Intestinal Epithelium Regeneration Boosted by NFAT5

NFAT5 deficiency reduces IEC survival and proliferation. We first found that NFAT5 protein, which is known to be expressed in various cell types, including those of the kidney, cancer cells, and immune cells (23), was also expressed in the intestinal epithelium of WT mice (Figure 1A). However, its expression was significantly downregulated in the intestinal epithelium of Nfat5-deficient (Nfat5+/–) mice, as shown by a comparative analysis of intestinal tissues from Nfat5+/– mice and WT littermates via immunofluorescence staining (Figure 1A). Given that complete KO of Nfat5 is lethal (24), we used haploinsufficient mice, in which a single Nfat5 allele is deleted. NFAT5 is required for the survival and proliferation of diverse types of cells, including kidney epithelial cells, cancer cells, T cells, and macrophages (15, 17, 19, 25). To determine the involvement of NFAT5 in IEC functions, we downregulated its expression in the human colorectal adenocarcinoma cell lines HT-29 and Caco-2 using siRNAs (Supplemental Figure 1, A and B; supplemental material available online with this article; https://doi.org/10.1172/JCI183093DS1). Under normal culture conditions, the NFAT5-knockdown cells had a significantly lower rate of cell proliferation than did the untransfected or scrambled siRNA–transfected cells (Figure 1, B and C, and Supplemental Figure 1, C and D). In the presence of endoplasmic reticulum (ER) stress inducers, such as thapsigargin and tunicamycin, and of apoptotic cell death inducers, such as sodium butyrate, NFAT5 knockdown rendered the cells significantly more susceptible to cell death (Figure 1D and Supplemental Figure 1E). Moreover, consistent with these results, 5-ethynyl-2′-deoxyuridine (EdU) pulse labeling and immunostaining for Ki-67 and cleaved caspase 3 revealed reduced IEC proliferation and a higher number of apoptotic cells in the colon epithelia of Nfat5+/– mice compared with their WT littermates (Figure 1, E–G). These findings indicate an intrinsic role of NFAT5 in IEC proliferation and survival.

Nfat5 knockdown accelerates experimental colitis in mice. On the basis of the in vitro results, we investigated the role of NFAT5 in the progression of intestinal mucosal injury. To this end, we used dextran sulfate sodium (DSS) to induce colitis in Nfat5-haploinsufficient (Nfat5+/–) mice and their WT (Nfat5+/+) littermates. The Nfat5+/– mice showed more severe body weight loss, had a higher disease activity index (DAI), and had shorter colons than did the control littermates (Figure 2, A and B). Consistent with these results, we observed a more severe pathology in the distal colons of Nfat5-deficient mice (Figure 2C). The colonic tissues of these mice also showed significantly higher mRNA and protein expression of proinflammatory cytokines, such as Il1b, Il6, Il17a, and Tnfa, than did the tissues of their WT counterparts (Figure 2, D–F). Nfat5 knockdown consistently led to a marked loss of proliferating epithelial cells and mucus-producing cells in mice with DSS-induced colitis (Figure 2, G and H), as determined by Ki-67 and Alcian blue staining, respectively. Together, these results suggest that NFAT5 contributed to protection against the progression of experimental colitis.

Both epithelial NFAT5 levels and gut microbiota influence the progression of DSS-induced colitis. Crosstalk between the gut microbiota and IECs is essential for maintaining gut homeostasis (2). To determine the role of the gut microbiota in the deterioration of colitis in Nfat5-deficient mice, we first compared the severity of DSS-induced colitis by evaluating weight loss and the DAI score for mice that were either housed separately or cohoused for more than 6 weeks. As expected, we noted more severe colitis symptoms in Nfat5-deficient mice than in their control littermates when they were housed separately (Supplemental Figure 2, A, B, and E). However, when cohoused, the 2 groups showed no significant differences (Supplemental Figure 2, C, D, and F). Similarly, mortality was higher for Nfat5+/– mice than for WT mice when housed separately, but this was almost completely reversed by cohousing the 2 groups (Supplemental Figure 2G). Consistent with this, the DSS-induced increase in gut permeability, as determined by a FITC-dextran permeability assay in Nfat5+/– mice, was completely restored through cohousing (Supplemental Figure 2, H and I). Collectively, the comparison of data between separately housed and cohoused mice suggests that gut microbiota is necessary for protection against DSS-induced colitis aggravated by Nfat5 deficiency.

