NFAT5 dictates crosstalk between intestinal epithelial regenerative capacity and microbiota in murine colitis models
Se-Hyeon Park, Dae Hee Cheon, Yu-Mi Kim, Yeji Choi, Yong-Joon Cho, Bong-Ki Hong, Sang-Hyun Cho, Mi-Na Kweon, Hyug Moo Kwon, Eugene B. Chang, Donghyun Kim, Wan-Uk Kim
Se-Hyeon Park, Dae Hee Cheon, Yu-Mi Kim, Yeji Choi, Yong-Joon Cho, Bong-Ki Hong, Sang-Hyun Cho, Mi-Na Kweon, Hyug Moo Kwon, Eugene B. Chang, Donghyun Kim, Wan-Uk Kim
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Research Article Gastroenterology Immunology

NFAT5 dictates crosstalk between intestinal epithelial regenerative capacity and microbiota in murine colitis models

Abstract

Hypertonic and hyperosmolar stimuli frequently pose challenges to the intestinal tract. Therefore, a resilient epithelial barrier is essential for maintaining gut homeostasis in the presence of osmotic perturbations. Nuclear factor of activated T cells 5 (NFAT5), an osmosensitive transcription factor, primarily maintains cellular homeostasis under hypertonic conditions. However, the osmoprotective role of NFAT5 in enterocyte homeostasis is poorly understood. Here, we demonstrate that NFAT5 was critical for the survival and proliferation of intestinal epithelial cells (IECs) and that its deficiency accelerated chemically induced or spontaneous colitis in mice. Mechanistically, NFAT5 promoted the survival of IECs and the renewal of intestinal stem cells, thereby regulating the production of mucus and antimicrobial compounds, including RegIII and lysozyme, which consequently shape the gut microbial composition to prevent colitis. Transcriptome analysis identified HSP70 as a key downstream target of NFAT5 in epithelial regeneration. Loss- and gain-of-function experiments involving HSP70 revealed that NFAT5 mitigated experimental colitis through IEC Hsp70, which protected stem cells from inflammation-induced injury and maintained barrier function. In conclusion, our study demonstrates what we believe to be a previously unknown role for NFAT5 in dictating the crosstalk between intestinal stem cells and the microbiota, underscoring the importance of the NFAT5/HSP70 axis in maintaining epithelial regeneration related to gut barrier function, balancing microbial composition, and subsequently preventing colitis progression.

Authors

Se-Hyeon Park, Dae Hee Cheon, Yu-Mi Kim, Yeji Choi, Yong-Joon Cho, Bong-Ki Hong, Sang-Hyun Cho, Mi-Na Kweon, Hyug Moo Kwon, Eugene B. Chang, Donghyun Kim, Wan-Uk Kim

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Figure 1

NFAT5 promotes the proliferation and survival of IECs.

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NFAT5 promotes the proliferation and survival of IECs. (A) Representativ...
(A) Representative images and a corresponding graph of immunofluorescence staining of ileal tissue from Nfat5+/+ and Nfat5+/– mice for NFAT5 (red) and E-cadherin (white). Nuclei are counterstained with DAPI (blue). Scale bars: 20 μm. (B and C) Proliferation of HT-29 cells transfected with siCtrl or siNFAT5 was assessed at the indicated time points using trypan blue exclusion (left), MTT (middle), and BrdU incorporation assays (right) (B). Representative images of Ki-67 immunocytochemistry and quantification of Ki-67+ cells after 48 hours of transfection with siCtrl or siNFAT5 are shown (C). Scale bars: 100 μm. (D) Flow cytometric analysis of annexin V and PI staining of HT-29 cells transfected with siCtrl or siNFAT5 for 48 hours, followed by 24 hours of treatment with thapsigargin, tunicamycin, or butyrate. The total frequency of apoptotic and necrotic cells is shown (annexin V–/PI–, live cells; annexin V–/PI+, necrotic cells; annexin V+/PI–, early apoptotic cells; annexin V+/PI+, late apoptotic cells). (E and F) Proliferating cells in distal colonic tissues of Nfat5+/+ and Nfat5+/– mice were assessed by EdU-incorporation assay (E) and Ki-67 IHC (F). EdU+ cells were detected after intraperitoneal injection of 1 mg EdU at the indicated time points. Representative images and quantified data are shown. Scale bars: 20 μm (E) and 50 μm (F). (G) Apoptotic cells were determined by cleaved caspase 3 IHC in proximal colonic tissues of Nfat5+/+ and Nfat5+/– mice after 3 days of DSS treatment. Representative images and quantification of cleaved caspase 3+ cells are shown. Scale bars: 100 μm (top panels) and 20 μm (bottom panels). Each dot represents an individual mouse, and the means are displayed as a line (A and E–G). Data are presented as the mean ± SD (B) and as a line indicating the mean (C). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Mann-Whitney U test (A, C, and E–G) and 2-way, repeated-measures ANOVA with Šídák’s multiple-comparison test (between cells transfected with siCtrl and siNFAT5; B and D). Data shown in A–G are representative of at least 2 independent experiments.