Evaluating the role of RNA binding protein CELF2 in modulating immune cells in colitis

Abstract

Inflammatory bowel disease (IBD) is a diverse set of inflammatory disorders that are primarily mediated by the immune system and affect the gastrointestinal tract. IBD is characterized by activated T cells and cytokine storm. In addition, there is activation of the innate immune system with significant infiltration of neutrophils, macrophages, and dendritic cells to the intestinal mucosa. Alternate splicing is induced by inflammation in macrophages and is important event for T cells activation. RNA binding proteins bind and form ribonucleoprotein (RNP) complexes and regulate mRNA stability, translation and alternate splicing. One such RNA binding protein involved in stability and alternate splicing is the CUG binding protein CELF2 or CUGBP2. CELF2 is a member of a family of proteins, termed CELF(CUG-BP and ETR-3-like factors), that interacts with CUG triplet repeats. Previously we have demonstrated that CELF2 binds to AU-rich sequences in the 3′untranslated region (3′UTR) of cyclooxygenase-2 (COX-2) mRNA with high affinity, and upon binding stabilizes the mRNA. The Cancer Genome Atlas (TCGA) analysis demonstrates that CELF2 expression is reduced in colon cancers in a stage specific manner. However, when we analyzed the expression of CELF2 in DSS colitis by GEO database we found increased expression of CELF2. We teased out the expression of CELF2 in the DSS colitis model by isolating the crypt epithelial and immune cell component of the colon. We used magnetic beads to isolate macrophages (F480+), neutrophils (LY6C +), dendritic cells (CD11c +), along with T cells like CD4+ T cells, CD8+ T cells and Tregs (CD4+, CD25+). We found that after DSS treatment the expression of CELF2 was reduced in the colon epithelial crypt cells but was significantly increased in the immune compartment. CELF2 was increased especially in macrophages and Tregs. This shows that CELF2 plays a role in inflammation by acting in the immune cell compartment. An important role of CELF2 is in alternate splicing of mRNA transcripts to give rise to various isoforms. As such we determined alternate splicing in Treg and macrophages. For this we choose FOXP3 mRNA alternate splicing. Two different alternative splicing events take place in Foxp3 transcript resulting in exclusion of exon 2 (FOXP3Δ2) and exon 7(FOXP3Δ7). FOXP3Δ2 lacks a NES in exon 2 and supports Treg cell-mediated suppression. FOXP3 exon 7 is part of the leucine zipper domain and is required for proper Treg cell function. We found increased splicing of both exon 2 and exon 7 in the Tregs isolated after DSS colitis. Based on these results, we conclude that RNA binding protein CELF2 plays an important role for alternative splicing in immune cell response in colitis.