Sympathoadrenerge Immunomodulation durch Regulation des Rezeptors für Advanced Glycation End Products (RAGE)

RAGE ist ein Multiligandenrezeptor und spielt eine maßgebliche Rolle in unterschiedlichen pathophysiologischen Prozessen. Dieser wurde initial vor allem mit diabetischen Spätkomplikationen und inflammatorischen sowie profibrotischen Prozessen in Verbindung gebracht. Die lösliche Isoform des Rezeptors, das sogenannte soluble RAGE (sRAGE) entsteht entweder durch Abspaltung des Rezeptors an der Zellmembran oder als Splicing-Variante durch aktive Sekretion. Das sRAGE wirkt als sog. Decoy-Rezeptor (englisch für „Köder“), indem es Liganden wie z.B. AGEs abfängt und somit die Signaltransduktion des membranständigen RAGE verhindert. Im Tiermodell hat die Zufuhr von sRAGE diabetische Spätkomplikationen auf makro- und mikrovaskulärer Ebene supprimieren können. sRAGE wird deshalb als protektiver Faktor angesehen. Eine Hochregulation der RAGE-Expression bzw. eine Herunterregulation des sRAGE wurden bereits für mehrere akute und chronische pathophysiologische Bedingungen beschrieben, die mit einer Aktivierung des sympathischen Nervensystems assoziiert sind wie zum Beispiel auch beim metabolischen Syndrom. Das metabolische Syndrom ist mit einem erhöhtem Sympathikotonus und einer subklinischen Inflammation assoziiert. Ein direkter Zusammenhang zwischen sympathoadrenergem System und dem RAGE/sRAGE-System wurde bisher noch nicht charakterisiert. Wir haben uns daher gefragt, ob durch Modulation des sympathischen Nervensystems auch das RAGE/sRAGE-System beeinflusst werden kann und führten hierfür in vivo und in vitro Experimente durch. Als in vivo Modell des metabolischen Syndroms wählten wir das Modell der spontan hypertensiven obesen Ratte (SHRob). Die obesen Ratten hatten einen Leptin-Rezeptor-Defekt, aufgrund dessen sie einen ausgeprägten Hypertonus, eine Insulinresistenz und eine Hyperinsulinämie entwickelten. Weiterhin wiesen sie eine moderate linksventrikuläre diastolische Dysfunktion und eine ausgeprägte Nephropathie auf. Sowohl afferente als auch efferente renale sympathische Nervenfasern, die netzartig um die Nierengefäße verlaufen und bis in die Adventitia reichen, sind an der Pathogenese und Progression der Hypertonie beteiligt. Aus tierexperimentellen und klinischen Untersuchungen ist bekannt, dass die Aktivität des gesamten sympathischen Nervensystems maßgeblich von diesen afferenten renalen Nervenfasern bestimmt wird. Eine Überaktivität des sympathischen Nervensystems führt neben der Blutdrucksteigerung unter anderem zu oben genannten Endorganschäden und Komorbiditäten. Wir haben uns die Methode der renalen Denervation, bei der die sympathischen Afferenzen und Efferenzen in der Adventitia unterdrückt werden, zunutze gemacht, um einen systemisch erniedrigten Katecholamin-Spiegel zu erzielen. Dabei haben wir vier Gruppen miteinander verglichen: normotensive, normalgewichtige Kontrollratten (Kontrolle), spontan hypertensive, normalgewichtige Ratten (SHR), sham-denervierte spontan hypertensive obese Ratten (SHRob) und spontan hypertensive obese Ratten, die renal denerviert wurden (SHRobRDN). Wir haben die RAGE- und sRAGE-Expression immunochemisch mittels Western Blot aus Gewebehomogenaten und die sRAGE-Expression im Serum dieses Rattenmodells untersucht. Auch hierbei konnten wir die oben beschriebene pro-inflammatorische Umkehr des RAGE/sRAGE-Gleichgewichtes in Herz- und Nierengewebe der obesen Ratten nachweisen. Auch die Expression der RAGE-Liganden CML und HMGB1 zeigte sich im Sinne einer pro-inflammatorischen Dysbalance in den obesen Ratten erhöht und konnte wie das RAGE/sRAGE-Verhältnis durch renale Denervation korrigiert werden. Ebenso führte die Reduktion des Katecholaminspiegels nach renaler Denervation zu einer Verbesserung der kardio-renalen Fibrose sowie kardiofunktioneller und renaler Funktionsparameter. Western Blot-Analysen aus Zellmembranpräparationen und Zellkulturüberständen weisen darauf hin, dass es sich um blutdruckunabhängige Effekte handeln könnte. Adulte kardiale Rattenfibroblasten, renale Fibroblasten, humane embryonale Nierenzellen, frisch isolierte Rattensplenozyten sowie humane periphere mononukleäre Zellen wurden zeitabhängig mit dem unselektiven beta-Adrenorezeptoragonisten Isoproterenol stimuliert. Dies führte zu einer stetigen Abnahme der sRAGE-Sekretion ins Zellkulturmedium und zu einer Zunahme der membranständigen RAGE-Expression. Des