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 8

The NFAT5/HSP70 axis prevents DSS-induced colitis in mice.

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The NFAT5/HSP70 axis prevents DSS-induced colitis in mice. (A and B) Rel...
(A and B) Relative expression levels of HSPA1B mRNA (A) as well as of HSP70 and NFAT5 protein (B) were assessed in HT-29 cells exposed to 100 mM NaCl (hyperosmotic stimuli) for the specified durations (A) or for 24 hours (B) following transfection with either control siRNA (siCtrl) or NFAT5-targeting siRNA (siNFAT5) for 48 hours. Gapdh mRNA and GAPDH protein served as internal controls. (C) The proliferation of HT-29 cells transfected with either siCtrl or HSP70-targeting siRNA (siHSP70) was evaluated at the indicated time points using MTT and BrdU incorporation assays. (D and E) Body weight changes and the DAI (D) were monitored over 14 days following initial DSS treatment in separately housed Hsp70WT Nfat5+/– and Hsp70IEC-TG Nfat5+/– mice. Representative images of the colons and their respective lengths (E) on day 14 are presented. (F) Representative IHC images and quantification of lysozyme+ cells in ileal tissues from separately housed Hsp70WT Nfat5+/– and Hsp70IEC-TG Nfat5+/– mice are shown. Scale bars: 50 μm. (G) 3D intestinal organoids were established by culturing small intestinal crypts isolated from separately housed Nfat5+/+, Nfat5+/–, and Hsp70IEC-TG Nfat5+/– mice in Matrigel for 5 days. Representative images of organoids, along with graphical analyses of their number and surface area, are presented. Scale bars: 200 μm. (H) Relative mRNA expression levels of TNFA, NFAT5, and HSPA1B were analyzed by qRT-PCR in HT-29 cells that were either left untreated or treated for 4 hours with fecal microbiota derived from Nfat5+/+ and Nfat5+/– mice. Data are presented as the mean ± SD (A and C), the mean ± SEM (D), a line indicating the mean (H), or as box-and-whiskers plots (minimum-to-maximum, median indicated by a line (G). Each dot represents an individual mouse, with the mean values indicated by lines. Data shown in A–H are representative of at least 2 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 2-way, repeated-measures ANOVA with Šídák’s multiple-comparison test (A and C [between cells transfected with siCtrl versus siNFAT5 or siHSP70], D, G, and H) and Mann-Whitney U test (E and F).