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Aim: Matrix metalloproteinase-9 (MMP9) plays a profibrotic role in renal fibrosis. Neutrophils produce MMP9 in many pathologic models. However, the effect of neutrophil on the progression of renal fibrosis and the relationship of MMP9 to the infiltration of neutrophils into the kidney remain unknown. Methods: The surgery of unilateral ureter obstruction (UUO) was performed in male C57BL/6 mice. Kidneys were collected for analyses on days 0, 1, 3, 5 or 7 following surgery. The inflammatory cells were analysed by flow cytometry. The mRNA and protein levels of renal fibrosis factor and inflammatory factor were measured by qRT-PCR, immumofluorescence and western blot analysis. Results: In a mouse kidney model of UUO, neutrophil infiltration significantly increased and neutrophil accumulation reached the highest level at 5 days after the injury. In the obstructed kidney, depleting neutrophils decreased the expression of inflammatory factors, inhibited the accumulation of macrophages including type 2 macrophages and suppressed renal fibrosis. Almost all neutrophils produced MMP9 at the early stage of kidney obstruction. MMP9 attracted neutrophils and inflammatory cells because inhibiting MMP9 suppressed the infiltration of neutrophils and other inflammatory cells and reduced renal fibrosis, regardless of using MMP9 neutralizing antibody or MMP9 inhibitor or different intervening periods of days (0-6, 0-3 or 3-6 were applied after kidney obstruction). Conclusion: MMP9 promotes neutrophil infiltration by increasing the inflammatory level, macrophage accumulation and renal fibrosis in the obstructed kidney.Inhibiting MMP9 or depleting neutrophils in the early stage of acute kidney injury can relieve the progression of kidney fibrosis. K E Y W O R D Sfibrosis, inflammation, kidney, macrophage, neutrophil 2 of 13 | WANG et Al.

T cell-mediated immunity in the intestine is stringently controlled to ensure proper immunity against pathogenic microbes and to prevent autoimmunity, a known cause of inflammatory bowel disease. However, precisely how T cells regulate intestine immunity remains to be fully understood. In this study, we found that mitogen-activated protein kinase kinase kinase 2 (MAP3K2) is required for the CD4 + T cell-mediated inflammation in the intestine. Using a T cell transfer colitis model, we found that MAP3K2-deficient naïve CD4 + T cells had a dramatically reduced ability to induce colitis compared to wild type T cells. In addition, significantly fewer IFN-γ-but more IL-17A-producing CD4 + T cells in the intestines of mice receiving MAP3K2deficient T cells than in those from mice receiving wild type T cells was observed. Interestingly, under well-defined in vitro differentiation conditions, MAP3K2-deficient naïve T cells were not impaired in their ability to differentiate into Th1, Th17 and Treg. Furthermore, the MAP3K2-regulated colitis severity was mediated by Th1 but not Th17 cells in the intestine. At the molecular level, we showed that MAP3K2-mediated Th1 cell differentiation in the intestine was regulated by IL-18 and required specific JNK activation. Together, our study reveals a novel regulatory role of MAP3K2 in intestinal T cell immunity via the IL-18-MAP3K2-JNK axis and may provide a novel target for intervention in T cell-mediated colitis.

The mammalian intestine is an organ that can be spatially defined by two axes: longitudinal and vertical. Such anatomical structure ensures the maintenance of a relatively immuno-quiescent and proliferation-promoting crypt for intestinal stem cell differentiation while actively warding off the invading intestinal microbes at the villus tip during digestion and nutrient absorption. Such behavior is achieved by the fine coordination among intestinal epithelial cells, intestinal mesenchymal stromal cells and tissue-resident immune cells like myeloid cells and lymphocytes. Among these cell types resided in the colon, intestinal mesenchymal stromal cells are considered to be the essential link between epithelium, vasculature, neuronal system, and hematopoietic compartment. Recent advancement of single cell and spatial transcriptomics has enabled us to characterize the spatial and functional heterogeneity of intestinal mesenchymal stromal cells. These studies reveal distinctive intestinal mesenchymal stromal cells localized in different regions of the intestine with diverse functions including but not limited to providing cytokines and growth factors essential for different immune cells and epithelial cells which predict niche formation for immune function from the villus tip to the crypt bottom. In this review, we aim to provide an overall view of the heterogeneity of intestinal mesenchymal stromal cells, the spatial distribution of these cells along with their interaction with immune cells and the potential regulatory cytokine profile of these cell types. Summarization of such information may enrich our current understanding of the immuno-regulatory functions of the newly identified mesenchymal stromal cell subsets beyond their epithelial regulatory function.

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, fibroblasts, myofibroblasts, smooth muscle cells, immune cells, and the recently identified cell type, telocytes. Telocytes are unique mesenchymal cells with long cytoplasmic processes, reaching a distance of tens to hundreds of microns from the cell body. Telocytes have recently emerged as an important intestinal stem cell niche component, providing Wnt proteins that are essential for stem and progenitor cell proliferation.Although protocols on how to isolate mesenchyme from the mouse intestine are available, it is not clear whether these procedures allow the efficient isolation of telocytes. Isolating telocytes efficiently requires special protocol adjustments that would allow dissociation of the strong cell-cell contact between telocytes and neighboring cells without affecting their viability. Here, available intestinal mesenchyme isolation protocols were adjusted to support the successful isolation and culture of mesenchyme containing a relatively high yield of viable single-cell telocytes.The obtained single-cell suspension can be analyzed by several techniques, such as immunostaining, cell sorting, imaging, and mRNA experiments. This protocol yields mesenchyme with sufficiently conserved antigenic and functional properties of telocytes, and can be used for several applications. For example, they can be used for co-culture with mouse-or human-derived organoids to support organoid growth with no growth factor supplementation, to better reflect the situation in the original tissue.

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