Weiteren zeigte sich durch die selektive Blockade des β1- bzw. β2-Adrenorezeptors, dass die RAGE-Expression über den β1-Adrenorezeptor und die sRAGE-Ausschüttung über den β2-Adrenorezeptor reguliert wird. Zusammenfassend kann man sagen, dass die sympathoadrenerge Aktivierung in vivo und in vitro zu einer RAGE-Überexpression und einer sRAGE-Reduktion führt. Dieses proinflammatorische Ungleichgewicht kann durch die Modulation des Sympathikus vermindert werden. Damit könnte das Fortschreiten von Endorganschäden wie Herz- und Niereninsuffizienz beim metabolischen Syndrom verlangsamt werden, was die Grundlage für prospektive klinische Untersuchungen legt.RAGE is a multi-ligand receptor and is involved in numerous pathophysiological processes. Initially it was described to be involved in diabetic late complications and pro-inflammatory and pro-fibrotic processes. The soluble isoform is either cleaved from the membrane bound receptor or is a splicing variant and secreted from the cytosol into the extracellular space/blood circulation. It acts as a decoy receptor by binding circulating RAGE ligands and thereby inhibiting the signal transduction of the membrane bound receptor. In animal models sRAGE administration prevented micro- and macrovascular diabetic late complications. Therefore, sRAGE is considered to be protective. RAGE-upregulation and sRAGE-downregulation was shown for acute and chronic pathophysiological processes with increased sympathetic activity such as the metabolic syndrome which is associated with increased sympathetic activity and subclinical inflammation. A direct relation between the sympathetic nervous system and the RAGE/sRAGE-system has not been characterized yet. The aim of this study was to figure out if the modulation of the sympathetic nervous system influences the RAGE/sRAGE-system in vivo and in vitro. The in vivo model for the metabolic syndrome was the spontaneously hypertensive obese rat. These animals had a leptin-receptor defect with insulin resistance and hyperinsulinemia. Furthermore, the rats showed moderate diastolic dysfunction and severe nephropathy. Afferent and efferent sympathetic nerves are located in the adventitia of renal arteries and are considered to be involved in the development and progression of arterial hypertension. Studies involving animal experiments and clinical studies showed a significant influence of the afferent renal nerves on the activity of the sympathetic nervous system. The overactivity of the sympathetic nervous system is associated with increased blood pressure, end-organ damage and comorbidities. For systemic reduction of catecholamine levels a surgical and chemical renal arterial denervation was performed in obese rats. We compared four groups: normotensive lean rats (Control), spontaneously hypertensive lean rats (SHR), spontaneously hypertensive obese rats with sham procedure (SHRob) and spontaneously hypertensive obese rats with renal denervation (SHRobRDN). RAGE- and sRAGE-expression in heart and renal tissue were assessed by western blot. We noted in all tissues a pro-fibrotic RAGE/sRAGE dysbalance in obese rats, while this dysbalance was corrected by renal denervation. We noted similar results regarding the RAGE ligands CML and HMGB1. We conclude that the modulation of the sympathetic nervous system led to a delayed progress of hypertension, renal and myocardial damage as shown by reduced cardio-renal fibrosis and improved cardio-functional and renal parameters. Western blot analysis of cell culture experiments indicate that the observed benefits could be independent of blood pressure reduction. Adult cardiac fibroblasts, renal fibroblasts, human embryonic kidney cells, fresh isolated rat splenocytes and human peripheral blood mononuclear cells were repeatedly stimulated with the β-adrenoreceptor agonist isoproterenol. We noted a reduction of sRAGE shedding into the cell culture medium, while membrane bound RAGE was upregulated. In conclusion, chronic sympathetic overactivity causes in vivo and in vitro RAGE upregulation and sRAGE downregulation. This pro-inflammatory dysbalance could be ameliorated by modulation of sympathetic nervous system via renal denervation. This could delay the progression of end organ damage such as renal and myocardial fibrosis in metabolic syndrome

Blockade of αEβ7 integrin suppresses accumulation of CD8+ and Th9 lymphocytes from patients with IBD in the inflamed gut in vivo

