mPGES-1-Mediated Production of PGE2 and EP4 Receptor Sensing Regulate T Cell Colonic Inflammation

PGE2 is a lipid mediator of the initiation and resolution phases of inflammation, as well as a regulator of immune system responses to inflammatory events. PGE2 is produced and sensed by T cells, and autocrine or paracrine PGE2 can affect T cell phenotype and function. In this study, we use a T cell-dependent model of colitis to evaluate the role of PGE2 on pathological outcome and T-cell phenotypes. CD4+ T effector cells either deficient in mPGES-1 or the PGE2 receptor EP4 are less colitogenic. Absence of T cell autocrine mPGES1-dependent PGE2 reduces colitogenicity in association with an increase in CD4+RORγt+ cells in the lamina propria. In contrast, recipient mice deficient in mPGES-1 exhibit more severe colitis that corresponds with a reduced capacity to generate FoxP3+ T cells, especially in mesenteric lymph nodes. Thus, our research defines how mPGES-1-driven production of PGE2 by different cell types in distinct intestinal locations impacts T cell function during colitis. We conclude that PGE2 has profound effects on T cell phenotype that are dependent on the microenvironment

Peritoneal Cavity Regulatory B Cells (B10 Cells) Modulate IFN- +CD4+ T Cell Numbers during Colitis Development in Mice

The spleen regulatory B cell subset with the functional capacity to express IL-10 (B10 cells) modulates both immune responses and autoimmune disease severity. However, the peritoneal cavity also contains relatively high frequencies of functionally-defined IL-10-competent B10 cells. In this study, peritoneal cavity B10 cells shared similar cell surface phenotypes with their spleen counterparts. However, peritoneal cavity B10 cells were 10-fold more frequent among B cells than occurred within the spleen, intestinal track or mesenteric lymph nodes and were present at higher proportions among the phenotypically-defined peritoneal B1a>B1b>B2 cell subpopulations. The development or localization of B10 cells within the peritoneal cavity was not dependent on the presence of commensal microbiota, T cells, IL-10 or B10 cell IL-10 production, or differences between their fetal liver or adult bone marrow progenitor cell origins. The BCR repertoire of peritoneal cavity B10 cells was diverse, as occurs in the spleen, and predominantly included germline-encoded VH and VL regions commonly found in either the conventional or B1 B cell compartments. Thereby, the capacity to produce IL-10 appears to be an intrinsic functional property acquired by clonally diverse B cells. Importantly, IL-10 production by peritoneal cavity B cells significantly reduced disease severity in spontaneous and induced models of colitis by regulating neutrophil infiltration, colitogenic CD4+ T cell activation and pro-inflammatory cytokine production during colitis onset. Thus, the numerically small B10 cell subset within the peritoneal cavity has regulatory function and is important for maintaining homeostasis within gastrointestinal tissues and the immune system

Nichtklassische pre-T-Zell Rezeptor vermittelte Signalwege waehrend der fruehen T-Zell Entwicklung

