Project description:Purpose: RNA-sequencing analysis of Th17 signature genes. The goals of this study are to compared the gene expression between wild type (WT) and lncRNA-GM-/- Th17 cells by RNA-seq analysise.
Project description:T-helper 17 (Th17) cells play a dual role in immunological responses, serving as essential components in tissue homeostasis and host defense against microbial pathogens, while also contributing to proinflammatory conditions and autoimmunity. While Transforming Growth Factor-beta 1 (TGFβ1) is pivotal for the differentiation of non-pathogenic Th17 cells, the role of TGFβ3 and Activin in steering Th17 cells toward a pathogenic phenotype has been acknowledged. However, the molecular mechanisms governing this dichotomy remain elusive. In this study, we demonstrate that the transcription factor Foxo1 is upregulated in a TGFβ1 dose-dependent manner, serving as a critical regulator that specifically modulates the fate of pathogenic Th17 cells. Analyses in both uveitis patients and an Experimental Autoimmune Uveitis (EAU) mouse model reveal a strong correlation between disease severity and diminished Foxo1 expression levels. Ectopic expression of Foxo1 selectively attenuates IL-17A production under pathogenic Th17-inducing conditions. Moreover, enhanced Foxo1 expression, triggered by TGFβ1 signaling, is implicated in fatty acid metabolism pathways that favor non-pathogenic Th17 differentiation. Our drug screening identifies several FDA-approved compounds capable of upregulating Foxo1. Collectively, our findings offer compelling evidence that Foxo1 serves as a molecular switch to specifically control pathogenic versus non-pathogenic Th17 differentiation in a TGFβ1-dependent manner. These insights suggest that targeting Foxo1 could be a promising therapeutic strategy for autoimmune diseases, offering efficacy without compromising immune homeostasis.
Project description:Tight regulation of Toll-like receptor (TLR)-mediated inflammatory responses is important in innate immunity. Here, we show that T cell death-associated gene 51 (TDAG51/PHLDA1) is a novel coactivator of the transcription factor FoxO1, regulating inflammatory mediator production in the lipopolysaccharide (LPS)-induced inflammatory response. TDAG51 induction by LPS stimulation was mediated by the TLR2/4 signaling pathway in bone marrow-derived macrophages (BMMs). LPS-induced inflammatory mediator production was significantly decreased in TDAG51-deficient BMMs. In TDAG51-deficient mice, LPS- or pathogenic Escherichia coli infection-induced lethal shock was reduced by decreasing serum proinflammatory cytokine levels. The recruitment of 14-3-3 to FoxO1 was competitively inhibited by the TDAG51-FoxO1 interaction, leading to blockade of FoxO1 cytoplasmic translocation and thereby strengthening FoxO1 nuclear accumulation. TDAG51/FoxO1 double-deficient BMMs showed significantly reduced inflammatory mediator production compared with TDAG51- or FoxO1-deficient BMMs. TDAG51/FoxO1 double deficiency protected mice against LPS- or pathogenic E. coli infection-induced lethal shock by weakening the systemic inflammatory response. Thus, these results indicate that TDAG51 acts as a coactivator of the transcription factor FoxO1, leading to strengthened FoxO1 activity in the LPS-induced inflammatory response.
Project description:Th17 cells are key players in autoimmune diseases. However, the roles of non-coding RNAs in Th17 cells are largely unknown. Here, we show that deletion of the Dicer gene specifically in Th17 cells protects from experimental autoimmune encephalomyelitis (EAE). Th17 cells highly express the miR-183/96/182 cluster (miR-183C), in response to IL-6/STAT3 signaling. Moreover, miR-183C regulates pathogenic cytokine expression during Th17 development. Furthermore, transcription factor Foxo1 is one of functional targets of miR-183C in Th17 cells: Foxo1 negatively regulates the pathogenicity of Th17 cells and miR-183C represses Foxo1 expression. Collectively, our results demonstrate one of crucial roles for miR-183C cluster in regulation of Th17 cell function in autoimmune diseases.
Project description:PAX3-FOXO1 is a fusion transcription factor that is the main driver of tumorigenesis leading to the development of alveolar rhabdomyosarcoma (aRMS). Since aRMS cells are addicted to PAX3-FOXO1 activity, the fusion protein also represents a major target for therapeutic interference, which is however challenging as transcription factors usually cannot be inhibited directly by small molecules. Hence, characterization of the biology of PAX3-FOXO1 might lead to the discovery of new possibilities for an indirect inhibition of its activity. Here, our goal was to characterize the proteomic neighborhood of PAX3-FOXO1 and to find candidates potentially affecting its activity and tumor cell viability. Towards this aim, we expressed BirA fused versions of PAX3-FOXO1 (N- and C-terminal) in HEK293T cells under presence of biotin. In the control setup, we expressed the BirA enzyme alone. After Streptavidin purification of biotinylated proteins, we performed mass spectrometry and quantified relative abundances compared to control conditions. This enabled us to determine PAX3-FOXO1 proximal proteins, which we investigated further in orthogonal endogenous systems.
Project description:In this study, we first analyzed the serological repertoire of anti-GM-CSF autoantibodies in a patients serum with autoimmune pulmonary alveolar proteinosis, using the trapped ion mobility spectrometry coupled with quadrupole time-of-flight (timsTOF) Pro mass spectrometry. The timsTOF Pro identified peptides that partially matched sequences in up to 96 percent of cDNA clones. Complementarity-determining region 3 (CDR3), is the most diverse in terms of sequence, was fully and partially detected in nine and 132 clones, respectively. Moreover, we confirmed one unique framework region 4 (FR4) and at least three unique across CDR3 to FR4 peptides via de novo peptide sequencing. This new technology may thus permit the comprehensive identification of polyclonal autoantibody structure.
Project description:Analysis of transcriptional changes upon silencing of endothelial VEGF. Because transcription factor Foxo1 levels increase under VEGF silencing, we hypothesized that Foxo1 transcriptional activity would be apparent in a whole-genome microarray. Double-silencing of VEGF+Foxo1 was also performed to determine which transcriptional changes are Foxo1-dependent in a VEGF-deficient background.
Project description:GM-CSF is involved in immune complex (IC)-mediated arthritis. However, little is known about what is the cellular source of GM-CSF and how it is regulated during IC-mediated inflammation. Using novel GM-CSF reporter mice, we show that NK cells produce GM-CSF during an IC-mediated model of inflammatory arthritis. NK cells promoted STIA in a GM-CSF-dependent manner, as deletion of NK cells and selective removal of GM-CSF production by NK cells abrogated disease. Furthermore, we show that myeloid cell activation by GM-CSF is restrained by induction of JAK/STAT checkpoint inhibitor cytokine-inducible SH2-containing protein, CIS. Myeloid cells from CIS-deficient mice had exaggerated responses to GM-CSF, and these mice develop exacerbated STIA. Our data suggest that tissue NK cells may amplify joint inflammation in arthritis via GM-CSF production and thus represent a novel target in IC-mediated pathology. Endogenous CIS provides a key brake on signaling through the GM-CSF receptor and strategies that boost its function may provide an alternative anti-inflammatory approach.