Next, to clarify the role of NFAT5 in IECs during DSS-induced colitis, we generated conditional-KO mice lacking Nfat5 specifically in IECs using the Cre/loxP system. As expected, we confirmed that in the Vil-Cre Nfat5fl/fl (Nfat5IEC-KO) mice, Nfat5 mRNA levels were reduced only in IECs and not in mesenteric lymph nodes, as compared with Nfat5fl/fl mice (Cre recombinase–negative mice) (Supplemental Figure 3A). Additionally, NFAT5 protein expression was also markedly diminished in the intestinal epithelium regardless of IEC subtype, thus confirming the depletion of Nfat5 specifically in IECs (Supplemental Figure 3, B–E). Similar to Nfat5+/– mice, we found that Nfat5IEC-KO mice had delayed replenishment of IECs, as evidenced by decreased EdU positivity in crypts at 4 hours and belatedly increased EdU positivity at 48 hours following intraperitoneal EdU injection (Supplemental Figure 3F). By contrast, WT mice had a higher number of EdU-stained cells in the crypts early on, with rapid migration toward the villi over time. These mice also exhibited reduced proliferation, as shown by a decrease in Ki-67+ IECs, and increased apoptosis, as indicated by elevated cleaved caspase 3+ IECs, confirming in vivo that epithelial Nfat5 deficiency repressed the proliferation and survival of intestinal epithelial cells (Supplemental Figure 3, G and H).

Moreover, Nfat5IEC-KO mice exhibited more severe DSS-induced colitis than did Nfat5fl/fl (Cre recombinase–negative) mice, as determined by body weight loss, DAI score, macroscopic colon shortening, and microscopic pathology results (Figure 3, A–C), demonstrating the critical role of epithelial NFAT5 in aggravating DSS-induced colitis. In contrast, consistent with the findings in Nfat5+/– and WT mice, there were no differences in the symptoms of DSS-induced colitis between the cohoused Nfat5IEC-KO and Nfat5fl/fl mice, suggesting the essential role of gut microbiota in protecting against Nfat5 deficiency–accelerated colitis (Figure 3, D–F). To further investigate this, we conducted fecal microbiota transplantation (FMT) experiments in Nfat5+/– and Nfat5+/+ mice. After separate housing in addition to distinct microbiome differences associated with NFAT5 expression, our findings from separate housing and cohousing experiments using FMT from Nfat5+/– mice into Nfat5+/– mice resulted in higher colitis severity than that observed in WT (Nfat5+/+) mice (Figure 3, G–I). This supports notion that the NFAT5 expression level in the host (recipients), which originates from genetic differences, determines the development of DSS-induced colitis. Meanwhile, the increased colitis severity observed in separately housed Nfat5+/– mice (Supplemental Figure 2A) was almost completely reversed by FMT using feces from WT donors (Figure 3J), suggesting that normal gut microbiota —presumably containing beneficial microbial components — can mitigate DSS-induced colitis in Nfat5-deficient recipients by counteracting pathobionts present in Nfat5+/– mice. In support of this notion, when transplanted into either WT or Nfat5+/– recipients, feces of Nfat5+/– mice were more effective at inducing DSS colitis than were feces from WT mice (Figure 3, K and L). Together, these observations suggest that both the gut microbiota and the integrity of IECs, compromised by Nfat5 deficiency, contribute to the progression of DSS-induced colitis.

