Characterization of TIRC7+ regulatory T cells in autoimmune diseases, Psoriatic Arthritis as an example

Einleitung: Im Rahmen dieser Dissertation wurden erstmalig humane TIRC7+ T regulatorische Zellen (Tregs) bei Psoriasis Arthritis (PsA) Patienten isoliert, expandiert und funktionell im Vergleich zur gesunden Kontrollgruppe untersucht. Um die TIRC7+ Treg aus den polyklonal expandierten Treg-Zellkulturen zu isolieren, erfolgte eine Evaluierung von verschiedenen monoklonalen anti-TIRC7 Antikörpern (mAbs) bezüglich der Bindung und funktionellen Eigenschaften in Proliferations- und Apoptose Assays auf Lymphozyten. Methodik: TIRC7+ Tregs wurden nach Expansion mittels magnet-aktivierter Zellsortierung (MACS) isoliert und mit Hilfe der Multicolor-Durchflusszytometrie phänotypisch charakterisiert. Die funktionelle Analyse der TIRC7+ Tregs erfolgte nach allogener Stimulation mittels Proliferations-Assays sowie der Bestimmung der IL-10 Zytokinproduktion anhand der ELISA Methode. Die Expressionsanalysen des TIRC7 auf Lymphozyten erfolgte mit Hilfe der Multicolor- Durchflusszytometrie. Analysen zur Wirkung der anti-TIRC7 Antikörper wurden in Form von gemischten Lymphozyten Reaktionen (MLRs) und Apoptose Assays durchgeführt. Ergebnisse: Die Ko-Inkubation des anti-TIRC7 mAb mit alloantigen-stimulierten T-Zellen zeigte eine Inhibierung der Proliferation der T-Zellen, jedoch keine antiproliferative Wirkung auf den Tregs. Dieser Antikörper eignete sich daher, um die TIRC7+ Tregs zu isolieren und funktionell zu analysieren. Die Gesamt-Treg-Population bei PsA Patienten war im Vergleich zu gesunden Kontrollprobanden nicht in der Lage die Proliferation und Effektorfunktionen der CD4+CD25- T-Zellen zu unterdrücken. TIRC7+ Tregs hingegen zeigten sowohl bei PsA Patienten als auch bei gesunden Probanden eine gesteigerte Suppressorfähigkeit. Phänotypische und auch funktionelle Analysen der TIRC7+ Tregs zeigten eine Korrelation der erhöhten suppressiven Kapazität mit der Produktion des IL-10. Dies konnte sowohl anhand durchflusszytometrischer Messungen als auch durch die Zytokinmessungen des IL-10 bestätigt werden. Schlussfolgerung: Im Vergleich zu gesunden Kontrollprobanden war bei PsA Patienten die Anzahl zirkulierender Tregs des peripheren Blutes erniedrigt. Ebenso war eine verminderte Suppressorfähigkeit dieser zu beobachten. Im Gegensatz dazu zeigten TIRC7+ Tregs eine substantielle Inhibition der Proliferation der Lymphozyten. Basierend auf unseren Ergebnissen, könnten sowohl TIRC7+ Tregs als auch der Einsatz des anti-TIRC7 (mAb), mit seiner selektiven proliferationshemmenden Wirkung auf den CD4+CD25- T-Zellen eine Alternative zu den bisherigen Therapiemöglichkeiten bei PsA Patienten darstellen. Um eindeutigere Aussagen über die bisher gemachten Beobachtungen zu treffen und um diese zu unterstützen, sind jedoch weiterführende Untersuchungen erforderlich.Introduction: The focus of this manuscript was the isolation and phenotypical and functional characterization of TIRC7+ regulatory T cells (Tregs) in patients with “Psoriatic Arthritis“ (PsA) compared with those of “Healthy Volunteers“. To isolate TIRC7+ Tregs, TIRC7 specific antibodies were evaluated, for capability of binding to TIRC7 and functional effects in T cells and Tregs. Methodology: TIRC7+ Tregs were sorted after in vitro expansion via MACS (Magnetic Activated Cell Sorting) and phenotypically analyzed for Treg-specific markers and IL-10 using flow cytometry. Functional analysis of TIRC7+ Tregs were performed after allogeneic stimulation using proliferation assays and determining the IL-10 expression via ELISA. Mixed Lymphocyte Reactions (MLRs) were utilized for the functional analysis of the monoclonal anti-TIRC7 antibodies (mAbs). Flow cytometric analysis were performed to determine the expression pattern of TIRC7 on different cell subsets. Results: The co-incubation with an anti-TIRC7 mAb of alloantigen stimulated T cells resulted in decreased proliferation, but no inhibitory effect of the same mAb on Tregs could be observed. Thus, this antibody was suitable to be used to select TIRC7+ Tregs in polyclonal expanded Treg cultures. In comparison to “Healthy Volunteers“ Tregs obtained from patients with PsA were not able to suppress the proliferation and effector function of CD4+CD25- responder T cells. Whereas TIRC7+ Tregs, both of patients with PsA and “Healthy Volunteers“ displayed a higher suppressive capacity. Phenotypical as well as functional analysis of TIRC7+ Tregs showed that the higher suppressive potential of TIRC7+ Tregs was linked to their ability of producing IL-10. These results were confirmed through flow cytometric analysis as well as by measurements of IL-10 via ELISA. Conclusions: The number of Tregs in the peripheral blood of patients with PsA was declined compared to “Healthy Volunteers”. We could also observe a substantial difference in terms of their suppressive capacity, which was decreased compared to ,,Healthy Volunteers”. Whereas TIRC7+ Tregs achieved substantial inhibition of the proliferative response in lymphocytes. Based on our findings, TIRC7+ Tregs as well as the anti-TIRC7 mAb, with its selective antiproliferative effect on CD4+CD25- responder T cells might be promising alternatives for the therapy of PsA. In order to be able to confirm our initial findings further research needs to be done

