of HIV susceptibility. No immune cell subsets measured were significantly different between groups. Inflammatory marker MCP-1 was significantly higher (adj p = .02), and sCD40L trended higher (adj p = .06) in vaginal cytobrushes of adults. Proteomic analysis of vaginal biopsies showed no significant (adj p < .05) protein or pathway differences between groups. Vaginal microbiomes were not significantly different between groups. No differences were observed between age groups in this PTM model, however, these animals may not reflect biological factors contributing to HIV risk such as those found in their human counterparts. This model is therefore not appropriate to explore human adolescent differences in HIV risk. Young women remain a key population at risk for HIV infection, and there is still a need for comprehensive assessment and intervention strategies for epidemic control of this uniquely vulnerable population.

Treatment with Commonly Used Antiretroviral Drugs Induces a Type I/III Interferon Signature in the Gut in the Absence of HIV Infection.

Tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) are used for HIV treatment and prevention. Previously, we found that topical rectal tenofovir gel caused immunological changes in the mucosa. Here, we assess the effect of oral TDF/FTC in three HIV pre-exposure prophylaxis trials, two with gastrointestinal and one with cervicovaginal biopsies. TDF/FTC induces type I/III interferon-related (IFN I/III) genes in the gastrointestinal tract, but not blood, with strong correlations between the two independent rectal biopsy groups (Spearman r = 0.91) and between the rectum and duodenum (r = 0.81). Gene set testing also indicates stimulation of the type I/III pathways in the ectocervix and of cellular proliferation in the duodenum. mRNA sequencing, digital droplet PCR, proteomics, and immunofluorescence confirm IFN I/III pathway stimulation in the gastrointestinal tract. Thus, oral TDF/FTC stimulates an IFN I/III signature throughout the gut, which could increase antiviral efficacy but also cause chronic immune activation in HIV prevention and treatment settings.

Intestinal damage precedes mucosal immune dysfunction in SIV infection.

HIV and pathogenic SIV infection are characterized by mucosal dysfunction including epithelial barrier damage, loss of Th17 cells, neutrophil infiltration, and microbial translocation with accompanying inflammation. However, it is unclear how and when these contributing factors occur relative to one another. In order to determine whether any of these features initiates the cycle of damage, we longitudinally evaluated the kinetics of mucosal and systemic T-cell activation, microbial translocation, and Th17 cell and neutrophil frequencies following intrarectal SIV infection of rhesus macaques. We additionally assessed the colon proteome to elucidate molecular pathways altered early after infection. We demonstrate increased T-cell activation (HLA-DR+) beginning 3-14 days post-SIV challenge, reduced peripheral zonulin 3-14 days post-SIV, and evidence of microbial translocation 14 days post-SIV. The onset of mucosal dysfunction preceded peripheral and mucosal Th17 depletion, which occurred 14-28 days post-SIV, and gut neutrophil accumulation was not observed. Proteins involved in epithelial structure were downregulated 3 days post-SIV followed by an upregulation of immune proteins 14 days post-SIV. These data demonstrate that immune perturbations such as Th17 loss and neutrophil infiltration occur after alterations to epithelial structural protein pathways, suggesting that epithelial damage occurs prior to widespread immune dysfunction.

Understanding mucosal and microbial functionality of the female reproductive tract by metaproteomics: Implications for HIV transmission.

The mucosal surface of the female genital tract contains physiological, immunological, and microbial components that collectively comprise a functioning "mucosal system" that is critical for reproductive health. Alterations or imbalances to any of these components can have significant consequences for susceptibility to sexually transmitted infections, such as HIV. In recent years the advent of advanced systems biology technologies, such as metaproteomics, has provided new toolsets to studying mucosal systems. Studies have linked an altered mucosal proteome to many HIV risk factors including mucosal inflammation, bacterial vaginosis, hormonal contraceptives, and reduced efficacy of antiretroviral drugs for HIV prevention. Herein we will discuss how metaproteomics has been used to study mucosal system components, including epithelial barriers, inflammation, and the microbiome, with a focus on what alterations may contribute to increased HIV transmission risk in women.

Differential Reovirus-Specific and Herpesvirus-Specific Activator Protein 1 Activation of Secretogranin II Leads to Altered Virus Secretion.