NFAT5 promotes intestinal barrier function through the regulation of gut microbiota. Next, we examined the effect of Nfat5 deficiency and gut microbes on intestinal barrier function to understand the mechanisms underlying Nfat5- and microbiota-mediated protection against colitis. Strikingly, we observed that the gut of Nfat5+/– mice became more permeable than that of WT mice, even in the absence of colitis, solely by raising the mice in separate cages according to their genotype (Figure 4A), indicating that a genetic defect in NFAT5 could impair the normal function of the physiological gut barrier. Interestingly, such an increase in gut permeability was substantially attenuated by cohousing Nfat5+/– and WT mice (Figure 4B), suggesting the additional involvement of gut microbiota in maintaining gut barrier function. In line with this, the mRNA and protein expression levels of zonula occludens 1 (ZO-1) (Tjp1) and occludin (Ocln), well-known tight-junction molecules (12), were also remarkably lower in Nfat5-lacking IECs of separately housed mice (Figure 4, C–E, and Supplemental Figure 4, A and B), a difference that was almost completely abolished by cohousing (Figure 4, F and G, and Supplemental Figure 4C). Moreover, when feces from Nfat5+/– mice were transplanted, the Nfat5-deficient gut still exhibited higher permeability than did the Nfat5-sufficient gut (Figure 4H). However, when feces from WT donors were transplanted, the Nfat5+/– recipient mice showed an improvement in gut permeability similar to that of WT recipient mice (Figure 4I). In parallel, FMT using feces from Nfat5+/+ mice significantly restored the expression of tight-junction proteins, such as ZO-1 and occludin, in the intestines of Nfat5+/– mice compared with FMT using feces from Nfat5+/– mice (Figure 4J and Supplemental Figure 4D). These data indicate that NFAT5 plays a pivotal role in preserving intestinal homeostasis and that microbiota alterations resulting from Nfat5 deficiency have a direct influence on gut barrier function.

To further support this assumption, we performed 16S ribosomal RNA amplicon sequencing to assess the microbial composition in feces and cecum contents obtained from Nfat5+/– and WT mice that were raised separately. The MiSeq system provided 310,555 and 287,009 qualified sequences (median: 23,132.5 and 25,414 reads per sample; range: 22,129-32,317 and 16,510-35,926) of 16S rRNA amplicons from fecal and cecal samples, respectively. The microbial taxa at the genus level were dominated by Muribaculaceae, an undefined taxon (belonging to the family Lachnospiraceae), and by Lactobacillus in the feces and an unidentified taxon (belonging to the family Lachnospiraceae), Muribaculaceae, and by the Lachnospiraceae NK4A136 group in the cecal contents (Figure 5A and Supplemental Figure 5A). There were no significant differences in species numbers or evenness in the feces between Nfat5+/– and WT mice, as determined by the observed features, Simpson, and Shannon indices (Figure 5B). However, according to the observed features index, the cecal contents of the Nfat5+/– mice had a higher total number of species than did those of WT mice (Supplemental Figure 5B). Of note, through principal coordinate analysis (PCOA), we observed clear and distinct clusters between Nfat5+/– and WT mice in both feces and cecal contents (Figure 5C and Supplemental Figure 5C), indicating that Nfat5 deficiency had a notable effect on the microbial composition of both feces and cecal contents. Given that alterations in the gut microbiota of Nfat5-deficient mice were detected in the small intestine prior to the arrival of luminal contents in the colon, these findings support the notion that dysbiosis may originate in the small intestine.

Given the aforementioned findings, we sought to identify the specific microbial components that differed between the 2 genotypes. We identified 21 microbial taxa and the top 10 genera using linear discriminant analysis (LDA) effect size (LEfSe) and random forest analysis, respectively (Figure 5, D and E). At the genus level, the Lachnospiraceae NK4A136 group as well as Ruminococcus and Faecalibacterium were enriched in the feces of WT mice, whereas Alistipes and Alloprevotella were dominant in the feces of Nfat5-deficient mice (Figure 5F and data not shown). Meanwhile, the order of Coriobacteriales was abundant in the feces of WT mice, whereas the species of uncultured Bacteroidales belonging to Alloprevotella was abundant in the feces of Nfat5+/– mice (Figure 5, F and G). Similarly, we identified significantly different taxa in the cecal contents of Nfat5+/– mice and WT mice (Supplemental Figure 5, D and E).

In summary, these findings demonstrate that Nfat5 deficiency altered the gut microbiota, presumably contributing to intestinal barrier dysfunction and increased susceptibility to DSS-induced colitis. Such microbial shifts appeared to stem from alterations in the small intestine.

