Publications by authors named "Sonja Reißig"

27 Publications

  • Page 1 of 1

Discovery of M5049: A Novel Selective Toll-Like Receptor 7/8 Inhibitor for Treatment of Autoimmunity.

J Pharmacol Exp Ther 2021 Mar 16;376(3):397-409. Epub 2020 Dec 16.

EMD Serono (a business of Merck KGaA, Darmstadt, Germany), Billerica, Massachusetts (J.V., A.T.B., M.P., A.P., A.D., T.J., E.T., N.T.M., S.F.Z., S.L.O., R.W., B.S.) and Merck KGaA, Darmstadt, Germany (S.R., D.M., P.H.).

Toll-like receptor (TLR) 7 and TLR8 are transmembrane receptors that recognize single-stranded RNA. Activation of these receptors results in immune cell stimulation and inflammatory cytokine production, which is normally a protective host response. However, aberrant activation of TLR7/8 is potentially pathogenic and linked to progression of certain autoimmune diseases such as lupus. Thus, we hypothesize that an inhibitor that blocks TLR7/8 would be an effective therapeutic treatment. Prior efforts to develop inhibitors of TLR7/8 have been largely unsuccessful as a result of the challenge of producing a small-molecule inhibitor for these difficult targets. Here, we report the characterization of M5049 and compound 2, molecules which were discovered in a medicinal chemistry campaign to produce dual TLR7/8 inhibitors with drug-like properties. Both compounds showed potent and selective activity in a range of cellular assays for inhibition of TLR7/8 and block synthetic ligands and natural endogenous RNA ligands such as microRNA and Alu RNA. M5049 was found to be potent in vivo as TLR7/8 inhibition efficaciously treated disease in several murine lupus models and, interestingly, was efficacious in a disease context in which TLR7/8 activity has not previously been considered a primary disease driver. Furthermore, M5049 had greater potency in disease models than expected based on its in vitro potency and pharmacokinetic/pharmacodynamic properties. Because of its preferential accumulation in tissues, and ability to block multiple TLR7/8 RNA ligands, M5049 may be efficacious in treating autoimmunity and has the potential to provide benefit to a variety of patients with varying disease pathogenesis. SIGNIFICANCE STATEMENT: This study reports discovery of a novel toll-like receptor (TLR) 7 and TLR8 inhibitor (M5049); characterizes its binding mode, potency/selectivity, and pharmacokinetic and pharmacodynamic properties; and demonstrates its potential for treating autoimmune diseases in two mouse lupus models. TLR7/8 inhibition is unique in that it may block both innate and adaptive autoimmunity; thus, this study suggests that M5049 has the potential to benefit patients with autoimmune diseases.
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http://dx.doi.org/10.1124/jpet.120.000275DOI Listing
March 2021

The metabolite BH4 controls T cell proliferation in autoimmunity and cancer.

Nature 2018 11 7;563(7732):564-568. Epub 2018 Nov 7.

IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria.

Genetic regulators and environmental stimuli modulate T cell activation in autoimmunity and cancer. The enzyme co-factor tetrahydrobiopterin (BH4) is involved in the production of monoamine neurotransmitters, the generation of nitric oxide, and pain. Here we uncover a link between these processes, identifying a fundamental role for BH4 in T cell biology. We find that genetic inactivation of GTP cyclohydrolase 1 (GCH1, the rate-limiting enzyme in the synthesis of BH4) and inhibition of sepiapterin reductase (the terminal enzyme in the synthetic pathway for BH4) severely impair the proliferation of mature mouse and human T cells. BH4 production in activated T cells is linked to alterations in iron metabolism and mitochondrial bioenergetics. In vivo blockade of BH4 synthesis abrogates T-cell-mediated autoimmunity and allergic inflammation, and enhancing BH4 levels through GCH1 overexpression augments responses by CD4- and CD8-expressing T cells, increasing their antitumour activity in vivo. Administration of BH4 to mice markedly reduces tumour growth and expands the population of intratumoral effector T cells. Kynurenine-a tryptophan metabolite that blocks antitumour immunity-inhibits T cell proliferation in a manner that can be rescued by BH4. Finally, we report the development of a potent SPR antagonist for possible clinical use. Our data uncover GCH1, SPR and their downstream metabolite BH4 as critical regulators of T cell biology that can be readily manipulated to either block autoimmunity or enhance anticancer immunity.
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http://dx.doi.org/10.1038/s41586-018-0701-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6438708PMC
November 2018

Alternative Splice Forms of CYLD Mediate Ubiquitination of SMAD7 to Prevent TGFB Signaling and Promote Colitis.

Gastroenterology 2019 02 10;156(3):692-707.e7. Epub 2018 Oct 10.

Institute for Molecular Medicine, University Medical Centre, Johannes Gutenberg University of Mainz, Mainz, Germany. Electronic address:

Background & Aims: The CYLD lysine 63 deubiquitinase gene (CYLD) encodes tumor suppressor protein that is mutated in familial cylindromatosus, and variants have been associated with Crohn disease (CD). Splice forms of CYLD that lack exons 7 and 8 regulate transcription factors and functions of immune cells. We examined the expression of splice forms of CYLD in colon tissues from patients with CD and their effects in mice.

