Helminth products modulate innate immune recognition of nucleic acids in systemic lupus erythematosus

Aim Current treatment of Systemic Lupus Erythematosus (SLE) is suboptimal and causes broad immunosuppression. Therapeutic use of helminths or helminth products has been suggested for autoimmune diseases such as SLE. In the present study, we evaluated possible immunomodulating effects of adult body fluid (ABF) from Ascaris suum on peripheral blood mononuclear cells (PBMCs) from SLE patients in an ex vivo setup. Methods PBMCs from SLE patients and healthy controls (HC) were isolated and stimulated ex vivo with ABF and Toll-like receptor agonists or activators of the stimulator of interferon genes (STING) or mitochondrial antiviral signaling protein (MAVS) pathways. After 24 h of incubation, the cytokine profile was analyzed using ELISA and Meso Scale Discovery techniques. Results ABF suppressed production of IL-6, TNF-α, CXCL10, and IL-10 by PBMCs from SLE patients and HCs following stimulation with specific agonists. ABF also reduced IFN-у production by stimulated PBMCs from HCs. Conclusions Our data show that ABF has an immunomodulatory effect on the production of key cytokines in the pathogenesis of SLE. These results suggest that ABF or ABF components hold potential as a novel treatment option for SLE

In vitro Studien zur molekularen Regulation der Expression von P-Selektin- Liganden in Th1 und Th2 Zellen, die wichtig für das Homing von CD4+ Zellen in entzündetes Gewebe sind

The proper localization of immune cells within the body is a prerequisite to effectively mount an immune response. P-selectin ligands (P-ligs) are oligosaccharide epitopes that support recruitment of T cells to the skin and inflamed tissue. P-ligs are synthesized by several glycosyltransferases, of which Core2- 1,6 glucosaminyltransferase-I (C2-GlcNAcT- I) and Fucosyltransferase-VII (FucT-VII) are crucial because T cells from mice deficient in the genes that encode them, gcnt1 and fut7, respectively, do not migrate to the site of immunization in classic delayed type hypersensitivity models. Initial and long-term molecular regulation of these genes and subsequently upregulation and maintenance of P-lig expression is not fully understood. Although P-lig has been observed on murine TH1 and TH2 cells in vivo [1] during inflammation, this has not been recapitulated in vitro for TH2 cells. In this thesis, the regulation of both gcnt1 and fut7 is characterized in TH1 and TH2 cells generated in vitro. In TH1 cells, we have shown that gcnt1 expression is induced and maintained by the transcription factors STAT4 and T-bet, respectively. Furthermore, using available ChIP- seq data, we observed that these transcription factors are implicated in the opening of the gcnt1 locus by promoting changes in histone methylation that affect its transcription. This is the first direct evidence of epigenetic regulation of P-lig that we have postulated in earlier studies [2]. This promotes long-term P-lig expression to give T cells a topographical migratory memory, which enables them to recirculate between blood and the tissue of first antigen encounter. For fut7, we have shown that initial activation in TH1 cells is induced by the transcription factors CREB and STAT5, the actions of which are inhibited by DNA methylation and the presence of RA, respectively. Unlike gcnt1, long-term expression of fut7 is therefore regulated by DNA methylation. In TH2 cells, we have shown that RA suppresses expression of both gcnt1 and fut7 through the nuclear receptor RAR. Minute amounts of RA are present in FCS-containing media, which prevents upregulation of P-lig in TH2 cells in vitro (in contrast to in vivo). Moreover, we have shown a reciprocal relationship between Il4 and Gcnt1 mRNA availability in mesenteric and peripheral lymph nodes (mLNs and pLNs) that might promote tight regulation of P-lig in mLNs. Finally, we have shown that the enhancer for gcnt1 in TH1 cells acts as a silencer in TH2 cells. This suggests that a complex interplay between epigenetic modifications, cytokine signaling, and microenvironmental factors control the molecular regulation/induction of gcnt1 and fut7, and subsequently P-lig on developing effector cells that are distinct in TH1 and TH2 in vitro generated cells.Die Lokalisierung von Immunzellen im Körper ist eine wichtige Voraussetzung für eine effektive Immunantwort. P-Selektin-Liganden (P-lig) sind Oligosaccharid-Epitope, die die Einwanderung von T-Zellen in die Haut und in andere entzündete Gewebe unterstützen. P-lig werden von verschiedenen Glycosyltransferasen synthetisiert. Vor allem die Core2- beta1,6 Glucosaminyltransferase-I (C2-GlcNAcT-I) und die Fucosyltransferase-VII (FucT- VII) sind essentiell. So hat sich im Modell der Hypersensitivität vom verzögerten Typ gezeigt, dass T-Zellen aus Mäusen, die diese Enzyme nicht exprimieren, nicht in der Lage sind, an den Ort der Entzündung zu migrieren. Es ist jedoch unklar, wie die Initiation der Expression beider Gene bzw deren Langzeitexpression und der daraus resultierenden Hochregulation der P-lig- Expression auf molekularer Ebene reguliert wird. Obwohl P-lig in vivo sowohl in TH1- und TH2-Zellen [1] exprimiert wird, konnte dies in vitro nur für TH1-Zellen gezeigt werden. In der vorliegenden Arbeit wurde die Regulation beider Gene, gcnt1 und fut7, sowohl für TH1 als auch für TH2-Zellen in vitro untersucht. In TH1-Zellen wird die gcnt1 Expression von STAT4 und T-bet induziert und aufrechterhalten. ChIP-seq Daten haben ergeben, dass diese Transkriptionsfaktoren in die öffnung des gcnt1 Locus involviert sind, indem sie änderungen der Histon-Methylierung hervorrufen und damit die Transkription beeinflussen. Das ist der erste direkte Beweis für epigenetische Regulation von P-lig, die wir in früheren Studien bisher nur postulieren konnten (78). Diese epigenetische Modifikation begünstigt Langzeit-P-lig-Expression, wodurch die T-Zellen ein topografisches Gedächtnis erhalten. Das ermöglicht ihnen, zwischen Blut und dem Gewebe zu rezirkulieren, in dem sie das erste Mal Antigen-Kontakt hatten. In TH1-Zellen wird die initiale Aktivierung des fut7-Gens durch die Transkriptionsfaktoren CREB und STAT5 induziert, was durch DNA-Methylierung und durch Anwesenheit von Retinolsäure (RA) inhibiert wird. Im Gegensatz zu gcnt1 wird daher die Langzeitexpression von fut7 durch DNA- Methylierung reguliert. In TH2 Zellen wurde gezeigt, dass RA die Expression beider Gene, gcnt1 und fut7, durch Bindung an den nukleären Rezeptor RARalpha unterdrückt. Es reichen bereits minimale Mengen an RA aus, um die P-lig Expression in TH2-Zellen zu verhindern. Das erklärt auch das Fehlen von P-lig auf TH2-Zellen in vitro, denn das FCS, was herkömmlichen Zellkulturmedien zugesetzt wird, enthält ebenfalls geringe Konzentrationen an RA. Außerdem haben wir in vivo eine reziproke Korrelation zwischen Il4 und Gcnt1 mRNA Verfügbarkeit in mesenterialen und peripheren Lymphknoten (mLN und pLN) nachgewiesen. Das könnte Ursache für die restriktive Regulation der P-lig Expression in mLN sein. Letztendlich haben wir gezeigt, dass der Enhancer für gcnt1 in TH1-Zellen als ein Silencer für TH2-Zellen fungiert. Das alles legt ein komplexes Zusammenspiel zwischen epigenetischen Modifikationen, Zytokinsignalen und Faktoren der Mikroumgebung nahe, die die molekulare Regulation/ Induktion von gcnt1 und fut7 kontrollieren. Dadurch wir die Expression von P-lig auf sich entwickelnden Effektorzellen gesteuert, die sich von in vitro generierten TH1- und TH2-Zellen unterscheiden