UNLABELLED: Viruses utilize host cell machinery for propagation and manage to evade cellular host defense mechanisms in the process. Much remains unknown regarding how the host responds to viral infection. We recently performed global proteomic screens of mammalian reovirus TIL- and T3D-infected and herpesvirus (herpes simplex virus 1 [HSV-1])-infected HEK293 cells. The nonenveloped RNA reoviruses caused an upregulation, whereas the enveloped DNA HSV-1 caused a downregulation, of cellular secretogranin II (SCG2). SCG2, a member of the granin family that functions in hormonal peptide sorting into secretory vesicles, has not been linked to virus infections previously. We confirmed SCG2 upregulation and found SCG2 phosphorylation by 18 h postinfection (hpi) in reovirus-infected cells. We also found a decrease in the amount of reovirus secretion from SCG2 knockdown cells. Similar analyses of cells infected with HSV-1 showed an increase in the amount of secreted virus. Analysis of the stress-activated protein kinase (SAPK)/Jun N-terminal protein kinase (JNK) pathway indicated that each virus activates different pathways leading to activator protein 1 (AP-1) activation, which is the known SCG2 transcription activator. We conclude from these experiments that the negative correlation between SCG2 quantity and virus secretion for both viruses indicates a virus-specific role for SCG2 during infection. IMPORTANCE: Mammalian reoviruses affect the gastrointestinal system or cause respiratory infections in humans. Recent work has shown that all mammalian reovirus strains (most specifically T3D) may be useful oncolytic agents. The ubiquitous herpes simplex viruses cause common sores in mucosal areas of their host and have coevolved with hosts over many years. Both of these virus species are prototypical representatives of their viral families, and investigation of these viruses can lead to further knowledge of how they and the other more pathogenic members of their respective families interact with the host. Here we show that secretogranin II (SCG2), a protein not previously studied in the context of virus infections, alters virus output in a virus-specific manner and that the quantity of SCG2 is inversely related to amounts of infectious-virus secretion. Herpesviruses may target this protein to facilitate enhanced virus release from the host.

Comparative proteomic analyses of two reovirus T3D subtypes and comparison to T1L identifies multiple novel proteins in key cellular pathogenic pathways.

Viruses induce changes in the host to facilitate replication and evade the immune response. These changes are reflected by the host's proteome, including differences in protein abundance. Focusing on up and down regulated proteins after a virus infects the cell will lead to a characterization of the host response to infection, and may give insight into how viruses modulate proteins to evade host defense responses. We previously used SILAC to examine host proteomic changes in protein abundance in HEK293 cells infected with reovirus type 1, strain Lang (T1L). For the present study, we extended this analysis by determining cell protein alterations induced by two different reovirus subtypes, a less pathogenic type 3 Dearing (T3D(F)) isolate, and a more pathogenic isolate named T3D(C) that is presently in clinical trials as an anti-cancer oncolytic agent. This comparison of host proteome regulation showed that T3D(C) had a more marked effect on DNA replication proteins, recombination and repair, as well as immunological, apoptotic, and survival cell functions. We also identified several proteins not previously identified in any virus infection; branched chain amino-acid transaminase 2 (BCAT), paternally expressed 10 (PEG10), target of myb1 (TOM1), histone cluster 2 H4b (HIST2H4B) and tubulin beta 4B (TUBB4B).

Quantification of the host response proteome after herpes simplex virus type 1 infection.

Viruses employ numerous host cell metabolic functions to propagate and manage to evade the host immune system. For herpes simplex virus type 1 (HSV1), a virus that has evolved to efficiently infect humans without seriously harming the host in most cases, the virus-host interaction is specifically interesting. This interaction can be best characterized by studying the proteomic changes that occur in the host during infection. Previous studies have been successful at identifying numerous host proteins that play important roles in HSV infection; however, there is still much that we do not know. This study identifies host metabolic functions and proteins that play roles in HSV infection, using global quantitative stable isotope labeling by amino acids in cell culture (SILAC) proteomic profiling of the host cell combined with LC-MS/MS. We showed differential proteins during early, mid and late infection, using both cytosolic and nuclear fractions. We identified hundreds of differentially regulated proteins involved in fundamental cellular functions, including gene expression, DNA replication, inflammatory response, cell movement, cell death, and RNA post-transcriptional modification. Novel differentially regulated proteins in HSV infections include some previously identified in other virus systems, as well as fusion protein, involved in malignant liposarcoma (FUS) and hypoxia up-regulated 1 protein precursor (HYOU1), which have not been identified previously in any virus infection.

Quantification of the host response proteome after mammalian reovirus T1L infection.

All viruses are dependent upon host cells for replication. Infection can induce profound changes within cells, including apoptosis, morphological changes, and activation of signaling pathways. Many of these alterations have been analyzed by gene arrays to measure the cellular "transcriptome." We used SILAC (stable isotope labeling by amino acids in cell culture), combined with high-throughput 2-D HPLC/mass spectrometry, to determine relative quantitative differences in host proteins at 6 and 24 hours after infecting HEK293 cells with reovirus serotype 1 Lang (T1L). 3,076 host proteins were detected at 6 hpi, of which 132 and 68 proteins were significantly up or down regulated, respectively. 2,992 cellular proteins, of which 104 and 49 were up or down regulated, respectively, were identified at 24 hpi. IPA and DAVID analyses indicated proteins involved in cell death, cell growth factors, oxygen transport, cell structure organization and inflammatory defense response to virus were up-regulated, whereas proteins involved in apoptosis, isomerase activity, and metabolism were down-regulated. These proteins and pathways may be suitable targets for intervention to either attenuate virus infection or enhance oncolytic potential.