Methods: We performed immunohistochemical analyses of colon tissues from patients with untreated CD and patients without inflammatory bowel diseases (controls). We obtained mice that expressed splice forms of CYLD (sCYLD mice) without or with SMAD7 (sCYLD/SMAD7 mice) from transgenes and CYLD-knockout mice (with or without transgenic expression of SMAD7) and performed endoscopic analyses. Colitis was induced in Rag1 mice by transfer of CD4 CD62L T cells from C57/Bl6 or transgenic mice. T cells were isolated from mice and analyzed by flow cytometry and quantitative real-time polymerase chain reaction and intestinal tissues were analyzed by histology and immunohistochemistry. CYLD forms were expressed in mouse embryonic fibroblasts, primary T cells, and HEK293T cells, which were analyzed by immunoblot, mobility shift, and immunoprecipitation assays.

Results: The colonic lamina propria from patients with CD was infiltrated by T cells and had higher levels of sCYLD (but not full-length CYLD) and SMAD7 than tissues from controls. Incubation of mouse embryonic fibroblasts and T cells with transforming growth factor β increased their production of sCYLD and decreased full-length CYLD. Transgenic expression of sCYLD and SMAD7 in T cells prevented the differentiation of regulatory T cells and T-helper type 17 cells and increased the differentiation of T-helper type 1 cells. The same effects were observed in colon tissues from sCYLD/SMAD7 mice but not in those from CYLD-knockout SMAD7 mice. The sCYLD mice had significant increases in the numbers of T-helper type 1 cells and CD44 CD62L memory-effector CD4 T cells in the spleen and mesenteric lymph nodes compared with wild-type mice; sCYLD/SMAD7 mice had even larger increases. The sCYLD/SMAD7 mice spontaneously developed severe colitis, with infiltration of the colon by dendritic cells, neutrophils, macrophages, and CD4 T cells and increased levels of Ifng, Il6, Il12a, Il23a, and Tnf mRNAs. Co-transfer of regulatory T cells from wild-type, but not from sCYLD/SMAD7, mice prevented the induction of colitis in Rag1 mice by CD4 T cells. We found increased levels of poly-ubiquitinated SMAD7 in sCYLD CD4 T cells. CYLD formed a nuclear complex with SMAD3, whereas sCYLD recruited SMAD7 to the nucleus, which inhibited the expression of genes regulated by SMAD3 and SMAD4. We found that sCYLD mediated lysine 63-linked ubiquitination of SMAD7. The sCYLD-SMAD7 complex inhibited transforming growth factor β signaling in CD4 T cells.

Conclusions: Levels of the spliced form of CYLD are increased in colon tissues from patients with CD. sCYLD mediates ubiquitination and nuclear translocation of SMAD7 and thereby decreases transforming growth factor β signaling in T cells. This prevents immune regulatory mechanisms and leads to colitis in mice.
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http://dx.doi.org/10.1053/j.gastro.2018.10.023DOI Listing
February 2019

Expression of IL-17F is associated with non-pathogenic Th17 cells.

J Mol Med (Berl) 2018 08 29;96(8):819-829. Epub 2018 Jun 29.

University Medical Center of the Johannes Gutenberg University Mainz, Institute for Molecular Medicine, 55131, Mainz, Germany.

IL-17A and IL-17F share the highest sequence homology of the IL-17 family and signal via the same IL-17RA/RC receptor heterodimer. To better explore the expression of these two cytokines, we used a double reporter mouse strain (IL-17 mice), where IL-17A expressing cells are marked by enhanced green fluorescent protein (eGFP) while red fluorescence protein (RFP) reports the expression of IL-17F. In steady state, we found that Th17 and γδ T cells only expressed IL-17A, while IL-17F expression was restricted to CD8 T cells (Tc17) and innate lymphoid cells (ILC type 3) of the gut. In experimental autoimmune encephalomyelitis, the vast majority of CNS-infiltrating Th17 cells expressed IL-17A but not IL-17F. In contrast, anti-CD3-induced, TGF-β-driven Th17 cells in the gut expressed both of these IL-17 cytokines. In line with this, in vitro differentiation of Th17 cells in the presence of IL-1β led primarily to IL-17A expressing T cells, while TGF-β induced IL-17F co-expressing Th17 cells. Our results suggest that expression of IL-17F is associated with non-pathogenic T cells, pointing to a differential function of IL-17A versus IL-17F.

Key Messages: Naïve mice: CD4 T cells and γδ T cells express IL-17A, and Tc17 cells express IL-17F. Gut ILC3 show differential expression of IL17A and F. Th17 differentiation with TGF-β1 induces IL-17A and F, whereas IL-1β induced cells expressing IL-17A. Th17 cells in EAE in CNS express IL-17A only. Gut Th17 cells induced by anti-CD3 express IL-17A and F together as skin γδ T cells of IMQ-treated mice.
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http://dx.doi.org/10.1007/s00109-018-1662-5DOI Listing
August 2018

PARP-1 protects against colorectal tumor induction, but promotes inflammation-driven colorectal tumor progression.

Proc Natl Acad Sci U S A 2018 04 9;115(17):E4061-E4070. Epub 2018 Apr 9.

Department of Toxicology, University Medical Center Mainz, 55131 Mainz, Germany;