Objective: Therapeutically targeting lymphocyte adhesion is of increasing relevance in IBD. Yet, central aspects of the action of anti-adhesion compounds are incompletely understood. We investigated the role of αEβ7 and α4β7 integrins and their blockade by vedolizumab and etrolizumab for trafficking of IBD T lymphocytes in an in vivo model of homing to and retention in the inflamed gut. Design: We explored integrin expression in IBD patients by flow cytometry and immunohistochemistry, while regulation of integrins was studied in T cell cultures. The functional relevance of integrins was assessed by adhesion assays and a recently established humanized mouse model in DSS-treated immunodeficient mice. Results: High expression of αEβ7 was noted on CD8+ and CD4+ Th9 cells, while α4β7 was expressed on CD8+, Th2 and Th17 cells. TCR stimulation and TGF-β were key inducers of αEβ7 on human T cells, while butyric acid suppressed αEβ7. In comparison to α4β7 blockade via vedolizumab, blockade of β7 via etrolizumab surrogate antibody superiorly reduced colonic numbers of CD8+ and Th9 cells in vivo after 3 hours, while no difference was noted after 0.5 hours. AEβ7 expression was higher on CD8+ T cells from IBD patients under vedolizumab therapy. Conclusion: AEβ7 is of key relevance for gut trafficking of IBD CD8+ T cells and CD4+ Th9 cells in vivo and mainly retention might account for this effect. These findings indicate that blockade of αEβ7 in addition to α4β7 may be particularly effective in intestinal disorders with expansion of CD8+ and Th9 cells such as IBD

Immunology of IL-12

As its first identified member, Interleukin-12 (IL-12) named a whole family of cytokines. In response to pathogens, the heterodimeric protein, consisting of the two subunits p35 and p40, is secreted by phagocytic cells. Binding of IL-12 to the IL-12 receptor (IL-12R) on T and natural killer (NK) cells leads to signaling via signal transducer and activator of transcription 4 (STAT4) and subsequent interferon gamma (IFN-γ) production and secretion. Signaling downstream of IFN-γ includes activation of T-box transcription factor TBX21 (Tbet) and induces pro-inflammatory functions of T helper 1 (TH1) cells, thereby linking innate and adaptive immune responses. Initial views on the role of IL-12 and clinical efforts to translate them into therapeutic approaches had to be re-interpreted following the discovery of other members of the IL-12 family, such as IL-23, sharing a subunit with IL-12. However, the importance of IL-12 with regard to immune processes in the context of infection and (auto-) inflammation is still beyond doubt. In this review, we will provide an update on functional activities of IL-12 and their implications for disease. We will begin with a summary on structure and function of the cytokine itself as well as its receptor and outline the signal transduction and the transcriptional regulation of IL-12 secretion. In the second part of the review, we will depict the involvement of IL-12 in immune-mediated diseases and relevant experimental disease models, while also providing an outlook on potential translational approaches

The α4β1 homing pathway is essential for ileal homing of Crohn's disease effector T cells in vivo

The precise mechanisms controlling homing of T effector (Teff) cells to the inflamed gut in Crohn’s disease (CD) are still unclear and clinical outcome data from inflammatory bowel disease (IBD) patients treated with the anti-α4β7 integrin antibody vedolizumab suggest differences between ulcerative colitis (UC) and CD. Methods: Expression of homing molecules was studied with flow cytometry and immunohistochemistry. Their functional role was investigated in in vitro adhesion assays and in a humanized mouse model of T cell homing to the inflamed gut in vivo. Results: Despite in vitro blockade of CD Teff adhesion to MAdCAM-1 and in contrast to previous oberservations in UC, anti-α4β7 treatment did not result in reduced Teff cell homing to the gut in vivo. However, the integrin α4β1 was expressed in higher levels on Teffs from CD patients compared with controls, while its expression in the peripheral blood declined and its expression in the intestine increased during the course of clinical vedolizumab treatment. Consistently, adhesion of CD Teffs to VCAM-1 was blocked by inhibition of α4 and α4β1 in vitro. Moreover, in vivo homing of CD Teffs to the inflamed ileum was reduced by inhibition of α4 and α4β1 integrins, but not α4β7 integrins. Conclusions: Our findings suggest that Teff cell homing to the ileum via the axis α4β1 – VCAM-1 is an essential and non-redundant pathway in CD in vivo possibly affecting efficacy of clinical treatment with anti-adhesion compounds

Nr4a1-dependent non-classical monocytes are important for macrophage-mediated wound healing in the large intestine

