Western diet induces Paneth cell defects through microbiome alterations and farnesoid X receptor and type I interferon activation

Abstract

Intestinal Paneth cells modulate innate immunity and infection. In Crohn's disease, genetic mutations together with environmental triggers can disable Paneth cell function. Here, we find that a western diet (WD) similarly leads to Paneth cell dysfunction through mechanisms dependent on the microbiome and farnesoid X receptor (FXR) and type I interferon (IFN) signaling. Analysis of multiple human cohorts suggests that obesity is associated with Paneth cell dysfunction. In mouse models, consumption of a WD for as little as 4 weeks led to Paneth cell dysfunction. WD consumption in conjunction with Clostridium spp. increased the secondary bile acid deoxycholic acid levels in the ileum, which in turn inhibited Paneth cell function. The process required excess signaling of both FXR and IFN within intestinal epithelial cells. Our findings provide a mechanistic link between poor diet and inhibition of gut innate immunity and uncover an effect of FXR activation in gut inflammation.

Keywords: cell-intrinsic; high fat diet; metabolism; microbiota; myeloid cells; obesity; organoids; transcriptomics.

Figure 1.. Obesity is associated with Paneth cell defects in humans and mice.

(A) In non-IBD patients, those with BMI≥25 (n=57) showed reduced percentage of normal Paneth cells compared to those with BMI<25 (n=34) (P=0.0129). Representative images of Paneth cells from patients with BMI<25 and BMI≥25 stained with HD5 immunofluorescence (green) are shown in (B). Scale bars: 10μm. (C) There was no difference in goblet cell density (P=0.6256) between the 2 groups. Compared to standard diet (SD)-fed mice (n=30), mice fed with Western diet (WD; n=41) for 8 weeks also resulted in (D) reduced percentage of normal Paneth cells (P<0.0001), and no significant changes in (E) goblet cell density/villus (P=0.1767), (F) neuroendocrine cells/villus (P=0.1193), or (G) crypt base proliferation (P=0.8982). (H) A time course study showed that 4 weeks of WD was sufficient to trigger Paneth cell defects (P<0.0001), whereas (I) WD only induced significant permeability change at 16 weeks (P=0.0022). (J) A 4-week washout period was sufficient to restore the percentage of normal Paneth cells (P=0.0198 compared to baseline). (H): n=3~10/group. (I): n=7/group. (J): baseline: n=5; 2 and 4wk: n=7; SD control: n=10. (A, C-G): Statistical analysis was performed by Mann-Whitney test. (H): Statistical analysis was performed by two-way ANOVA. (I, J): Statistical analysis was performed by Kruskal-Wallis test. *: P<0.05; **: P<0.01; ****: P<0.0001. Error bars represent standard deviations.

Figure 2.. Activated FXR and type I IFN signaling in WD-associated Paneth cell defects in mice.

(A) A heat map showing upregulated Fxr-associated genes in full-thickness ileum from WD-fed mice and their level of contribution to specific themes in the data set. (B) RT-qPCR of Fxr target gene Fgf15 showed that WD treatment resulted in induction of Fgf15 in the ileum (n=5/group; P=0.0079). By mining publically-available dataset GSE74101 (FXR agonist PX20606 treatment in WT mice), we found that FXR activation reduced Paneth cell-associated (C) Defa6 (adjusted P=0.002) and (D) Reg3g (adjusted P=0.009) expression, without significantly altering the expression of (E) gob let cell-specific gene Muc2 (adjusted P=0.1143). PX20606 treatment also increased expression of (F) Irf7 (adjusted P=0.01) and (G) Oas1 (adjusted P=0.03). (H) Ileum from WD-fed mice showed enhanced expression of Oas1 (P=0.0079). (I) WD-fed mice showed higher serum type I IFN activity (P=0.0159). (C-G): Benjamini & Hochberg adjustment to the P value was applied (n=4/group). (B, H, I): Statistical analysis was performed by Mann-Whitney test (n=5/group). (B-H): Error bars represent standard deviations.

Figure 3.. WD-DCA-mediated FXR activation induces Paneth cell defects.

(A) WT mice pretreated with FXR agonist GW4064 showed worse mortality after Salmonella Typhimurium infection compared to non-GW4064 treated or treated with GW4064 at the onset of infection (n=10/group; P=0.0035). (B) GW4064 pretreatment increased the mortality of Fxr^+/+ mice after Salmonella Typhimurium infection but not in Fxr^−/− mice (n=5/group; P=0.0084). Distal ileum from WD-fed mice had higher levels of (C) DCA (P<0.0001) and (D) lithocholic acid (LCA; P=0.0021). n=10/group. (E) Reduction of DCA from WD-fed mice by bile acid sequestrant cholestyramine prevented Paneth cell defects (P<0.0001). Total n: no cholestyramine: n=6, with cholestyramine: n=14. (F) Administration of DCA but not LCA in SD-fed mice recapitulated WD-induced Paneth cell defects (P<0.0001). Total n: SD, WD, DCA: n=15/group, LCA: n=10. (G) Fxr^−/− mice and littermates (Fxr^+/+) were treated with SD, WD, SD+DCA, or SD+GW4064, and analyzed for Paneth cell morphology. While WD, DCA, and GW4064 all induced Paneth cell defects in the Fxr^+/+ mice (P=0.0005), the Fxr^−/− mice did not develop this phenotype (P=0.1257). n: 4–14/group. (H) WT, WD-fed mice treated with FXR antagonist guggelsterone did not develop Paneth cell defects (P=0.0286). n=4/group. (A, B): Kaplan-Meier curve analysis was performed with Logrank test. (C-H): Statistical analysis between 2 groups was performed by Mann-Whitney test. Statistical analysis between more than 2 groups was performed by Kruskal-Wallis test. *: P<0.05; **: P<0.01; ***: P<0.001. ****: P<0.0001. Error bars represent standard deviations.