Nfat5 deficiency impairs IEC regeneration and reduces goblet and Paneth cells, leading to gut barrier dysfunction. It is widely acknowledged that the small intestine — particularly goblet cells and Paneth cells — plays a crucial role in pathogen inhibition through direct antimicrobial action. Meanwhile, the large intestine contributes to the limitation of pathogen growth by fostering competition with beneficial microbes and generating metabolic byproducts (26). Importantly, Paneth cells are absent in the colon but are primarily found in the small intestine, where they play a crucial role in regulating the gut microbiome by secreting antimicrobial peptides, such as Reg3β and 3γ, defensins, and lysozyme. The effects of NFAT5 on microbial composition prompted us to investigate how its deficiency alters this composition. To this end, we focused on 2 significant factors — mucus and antimicrobial compounds secreted by IECs, which are necessary for maintaining gut barrier function (4). We stained intestinal tissues with Alcian blue and an anti–mucin 2 antibody to detect mucin 2, the key macromolecular component of mucus, in goblet cells — the primary mucin-producing cells (27). The results showed that the colon and ileum of Nfat5+/– mice had fewer goblet cells than did those of their WT littermates (Figure 6A and Supplemental Figure 6, A and B). Concurrently, mucin 2 (Muc2) mRNA expression levels in IECs of the colon and ileum were lower in Nfat5+/– mice than in WT mice (Figure 6B). Next, we assessed mRNA levels of the antimicrobial compounds and found that RegIIIγ (Reg3g), defensin α5 (Defa5), and lysozyme 1 (Lyz1) mRNA expression levels were all significantly downregulated in the ileal epithelial cells of Nfat5+/– mice (Figure 6C). Reg3b mRNA expression tended to decrease, but this was not statistically significant (Figure 6C). In parallel, the number of Paneth cells (lysozyme+), the primary producers of antimicrobial compounds, was markedly reduced in the Nfat5-deficient ileum compared with that in WT controls (Figure 6D). Likewise, the number of mucin 2+ goblet cells and Muc2 mRNA expression levels were lower in the IECs of Nfat5IEC-KO mice than in those of Nfat5fl/fl mice (Figure 6, E and F). The decreases in Lyz1 mRNA expression and Paneth cell numbers were similarly reproduced in Nfat5IEC-KO mice (Figure 6, G and H). In summary, we demonstrated that Nfat5 deficiency reduced the number of cells responsible for producing mucin and antimicrobial compounds, which may explain the gut barrier dysfunction and altered microbial composition observed in Nfat5+/– mice.

In addition to specific epithelial cell subtypes, such as goblet and Paneth cells, our findings in Figure 1, E and F, suggest that NFAT5 regulates overall IEC proliferation. ISCs continuously proliferate and differentiate to replace the old epithelium (6). We therefore suspected that the reduction in the number of cells secreting mucin and antimicrobial compounds, as well as overall IEC proliferation, stems from Nfat5 deficiency–related dysfunction of ISCs. The data showed that Nfat5 depletion significantly reduced the mRNA expression of ISC-related genes, such as EphB2 and Lgr5, as well as the number of OLFM4+ cells, a marker protein of ISCs, in ileal epithelial cells (Supplemental Figure 6, C and D), indicating NFAT5-mediated regulation of ISCs. To ascertain the functional difference in ISCs between Nfat5+/– and WT mice, we established organoids from small intestinal crypts. The budding organoids derived from Nfat5+/– mice were noticeably fewer in quantity and smaller in size and budding than were those from WT mice (Supplemental Figure 6, E–G), indicative of a defect in the self-renewal and differentiation capacity of NFAT5-depleted ISCs. Intriguingly, the defect in budding organoid formation was still observed in Nfat5-deficient mice even after cohousing (Supplemental Figure 6H), indicating that NFAT5 controlled the regenerative capacity of IECs regardless of gut microbiota. We observed NFAT5-mediated regulation of epithelial regenerative capacity in mice with conditional deletion of epithelial Nfat5 (Figure 6, I–L). As shown in Figure 6, I and J, the levels of Lgr5 and Olfm4 mRNA and the number of OLFM4+ cells were substantially reduced in the ileal epithelium of Nfat5IEC-KO mice. Organoids from Nfat5IEC-KO mice were also markedly fewer and smaller than were those from Nfat5fl/fl mice, regardless of gut microbe composition (Figure 6, K and L).