Colorectal cancer (CRC) is one of the most common tumor entities, which is causally linked to DNA repair defects and inflammatory bowel disease (IBD). Here, we studied the role of the DNA repair protein poly(ADP-ribose) polymerase-1 (PARP-1) in CRC. Tissue microarray analysis revealed PARP-1 overexpression in human CRC, correlating with disease progression. To elucidate its function in CRC, PARP-1 deficient (PARP-1) and wild-type animals (WT) were subjected to azoxymethane (AOM)/ dextran sodium sulfate (DSS)-induced colorectal carcinogenesis. Miniendoscopy showed significantly more tumors in WT than in PARP-1 mice. Although the lack of PARP-1 moderately increased DNA damage, both genotypes exhibited comparable levels of AOM-induced autophagy and cell death. Interestingly, miniendoscopy revealed a higher AOM/DSS-triggered intestinal inflammation in WT animals, which was associated with increased levels of innate immune cells and proinflammatory cytokines. Tumors in WT animals were more aggressive, showing higher levels of STAT3 activation and cyclin D1 up-regulation. PARP-1 animals were then crossed with -methylguanine-DNA methyltransferase (MGMT)-deficient animals hypersensitive to AOM. Intriguingly, PARP-1/MGMT double knockout (DKO) mice developed more, but much smaller tumors than MGMT animals. In contrast to MGMT-deficient mice, DKO animals showed strongly reduced AOM-dependent colonic cell death despite similar -methylguanine levels. Studies with PARP-1 cells provided evidence for increased alkylation-induced DNA strand break formation when MGMT was inhibited, suggesting a role of PARP-1 in the response to -methylguanine adducts. Our findings reveal PARP-1 as a double-edged sword in colorectal carcinogenesis, which suppresses tumor initiation following DNA alkylation in a MGMT-dependent manner, but promotes inflammation-driven tumor progression.
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http://dx.doi.org/10.1073/pnas.1712345115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5924876PMC
April 2018

Wheat amylase-trypsin inhibitors exacerbate intestinal and airway allergic immune responses in humanized mice.

J Allergy Clin Immunol 2019 01 21;143(1):201-212.e4. Epub 2018 Mar 21.

Department of Dermatology, University Medical Center, Johannes Gutenberg University, Mainz, Germany; Research Center for Immunotherapy, University Medical Center, Johannes Gutenberg University, Mainz, Germany.

Background: Amylase-trypsin inhibitors (ATIs) in wheat and related cereals are potent activators of myeloid innate immune cells via engagement of TLR4. Furthermore, ATIs have been shown to serve as adjuvants in experimental intestinal inflammatory diseases.

Objective: The aim of this study was to analyze whether ATIs are also modifiers of allergic inflammation.

Methods: Therefore, CD4 T cells from donors sensitized to grass or birch pollen were stimulated with autologous allergen-pulsed dendritic cells in the presence or absence of ATIs or the control storage protein zein from corn. To analyze allergen-induced gut and lung inflammation, immunodeficient mice were engrafted with PBMCs from these allergic donors plus the respective allergen, and fed with selected diets. Three weeks later, inflammation was induced by rectal or intranasal allergen challenge and monitored by mini endoscopy or airway hyperreactivity, respectively.

Results: Allergen-specific T-cell proliferation and cytokine production was significantly exacerbated by ATIs and not by zein. In vivo, allergen-specific human IgE level was strongly elevated in sera of mice receiving an ATI-containing diet compared with mice that were fed gluten-free and thus ATI-free diet. Importantly, allergen-induced IgE-dependent colitis and airway hyperreactivity were also enhanced in ATI-fed mice. Gut inflammation was further increased in mice receiving an additional ATI injection and even detectable in the absence of the aeroallergen, whereas zein had no such effect. Injection of anti-human TLR4 mAbs or the anti-human IgE mAb omalizumab completely abolished ATI-induced allergic inflammation.

Conclusions: These results underline that wheat ATIs are important nutritional activators and adjuvants of allergy, which might be exploited for nutritional therapeutic strategies.
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http://dx.doi.org/10.1016/j.jaci.2018.02.041DOI Listing
January 2019

Balanced Bcl-3 expression in murine CD4 T cells is required for generation of encephalitogenic Th17 cells.

Eur J Immunol 2017 08 29;47(8):1335-1341. Epub 2017 Jun 29.

Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany.

The function of NF-κB family members is controlled by multiple mechanisms including the transcriptional regulator Bcl-3, an atypical member of the IκB family. By using a murine model of conditional Bcl-3 overexpression specifically in T cells, we observed impairment in the development of Th2, Th1, and Th17 cells. High expression of Bcl-3 promoted CD4 T-cell survival, but at the same time suppressed proliferation in response to TCR stimulation, resulting in reduced CD4 T-cell expansion. As a consequence, T-cell-specific overexpression of Bcl-3 led to reduced inflammation in the small intestine of mice applied with anti-CD3 in a model of gut inflammation. Moreover, impaired Th17-cell development resulted in the resistance of Bcl-3 overexpressing mice to EAE, a mouse model of multiple sclerosis. Thus, we concluded that fine-tuning expression of Bcl-3 is needed for proper CD4 T-cell development and is required to sustain Th17-cell mediated pathology.
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http://dx.doi.org/10.1002/eji.201746933DOI Listing
August 2017

Elevated levels of Bcl-3 inhibits Treg development and function resulting in spontaneous colitis.

Nat Commun 2017 04 28;8:15069. Epub 2017 Apr 28.

Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg, University of Mainz, Obere Zahlbarer Str 67, 55131 Mainz, Germany.

Bcl-3 is an atypical NF-κB family member that regulates NF-κB-dependent gene expression in effector T cells, but a cell-intrinsic function in regulatory T (Treg) cells and colitis is not clear. Here we show that Bcl-3 expression levels in colonic T cells correlate with disease manifestation in patients with inflammatory bowel disease. Mice with T-cell-specific overexpression of Bcl-3 develop severe colitis that can be attributed to defective Treg cell development and function, leading to the infiltration of immune cells such as pro-inflammatory γδT cells, but not αβ T cells. In Treg cells, Bcl-3 associates directly with NF-κB p50 to inhibit DNA binding of p50/p50 and p50/p65 NF-κB dimers, thereby regulating NF-κB-mediated gene expression. This study thus reveals intrinsic functions of Bcl-3 in Treg cells, identifies Bcl-3 as a potential prognostic marker for colitis and illustrates the mechanism by which Bcl-3 regulates NF-κB activity in Tregs to prevent colitis.
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http://dx.doi.org/10.1038/ncomms15069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5414353PMC
April 2017

EBI2 Is Highly Expressed in Multiple Sclerosis Lesions and Promotes Early CNS Migration of Encephalitogenic CD4 T Cells.