Figure 4.. Fxr signaling in Paneth cells is essential for Western diet (WD)-mediated Paneth cell defects.

(A) WD, DCA, and GW4064 triggered Paneth cell defects in Fxr^fl/fl mice, but the effects were abrogated in the Fxr^ΔIEC mice (P=0.0005). Total n=5~13/group. (B) Ileal organoids derived from Fxr^−/− and Fxr^+/+ mice showed differential responses to DCA and GW4064. Whereas DCA and GW4064 both reduced the percentages of Paneth cells/organoid in the Fxr^+/+ organoids, such an effect was abrogated in the Fxr^−/− organoids (P=0.0036). (C) WD-mediated Paneth cell defects were abrogated in the Fxr^ΔPC mice (P=0.0006). Total n=7/group. (D) Ileal organoids derived from the Fxr^ΔPC mice did not show reduced percentages of Paneth cells/organoid when treated with DCA or GW4064. (B, D): Results were from 3 independent experiments, with each experiment containing 20 organoids/group. (A-D) Statistical analysis between 2 groups was performed by Mann-Whitney test. Statistical analysis between more than 2 groups was performed by Kruskal-Wallis test. *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001. Error bars represent standard deviations.

Figure 5.. The role of microbiota in WD-mediated Paneth cell defects.

(A) Mucosal microbiome from WD-fed mice was enriched with BaiCD operon (n=8/group; P=0.0281). (B) The amount of BaiCD operon in the microbiota was diminished after vancomycin treatment (n=5/group; P=0.0079). (C) Vancomycin treatment prevented WD-mediated Paneth cell defects in SPF housed mice (n=10/group; P<0.0001). (D) Germ-free mice (GF) fed with WD and gavaged with C. scindens showed Paneth cell defects compared to those gavaged with PBS (P=0.0003). Total n: PBS: 7, C. scindens: 8. (E) GF mice fed with SD or WD without cecal content transfer did not develop Paneth cell defects (n=10/group; P=0.4309). Statistical analysis was performed by Mann-Whitney test. *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001. Error bars represent standard deviations.

Figure 6.. Type I IFN mediates WD-associated Paneth cell defects.

(A) Administration of GW4064 in WT, SD-fed mice showed induction of type I IFN (Ctl: n=5; GW4064: n=10; P=0.0193). This was associated with enhanced expression of type I IFN associated genes (B) Irf1 and (C) Irf9 in the crypt base compartment. (D) Treatment with anti-Ifnar1 (MAR1–5A3) but not isotype control antibody (GIR208) prevented WD-induced Paneth cell defects (n=5/group; P=0.0079). (E) Ifnar^−/− mice were protected from WD-associated Paneth cell defects (Ifnar^+/+: n=13, Ifnar^−/−: n=14; P<0.0001). (F) Stat1^ΔIEC mice fed with WD did not develop Paneth cell defects (P=0.0238). Total n: Stat1^fl/fl: n=3; Stat1^ΔIEC: n=6. (G) Stat1^ΔIEC mice fed with WD maintained high Fgf15 expression compared to the Stat1^fl/fl mice (P=0.1). (H) Fxr^ΔIEC mice fed with WD did not show abrogation of type I IFN induction (P=0.6523). Total n: Fxr^fl/fl: n=8; Fxr^ΔIEC: n=6. (I) GW-4064-treated mice did not show increased lamina propria F4/80+ cells (P=0.6560). (J) WD-fed WT mice treated with clodronate were protected from Paneth cell defects (P=0.0031). Total n: Ct: n=10; clodronate: n=6. (K) DCA treatment induced type I IFN activity in macrophages in vitro (P<0.0001). n=4/group. (L) Fxr^ΔMye mice fed with WD showed abrogation of type I IFN induction (P=0.0193). Total n: Fxr^fl/fl: n=3; Fxr^ΔMye: n=4. (M) Fxr^ΔMye mice were protected from WD-mediated Paneth cell defects (P=0.0033). Total n: Fxr^fl/fl: n=15; Fxr^ΔMye: n=16. Statistical analysis for all panel was performed by Mann-Whitney test. *: P<0.05; **: P<0.01, ****: P<0.0001. Error bars represent standard deviations.

Figure 7.. Proposed model of how WD triggers Paneth cell defects.

Our current study shows that potent environmental factors (such as WD consumption) could induce Paneth cell defects without host genetic susceptibility. WD induces FXR signaling that directly target Paneth cells, and also induces type I IFN production in myeloid cells such as macrophages. Both FXR and type I IFN signaling are required to trigger Paneth cell defects.