Collectively, our results substantiate the idea that NFAT5 plays a role in regulating the self-renewal of ISCs and their differentiation into goblet and Paneth cells, ultimately promoting the production of mucus and antimicrobial compounds. These cascades could be critical mechanisms underlying NFAT5-mediated regulation of gut microbe composition.

HSP70 promotes the survival and proliferation of IECs and protects against DSS-induced colitis as an NFAT5 target gene. Our in vitro and in vivo data demonstrate that NFAT5 was essential for the survival and proliferation of IECs, although the underlying molecular mechanisms remain unclear. To understand the mechanism(s) involved, we performed microarray-based global gene expression profiling of IECs from Nfat5 conditional-KO mice. In a comparison of Nfat5IEC-KO and Nfat5fl/fl mice, we identified 1,014 (314 upregulated and 700 downregulated genes) and 237(113 upregulated and 124 downregulated genes) differentially expressed genes (DEGs) in the small and large IECs, respectively (Figure 7, A and B). Functional enrichment analysis demonstrated that gene ontology biological process (GOBP) terms related to cell survival and proliferation, including mitotic cell-cycle-phase transition, positive regulation of the cell-cycle process, DNA replication, response to unfolded protein, and intrinsic regulation of the apoptotic pathway, were substantially enriched by DEGs (Figure 7C), which is in parallel with the data in Figure 1 and Supplemental Figure 1. To further characterize the physiological relevance of NFAT5 in IECs, we defined a set of 524 downregulated DEGs (redundant genes excluded) that were markedly enriched in the small IECs of Nfat5IEC-KO mice as the “NFAT5 signature” and compared it with the single-cell RNA-Seq (scRNA-Seq) data from human intestinal tissues, extracted from a public database (28). A total of 18 cell types were identified from the scRNA-Seq data on intestinal tissues (Supplemental Figure 7A). When the NFAT5 signature score was calculated for each cell type, we found that high signature scores (z score ≥0.2) were concentrated in the stem cells, Paneth cells, and transit-amplifying cells involved in epithelial repair (Figure 7D), supporting the decisive role of the NFAT5 signature in epithelial repair and regeneration.

Next, we assessed the genes that are specifically involved in the NFAT5-mediated regulation of IEC survival and proliferation. As shown in the volcano plot in Figure 7A, the Hspa1b gene, a member of the inducible 70 kDa HSP70 family, was the top-ranked downregulated gene. In another experiment using gene expression profiles in IECs of Nfat5+/– versus WT mice, we also found Hspa1b to be the top-ranked downregulated gene (Supplemental Figure 7B). HSP70 is a critical regulator of epithelial cell integrity, and aberrations in its expression can increase the severity of DSS colitis (29). Furthermore, under hypertonic conditions, NFAT5 promotes the transcription of Hspa1b through direct binding to its promoter (30). On the basis of our volcano plot analysis and previous reports (29, 30), we postulated that HSP70 primarily mediates the NFAT5-dependent survival, proliferation, and regenerative capacity of IECs and the progression of colitis. To prove this assumption, we first confirmed a significant decrease in Hspa1b mRNA and HSP70 protein expression in IECs of Nfat5+/– and Nfat5IEC-KO mice using quantitative PCR (qPCR) analysis and IHC, respectively (Figure 7, E and F, and Supplemental Figure 7, C and D). Moreover, we found that NFAT5 knockdown using siRNAs mitigated the expression of HSPA1B mRNA and HSP70 protein induced by the hypertonic NaCl stimulus in HT-29 cells (Figure 8, A and B), which is consistent with the findings in a previous report (30). Notably, HSP70 knockdown using siRNA substantially reduced the survival and proliferation of HT-29 cells, as assessed by MTT and BrdU incorporation assays (Figure 8C and Supplemental Figure 8, A and B).

