No description available.

Chronic inflammation is associated with the pathophysiology of obstetrical disorders (e.g. preterm birth [PTB]) and gynecological diseases (e.g. endometriosis); however, the exact mechanism(s) for these conditions are unknown. Numerous immunological conditions and disease states (e.g. inflammatory bowel disease, Crohn’s disease, systemic lupus erythematomus) also disrupt the microbiome homeostasis by inducing a number of changes in the microbial flora when compared to that of healthy individuals. Furthermore, the gastrointestinal (GI) microbiome is one of the principal regulators of circulating estrogens which are known to directly impact the female reproductive disorders endometriosis and PTB. Thus, an alteration of microbial species could indicate a shift in immune balance from homeostatic to pro-inflammatory, and an aberrant estrogen metabolism that precipitates the development of disease stages in endometriosis and/or PTB. The Braundmeier-Fleming lab has developed a systems biology model that investigates the interactions between the immune system, microbial dynamics (in the GI and reproductive system) and estrogen metabolism, in women, as a potential diagnostic tool for endometriosis and PTB. This dissertation, therefore, examined how inflammation triggered by female reproductive disorders (endometriosis or PTB) alter the systemic and localization immune responses, the microbial communities in the urogenital (UG), peritoneal and GI mucosal epithelium, as well as levels of excreted conjugated estrogen. The first specific hypothesis is that inflammation associated with endometriosis alters microbial dynamics and functions that are distinct from those of non-diseased patients. Preliminary data indicated that reproductive tract microbial communities from patients with endometriosis are unique when compared to non-disease patients. Therefore, the central aims of this study are to identify the immune and microbial profiles of patients diagnosed with endometriosis and determine if an alteration of these profiles impact estrogen signaling, thus driving disease pathogenesis. Additionally, I hypothesized that surgery or hormonal therapy will temporarily restore the microbiome and estrogen levels of patients with endometriosis. Differences in systemic (blood) regulatory T cell (Treg) and T-helper 17 (Th17) cell populations (tolerant and inflammatory, respectively) were measured by flow cytometry, and the immune mediators was measured by serum cytokine levels via 10-plex-ELISA kits. Immunohistochemistry was used to identify resident Th17/Treg immune cell distribution within the endometrium and ectopic endometriotic lesions, and RORγt+/FOXP3+ transcripts within these same tissues were analyzed by real-time-qPCR. We implemented high-throughput non genomic sequencing targeting bacterial-V4 16S rRNA and robust bioinformatics analyses to characterize microbial composition/diversity within the GI (fecal swab), vaginal (vaginal swab), and UG (urine) cavities. Alterations in estrogen metabolism, parent estrogens and metabolites, in urine were analyzed via LC-MS/MS. Patients with endometriosis exhibit 1) systemic and localized inflammation within ectopic and endometrial tissues, 2) altered GI/UG microbial dynamics, 3) aberrant levels of endogenous estrogen and estrogen metabolites, 4) dampened inflammation (caused by disease) due to hormonal therapy, 5) altered bacteria populations in the gut and vaginal canal of patients with endometriosis due to hormonal therapy treatment, and 6) increased post-surgical variability in microbial community dynamics. The second specific aims examined the hypothesis that induction of endometriosis in baboons (P. Anubis) results in chronic systemic and tissue specific inflammation through regulation of Th17 and Treg populations. Further, the induction of endometriosis altered GI/UG/peritoneal cavity microbial communities that are distinct from non-diseased animals. Utilizing a non-human primate animal model of induced endometriosis allowed us to characterize factors involved at the early onset of endometriosis and throughout the disease progression. We collected samples from 8 baboons at pre-inoculation (no evidence of disease) and at 3, 6, 9, and 15 months post-induction of the disease. We found that the induction of endometriosis decreased peripheral Tregs cells while Th17 cells increased at all post-induction collections with reduced ratio of total Tregs to Th17 cells indicating systemic inflammation. Microbial community diversities as well as abundances at each sample site (GI, UG [vagina, urine] tracts and peritoneal cavity) were also altered at post-induction. These results therefore suggest that induction of endometriosis in non-human primates caused an inflammatory shift. Disease induction also resulted in altered vaginal, urinary and fecal microbial profiles, which may drive inflammation through the production of inflammatory mediators. The last specific aims studied the hypothesis that patients who deliver preterm have a systemic and placental inflammatory phenotype and abnormal estrogen levels during pregnancy that are distinct from those of patients with term delivery. Biological samples were collected at 8-12 weeks, 20-24 weeks, 32-36 weeks, at delivery and 6 weeks postpartum. Subjects with PTB showed signs of systemic inflammation with an elevation in Th17:Treg ratio, greater Th17 and lower levels of natural Tregs during the 2nd trimester, and lower inducible Tregs during the 3rd trimester and at delivery. Placental tissues from subjects with PTB also had an inflammatory immune phenotype (higher Th17) within the decidua basalis and maternal-fetal interface. Immunological shifts from tolerant to inflammatory were observed in both patient groups, but these shifts occurred early in gestation for subjects with PTB and at a later gestational age for subjects delivering at term. Levels of conjugated parent estrogens and estrogen metabolites were reduced in subjects with PTB, indicative of an abnormal production of estrogen. These analyses gave us a better understanding of the inflammatory cascade with estrogen metabolism associated with pregnancy, and how these effects are correlated with premature labor. The data from this study suggest that the levels of endogenous estrogen and estrogen metabolites of estrogen metabolism were abnormal in PTB and endometriosis disease models of inflammation compared to their respective controls. In the human and non-human primate model of endometriosis studies, we observed that both patients and baboons with endometriosis had systemic and resident inflammatory phenotypes and an alteration in mucosal microbial community dynamics compared to their respective controls. All together, our long-term goal is to identify factors from the microbiome and/or the immune system that would allow us to have early non-invasive diagnostics for endometriosis or to predict which mothers are most at risk to encounter PTB. Furthermore, it would allow us to determine whether the mucosal microbiome may be a good indicator of immune stress, and if alternative therapies can alter microbial community dynamics—thereby eliminating immune stress associated with female reproductive diseases. These findings may have a substantial impact on the obstetrical care and management of patients with endometriosis and women at risk for PTB, as well as provide evidence to support the development of novel therapeutics to treat these diseases.

