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

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 14-3-3ß. 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.

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

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 ß-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.

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

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

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

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 pain1,2. 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.

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

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.

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

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 O6-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 O6-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 O6-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.

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

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 ß 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 CD44high CD62Llow 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 ß 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 ß signaling in T cells. This prevents immune regulatory mechanisms and leads to colitis in mice.

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

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.

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

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.

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

Cylindromatosis (CYLD) is a ubiquitously expressed deubiquitinating enzyme which removes activating ubiquitin residues from important signaling molecules of the NF-κB pathway. In CYLDex7/8 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 CYLDex7/8 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 (CD4+CYLDex7/8) 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 CD4+CYLDex7/8 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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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-ß 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-ß-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.

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

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.

Myeloid cell-derived interleukin-6 induces vascular dysfunction and vascular and systemic inflammation.

Aims: The cytokine interleukin-6 (IL-6) plays a central role in the inflammation cascade as well as cardiovascular disease progression. Since myeloid cells are a primary source of IL-6 formation, we aimed to generate a mouse model to study the role of myeloid cell-derived IL-6 in vascular disease. Methods and results: Interleukin-6-overexpressing (IL-6OE) mice were generated and crossed with LysM-Cre mice, to generate mice (LysM-IL-6OE mice) overexpressing the cytokine in myeloid cells. Eight- to 12-week-old LysM-IL-6OE mice spontaneously developed inflammatory colitis and significantly impaired endothelium-dependent aortic relaxation, increased aortic reactive oxygen species (ROS) formation, and vascular dysfunction in resistance vessels. The latter phenotype was associated with decreased survival. Vascular dysfunction was accompanied by a significant accumulation of neutrophils, monocytes, and macrophages in the aorta, increased myeloid cell reactivity (elevated ROS production), and vascular fibrosis associated with phenotypic changes in vascular smooth muscle cells. In addition to elevated Mcp1 and Cxcl1 mRNA levels, aortae from LysM-IL-6OE mice expressed higher levels of inducible NO synthase and endothelin-1, thus partially accounting for vascular dysfunction, whereas systemic blood pressure alterations were not observed. Bone marrow (BM) transplantation experiments revealed that vascular dysfunction and ROS formation were driven by BM cell-derived IL-6 in a dose-dependent manner. Conclusion: Mice with conditional overexpression of IL-6 in myeloid cells show systemic and vascular inflammation as well as endothelial dysfunction. A decrease in circulating IL-6 levels by replacing IL-6-producing myeloid cells in the BM improved vascular dysfunction in this model, underpinning the relevant role of IL-6 in vascular disease.

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

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.

Mutated cylindromatosis gene affects the functional state of dendritic cells.

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.