Nuclear Receptors Regulate Intestinal Inflammation in the Context of IBD

Gastrointestinal (GI) homeostasis is strongly dependent on nuclear receptor (NR) functions. They play a variety of roles ranging from nutrient uptake, sensing of microbial metabolites, regulation of epithelial intestinal cell integrity to shaping of the intestinal immune cell repertoire. Several NRs are associated with GI pathologies; therefore, systematic analysis of NR biology, the underlying molecular mechanisms, and regulation of target genes can be expected to help greatly in uncovering the course of GI diseases. Recently, an increasing number of NRs has been validated as potential drug targets for therapeutic intervention in patients with inflammatory bowel disease (IBD). Besides the classical glucocorticoids, especially PPARγ, VDR, or PXR-selective ligands are currently being tested with promising results in clinical IBD trials. Also, several pre-clinical animal studies are being performed with NRs. This review focuses on the complex biology of NRs and their context-dependent anti- or pro-inflammatory activities in the regulation of gastrointestinal barrier with special attention to NRs already pharmacologically targeted in clinic and pre-clinical IBD treatment regimens

Nuclear orphan receptor NR2F6 directly antagonizes NFAT and RORγt binding to the Il17a promoter

AbstractInterleukin-17A (IL-17A) is the signature cytokine produced by Th17 CD4+ T cells and has been tightly linked to autoimmune pathogenesis. In particular, the transcription factors NFAT and RORγt are known to activate Il17a transcription, although the detailed mechanism of action remains incompletely understood. Here, we show that the nuclear orphan receptor NR2F6 can attenuate the capacity of NFAT to bind to critical regions of the Il17a gene promoter. In addition, because NR2F6 binds to defined hormone response elements (HREs) within the Il17a locus, it interferes with the ability of RORγt to access the DNA. Consistently, NFAT and RORγt binding within the Il17a locus were enhanced in Nr2f6-deficient CD4+ Th17 cells but decreased in Nr2f6-overexpressing transgenic CD4+ Th17 cells. Taken together, our findings uncover an example of antagonistic regulation of Il17a transcription through the direct reciprocal actions of NR2F6 versus NFAT and RORγt

Regulation of Lymphatic GM-CSF Expression by the E3 Ubiquitin Ligase Cbl-b

Genome-wide association studies as well as lymphatic expression analyses have linked both Cbl-b and GM-CSF to human multiple sclerosis as well as other autoimmune diseases. Both Cbl-b and GM-CSF have been shown to play a prominent role in the development of murine encephalomyelitis; however, no functional connection between the two has yet been established. In this study, we show that Cblb knockout mice demonstrated significantly exacerbated severity of experimental autoimmune encephalomyelitis (EAE), augmented T cell infiltration into the central nervous system (CNS) and strongly increased production of GM-CSF in T cells in vitro and in vivo.GM-CSF neutralization demonstrated that the increased susceptibility of Cblb−/− mice to EAE was dependent on GM-CSF. Mechanistically, p50 binding to the GM-CSF promoter and the IL-3/GM-CSF enhancer element “CNSa” was strongly increased in nuclear extracts from Cbl-b-deficient T cells. This study suggests that Cbl-b limits autoimmunity by preventing the pathogenic effects of GM-CSF overproduction in T cells

Protein kinase C theta is dispensable for suppression mediated by CD25+CD4+ regulatory T cells.

The activation of conventional T cells upon T cell receptor stimulation critically depends on protein kinase C theta (PKCθ). However, its role in regulatory T (Treg) cell function has yet to be fully elucidated. Using siRNA or the potent and PKC family-selective pharmacological inhibitor AEB071, we could show that murine Treg-mediated suppression in vitro is independent of PKCθ function. Likewise, Treg cells of PKCθ-deficient mice were fully functional, showing a similar suppressive activity as wild-type CD25+CD4+ T cells in an in vitro suppression assay. Furthermore, in vitro-differentiated wild-type and PKCθ-deficient iTreg cells showed comparable Foxp3 expression as well as suppressive activity. However, we observed a reduced percentage of Foxp3+CD25+ CD4+ T cells in the lymphatic organs of PKCθ-deficient mice. Taken together, our results suggest that while PKCθ is involved in Treg cell differentiation in vivo, it is dispensable for Treg-mediated suppression

Cbl-b mediates TGF\uce\ub2 sensitivity by downregulating inhibitory SMAD7 in primary T cells

T cell-intrinsic transforming growth factor \u3b2 (TGF\u3b2) receptor signaling plays an essential role in controlling immune responses. The RING-type E3 ligase Cbl-b has been shown to mediate the sensitivity of T cells to TGF\u3b2; however, the mechanism underlying this process is unknown. This study shows that SMAD7, an established negative regulator of TGF\u3b2 receptor (TGF\u3b2R) signaling, is a key downstream effector target of Cbl-b. SMAD7 protein levels, but not SMAD7 mRNA levels, are upregulated in cblb-/- T cells. Cbl-b directly interacts with and ubiquitinates SMAD7, suggesting that Cbl-b posttranscriptionally regulates SMAD7. In support of this notion, concomitant genetic loss of SMAD7 in cblb-/- mice restored TGF\u3b2 sensitivity on T cell cytokine responses and abrogated the tumor rejection phenotype of cblb-/- mice. These results demonstrate an essential and non-redundant role for Cbl-b in controlling TGF\u3b2R signaling by directly targeting SMAD7 for degradation during T cell responses in vitro and in vivo

