Conditional mutagenesis using site-specific recombinases is widely used in transgenic mouse models; however, its efficiency in vitro is limited by poor tamoxifen activity. Here, we present an optimized in vitro system for Cre-mediated loxP/loxP deletion in primary colonic fibroblasts. First, we describe steps for isolating, culturing, and monitoring murine colonic fibroblasts. Second, we detail procedures for inducing recombination using transducible TAT-Cre recombinase, enabling controlled gene activation or deletion in vitro. For complete details on the use and execution of this protocol, please refer to Chulkina et al.1.

Telocytes, a novel mesenchymal cell population, are characterized by their distinctive long and slender projections known as telopodes and have garnered significant interest since their formal introduction to the literature in 2010. These cells have been identified in various tissues, including the gastrointestinal (GI) tract, where they are suggested to play important roles in maintaining structural integrity, immune modulation, and barrier function. Inflammatory bowel diseases (IBD), which include Crohn’s disease (CD) and ulcerative colitis (UC), are characterized by chronic inflammation and fibrosis. While limited information is available on the fate of telocytes in this group of diseases, it has been suggested that loss/plasticity of telocytes can be among the key factors contributing to their pathogenesis. This review focuses on the current understanding of telocytes, their structural features, and their distribution within the GI tract under gut homeostasis and IBD. We also discuss the roles of these cells in immune regulation and intestinal repair. We highlight evidence implicating telocytes in the pathogenesis of IBD and other chronic inflammatory diseases that share similar pathophysiological processes with IBD. Lastly, we discuss the current challenges in gut telocyte biology and the potential therapeutic implications of telocytes in IBD.

The development of Inflammatory Bowel Disease (IBD) involves increases in the inflammatory immune response to dysbiotic gut microbiota in genetically-susceptible individuals. Bacterial sensing by Toll-like receptors and downstream MyD88 signaling is critical for maintaining intestinal homeostasis and is involved in the pathogenesis of intestinal inflammation relevant to IBD. Mesenchymal stromal cells, particularly fibroblasts, have recently emerged as key regulators of macrophages. MyD88 is suggested to shape fibroblast responses in the intestinal microenvironment and may be involved in macrophage activity. However, the role of fibroblast-restricted MyD88 signaling in regulating macrophages host defense in the gut, and its role in IBD development remains unclear. Thus, the objective of this study was to examine the role of fibroblast-intrinsic MyD88 signaling in the development of intestinal inflammation relevant to IBD and in the regulation of antimicrobial activity in macrophages. Fibroblast-specific MyD88-conditional KO mice (MyD88KO-Fib) in wild type, Rag1KO, and IL-10KO backgrounds were used in this study. Bulk RNAseq and 16sRNAseq were used to analyze changes in the mucosal transcriptome and microbiome composition in MyD88KO-Fib mice, respectively. Changes in the activity of myeloid cells were analyzed by flow cytometry. MyD88-dependent interactions between fibroblasts and macrophages were analyzed in co-culture. We found that mice lacking MyD88 in fibroblasts showed a decrease in the colonic antimicrobial defense, developing dysbiosis and aggravated DSS-induced colitis. These pathological changes were associated with an accumulation of Arginase1+ macrophages with low antimicrobial defense capability. These processes did not require mature B and T cells. Furthermore, depletion of MyD88 signaling in colonic fibroblasts prior to the onset of chronic colitis in murine model of IL-10KO colitis led to increased infiltration of myeloid cells and increased transcription of proinflammatory factors in colonic mucosa. Priming of bone marrow-derived monocytes with colonic fibroblasts lacking MyD88 resulted in the generation of macrophages with low production of antimicrobial molecules and decreased bactericidal function. Mechanistically, the production of IL6 and CCL2 downstream of MyD88 was critically involved in the fibroblast-mediated support of macrophage-intrinsic antimicrobial function. Additionally, IL-6/CCL2 neutralization resulted in the generation of macrophages with decreased production of antimicrobial peptide cathelicidin and impaired bacterial clearance. Thus, fibroblast-intrinsic MyD88 signaling contributes to colonic homeostasis by regulating macrophage-mediated host defense, and its disruption drives the severity of IBD-relevant colitis.

Three novel therapies have shown promise in managing moderate-to-severe ulcerative colitis (UC): fecal microbiota transplantation (FMT), mesenchymal stem cell (MSC) therapy, and targeting the JAK/STAT signaling pathway. Individually, these treatment approaches face issues with either safety concerns or limited efficacy due to disease relapses. Yet, the development of better strategies is hampered by limited knowledge about the cellular mechanisms driving chronic inflammation and disease relapse in UC. In UC, the number of aberrantly differentiated tissue resident MSCs in the colonic mucosa is increased. The mechanisms behind this abnormal MSC differentiation and how they affect MSC therapy in UC remain unclear. JAK2 signaling is involved in the differentiation of bone marrow derived (BM)-MSCs and previously we observed that stimulation of BM-MSCs with UC relevant microbial ligand LPS upregulated JAK2. We hypothesized that dysbiotic microbial ligands in UC promote aberrant MSC differentiation through JAK2 activation, reducing the efficacy of MSC therapy. Mesenchymal progenitor cells were isolated from human UC tissue. Wild-type and MyD88 mesenchymal lineage conditional knockout mice (Grem1CreMyD88KO) were used in two models of experimental colitis (TNBS-induced and oxazolone-induced colitis). Murine FMT and syngeneic MSC transplant were also used in these studies. Analysis of publicly available single-cell RNA seq data showed increased numbers of mesenchymal cells co-expressing stem cell (POU5F1) and progenitor markers (Grem1), along with JAK2, in inflamed colon tissue from UC patients. Bulk RNA seq analysis of healthy colonic mesenchymal cells from normal tissue stimulated with the Gram-negative bacteria-derived TLR4 ligand, LPS, revealed upregulation of inflammatory genes IL-6, IL-33, PD-L1, and JAK2 signaling. Deletion of MyD88 in MSCs during the acute phase of TNBS-induced colitis improved symptoms and decreased UC-relevant inflammation concomitantly with downregulation of JAK2. In the oxazolone-induced model of colitis, depletion of dysbiotic microbiota with antibiotics and subsequent FMT resulted in reduced JAK2 expression in the colonic mucosa, and when combined with MSC therapy shown superior efficacy in improvement of clinical sign of colitis, improvement of colonic tissue architecture and reduction of UC-relevant inflammatory responses when compared to FMT or MSCs therapy alone. Dysbiotic microbial ligands in UC contribute to the generation of abnormally differentiated MSCs with active JAK2 signaling and downstream molecules supporting inflammation in UC and this process involves MSC intrinsic MyD88 signaling. Further, our data suggests depletion of the dysbiotic microbiota in combination of FMT/MSCs therapy could counteract this effect and may offer a more effective approach to treating UC.