β-(1,3)-Glucan is present in mould cell walls and frequently detected in house dust mite (HDM) faeces. β-Glucan exposure is thought to be associated with pulmonary allergic inflammation in mouse and man, although the published data are inconsistent. Here, we show that highly purified β-glucan exacerbates HDM-induced eosinophilic, T helper 2 type airway responses by acting as an adjuvant, promoting activation, proliferation and polarisation of HDM-specific T cells (1-Derβ T cells). We therefore provide definitive evidence that β-glucan can influence allergic pulmonary inflammation.

Vitamin A and the T helper 2 cytokines IL-4 and IL-13 play important roles in the induction of mucin gene expression and mucus hypersecretion. However, the effects of these agents on enzymes responsible for mucin glycosylation have received little attention. Here, we report the upregulation of core 2 β1,6 N-acetylglucosaminyltransferase (C2GnT) activity both by all-trans retinoic acid (RA) and by IL-4 and IL-13 in the H292 airway epithelial cell line. Northern blotting analysis showed that the M isoform of C2GnT, which is expressed in mucus-secreting tissues and can form all mucin glycan β1,6-branched structures, including core 2, core 4, and blood group I antigen, was upregulated by both RA and IL-4/13. The L isoform, which forms only the core 2 structure, was moderately upregulated by IL-4/13 but not by RA. Enhancement of the M isoform of C2GnT by RA was abolished by an inhibitor, of RA receptor α, implicating RA receptor α in the effect of RA. Likewise, an inhibitor of the Janus kinase 3 pathway blocked the enhancing effects of IL-4/13 on the L and M isoforms of C2GnT, suggesting a role of this pathway in the upregulation of these two C2GnTs by these cytokines. Taken together, the results suggest that IL-4/13 T helper 2 cytokines and RA can alter the activity of enzymes that synthesize branching mucin carbohydrate structure in airway epithelial cells, potentially leading to altered mucin carbohydrate structure and properties.

B cells get help in the antibody response by presenting processed antigen to helper T cells. We asked whether the antigen presenting B cell must induce T helper functions before receiving help, or whether B cell activation is a direct consequence of T cell antigen recognition on the B cell surface. Although antigen-dependent increases in B cell c-myc expression occur as early as two hours after conjugation, the B cell response depends on induction of a contact-dependent helper function in the T cell, which is inhibitable by cyclosporin A. Induction but not delivery of contact help is blocked by anti-class II MHC antibody, indicating that the delivery of T cell help is not Ag dependent or MHC restricted. Also, contact with activated helper T cells induces a different pattern of immediate early gene expression from signals transduced through the B cell antigen receptor. Egr-1 is rapidly upregulated in response to mitogenic signals induced by receptor crosslinking on murine B lymphocytes, and its expression closely correlates with B cell proliferation in several models of B cell activation and tolerance. We compared egr-1 expression during B cell stimulation with Fab'2 and IgG anti-Ig, since it is known that Fab'2 anti-Ig is mitogenic while IgG is not, due to a dominant inhibitory effect of crosslinking the B cell FcγRII to membrane Ig. While mitogenic doses of Fab'2 anti-Ig induce large and rapid increases in egr-1 expression, intact anti-Ig results in only small increases in egr-1 mRNA, comparable to that seen with submitogenic concentrations of Fab'2 anti-Ig. However, when IL-4 is added as a comitogen to induce B cell proliferation with submitogenic concentrations of Fab'2 anti-Ig or IgG anti-Ig, no concomitant increases in egr-1 are observed. The regulation of egr-1 therefore, is similar to that of c-myc in this system, since neither correlates with IL-4 induced DNA synthesis.