A previous study reported that IEC-specific Hsp70-Tg mice were more resistant to inflammatory colitis (29). Thus, we performed gain-of-function experiments by crossbreeding Villin1 promoter–mediated Hsp70-Tg (Hsp70IEC-TG) mice with Nfat5+/– mice (Supplemental Figure 8, C and D) and challenging them with DSS. As shown in Figure 8, D and E, Nfat5+/– mice with specific overexpression of Hsp70 in IECs (Hsp70IEC-TG Nfat5+/–) had less severe colitis than did Nfat5+/– mice without Hsp70 overexpression (Hsp70WT Nfat5+/–), demonstrating that epithelial HSP70 contributed to the attenuation of DSS-induced colitis accelerated by Nfat5 deficiency. Moreover, the overexpression of Hsp70 reversed the loss of Paneth cells observed in Nfat5-deficient mice (Figure 8F), and the decrease in organoids caused by Nfat5 deficiency was almost completely restored by forced expression of Hsp70 in IECs (Figure 8G), suggesting the involvement of the NFAT5/HSP70 axis in ISC self-renewal and differentiation capacity. Notably, the expression of NFAT5 and HSPA1B mRNAs in HT-29 cells was induced to significant levels by adding WT feces, whereas Nfat5+/– mice feces did not show upregulation of these mRNAs (Figure 8H). Meanwhile, Tnfa mRNA expression as a control increased significantly with the treatment of feces from both WT and Nfat5+/– mice, with no significant difference between the 2 groups (Figure 8H). These findings suggest that the altered gut microbe composition caused by Nfat5 deficiency renders IECs less protective, thereby explaining the protection against colitis progression in Nfat5+/– mice by FMT of WT feces shown in Figure 3J.

In summary, these data suggest that, as a direct target molecule of NFAT5, HSP70 mediated the NFAT5-dependent survival, proliferation, and regenerative capacity of IECs, consequently preventing the progression of experimental colitis.

Epithelial NFAT5 levels and the gut microbiota contribute to the development of spontaneous colitis in mice. Although DSS-induced colitis is widely used in experimental colitis, it does not involve T or B cells, unlike human IBD (31). In contrast, Il10-deficient mice develop spontaneous colitis with increased Th1 and Th17 responses and abundant production of proinflammatory cytokines, mimicking human IBD pathogenesis (32). To confirm the main findings of this study in another model of experimental colitis that more closely resembles the development of human IBD, we mated Il10-homozygous KO (Il10–/–) mice with Nfat5-heterozygous KO (Nfat5+/–) mice (Supplemental Figure 9) and compared the development of spontaneous colitis between the Nfat5+/– and Nfat5+/+ littermates. The experiments ended before any difference in body weight was observed (data not shown) because severe rectal prolapse was observed in some Il10 and Nfat5 double-KO (Il10–/– Nfat5+/–) mice. Nonetheless, compared with the Il10–/– Nfat5+/+ mice, the Il10–/– Nfat5+/– mice had higher DAI scores, more frequent rectal prolapse, and more severe shortening and microscopic pathology of the colon (Figure 9, A–C). Importantly, these differences disappeared when the mice were cohoused (Figure 9, D–F), which concurs with the cohousing data observed in the DSS-induced colitis model (Supplemental Figure 2), suggesting that the gut microbiota is critical for NFAT5-dependent protection against inflammatory colitis in this spontaneous model as well.

To further determine the role of epithelial NFAT5 in spontaneous colitis, we generated conditional-KO Il10–/– Nfat5IEC-KO and their Il10–/– Nfat5fl/fl mice. As expected, these mice, with conditional deletion of Nfat5 in the IECs exhibited more severe colitis with higher DAI and histological scores and rectal prolapse (Figure 9, G–I). Despite being IL-10 deficient, Nfat5 depletion suppressed the expression levels of Hspa1b mRNA and HSP70 protein and diminished the number of goblet (mucin 2+) and Paneth (lysozyme+) cells (Figure 10, A–E). Moreover, budding organoids from Il10–/– Nfat5+/– mice were significantly fewer in number and smaller in size than were those from Il10–/– Nfat5+/+ mice (Figure 10F), confirming NFAT5-dependent regulation of IEC regeneration.