IntroductionMacrophages play an important role in intestinal wound healing. However, the trajectories from circulating monocytes to gut macrophages are incompletely understood.MethodsTaking advantage of mice depleted for non-classical monocytes due to deficiency for the transcription factor Nr4a1, we addressed the relevance of non-classical monocytes for large intestinal wound healing using flow cytometry, in vivo wound healing assays and immunofluorescence.ResultsWe show that wound healing in Nr4a1-deficient mice is substantially delayed and associated with reduced peri-lesional presence of macrophages with a wound healing phenotype.DiscussionOur data suggest that non-classical monocytes are biased towards wound healing macrophages. These insights might help to understand, how targeting monocyte recruitment to the intestine can be used to modulate intestinal macrophage functions

Case Report: IBD-like colitis following CAR T cell therapy for diffuse large B cell lymphoma

Chimeric antigen receptor (CAR) T cell therapy has become a new mainstay in the treatment of several hematologic malignancies, but the spectrum of associated complications is still incompletely defined. Here, we report the case of a 70-year-old female patient treated with tisagenlecleucel for diffuse large B cell lymphoma (DLBCL), who developed chronic diarrhea with characteristics of inflammatory bowel disease (IBD)-like colitis. CAR T cells were substantially enriched in the colon lamina propria and other diagnoses were ruled out. Thus, we conclude that IBD-like colitis in this patient was associated to CAR T cell therapy and needs to be considered as a rare potential complication

Differential effects of α4β7 and GPR15 on homing of effector and regulatory T cells from patients with UC to the inflamed gut in vivo

Objective: Gut homing of lymphocytes via adhesion molecules has recently emerged as new target for therapy in inflammatory bowel diseases. We aimed to analyze the in vivo homing of effector (Teff) and regulatory (Treg) T cells to the inflamed gut via α4β7 and GPR15. Design: We assessed the expression of homing receptors on T cells in peripheral blood and inflamed mucosa. We studied the migration pattern and homing of Teff and Treg cells to the inflamed gut using intravital confocal microscopy and FACS in a humanized mouse model in DSS-treated NSG (NOD.Cg-Prkdcscid-Il2rgtm1Wjl/SzJ) mice. Results: Expression of GPR15 and α4β7 was significantly increased on Treg rather than Teff cells in peripheral blood of patients with ulcerative colitis (UC) as compared to Crohn´s disease and controls. In vivo analysis in a humanized mouse model showed augmented gut homing of UC Treg cells as compared to controls. Moreover, suppression of UC (but not control) Teff and Treg cell homing was noted upon treatment with the α4β7 antibody vedolizumab. In contrast, siRNA blockade of GPR15 had only effects on homing of Teff cells but did not affect Treg homing in UC. Clinical vedolizumab treatment was associated with marked expansion of UC Treg cells in peripheral blood. Conclusion: α4β7 rather than GPR15 is crucial for increased colonic homing of UC Treg cells in vivo, while both receptors control UC Teff homing. Vedolizumab treatment impairs homing of UC Treg cells leading to their accumulation in peripheral blood with subsequent suppression of systemic effector T cell expansion

Serum levels of cytokines in water buffaloes experimentally infected with Fasciola gigantica

Fasciola gigantica infection in water buffaloes causes significant economic losses especially 27 in developing countries. Although modulation of the host immune response by cytokine 28 neutralization or vaccination is a promising approach to control infection with this parasite, our 29 understanding of cytokine's dynamic during F. gigantica infection is limited. To address this, 30 we quantified the levels of serum cytokines produced in water buffaloes following experimental 31 infection with F. gigantica. Five buffaloes were infected via oral gavage with 500 viable F. 32 gigantica metacercariae and blood samples were collected from buffaloes one week before 33 infection and for 13 consecutive weeks thereafter. The levels of 10 cytokines in serum samples 34 were simultaneously determined using ELISA. F. gigantica failed to elicit the production of 35 various pro-inflammatory cytokines, including interleukin-1β (IL-1β), IL-2, IL-6, IL-12, and 36 IFN-γ. On the other hand, evidence of a Th2 type response was detected, but only early in the 37 course of parasite colonization and included modest increase in the levels of IL-10 and IL-13. 38 The results also revealed suppression of the immune responses as a feature of chronic F. 39 gigantica infection in buffaloes. Taken together, F. gigantica seems to elicit a modest Th2 40 response at early stage of infection in order to downregulate harmful Th1- and Th17-type 41 inflammatory responses in experimentally infected buffaloes. The full extent of anti-F. 42 gigantica immune response and its relation to pathogenesis requires further study