Cell Rep 2017 01;18(5):1270-1284

Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, 55131 Mainz, Germany. Electronic address:

Arrival of encephalitogenic T cells at inflammatory foci represents a critical step in development of experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis. EBI2 and its ligand, 7α,25-OHC, direct immune cell localization in secondary lymphoid organs. CH25H and CYP7B1 hydroxylate cholesterol to 7α,25-OHC. During EAE, we found increased expression of CH25H by microglia and CYP7B1 by CNS-infiltrating immune cells elevating the ligand concentration in the CNS. Two critical pro-inflammatory cytokines, interleukin-23 (IL-23) and interleukin-1 beta (IL-1β), maintained expression of EBI2 in differentiating Th17 cells. In line with this, EBI2 enhanced early migration of encephalitogenic T cells into the CNS in a transfer EAE model. Nonetheless, EBI2 was dispensable in active EAE. Human Th17 cells do also express EBI2, and EBI2 expressing cells are abundant within multiple sclerosis (MS) white matter lesions. These findings implicate EBI2 as a mediator of CNS autoimmunity and describe mechanistically its contribution to the migration of autoreactive T cells into inflamed organs.
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http://dx.doi.org/10.1016/j.celrep.2017.01.020DOI Listing
January 2017

Monitoring of Chemically Induced Colitis.

Methods Mol Biol 2017 ;1559:297-309

Department of Medicine 1, University of Erlangen-Nuremberg, Campus for medical research, Hartmannstr.14, 91052, Erlangen, Germany.

Inflammation is a common symptom of inflammatory bowel disease (IBD). Actually, many experimental models of colitis exist and try to mimic the human situation in order to understand the pathogenesis of Crohn's disease and ulcerative colitis. These experimental models of inflammation can be characterized by specific parameters, which illustrate the proceeding inflammatory process. By use of these models potentially new reagents for improved therapeutic approaches can be analyzed. Here, we describe the TNBS-mediated colitis model and specify different parameters for the detailed characterization of the inflammatory process in experimental colitis models.
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http://dx.doi.org/10.1007/978-1-4939-6786-5_20DOI Listing
January 2018

Cylindromatosis (Cyld) gene mutation in T cells promotes the development of an IL-9-dependent allergic phenotype in experimental asthma.

Cell Immunol 2016 10 25;308:27-34. Epub 2016 Jun 25.

Institute for Immunology and Research Center for Immunotherapy, University Medical Center of the Johannes Gutenberg-University Mainz, Germany; Institute of Pathology, Saarland University Medical Center, Homburg, Germany. Electronic address:

Cylindromatosis (CYLD) is a ubiquitously expressed deubiquitinating enzyme which removes activating ubiquitin residues from important signaling molecules of the NF-κB pathway. In CYLD transgenic mice, a naturally occurring short isoform (sCYLD) is overexpressed in the absence of full length CYLD, leading to excessive NF-κB activity. Herein, we investigated the impact of the CYLD mutation selectively in T cells on the development of experimental allergic airway disease induced by sensitization and challenge with ovalbumin. Compared with their wildtype littermates, mice bearing the T cell-specific mutation (CD4CYLD) display stronger eosinophilia and mucus production in the lungs and higher IgE serum levels. The reason for these observations is excessive production of T cell-derived IL-9, a cytokine to whom allergy-promoting properties were ascribed. Consequently, blockade of IL-9 in CD4CYLD mice alleviates the development of disease symptoms. Thus, by polarization of the T cell cytokine response, sCYLD can favor the development of allergic airway disease.
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http://dx.doi.org/10.1016/j.cellimm.2016.06.003DOI Listing
October 2016

Epicutaneous and Oral Low-Zone Tolerance Protects from Colitis in Mice.

J Invest Dermatol 2016 09 17;136(9):1831-1839. Epub 2016 May 17.

Department of Dermatology, University Medical Center, Johannes Gutenberg-University of Mainz, Mainz, Germany. Electronic address:

Tolerance to environmental antigens that encounter the organism at interfaces like skin or gut prevents deleterious systemic immune responses. The aim of this study was to analyze whether and how low doses of haptens, by entry through the skin or gastrointestinal tract, affect the outcome of the predominantly Th1/Th17-mediated 2,4,6-trinitro-benzenesulfonic acid-induced colitis, which mimics an autoimmune bowl disease in man. Epicutaneous and oral applications of low doses of the allergen resulted in the induction of low-zone tolerance (LZT) and protected from colitis development, demonstrated by a significantly reduced inflammatory response of the gut in vivo. In line with this observation, we found a significantly diminished Th1/Th17-mediated T cell response and reduced T cell proliferation after both tolerance regimes, indicating that epicutaneous LZT is just as well efficient as oral tolerance in prevention of a gut-associated inflammatory immune response. Use of a second, unrelated hapten for LZT induction revealed an antigen-specific tolerance mechanism. Intriguingly, in the absence of hapten-activated CD4(+)CD25(+)Foxp3(+) regulatory T cells and IL-10, epicutaneous and oral LZT failed to abrogate the development of the intestinal inflammation. In conclusion, this study highlights in particular epicutaneous immunotherapies in the form of LZT through activation of CD4(+)CD25(+)Foxp3(+) regulatory T cells as treatment strategies for inflammatory, allergic, or autoimmune diseases.
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http://dx.doi.org/10.1016/j.jid.2016.04.037DOI Listing
September 2016

DNA repair by MGMT, but not AAG, causes a threshold in alkylation-induced colorectal carcinogenesis.