T cells undergoing maturation in the thymus need to receive a pre-TCR-delivered signal to undergo ƒÒ-selection and develop further properly. The pre-TCR is composed of a constitutively expressed pre-Talpha (pTa) chain and a de novo expressed TCRbeta chain. A productive recombination of the TCRbeta chain is mandatory to overcome the first checkpoint that controls T cells survival and transition from double-negative (DN) 3 to DN4 stages. However, in a very low but consistent extent, DN3 thymocytes lacking a canonical pre-TCR can differentiate to the double positive (DP) and CD4+ or CD8+ single positive (SP) stages. Nevertheless, pTa- and pTaxTCRa-deficient mice denote a very similar phenotype in thymus, displaying few DP thymocytes, while only TCRbeta-/- x TCRdelta-/- mice depict a phenotype comparable to RAG-/- mice. Control of apoptosis during T cell maturation demands strict regulation of survival and cell-cycle proteins. The ubiquitin-proteasome machinery is in great extent responsible for protein degradation in mammalian cells. Inhibition of the proteasomal enzymatic activity can lead to dramatic changes in protein homeostasis and lead to cell death. Important mechanisms of proteasome inhibition-mediated cell death are the impairment of NF-kappaB activation and induction of the terminal unfolded protein response (UPR). However, little is known about proteasomal activity in early developing thymocytes. The aims of this thesis were to elucidate apoptotic mechanisms shared by unconventional pre-TCR signals and altered proteasomal activity during early T cell development. In the first part of this thesis it could be demonstrated that in the absence of pTa and TCRa chains, DN thymocytes have the potential to develop into DP and SP thymocytes. This survival was accompanied by increased Ca2+ influx and NF-kappaB activity. It could also be described that unconventional pre-TCR signaling affects alphabeta vs. gammadelta T cell proportions. Finally, the data suggest that there may be additional factors in vivo that explain the overcome of beta-selection in the absence of pTa and TCRa chains which are not dependent on Notch or IL-7 signaling. The effect of proteasomal inhibition during T cell development was investigated in the second part of this work. In vivo treatment with bortezomib resulted in a dramatic decrease of thymocytes. The decreased numbers of developing thymocytes was detected 24h after a single injection of bortezomib and was accompanied by strongly increased caspase 3/7 activities. Thymocytes were almost completely depleted at day three, with complete recovery within two weeks. This impairment of lymphopoiesis did correlate with induction of Hsp70 and CHOP, indicating activation of ER-stress and triggering of a pro-apoptotic terminal UPR. However, we could not detect a clear direct implication of pre-TCR or TCR signaling during induction of apoptosis by proteasome inhibition. In summary, this thesis evidences that thymocytes lacking a classical pre-TCR can survive and differentiate to DP and SP stages. Besides, signaling through a pTa-less TCR is enough to achieve Ca2+ influx and NF-kappaB activation, which may explain the survival till DP and SP stages. In addition, it was demonstrated that the mechanisms underlying cell death of thymocytes in conditions of proteasome inhibition are in great extent independent of NF-kappaB activation and pre-TCR or TCR signaling, rather dependent on triggering of the pro-apoptotic terminal UPRT-Zellen, die sich im Thymus entwickeln, benoetigen ein prae-T Zell Rezeptor-vermitteltes (pre-TZR) Signal zur beta-Selektion und Weiterdifferenzierung. Der prae-TZR besteht aus einer konstitutiv exprimierten prae-Talpha (pTa) Kette gepaart mit einer de novo exprimierten TZRbeta Kette. Eine produktive Rekombination der TZRbeta-Kette ist notwendig fuer das Ueberleben und die Entwicklung von Thymozyten vom doppelt negative (DN) 3 zum DN4 Stadium. Trotz Fehlen des klassischen prae-TZR Signals koennen einige DN3 bis zum CD4+/CD8+ (DP) und CD4+ oder CD8+ single positive (SP) Stadium differenzieren. Allerdings weisen pTa-defiziente und pTa-/TZRa-doppeltdefiziente Maeuse einen sehr aehnlich Phaenotyp auf, d.h. einige CD4+CD8+-Thymozyten sind nachweisbar, waehrend TZRbeta-/- x TZRdelta-/--Maeuse, aehnlich wie RAG-/- Mause, keine CD4+CD8+-Zellen mehr besitzen. Der Apoptoseprozess benoetigt waehrend der T-Zell-Entwicklung eine strenge Regulation von Proteinen, die fuer das Ueberleben und die Zellzyklusregulation wichtig sind. Das Ubiquitin-Proteasom-System ist fuer die Degradierung von Proteinen in Saeugetierzellen verantwortlich. Die Hemmung der enzymatischen Aktivitaet des Proteasoms kann zu drastischen Veraenderungen in der Proteinhomeostase und somit zum Zelltod fuehren. Verantwortlich fuer den Zelltod als Folge der Proteasominhibition werden in erster Linie eine verminderte NF-kappaB-Aktivitaet und Induktion der Unfolded Protein Response (UPR) verantwortlich gemacht. Es ist jedoch sehr wenig bekannt ueber die Rolle der Proteasom-Aktivierung waehrend der T-Zell Entwicklung. Ziel dieser Arbeit war es, gemeinsame Mechanismen des Zelltodes durch unkonventionelle prae-TZR-Signale und veraenderte Proteasom-Aktivitaet in frueheren Stadien der T-Zell Entwicklung aufzuklaeren. Im ersten Teil dieses Arbeit wurde gezeigt, dass DN Thymozyten von pTa-/- und pTa-/- x TZRa-/--Maeusen in der Lage sind, sich ohne ein klassisches prae-TZR-vermitteltes Signal in DP und SP Thymozyten zu entwickeln. In diesen Ko-Thymozyten konnte sowohl Ca2+-Einstrom als auch Aktivierung des Transkriptionsfaktors NF-kappaB nachgewiesen werden. Ausserdem zeigte sich, dass ein unkonventionelles prae-TZR-Signal das Verhaeltnis von alphabeta- zu gammadelta-T-Zellen beeinflusst. Ferner fanden sich Hinweise, dass in vivo neben IL-7 und Notch weitere Faktoren bei der beta-Selektion eine Rolle spielen. Im zweiten Teil der Arbeit wurde der Effekt von Proteasom-Inhibitoren auf die Thymozytenentwicklung untersucht. Behandlung mit Bortezomib fuehrte in Wt-Maeusen zu einer drastischen Abnahme der Thymozyten nach 24 Std. Zu diesem Zeitpunkt konnte ausserdem Caspase 3/7-Aktivierung nachgewiesen werden. Drei Tage nach Behandlung waren die Thymozyten extrem reduziert, nach zwei Wochen hatte sich die Zellzahl jedoch vollstaendig regeneriert. Die Abnahme der Thymozyten korrelierte mit der Induktion von Komponenten der ER-Stress-Antwort wie Hsp70 und CHOP, wobei die Expression von CHOP auf die Aktivierung der pro-apopototischen/terminalen UPR hinweist. Somit wurde gezeigt, dass Thymozyten ohne pTa- und TZRa-Expression sich zu alphabeta-aehnlichen DP und SP Thymozyten weiterentwickeln koennen. Ausserdem war das unkonventionelle prae-TZR-Signal ausreichend fuer die NF-kappaB-Aktivierung und den Ca2+-Einstrom und konnte somit die weitere Thymozytendifferenzierung vermitteln. Weiterhin wurde gezeigt, dass der durch Proteasom-Inhibition induzierte Zelltod mit Aktivierung der terminalen UPR einhergeht und auch in Reifungsstadien auftritt, die weitgehend unabhaengig von NF-kappaB und TZR-Signalen sind