In vitro induction of Entamoeba gingivalis cyst-like structures from trophozoites in response to antibiotic treatment

Background: Entamoeba gingivalis (E. gingivalis) is an anaerobic protozoan that is strongly associated with inflamed periodontal pockets. It is able to invade the mucosal epithelium of the human host, where it can feed on epithelial cells and elicit a severe innate immune response. Unlike other Entamoeba species, it is considered that E. gingivalis cannot form cysts, because it is a non-infectious protozoan. The lack of encystation capability would make it susceptible to periodontal treatment. However, it is not clear how the human host becomes infected with E. gingivalis trophozoites. We investigated the ability of E. gingivalis to encapsulate in response to an unfavorable environment in vitro. Methods: Different strains of E. gingivalis, isolated from inflamed periodontal pocket samples, were cultured for 8 days in the presence or absence of the antimicrobials amoxycillin and metronidazole. To reveal cyst formation, we investigated the morphology and ultrastructure of the amoeba by light, fluorescence, transmission and scanning electron microscopy. We also used the fluorescent dye calcofluor white M2R to demonstrate chitin present in the cyst wall. Results: We observed exocysts and an intra-cystic space separating the encapsulated trophozoite from the environment. Remarkably, cysts showed a smooth surface, polygonal edges and smaller size compared to free-living trophozoites. In addition, encapsulated trophozoites that detached from the cyst wall had a dense cytoplasma without phagocytic vesicles. The cyst walls consisted of chitin as in other Entamoba species. The encapsulated trophozoids were mononuclear after antibioticinduced encapsulation. Discussion: We conclude that E. gingivalis cyst formation has significant implications for dissemination and infection and may explain why established treatment approaches often fail to halt periodontal tissue destruction during periodontitis and peri-implantitis

In vitro induction of Entamoeba gingivalis cyst-like structures from trophozoites in response to antibiotic treatment

Background: Entamoeba gingivalis (E. gingivalis) is an anaerobic protozoan that is strongly associated with inflamed periodontal pockets. It is able to invade the mucosal epithelium of the human host, where it can feed on epithelial cells and elicit a severe innate immune response. Unlike other Entamoeba species, it is considered that E. gingivalis cannot form cysts, because it is a non-infectious protozoan. The lack of encystation capability would make it susceptible to periodontal treatment. However, it is not clear how the human host becomes infected with E. gingivalis trophozoites. We investigated the ability of E. gingivalis to encapsulate in response to an unfavorable environment in vitro. Methods: Different strains of E. gingivalis, isolated from inflamed periodontal pocket samples, were cultured for 8 days in the presence or absence of the antimicrobials amoxycillin and metronidazole. To reveal cyst formation, we investigated the morphology and ultrastructure of the amoeba by light, fluorescence, transmission and scanning electron microscopy. We also used the fluorescent dye calcofluor white M2R to demonstrate chitin present in the cyst wall. Results: We observed exocysts and an intra-cystic space separating the encapsulated trophozoite from the environment. Remarkably, cysts showed a smooth surface, polygonal edges and smaller size compared to free-living trophozoites. In addition, encapsulated trophozoites that detached from the cyst wall had a dense cytoplasma without phagocytic vesicles. The cyst walls consisted of chitin as in other Entamoba species. The encapsulated trophozoids were mononuclear after antibioticinduced encapsulation. Discussion: We conclude that E. gingivalis cyst formation has significant implications for dissemination and infection and may explain why established treatment approaches often fail to halt periodontal tissue destruction during periodontitis and peri-implantitis.Peer Reviewe