Fibroblasts that reside in the gut mucosa are among the key regulators of innate immune cells, but their role in the regulation of the defense functions of macrophages remains unknown. MyD88 is suggested to shape fibroblast responses in the intestinal microenvironment. We found that mice lacking MyD88 in fibroblasts showed a decrease in the colonic antimicrobial defense, developing dysbiosis and aggravated dextran sulfate sodium (DSS)-induced colitis. These pathological changes were associated with the accumulation of Arginase 1+ macrophages with low antimicrobial defense capability. Mechanistically, the production of interleukin (IL)-6 and CCL2 downstream of MyD88 was critically involved in fibroblast-mediated support of macrophage antimicrobial function, and IL-6/CCL2 neutralization resulted in the generation of macrophages with decreased production of the antimicrobial peptide cathelicidin and impaired bacterial clearance. Collectively, these findings revealed a critical role of fibroblast-intrinsic MyD88 signaling in regulating macrophage antimicrobial defense under colonic homeostasis, and its disruption results in dysbiosis, predisposing the host to the development of intestinal inflammation.

Background CD4+ T cells contribute to chronic inflammation and fibrosis in inflammatory bowel disease (IBD), but the cellular mechanisms remain elusive. We have found that the mitogen-activated protein kinase 2 (MK2) pathway plays a major role in inflammation and overall pathology in IBD. Thus, here, we examined the role of MK2 in regulating CD4+ T cell responses in IBD models. Methods Interleukin-10 (IL-10) knockout (KO) mice treated with MK2 inhibitors (MK2i) and CD4-specific MK2 knockdown mice treated with chronic dextran sodium sulfate (DSS) treatments were used to examine inflammation and fibrosis by multiplex array, gene expression, flow cytometry, and histology. Human tissues were treated with MK2i to examine Th1 and Th17 markers. Results IL-10 KO mice treated with MK2i therapeutically showed significantly reduced interferon gamma (IFNγ) and interleukin-17A (IL-17A) and a significantly reduced number of IFNγ+ and IL-17A+ producing CD4+ T cells by flow cytometry. To investigate the direct role of MK2 in CD4+ T cells during IBD, we utilized CD4-specific MK2 knockdown mice in chronic DSS colitis. A decrease in colonic inflammation, IFNγ and IL-17, pro-fibrotic genes, and extracellular matrix deposition was observed in mice with MK2 knockdown in CD4+ T cells compared to control mice. Additionally, IL-17A and IFNγ directly regulated the expression of fibrosis genes in colon tissues. Conclusions The MK2 pathway regulates inflammatory CD4+ T cells and fibrosis in IBD models and is a potential therapeutic target.

Background Mesenchymal stromal cells are suggested to play a critical role in Crohn's disease [CD]-associated fibrosis. MAPKAPK2 [MK2] has emerged as a potential therapeutic target to reduce inflammation in CD. However, the cell-specific pattern of phospho-MK2 activation and its role in CD-associated fibrosis are unknown. The objectives of this study were to evaluate cell-specific changes in MK2 activity between predominantly inflammatory CD vs CD with fibrotic complications and define the role of stromal cell-specific MK2 activation in CD-associated fibrosis.Methods CD tissue, CD tissue-derived mesenchymal stromal cells known as myo-/fibroblasts [CD-MFs], and fibroblast-specific MK2 conditional knockout [KO] mice were used.Results In the inflamed area of predominantly inflammatory CD, high MK2 activity was equally distributed between mesenchymal and haematopoietic cells. By contrast, in CD with fibrotic complications, high MK2 activity was mostly associated with mesenchymal stromal cells. Using ex vivo CD tissue explants and an IL-10KO murine colitis model, we demonstrated that pro-fibrotic responses are significantly reduced by treatment with the MK2 inhibitor PF-3644022. Inhibition of MK2 activity in primary cultures of CD-MFs significantly reduced basal and TGF-beta 1-induced profibrotic responses. Using fibroblast-specific MK2 knockout mice in chronic dextran saline sulphate colitis, we demonstrated that fibroblast intrinsic MK2 signalling is among the key processes involved in the chronic inflammation-induced profibrotic responses.Conclusions Our data suggest that activation of MK2 within fibroblasts contributes to the chronic inflammation-induced fibrosis in CD and that targeting MK2 has potential for the development of novel therapeutic approaches for fibrosis in CD. Graphical Abstract