Nonsteroidal Anti-inflammatory Drugs Alter the Microbiota and Exacerbate Clostridium difficile Colitis while Dysregulating the Inflammatory Response

Clostridium difficile infection (CDI) is a spore-forming anaerobic bacterium and leading cause of antibiotic-associated colitis. Epidemiological data suggest that use of nonsteroidal anti-inflammatory drugs (NSAIDs) increases the risk for CDI in humans, a potentially important observation given the widespread use of NSAIDs. Prior studies in rodent models of CDI found that NSAID exposure following infection increases the severity of CDI, but mechanisms to explain this are lacking. Here we present new data from a mouse model of antibiotic-associated CDI suggesting that brief NSAID exposure prior to CDI increases the severity of the infectious colitis. These data shed new light on potential mechanisms linking NSAID use to worsened CDI, including drug-induced disturbances to the gut microbiome and colonic epithelial integrity. Studies were limited to a single NSAID (indomethacin), so future studies are needed to assess the generalizability of our findings and to establish a direct link to the human condition.Clostridium difficile infection (CDI) is a major public health threat worldwide. The use of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with enhanced susceptibility to and severity of CDI; however, the mechanisms driving this phenomenon have not been elucidated. NSAIDs alter prostaglandin (PG) metabolism by inhibiting cyclooxygenase (COX) enzymes. Here, we found that treatment with the NSAID indomethacin prior to infection altered the microbiota and dramatically increased mortality and the intestinal pathology associated with CDI in mice. We demonstrated that in C. difficile-infected animals, indomethacin treatment led to PG deregulation, an altered proinflammatory transcriptional and protein profile, and perturbed epithelial cell junctions. These effects were paralleled by increased recruitment of intestinal neutrophils and CD4+ cells and also by a perturbation of the gut microbiota. Together, these data implicate NSAIDs in the disruption of protective COX-mediated PG production during CDI, resulting in altered epithelial integrity and associated immune responses