Clinical characteristics of idiosyncratic drug reactions (IDRs) suggest that they are immune mediated. Penicillamine-induced autoimmunity in Brown Norway rats was used as a tool for mechanistic studies of this type of IDR. It has been shown that T helper 17 (Th17) cells play a central role in many types of autoimmune diseases. This study was designed to test whether Th17 cells are involved in the pathogenesis of penicillamine-induced autoimmunity. In sick animals, interleukin (IL) 6 and transforming growth factor-β1, known to be driving forces of Th17 differentiation, were consistently increased following penicillamine treatment. IL-17 and IL-22, characteristic cytokines produced by Th17 cells, were increased in sick animals. Furthermore, the percentage of IL-17-producing CD4 T cells was significantly increased, but only in sick animals. Retinoic acid, which has been reported to inhibit Th17 cell development, made the autoimmunity worse, increased IL-6 production, and did not decrease the number of Th17 cells. An infiltration of CD8 cytotoxic T cells in the liver suggests that they may be the key player in causing liver toxicity induced by D-penicillamine. Drug-induced liver injury (DILI) is one of the major causes of morbidity, mortality, and drug candidate failure. Recently, it has been suggested that Th17 cells may play an active role in inflammatory human liver diseases. In a study of patients being treated with isoniazid, some patients developed mild liver injury. The percentage of Th17 cells in the blood of these patients significantly increased when the ALT increased, and this suggests that they play a role in the mechanism of this liver injury. Furthermore, IL-10-producing T cells also increased and this may have prevented the development of severe liver injury. In another study, two hours after treatment of mice with acetaminophen there was a significant increase in Th17 cells in the liver. This rapid response suggests that Th17 cells can be part of the innate immune response to liver injury. Our data provided evidence that Th17 cells are involved in both “toxic” and idiosyncratic liver toxicity. This pathway could be a new target for the therapeutic interventions to treat DILI.

Clinical characteristics of idiosyncratic drug reactions (IDRs) suggest that they are immune mediated. Penicillamine-induced autoimmunity in Brown Norway rats was used as a tool for mechanistic studies of this type of IDR. It has been shown that T helper 17 (Th17) cells play a central role in many types of autoimmune diseases. This study was designed to test whether Th17 cells are involved in the pathogenesis of penicillamine-induced autoimmunity. In sick animals, interleukin (IL) 6 and transforming growth factor-β1, known to be driving forces of Th17 differentiation, were consistently increased following penicillamine treatment. IL-17 and IL-22, characteristic cytokines produced by Th17 cells, were increased in sick animals. Furthermore, the percentage of IL-17-producing CD4 T cells was significantly increased, but only in sick animals. Retinoic acid, which has been reported to inhibit Th17 cell development, made the autoimmunity worse, increased IL-6 production, and did not decrease the number of Th17 cells. An infiltration of CD8 cytotoxic T cells in the liver suggests that they may be the key player in causing liver toxicity induced by D-penicillamine. Drug-induced liver injury (DILI) is one of the major causes of morbidity, mortality, and drug candidate failure. Recently, it has been suggested that Th17 cells may play an active role in inflammatory human liver diseases. In a study of patients being treated with isoniazid, some patients developed mild liver injury. The percentage of Th17 cells in the blood of these patients significantly increased when the ALT increased, and this suggests that they play a role in the mechanism of this liver injury. Furthermore, IL-10-producing T cells also increased and this may have prevented the development of severe liver injury. In another study, two hours after treatment of mice with acetaminophen there was a significant increase in Th17 cells in the liver. This rapid response suggests that Th17 cells can be part of the innate immune response to liver injury. Our data provided evidence that Th17 cells are involved in both “toxic” and idiosyncratic liver toxicity. This pathway could be a new target for the therapeutic interventions to treat DILI.

La nature du lien qui unit les deux aspects du système immunitaire, que sont la défense de l’hôte contre les agressions extérieures et la genèse des maladies auto- immunes, n’a pas été élucidée. L’axe IL-23/Th17 joue un rôle dans les deux versants, ce qui le place en bonne position pour être un potentiel chaînon manquant. Objectif : connaître l’implication de cet axe dans un modèle infectieux, Mycoplasma hominis, et un modèle de maladie auto-immune, la sclérodermie systémique (ScS), dans lesquels il n’a pas encore été étudié. Les lipoprotéines membranaires de M. hominis sont capables d’induire une maturation des cellules dendritiques humaines. Elle s’accompagne d’une sécrétion d’IL-23 variable selon l’origine clinique des isolats, via TLR2, et d’une polarisation vers la voie Th17. Nous avons observé une augmentation de la fréquence des cellules Th17 et Th22 dans le sang périphérique des patients ScS, potentialisée par l’iloprost, via entre autres la production monocytaire d’IL-23. Dans la peau des patients ScS, il existe une augmentation des cellules produisant l’IL-17 inversement corrélé au score de fibrose cutanée. In vitro, l’IL-17 est capable d’inhiber partiellement l’expression d’α-SMA induite par le TGF-ß et d’induire la sécrétion de MMP1 par des fibroblastes dermiques humains. L’axe IL-23/IL-17 et les cellules Th17 jouent un rôle dans la défense contre M. hominis et dans la physiopathologie de la ScS. / Relationship between both aspects of the immune system, ie host defense against external aggression and genesis of autoimmune diseases, has not been elucidated. IL-23/Th17 axis plays a role in both sides, which puts him in a good position to be a potential missing link. Objective: To understand the implication of this axis in a model of infection, Mycoplasma hominis, and a model of autoimmune disease, systemic sclerosis (SSc), in which it has not yet been studied.
The membrane lipoproteins of M. hominis are capable of inducing human dendritic cell maturation. It occurs along with an IL-23 secretion changing with the clinical origin of isolates, via TLR2, and a T cell polarization towards Th17. Then we observed an increase in the Th17 and Th22 cell frequency in peripheral blood of SSc patients, further enhanced by iloprost via monocyte production of IL-23 among others. In the skin of SSc patients, we showed an increase in IL-17-producing cells with an inverse correlation to the skin fibrosis score. In vitro, IL-17 is able to partially inhibit the expression of α-SMA induced by TGF-ß and to induce the secretion of MMP1 in human dermal fibroblasts. The IL-23/IL-17 axis and Th17 cells play a role in defense against M. hominis and in the pathogenesis of SSc.

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.