Imprinting of Skin/Inflammation Homing in CD4+ T Cells Is Controlled by DNA Methylation within the Fucosyltransferase 7 Gene

E- and P-selectin ligands (E- and P-ligs) guide effector memory T cells into skin and inflamed regions, mediate the inflammatory recruitment of leukocytes, and contribute to the localization of hematopoietic precursor cells. A better understanding of their molecular regulation is therefore of significant interest with regard to therapeutic approaches targeting these pathways. In this study, we examined the transcriptional regulation of fucosyltransferase 7 (FUT7), an enzyme crucial for generation of the glycosylated E- and P-ligs. We found that high expression of the coding gene fut7 in murine CD4(+) T cells correlates with DNA demethylation within a minimal promoter in skin/inflammation-seeking effector memory T cells. Retinoic acid, a known inducer of the gut-homing phenotype, abrogated the activation-induced demethylation of this region, which contains a cAMP responsive element. Methylation of the promoter or mutation of the cAMP responsive element abolished promoter activity and the binding of CREB, confirming the importance of this region and of its demethylation for fut7 transcription in T cells. Furthermore, studies on human CD4(+) effector memory T cells confirmed demethylation within FUT7 corresponding to high FUT7 expression. Monocytes showed an even more extensive demethylation of the FUT7 gene whereas hepatocytes, which lack selectin ligand expression, exhibited extensive methylation. In conclusion, we show that DNA demethylation within the fut7 gene controls selectin ligand expression in mice and humans, including the inducible topographic commitment of T cells for skin and inflamed sites

Evidence for Heterodimerization and Functional Interaction of the Angiotensin Type 2 Receptor and the Receptor MAS

The angiotensin type 2 receptor (AT2R) and the receptor MAS are receptors of the protective arm of the renin-angiotensin system. They mediate strikingly similar actions. Moreover, in various studies, AT2R antagonists blocked the effects of MAS agonists and vice versa. Such cross-inhibition may indicate heterodimerization of these receptors. Therefore, this study investigated the molecular and functional interplay between MAS and the AT2R. Molecular interactions were assessed by fluorescence resonance energy transfer and by cross correlation spectroscopy in human embryonic kidney-293 cells transfected with vectors encoding fluorophore-tagged MAS or AT2R. Functional interaction of AT2R and MAS was studied in astrocytes with CX3C chemokine receptor-1 messenger RNA expression as readout. Coexpression of fluorophore-tagged AT2R and MAS resulted in a fluorescence resonance energy transfer efficiency of 10.8 ± 0.8%, indicating that AT2R and MAS are capable to form heterodimers. Heterodimerization was verified by competition experiments using untagged AT2R and MAS. Specificity of dimerization of AT2R and MAS was supported by lack of dimerization with the transient receptor potential cation channel, subfamily C-member 6. Dimerization of the AT2R was abolished when it was mutated at cysteine residue 35. AT2R and MAS stimulation with the respective agonists, Compound 21 or angiotensin-(1-7), significantly induced CX3C chemokine receptor-1 messenger RNA expression. Effects of each agonist were blocked by an AT2R antagonist (PD123319) and also by a MAS antagonist (A-779). Knockout of a single of these receptors made astrocytes unresponsive for both agonists. Our results suggest that MAS and the AT2R form heterodimers and that-at least in astrocytes-both receptors functionally depend on each other