Paneth cell-derived iNOS is required to maintain homeostasis in the intestinal stem cell niche

Abstract

Background: Mammalian intestinal epithelium constantly undergoes rapid self-renewal and regeneration sustained by intestinal stem cells (ISCs) within crypts. Inducible nitric oxide synthase (iNOS) is an important regulator in tissue homeostasis and inflammation. However, the functions of iNOS on ISCs have not been clarified. Here, we aimed to investigate the expression pattern of inducible nitric oxide synthase (iNOS) within crypts and explore its function in the homeostatic maintenance of the ISC niche.

Methods: Expression of iNOS was determined by tissue staining and qPCR. iNOS^-/- and Lgr5 transgenic mice were used to explore the influence of iNOS ablation on ISC proliferation and differentiation. Enteroids were cultured to study the effect of iNOS on ISCs in vitro. Ileum samples from wild-type and iNOS^-/- mice were collected for RNA-Seq to explore the molecular mechanisms by which iNOS regulates ISCs.

Results: iNOS was physiologically expressed in Paneth cells. Knockout of iNOS led to apparent morphological changes in the intestine, including a decrease in the small intestine length and in the heights of both villi and crypts. Knockout of iNOS decreased the number of Ki67⁺ or BrdU⁺ proliferative cells in crypts. Loss of iNOS increased the number of Olfm4⁺ ISCs but inhibited the differentiation and migration of Lgr5⁺ ISCs in vivo. iNOS depletion also inhibited enteroid formation and the budding efficiency of crypts in vitro. Moreover, iNOS deficiency altered gluconeogenesis and the adaptive immune response in the ileum transcriptome.

Conclusion: Paneth cell-derived iNOS is required to maintain a healthy ISC niche, and Knockout of iNOS hinders ISC function in mice. Therefore, iNOS represents a potential target for the development of new drugs and other therapeutic interventions for intestinal disorders.

Keywords: Differentiation; Intestinal stem cell; Paneth cell; Proliferation; iNOS.

Fig. 1

Distribution and localization of iNOS in the small intestine of mice. A Representative IHC staining images of iNOS in different intestinal segments from wild-type mice. Bar = 100 μm. B, C Quantification of the staining intensity of iNOS and the number of iNOS positive cells in different segments. *P < 0.05, **P < 0.01, ***P < 0.001. D qPCR analysis of iNOS mRNA levels in different intestinal segments. The ileum had the highest level of iNOS mRNA, and the differences in iNOS expression between the duodenum vs. Jejunum, Duodenum vs. Colon and Jejunum vs. Ileum were not significant (P > 0.05). *P < 0.05, **P < 0.01, n = 3. E qPCR analysis of iNOS mRNA expression in the whole small intestine and crypts. F Lgr5-tdTomato (red), iNOS (green) and DAPI (blue) positive signals at the cryptal base of Lgr5-tdTomato mice. G Lysozyme granules (red), iNOS (green) and β-catenin (purple) positive signals at the crypt base of wild-type mice. H Cultured wild-type enteroid was stained by lysozyme (red), iNOS (green) and DAPI (blue) at day 4. Bar = 20 μm

Fig. 2

iNOS deletion impaired the morphology of the intestine. A Gross images of gastrointestinal tract samples from the control and iNOS^−/− groups. B The length of the small intestine (SI) of iNOS^−/− group mice was significantly shorter than that of control group mice (n = 3). C There was no difference in the length of colons from the control and iNOS^−/− groups (n = 3). D Representative images of hematoxylin and eosin staining in the ileum for the control group and iNOS^−/− mice. Bar = 50 μm. E Statistical analysis of villus height, crypt depth, crypt width and crypt density between the control group and iNOS^−/− group. n.s: P > 0.05, *P < 0.05, ****P < 0.0001, n = 3

Fig. 3

Knockout of iNOS changed the proliferative status within intestinal crypts. A Representative images of immunohistochemical staining for Ki67, BrdU and pHH3 in the crypts of the mouse ileum. Tissues were collected from the control group and iNOS^−/− group mice at 1.5 h after BrdU injection. Bar = 50 μm. B Quantification of cells positive for Ki67, BrdU, and pHH3 staining and statistical analysis between the control and iNOS^−/− groups. ***P < 0.001, ****P < 0.0001, n = 5

Fig. 4

iNOS deletion inhibited the quantity and differentiation of AISCs in the intestinal epithelium. A Immunohistochemistry staining of Olfm4 in ileum tissues from the control group and iNOS^−/− group. Bar = 50 μm. B Statistical analysis of Olfm4-positive cells between the two groups in Fig. 4A. C qPCR analysis of the mRNA level of Olfm4 gene in ileum samples. *: P < 0.05. D Confocal images of tdTomato (red), BrdU (green) and DAPI (blue) positive signals in Lgr5-tdTomato and Lgr5-tdTomato; iNOS^−/− littermate mice. Bar = 100 μm. E Comparison of tdTomato-positive cells in villus or crypts in the control and iNOS^−/− groups. F Respective analysis of BrdU⁺ cells in the lower 1/3 or the upper 2/3 of crypts. n.s: P > 0.05, **P < 0.01, ****P < 0.0001, n = 3

Fig. 5

iNOS deficiency inhibited the growth of enteroids. A Representative results for the growth of enteroids derived from wild-type and iNOS^−/− mice on days 1, 3, and 7 after seeding. Bar = 200 μm. B The enteroid formation efficiency of the iNOS^−/− group was clearly decreased compared to that of the control group at 24 h after culture. C The budding numbers of enteroids between the control group and iNOS^−/− group were divided into 0, 1–3 or > 3 per organoid. iNOS^−/− group had less budding capacity. D RNA samples from the enteroids of the two groups at Day 4 were used for qPCR analysis of Lgr5, MKi67, Lyz2 and Wnt3a. n.s: P > 0.05, *P < 0.05, n = 3. E Enteroids derived from the control and iNOS^−/− groups in Lgr5-tdTomato mice on day 1 after seeding. Bar = 50 μm. F Cultured enteroids were treated with 1400W, and the growth was significantly inhibited. Bar = 200 μm. G Areas of enteroids in the two groups. *P < 0.05

Fig. 6

Knockout of iNOS affected the differentiation pattern in the intestinal epithelium. A Staining of absorptive epithelial cells (FABP1), goblet cells (Alcian blue), Tuft cells (DCLK1), enteroendocrine cells (Chromogranin A) and Paneth cells (lysozyme) between the control group and iNOS^−/− group. Bar = 100 μm. B Statistical analysis of different subpopulations of IECs in Fig. 6A. n.s: P > 0.05, **P < 0.01, ****P < 0.0001, n = 5

Fig. 7

iNOS deficiency altered gluconeogenesis and the adaptive immune response in the ileum transcriptome. A Volcano plot depicting transcriptomics data with dotted line marking P = 0.05 on y-axis and fold change of greater than 1 on X-axis. The top ten gene names are shown (red: upregulated, blue: downregulated). B Gene Ontology analyses of RNA-seq data showed significant changes in pathways in the ileum from the control and iNOS^−/− groups. C Kyoto Encyclopedia of Genes and Genomes enrichment analysis (upregulated and downregulated differential) bubble plot. The X-axis is the enrichment score, and the y-axis is the pathway information of the top 20. The larger the bubble, the greater the number of differential proteins contained in the entry. When the color of bubbles changes from red to yellow and blue, the p value decreases. D GSEA of genes upregulated or downregulated in association with iNOS depletion