Carcinogenesis 2015 Oct 4;36(10):1235-44. Epub 2015 Aug 4.

Institute of Molecular Medicine, University Medical Center Mainz, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany.

Epidemiological studies indicate that N-nitroso compounds (NOC) are causally linked to colorectal cancer (CRC). NOC induce DNA alkylations, including O (6)-methylguanine (O (6)-MeG) and N-methylated purines, which are repaired by O (6)-MeG-DNA methyltransferase (MGMT) and N-alkyladenine-DNA glycosylase (AAG)-initiated base excision repair, respectively. In view of recent evidence of nonlinear mutagenicity for NOC-like compounds, the question arises as to the existence of threshold doses in CRC formation. Here, we set out to determine the impact of DNA repair on the dose-response of alkylation-induced CRC. DNA repair proficient (WT) and deficient (Mgmt (-/-), Aag (-/-) and Mgmt (-/-)/Aag (-/-)) mice were treated with azoxymethane (AOM) and dextran sodium sulfate to trigger CRC. Tumors were quantified by non-invasive mini-endoscopy. A non-linear increase in CRC formation was observed in WT and Aag (-/-) mice. In contrast, a linear dose-dependent increase in tumor frequency was found in Mgmt (-/-) and Mgmt (-/-)/Aag (-/-) mice. The data were corroborated by hockey stick modeling, yielding similar carcinogenic thresholds for WT and Aag (-/-) and no threshold for MGMT lacking mice. O (6)-MeG levels and depletion of MGMT correlated well with the observed dose-response in CRC formation. AOM induced dose-dependently DNA double-strand breaks in colon crypts including Lgr5-positive colon stem cells, which coincided with ATR-Chk1-p53 signaling. Intriguingly, Mgmt (-/-) mice displayed significantly enhanced levels of γ-H2AX, suggesting the usefulness of γ-H2AX as an early genotoxicity marker in the colorectum. This study demonstrates for the first time a non-linear dose-response for alkylation-induced colorectal carcinogenesis and reveals DNA repair by MGMT, but not AAG, as a key node in determining a carcinogenic threshold.
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http://dx.doi.org/10.1093/carcin/bgv114DOI Listing
October 2015

The ubiquitin-specific protease USP8 is critical for the development and homeostasis of T cells.

Nat Immunol 2015 Sep 27;16(9):950-60. Epub 2015 Jul 27.

1] Institute of Neuropathology, University of Freiburg, Freiburg, Germany. [2] Leibniz Institute for Molecular Pharmacology, Berlin, Germany.

The modification of proteins by ubiquitin has a major role in cells of the immune system and is counteracted by various deubiquitinating enzymes (DUBs) with poorly defined functions. Here we identified the ubiquitin-specific protease USP8 as a regulatory component of the T cell antigen receptor (TCR) signalosome that interacted with the adaptor Gads and the regulatory molecule 14-3-3β. Caspase-dependent processing of USP8 occurred after stimulation of the TCR. T cell-specific deletion of USP8 in mice revealed that USP8 was essential for thymocyte maturation and upregulation of the gene encoding the cytokine receptor IL-7Rα mediated by the transcription factor Foxo1. Mice with T cell-specific USP8 deficiency developed colitis that was promoted by disturbed T cell homeostasis, a predominance of CD8(+) γδ T cells in the intestine and impaired regulatory T cell function. Collectively, our data reveal an unexpected role for USP8 as an immunomodulatory DUB in T cells.
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http://dx.doi.org/10.1038/ni.3230DOI Listing
September 2015

Activated glycoprotein A repetitions predominant (GARP)-expressing regulatory T cells inhibit allergen-induced intestinal inflammation in humanized mice.

J Allergy Clin Immunol 2015 Jul;136(1):159-68

Department of Dermatology, University Hospital Erlangen, University Erlangen-Nürnberg, Erlangen, Germany. Electronic address:

Background: Recently, we developed a humanized mouse model of allergen-induced IgE-dependent gut inflammation in PBMC-engrafted immunodeficient mice.

Objective: In the present study, we wanted to investigate the role of regulatory T (Treg) cells and their activation status in this model.

Methods: Nonobese diabetic-severe combined immunodeficiency-γc(-/-) mice were injected intraperitoneally with human PBMCs from allergic donors together with the respective allergen or NaCl as control in the presence or absence of different concentrations of CD4(+)CD25(+) Treg cells of the same donor. After an additional allergen boost 1 week later, mice were challenged with the allergen rectally on day 21 and gut inflammation was monitored by a high-resolution video mini-endoscopic system evaluating translucency, granularity, fibrin production, vascularity, and stool.

Results: Allergen-specific human IgE in mouse sera, which was detectable only in PBMC plus allergen-treated mice, was strongly inhibited by coinjection of Treg cells at a ratio of at least 1:10. Consequently, the presence of Treg cells significantly decreased IgE-dependent allergen-induced gut inflammation after rectal allergen challenge. In addition, Treg cells reduced allergen-specific proliferation and cytokine production of recovered human CD4(+) T cells in vitro. Activation of Treg cells before injection further increased all inhibitory effects. Prevention of gut inflammation also occurred by the administration of glycoprotein A repetitions predominant, a molecule expressed by activated Treg cells, whereas its blockade completely abrogated inhibition by Treg cells.

