TRIM33 deficiency in monocytes and macrophages impairs resolution of colonic inflammation.

BACKGROUND: Mature myeloid cells play a crucial role in Crohn's disease (CD) but the molecular players that regulate their functions in CD are not fully characterized. We and others have shown that TRIM33 is involved in the innate immune response and in the inflammatory response but TRIM33 role in intestinal inflammation is not known. In this study, we investigated the role of TRIM33 in myeloid cells during dextran sulfate sodium (DSS)-induced colitis. METHODS: We study the role of TRIM33 during DSS-induced colitis which mimics intestinal inflammation using mice deleted for Trim33 only in mature myeloid cells (Trim33-/- mice) FINDINGS: We first show that Trim33 mRNA level is decreased in CD patient's blood monocytes suggesting a role of TRIM33 in CD. Using Trim33-/- mice, we show that these mice display an impaired resolution of colonic inflammation with an increased number of blood and colon monocytes and a decreased number of colonic macrophages. Trim33-/- monocytes are less competent for recruitment and macrophage differentiation. Finally, during resolution of inflammation, Trim33-/- colonic macrophages display an impaired M1/M2 switch and express a low level of membrane-bound TNF that is associated with an increased number of colonic neutrophils. INTERPRETATION: Our study shows an important role of TRIM33 in monocytes/macrophages during DSS-induced colitis and suggests that the decreased expression of TRIM33 in CD patient's blood monocytes might not be a consequence but might be involved in CD progression. FUND: La Ligue contre le Cancer (équipe labelisée), INSERM, CEA, Université Paris-Diderot, Université Paris-Sud.

Macrophage production and activation are dependent on TRIM33.

The tripartite motif (TRIM) family of proteins plays important roles in innate immunity and antimicrobial infection. None of these proteins has been shown to directly regulate transcription of genes in monocyte/macrophage except TRIM33 that we have recently shown to be a macrophage specific transcriptional inhibitor of Ifnb1. Using ChIP-seq analyses, we now report that TRIM33 is bound to two fold more genes in immature than in mature myeloid cell lines. When located near the same genes, TRIM33 is bound to different sequences in the two cell lines suggesting a role of TRIM33 in both immature and mature myeloid cells. Accordingly, expression of TRIM33 in immature myeloid cells is necessary for efficient production of small peritoneal macrophages, monocytes and bone marrow derived macrophage (BMDM) and TRIM33 targets a subset of genes involved in the inflammatory response only in mature myeloid cells. Functionally, this targeting is associated with impaired repression of pathways regulating the late phases of lipopolysaccharide (LPS) activation of BMDM and a high sensitivity to LPS in vivo when the trim33 gene is inactivated in mature myeloid cells. These findings pinpoint TRIM33 as an important transcriptional actor of monocyte/macrophage mediated inflammation.

TRIM33 switches off Ifnb1 gene transcription during the late phase of macrophage activation.

Despite its importance during viral or bacterial infections, transcriptional regulation of the interferon-ß gene (Ifnb1) in activated macrophages is only partially understood. Here we report that TRIM33 deficiency results in high, sustained expression of Ifnb1 at late stages of toll-like receptor-mediated activation in macrophages but not in fibroblasts. In macrophages, TRIM33 is recruited by PU.1 to a conserved region, the Ifnb1 Control Element (ICE), located 15 kb upstream of the Ifnb1 transcription start site. ICE constitutively interacts with Ifnb1 through a TRIM33-independent chromatin loop. At late phases of lipopolysaccharide activation of macrophages, TRIM33 is bound to ICE, regulates Ifnb1 enhanceosome loading, controls Ifnb1 chromatin structure and represses Ifnb1 gene transcription by preventing recruitment of CBP/p300. These results characterize a previously unknown mechanism of macrophage-specific regulation of Ifnb1 transcription whereby TRIM33 is critical for Ifnb1 gene transcription shutdown.

Identification of TMEM131L as a novel regulator of thymocyte proliferation in humans.

In this study, we identify transmembrane protein 131-like (TMEM131L) as a novel regulator of thymocyte proliferation and demonstrate that it corresponds to a not as yet reported inhibitor of Wnt signaling. Short hairpin RNA-mediated silencing of TMEM131L in human CD34(+) hematopoietic progenitors, which were then grafted in NOD-SCID/IL-2rγ(null) mice, resulted in both thymocyte hyperproliferation and multiple pre- and post-ß-selection intrathymic developmental defects. Consistent with deregulated Wnt signaling, TMEM131L-deficient thymocytes expressed Wnt target genes at abnormally high levels, and they displayed both constitutive phosphorylation of Wnt coreceptor LRP6 and ß-catenin intranuclear accumulation. Using T cell factor reporter assays, we found that membrane-associated TMEM131L inhibited canonical Wnt/ß-catenin signaling at the LRP6 coreceptor level. Whereas membrane-associated TMEM131L did not affect LRP6 expression under basal conditions, it triggered lysosome-dependent degradation of its active phosphorylated form following Wnt activation. Genetic mapping showed that phosphorylated LRP6 degradation did not depend on TMEM131L cytoplasmic part but rather on a conserved extracellular domain proximal to the membrane. Collectively, these data indicate that, during thymopoiesis, stage-specific surface translocation of TMEM131L may regulate immature single-positive thymocyte proliferation arrest by acting through mixed Wnt-dependent and -independent mechanisms.

AF1q/MLLT11 regulates the emergence of human prothymocytes through cooperative interaction with the Notch signaling pathway.

The mechanisms regulating the emergence of BM prothymocytes remain poorly characterized. Genome-wide transcriptome analyses looking for genes expressed in human prothymocytes led to the identification of AF1q/MLLT11 as a candidate gene conceivably involved in this process. Analysis of AF1q protein subcellular localization and intracellular trafficking showed that despite pronounced karyophily, it was subjected to constitutive nuclear export followed by ubiquitin-mediated degradation in the centrosomal area. Using in vitro assays based on either forced expression or shRNA-mediated silencing of AF1q, we provide evidence that the protein promotes T- over B-cell differentiation in multipotent hematopoietic progenitors. At the molecular level, AF1q confers to multipotent progenitors an increased susceptibility to Delta-like/Notch-mediated signaling. Consistent with these findings, enforced AF1q expression in humanized mice fosters the emergence of BM CD34(+)CD7(+) prothymocytes, enhances subsequent thymus colonization, and accelerates intrathymic T-cell development. In contrast, AF1q silencing provokes a global shift of BM lymphopoiesis toward the B-cell lineage, hinders prothymocyte development, inhibits thymus colonization, and leads to intrathymic accumulation of B cells. Our results indicate that AF1q cooperates with the Notch signaling pathway to foster the emergence of BM prothymocytes and drive subsequent intrathymic specification toward the T-cell lineage.