Lasp1 misexpression influences chondrocyte differentiation in the vertebral column

The mouse mutant wavy tail Tg(Col1a1-lacZ)304ng was created through transgene insertion and exhibits defects of the vertebral column. Homozygous mutant animals have compressed tail vertebrae and wedge-shaped intervertebral discs, resulting in a meandering tail. Delayed closure of lumbar neural arches and lack of processus spinosi have been observed; these defects become most prominent during the transition from cartilage to bone. The spina bifida was resistant to folic acid treatment, while retinoic acid administration caused severe skeletal defects in the mutant, but none in wild type control animals. The transgene integrated at chromosome 11 band D, in an area of high gene density. The insertion site was located between the transcription start sites of the RpI23 and Lasp1 genes. LASP1 (an actin binding protein involved in cell migration and survival) was found to be produced in resting and hypertrophic chondrocytes in the vertebrae. In mutant vertebrae, temporal and spatial misexpression of Lasp1 was observed, indicating that alterations in Lasp1 transcription are most likely responsible for the observed phenotype. These data reveal a yet unappreciated role of Lasp1 in chondrocyte differentiation during cartilage to bone transition

A role for the nuclear receptor NR2F6 in peritoneal B cell homeostasis

B cells are key mediators of humoral immunity. Mature B cells fall into various sub-classes that can be separated by their ontogeny, expression of cell surface markers, anatomical location, and function. B1 subsets play important roles in natural immunity and constitute the majority of B cells in newborns. In the adult, B1 cells predominate in the pleural and peritoneal cavities, while the mature B2 follicular subset makes up the major fraction of B cells in lymphoid tissue, although important subsets of antibody-secreting B1 cells are also present at these sites. B1 cells are the main producers of natural IgM but can also contribute to elimination of some pathogens, while B2 cells primarily mediate response to foreign antigens. The differential molecular underpinning of the B1 and B2 subsets remains incompletely understood. Here we demonstrate that germline-deficiency of the orphan nuclear receptor NR2F6 causes a partial loss of B1b and B2 B cells in the peritoneum while leaving peritoneal B1a cells unaltered. A competitive bone marrow chimera in Nr2f6(+/+) host mice produced similar numbers of Nr2f6(+/+) and Nr2f6(-/-) peritoneal B1b and B2 cells. The proliferation of Nr2f6(-/-) peritoneal B cells was not altered, while the migration marker CXCR5 was reduced on all subsets but Beta7-integrin was reduced only on peritoneal B1b and B2 cells. Similarly, B1b and B2 but not B1a cells, exhibited significantly reduced survival

PKCθ/β and CYLD are antagonistic partners in the NFκB and NFAT transactivation pathways in primary mouse CD3+ T lymphocytes.

In T cells PKCθ mediates the activation of critical signals downstream of TCR/CD28 stimulation. We investigated the molecular mechanisms by which PKCθ regulates NFκB transactivation by examining PKCθ/β single and double knockout mice and observed a redundant involvement of PKCθ and PKCβ in this signaling pathway. Mechanistically, we define a PKCθ-CYLD protein complex and an interaction between the positive PKCθ/β and the negative CYLD signaling pathways that both converge at the level of TAK1/IKK/I-κBα/NFκB and NFAT transactivation. In Jurkat leukemic T cells, CYLD is endoproteolytically processed in the initial minutes of stimulation by the paracaspase MALT1 in a PKC-dependent fashion, which is required for robust IL-2 transcription. However, in primary T cells, CYLD processing occurs with different kinetics and an altered dependence on PKC. The formation of a direct PKCθ/CYLD complex appears to regulate the short-term spatial distribution of CYLD, subsequently affecting NFκB and NFAT repressional activity of CYLD prior to its MALT1-dependent inactivation. Taken together, our study establishes CYLD as a new and critical PKCθ interactor in T cells and reveals that antagonistic PKCθ/β-CYLD crosstalk is crucial for the adjustment of immune thresholds in primary mouse CD3(+) T cells

PKCθ is dispensable for iTreg cell differentiation and function.

<p>(A) Naïve CD4⁺ T cells isolated from PKCθ^+/+ and PKCθ^-/- mice were differentiated <i>in vitro</i> under iTreg-inducing conditions (IL-2/TGF-β) and analyzed for Foxp3 and CD25 expression by flow cytometry on day 5 of culture. Representative FACS dot plots of Foxp3 and CD25 staining together with summarizing bar graphs are shown (n = 8). Statistical significance was determined using the Mann-Whitney U test. (B) The suppressive capacity of PKCθ^+/+ and PKCθ^-/- iTreg cells was analyzed in co-cultures with CFSE-labeled CD25^-CD4⁺ T cells (Tresp) stimulated with APCs and anti-CD3 antibodies. Representative histograms of CFSE staining (gated on CFSE⁺ CD4⁺ 7AAD^- cells) are depicted together with summarizing bar graphs of 4 independent experiments. Statistical analysis was determined using Friedman test together with Dunn´s multiple comparison test. ns = not significant.</p