Conclusions: These results demonstrate that allergen-specific gut inflammation in human PBMC-engrafted mice can be avoided by enhancing the numbers or activity of autologous Treg cells, which is of great interest for therapeutic intervention of allergic diseases of the intestine.
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http://dx.doi.org/10.1016/j.jaci.2015.04.020DOI Listing
July 2015

The deubiquitinating enzyme CYLD regulates the differentiation and maturation of thymic medullary epithelial cells.

Immunol Cell Biol 2015 Jul 20;93(6):558-66. Epub 2015 Jan 20.

Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University of Mainz, Mainz, Germany.

The cross talk between thymocytes and the thymic epithelium is critical for T-cell development and the establishment of central tolerance. Medullary thymic epithelial cells (mTECs) are located in the thymic medulla and mediate the elimination of self-reactive thymocytes, thereby preventing the onset of autoimmunity. Previous studies identified the deubiquitinating enzyme CYLD as a critical regulator of T-cell development by activating proximal T-cell receptor signaling during the transition of double-positive to single-positive thymocytes. Here we evaluated the impact of the naturally occurring short-splice variant of the cyld gene (sCYLD) on the development and maturation of mTECs. We found that thymi of CYLD(ex7/8) mice, solely expressing sCYLD, displayed a reduced number of mature mTECs caused by a developmental block during the transition of immature to mature mTECs. Further, we could demonstrate an impaired negative selection of thymocytes in these mice. Our data demonstrate that inefficient negative selection in the thymus of CYLD(ex7/8) mice result from a defect in mTEC maturation.
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http://dx.doi.org/10.1038/icb.2014.122DOI Listing
July 2015

Protein kinase CK2 enables regulatory T cells to suppress excessive TH2 responses in vivo.

Nat Immunol 2015 Mar 19;16(3):267-75. Epub 2015 Jan 19.

Institute for Immunology, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany.

The quality of the adaptive immune response depends on the differentiation of distinct CD4(+) helper T cell subsets, and the magnitude of an immune response is controlled by CD4(+)Foxp3(+) regulatory T cells (Treg cells). However, how a tissue- and cell type-specific suppressor program of Treg cells is mechanistically orchestrated has remained largely unexplored. Through the use of Treg cell-specific gene targeting, we found that the suppression of allergic immune responses in the lungs mediated by T helper type 2 (TH2) cells was dependent on the activity of the protein kinase CK2. Genetic ablation of the β-subunit of CK2 specifically in Treg cells resulted in the proliferation of a hitherto-unexplored ILT3(+) Treg cell subpopulation that was unable to control the maturation of IRF4(+)PD-L2(+) dendritic cells required for the development of TH2 responses in vivo.
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http://dx.doi.org/10.1038/ni.3083DOI Listing
March 2015

Interleukin 17 drives vascular inflammation, endothelial dysfunction, and arterial hypertension in psoriasis-like skin disease.

Arterioscler Thromb Vasc Biol 2014 Dec 23;34(12):2658-68. Epub 2014 Oct 23.

From the Institute for Molecular Medicine (S.K., A.L.C., R.S., N.Y., A.N., S.R., A.W.), Department of Medicine 2 (S.K., M.O., D.M., M.K., P. Wild, A.D., P. Wenzel, T.M.), Department of Dermatology (J.W., L.K., E.v.S.), Department of Pharmacology (H.L., Z.W.), Institute for Pathology (C.B.), Center for Thrombosis and Hemostasis (P. Wild, P. Wenzel, T.M.), German Center for Cardiovascular Research (DZHK), partner site RhineMain (A.U., P.W.), and Department of Medicine 3 (M.R.), University Medical Center of the Johannes-Gutenberg University of Mainz, Germany; Department of Internal Medicine I, University Hospital Erlangen, Germany (M.W., M.F.N.); Institute of Anatomy, University of Leipzig, Germany (C.P., I.B.); Institute for Biochemistry, Christian-Albrechts-University of Kiel, Germany (S.R.-J.); Institute of Biochemistry and Molecular Biology II, Medical Faculty, Heinrich-Heine University, Düsseldorf, Germany (J.S.).

Objective: Interleukin (IL)-17A is regarded as an important cytokine to drive psoriasis, an inflammatory skin disease marked by increased cardiovascular mortality. We aimed to test the hypothesis that overproduction of IL-17A in the skin leading to dermal inflammation may systemically cause vascular dysfunction in psoriasis-like skin disease.

Approach And Results: Conditional overexpression of IL-17A in keratinocytes caused severe psoriasis-like skin inflammation in mice (K14-IL-17A(ind/+) mice), associated with increased reactive oxygen species formation and circulating CD11b(+) inflammatory leukocytes in blood, with endothelial dysfunction, increased systolic blood pressure, left ventricular hypertrophy, and reduced survival compared with controls. In K14-IL-17A(ind/+) mice, immunohistochemistry and flow cytometry revealed increased vascular production of the nitric oxide/superoxide reaction product peroxynitrite and infiltration of the vasculature with myeloperoxidase(+)CD11b(+)GR1(+)F4/80(-) cells accompanied by increased expression of the inducible nitric oxide synthase and the nicotinamide dinucleotide phosphate (NADPH) oxidase, nox2. Neutrophil depletion by anti-GR-1 antibody injections reduced oxidative stress in blood and vessels. Neutralization of tumor necrosis factor-α and IL-6 (both downstream of IL-17A) reduced skin lesions, attenuated oxidative stress in heart and blood, and partially improved endothelial dysfunction in K14-IL-17A(ind/+) mice.