Indiana University-Purdue University Indianapolis (IUPUI) / Human immunodeficiency virus (HIV) primarily infects T helper (Th) cells. Decrease in the number of Th cells is the hallmark of HIV infection. Latent reservoirs of human immunodeficiency virus (HIV) are the leading barrier towards eradication of HIV infection. T Follicular helper (Tfh) cells are a subset of Th cells that function to provide aid to B cells for their maturation, affinity selection and antibody class switch. Several studies have shown that Tfh cells are a major reservoir of latent as well as productive hiv infection. But in contrast to the fate of other Th cell subsets, the frequency of Tfh cells was shown to have increased during HIV infection which could not be attributed to their reduced susceptibility to HIV infection. The hypothesis was that Tfh cells possess a unique metabolic phenotype that protects them from HIV induced cell death. Transcriptome analysis of Th subsets from human donors and showed that Tfh cells rely less on glycolysis for their energetic requirements and instead have increased transcription of fatty acid synthesis genes. This finding was corroborated by seahorse extracellular flux assay. The results shoId that glycolysis was not essential for Tfh cell differentiation in-vitro. The observed increase in Tfh cell frequency could not be attributed to increased Tfh differentiation upon HIV infection since HIV infection inhibited the differentiation of both non-Tfh and Tfh cells. The results found that bypassing the glycolytic pathway by providing Tfh cells with Galactose in the medium protected ex-vivo infected primary tonsillar cells from HIV induced cell death. This protection could be partly explained by the induction of Baculovirus IAP repeat containing 5 (BIRC5) when the cells utilized Galactose instead of Glucose. The studies together show that Tfh cells have an oxidative metabolic phenotype which protects them from HIV induced cell death in part by induction of BIRC5 expression.

No description available.

No description available.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / The differentiation of naïve CD4+ T cells into subsets of T helper cells (Th) is an essential process that impacts host defense and the pathogenesis of immunemediated diseases. Signal transducers and activators of transcription (STAT) proteins, activated downstream of instructive cytokines, dictate and perpetuate the lineage decision of Th cells through both positive and negative effects. This is accomplished by regulating transcription factors, surface receptors and promoting epigenetic changes in gene expression through chromatin remodeling. Transforming growth factor-β1 (TGF-β1) can induce Foxp3 in developing Th cells and these Foxp3-expressing adaptive T regulatory cells (aTregs) are able to suppress inflammation in vitro and in vivo. To define the mechanism by which STAT proteins regulate Th cell pro- and anti-inflammatory phenotypes, we examined T cells deficient in Stat3, Stat4, and Stat6 as well as T cells expressing two STAT4 isoforms after being cultured in the presence or absence of TGF-β1 and cytokines known to be instructive in Th cell development. The negative effects of STAT proteins are demonstrated by our results indicating STAT3, STAT4 and STAT6 proteins activated downstream of the instructive cytokines IL- 6, IL-12 and IL-4, respectively, negatively regulate the development of TGF-β induced Foxp3 and aTreg development. STAT3, STAT4, and STAT6 utilize a vi Mark H. Kaplan, Ph.D., Chair common mechanism to inhibit aTreg generation by inhibiting STAT5, a positive regulator of Foxp3 expression, from binding to the Foxp3 gene. STAT proteins positively effecting inflammatory immunity are demonstrated by our analysis of STAT4 isoforms and their ability to regulate the production of proinflammatory cytokines downstream of IL-12. STAT4β, a STAT4 splice isoform that lacks a Cterminal domain, and STAT4α, a full-length isoform are both capable of mediating inflammatory cell development. However, STAT4β promotes greater inflammation in vivo than STAT4α independent of its ability to repress Foxp3. Instead, the inflammation correlates with STAT4 isoform-dependent expression of inflammatory cytokines. Thus, cytokine-stimulated STAT proteins orchestrate T helper cell pro- and anti-inflammatory cell phenotypes.