Conclusions: Dermal overexpression of IL-17A induces systemic endothelial dysfunction, vascular oxidative stress, arterial hypertension, and increases mortality mainly driven by myeloperoxidase(+)CD11b(+)GR1(+)F4/80(-) inflammatory cells. Depletion of the GR-1(+) immune cells or neutralization of IL-17A downstream cytokines by biologicals attenuates the vascular phenotype in K14-IL-17A(ind/+) mice.
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http://dx.doi.org/10.1161/ATVBAHA.114.304108DOI Listing
December 2014

Isolation of T cells from the gut.

Methods Mol Biol 2014 ;1193:21-5

Institute for Molecular Medicine, University Medical Center, Johannes Gutenberg-University, Obere Zahlbacher Str. 67, 55131, Mainz, Germany,

The lymphocytes of epithelial and lamina proprial compartments of the intestine are phenotypically and functionally distinct and serve a wide range of functions in the intestinal mucosa like regulating intestinal homeostasis, maintaining epithelial barrier function as well as regulating adaptive and innate immune responses. To analyze the role of these cells in different disease states, it is necessary to isolate pure cell populations of the intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) of the gut. In this protocol we describe a method to isolate T cells from IEL and LPL, which can be used for further investigations like comparative studies of mRNA expression, cell proliferation assay, or protein analysis.
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http://dx.doi.org/10.1007/978-1-4939-1212-4_3DOI Listing
April 2015

Overexpression of Bcl-3 inhibits the development of marginal zone B cells.

Eur J Immunol 2014 Feb 18;44(2):545-52. Epub 2013 Nov 18.

Institute for Molecular Medicine, Johannes Gutenberg-University of Mainz, Mainz, Germany.

The transcription factor Bcl-3 functions as a proto-oncogene via regulation of cell proliferation and apoptosis. Bcl-3 is an atypical member of the IκB family and plays a central role in the immune response through interactions with the NF-κB subunits p50 and p52. To investigate the impact of Bcl-3 on B-cell maturation and regulation, we generated mice that overexpress Bcl-3 specifically in B cells. Interestingly, these mice lack marginal zone B cells and exhibit a significant reduction in the number of B-1 B cells. Further, B cells from these mice are impaired in their proliferative capacity. Our data demonstrate that the overexpression of the transcription factor Bcl-3 inhibits germinal center formation, marginal zone B-cell development, and affects the B-1 B-cell compartment.
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http://dx.doi.org/10.1002/eji.201343655DOI Listing
February 2014

IL-6 regulates neutrophil microabscess formation in IL-17A-driven psoriasiform lesions.

J Invest Dermatol 2014 Mar 25;134(3):728-735. Epub 2013 Sep 25.

Institute for Molecular Medicine, University Medical Center of the Johannes-Gutenberg University of Mainz, Mainz, Germany. Electronic address:

The lack of a generally accepted animal model for human psoriasis has hindered progress with respect to understanding the pathogenesis of the disease. Here we present a model in which transgenic IL-17A expression is targeted to the skin in mice, achievable after crossing our IL-17A(ind) allele to the K14-Cre strain. K14-IL-17A(ind/+) mice invariably develop an overt skin inflammation bearing many hallmark characteristics of human psoriasis including dermal infiltration of effector T cells, formation of neutrophil microabscesses, and hyperkeratosis. IL-17A expression in the skin results in upregulated granulopoiesis and migration of IL-6R-expressing neutrophils into the skin. Neutralization of IL-6 signaling efficiently reduces the observed pathogenesis in skin of IL-17A-overexpressing mice, with marked reductions in epidermal neutrophil abscess formation and epidermal thickening. Thus, IL-6 functions downstream of IL-17A to exacerbate neutrophil microabscess development in psoriasiform lesions.
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http://dx.doi.org/10.1038/jid.2013.404DOI Listing
March 2014

The tumor suppressor CYLD controls the function of murine regulatory T cells.

J Immunol 2012 Nov 12;189(10):4770-6. Epub 2012 Oct 12.

Institute for Molecular Medicine, Johannes Gutenberg-University of Mainz, 55131 Mainz, Germany.

CYLD was originally identified as a tumor suppressor gene mutated in familial cylindromatosis, an autosomal dominant predisposition to multiple benign neoplasms of the skin known as cylindromas. The CYLD protein is a deubiquitinating enzyme that acts as a negative regulator of NF-κB and JNK signaling through its interaction with NEMO and TNFR-associated factor 2. We have previously described a novel mouse strain that expresses solely and excessively a naturally occurring splice variant of CYLD (CYLD(ex7/8)). In this study, we demonstrate that CYLD plays a critical role in Treg development and function. T cells of CYLD(ex7/8) mice had a hyperactive phenotype manifested by increased production of inflammatory cytokines and constitutive activation of the NF-κB pathway. Furthermore, the amount of Foxp3(+) regulatory T cells in these mice was markedly enhanced in thymus and peripheral organs. Importantly, these regulatory T cells displayed decreased expression levels of CD25 and CTLA-4 associated with impaired suppressive capacity. Hence, our data emphasize an essential role of CYLD in maintaining T cell homeostasis as well as normal T regulatory cell function, thereby controlling abnormal T cell responses.
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http://dx.doi.org/10.4049/jimmunol.1201993DOI Listing
November 2012

Dependence on nuclear factor of activated T-cells (NFAT) levels discriminates conventional T cells from Foxp3+ regulatory T cells.

Proc Natl Acad Sci U S A 2012 Oct 18;109(40):16258-63. Epub 2012 Sep 18.

Department of Molecular Pathology, Institute of Pathology, Julius Maximilians-University Würzburg, 97080 Würzburg, Germany.

Several lines of evidence suggest nuclear factor of activated T-cells (NFAT) to control regulatory T cells: thymus-derived naturally occurring regulatory T cells (nTreg) depend on calcium signals, the Foxp3 gene harbors several NFAT binding sites, and the Foxp3 (Fork head box P3) protein interacts with NFAT. Therefore, we investigated the impact of NFAT on Foxp3 expression. Indeed, the generation of peripherally induced Treg (iTreg) by TGF-β was highly dependent on NFAT expression because the ability of CD4(+) T cells to differentiate into iTreg diminished markedly with the number of NFAT family members missing. It can be concluded that the expression of Foxp3 in TGF-β-induced iTreg depends on the threshold value of NFAT rather than on an individual member present. This is specific for iTreg development, because frequency of nTreg remained unaltered in mice lacking NFAT1, NFAT2, or NFAT4 alone or in combination. Different from expectation, however, the function of both nTreg and iTreg was independent on robust NFAT levels, reflected by less nuclear NFAT in nTreg and iTreg. Accordingly, absence of one or two NFAT members did not alter suppressor activity in vitro or during colitis and transplantation in vivo. This scenario emphasizes an inhibition of high NFAT activity as treatment for autoimmune diseases and in transplantation, selectively targeting the proinflammatory conventional T cells, while keeping Treg functional.
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http://dx.doi.org/10.1073/pnas.1203870109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3479579PMC
October 2012

An alternative pathway of imiquimod-induced psoriasis-like skin inflammation in the absence of interleukin-17 receptor a signaling.

J Invest Dermatol 2013 Feb 6;133(2):441-51. Epub 2012 Sep 6.

Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany.

Topical application of imiquimod (IMQ) on the skin of mice induces inflammation with common features found in psoriatic skin. Recently, it was postulated that IL-17 has an important role both in psoriasis and in the IMQ model. To further investigate the impact of IL-17RA signaling in psoriasis, we generated IL-17 receptor A (IL-17RA)-deficient mice (IL-17RA(del)) and challenged these mice with IMQ. Interestingly, the disease was only partially reduced and delayed but not abolished when compared with controls. In the absence of IL-17RA, we found persisting signs of inflammation such as neutrophil and macrophage infiltration within the skin. Surprisingly, already in the naive state, the skin of IL-17RA(del) mice contained significantly elevated numbers of Th17- and IL-17-producing γδ T cells, assuming that IL-17RA signaling regulates the population size of Th17 and γδ T cells. Upon IMQ treatment of IL-17RA(del) mice, these cells secreted elevated amounts of tumor necrosis factor-α, IL-6, and IL-22, accompanied by increased levels of the chemokine CXCL2, suggesting an alternative pathway of neutrophil and macrophage skin infiltration. Hence, our findings have major implications in the potential long-term treatment of psoriasis by IL-17-targeting drugs.
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http://dx.doi.org/10.1038/jid.2012.318DOI Listing
February 2013

A20 deficiency in B cells enhances B-cell proliferation and results in the development of autoantibodies.

Eur J Immunol 2011 Mar 10;41(3):595-601. Epub 2011 Feb 10.

Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University of Mainz, Mainz, Germany.

A20/TNFAIP3 is an ubiquitin-editing enzyme, important for the regulation of the NF-κB pathway. Mutations in the TNFAIP3 gene have been linked to different human autoimmune disorders. In human B-cell lymphomas, the inactivation of A20 results in constitutive NF-κB activation. Recent studies demonstrate that in mice the germline inactivation of A20 leads to early lethality, due to inflammation in multiple organs of the body. In this report, we describe a new mouse strain allowing for the tissue-specific deletion of A20. We show that B-cell-specific deletion of A20 results in a dramatic reduction in marginal zone B cells. Furthermore, A20-deficient B cells display a hyperactive phenotype represented by enhanced proliferation upon activation. Finally, these mice develop higher levels of serum immunoglobulins, resulting in an excessive production of self-reactive autoantibodies.
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http://dx.doi.org/10.1002/eji.201041313DOI Listing
March 2011

Mutated cylindromatosis gene affects the functional state of dendritic cells.

Eur J Immunol 2010 Oct;40(10):2848-57

Clinical Research Unit Allergology, Department of Dermatology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany.

Cylindromatosis gene (CYLD) is a ubiquitously expressed deubiquitinating enzyme, which interacts with members of the NF-κB signaling pathway and attenuates NF-κB and JNK signaling. Here, we report that DC derived from transgenic mice, which solely express a naturally occurring CYLD isoform (CYLD(ex7/8)), display a higher content of nuclear RelB and express elevated levels of NF-κB family members as well as of known NF-κB-target genes comprising costimulatory molecules and pro-inflammatory cytokines, as compared with WT DC. Accordingly, unstimulated CYLD(ex7/8) DC exhibited a significantly higher primary allogenic T-cell stimulatory capacity than WT DC and exerted no tolerogenic activity. Transduction of unstimulated CYLD(ex7/8) DC with relB-specific shRNA reduced their T-cell stimulatory capacity. Treatment with the synthetic glucocorticoid dexamethasone known to inhibit NF-κB and AP-1 activity reverted the pro-immunogenic phenotype and function of CYLD(ex7/8) DC and re-established their tolerogenic function. DC derived from CYLD knockout mice showed no functional alterations compared with WT DC. Therefore, although complete loss of CYLD may be compensated for by other endogenous NF-κB inhibitors, CYLD(ex7/8) acts in a dominant negative manner. Our findings raise the question of whether genetic defects associated with increased NF-κB activity may result in disturbed maintenance of peripheral tolerance.
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http://dx.doi.org/10.1002/eji.200939285DOI Listing
October 2010