Intestinal IL-22RA1 signaling regulates intrinsic and systemic lipid and glucose metabolism to alleviate obesity-associated disorders

Gaudino, Stephen J.; Singh, Ankita; Huang, Huakang; Padiadpu, Jyothi; Jean-Pierre, Makheni; Kempen, Cody; Bahadur, Tej; Shiomitsu, Kiyoshi; Blumberg, Richard; Shroyer, Kenneth R.; Beyaz, Semir; Shulzhenko, Natalia; Morgun, Andrey; Kumar, Pawan

doi:10.1038/s41467-024-45568-6

Download PDF

Article
Open access
Published: 21 February 2024

Intestinal IL-22RA1 signaling regulates intrinsic and systemic lipid and glucose metabolism to alleviate obesity-associated disorders

Nature Communications volume 15, Article number: 1597 (2024) Cite this article

2925 Accesses
5 Altmetric
Metrics details

Subjects

Abstract

IL-22 is critical for ameliorating obesity-induced metabolic disorders. However, it is unknown where IL-22 acts to mediate these outcomes. Here we examine the importance of tissue-specific IL-22RA1 signaling in mediating long-term high fat diet (HFD) driven metabolic disorders. To do so, we generated intestinal epithelium-, liver-, and white adipose tissue (WAT)-specific Il22ra1 knockout and littermate control mice. Intestinal epithelium- and liver-specific IL-22RA1 signaling upregulated systemic glucose metabolism. Intestinal IL-22RA1 signaling also mediated liver and WAT metabolism in a microbiota-dependent manner. We identified an association between Oscillibacter and elevated WAT inflammation, likely induced by Mmp12 expressing macrophages. Mechanistically, transcription of intestinal lipid metabolism genes is regulated by IL-22 and potentially IL-22-induced IL-18. Lastly, we show that Paneth cell-specific IL-22RA1 signaling, in part, mediates systemic glucose metabolism after HFD. Overall, these results elucidate a key role of intestinal epithelium-specific IL-22RA1 signaling in regulating intestinal metabolism and alleviating systemic obesity-associated disorders.

Microbiota in health and diseases

Article Open access 23 April 2022

Fasting-mimicking diet causes hepatic and blood markers changes indicating reduced biological age and disease risk

Article Open access 20 February 2024

A lipidome landscape of aging in mice

Article 12 April 2024

Introduction

Interleukin (IL)−22 is an IL-10 family cytokine that is predominantly produced by Th17 cells and type 3 innate lymphoid cells. IL-22 signals through its receptor complex (IL-22RA1/IL-10Rβ) which is expressed throughout the body including by the intestines, liver, and adipose tissues^1,2. Previous studies have shown that IL-22 is protective against inflammatory and metabolic disorders^3,4,5. IL-22 particularly plays a critical role in maintaining homeostatic properties in the intestine by signaling to an assortment of unique cell types. The intestinal epithelium consists of crypt/villus structures that are comprised of absorptive and secretory cell lineages. Leucine-rich repeat-containing G protein coupled-receptor 5 (Lgr5)⁺ intestinal stem cells (ISCs) reside at the bottom of the crypt where they replicate. As ISCs move toward the villus, they develop into progenitor cells which can differentiate into either absorptive enterocytes or secretory (Paneth, goblet, endocrine, tuft) cell types. Paneth cells particularly play a key role in secreting antimicrobial proteins (including α-defensins and lysozyme) that maintain a homeostatic microbiota as well as growth factors that maintain the Lgr5⁺ ISC niche^6,7. IL-22 acts on Lgr5⁺ ISCs to regulate epithelial regeneration, goblet cells to mediate mucous secretion, and Paneth cells and other enterocytes to regulate antimicrobial peptide production^{1,3,8,9,10,11}.

The rise of obesity and its associated metabolic disorders has become an issue of great concern in recent years and can result in debilitating health disorders¹². IL-22 plays a beneficial role in ameliorating various metabolic disorders^4,13. After a prolonged high-fat diet (HFD), Il22RA1^−/− mice displayed increased weight gain and impaired clearance of glucose from their blood⁴. Furthermore, wildtype mice fed a HFD and intraperitoneally injected with IL-22-Fc, a fusion protein of human IL-22 and mIgG2a, displayed improved glucose clearance and decreased weight gain, adiposity, and hepatic lipid levels compared to their IgG-injected counterparts⁴. However, studies utilizing Il22^−/− mice or models that regulate endogenous IL-22 levels did not observe differences in glucose clearance when compared to their controls^4,14. This suggests that IL-22 may act in a context-dependent manner and highlights the importance of tissue-specific IL-22RA1 signaling in regulating metabolism. In addition, IL-22 may differentially mediate systemic inflammatory responses. IL-22 reduces liver damage during murine injury models but increases inflammation in adipose tissue from patients with obesity and type II diabetes^15,16,17.

While studies have indicated a beneficial role for IL-22 in ameliorating the onset of metabolic disorders, key questions remain. Primarily, it remains unknown which tissue types (liver, intestines, or adipose) IL-22 specifically acts on to mediate these outcomes. It is important to note that previous studies evaluating the role of IL-22 in mediating metabolic disorders have relied solely on the use of global knockouts of the cytokine or its receptor or systemic treatment and/or depletion of IL-22. While these models help determine the global effects that IL-22 has on mediating metabolism, it is difficult to dissect the tissue-specific functions regulated by IL-22. Elucidating the tissue-specific roles of IL-22 is vital to understand how this cytokine mediates key metabolic and inflammatory functions that influence disease etiology as well as for the development of more targeted therapeutics.

To address this question, we generated tissue-specific Il22ra1 knockout and littermate control mice of the intestinal epithelium (Il22ra1^fl/fl;Villin-cre), liver (Il22ra1^fl/fl;Albumin-cre), and white adipose tissue (WAT; Il22ra1^fl/fl;Adiponectin-cre). Overall, we elucidate an important regulatory role of intestinal IL-22RA1 signaling in mediating systemic glucose and lipid metabolism after diet-induced obesity.

Results

Systemic IL-22 differentially regulates lipid metabolism in the WAT, liver, and small intestine

We first examined what general metabolic effects may be regulated by IL-22. Importantly, we recapitulated data showing that Il22^‒/‒ mice display increased weight gain and similar glucose clearance when compared to wildtype (C57BL/6J) control mice after long-term HFD (Fig. 1A, B).

**Fig. 1: IL-22 differentially regulates systemic lipid metabolism.**

Next, we wanted to observe what effects IL-22 solely exerts under homeostatic conditions across different metabolic tissues. To do so, C57BL/6 wildtype mice (Jackson Laboratory) were fed a chow diet to recapitulate homeostatic conditions and minimize the effects that more inflammatory diets may have on gene expression. We then intraperitoneally injected mice with either 0 or 80 μg of IL-22.Fc and harvested tissues after 24 h. This dose was used based on our previous publication and is high enough to broadly induce the expression of IL-22-regulated genes to assess its general effects on systemic tissues under homeostatic conditions⁸. IL-22 has been shown to influence functions of the WAT, liver, and intestines. We confirmed that these tissues all express Il22ra1 and that IL-22 treatment did not regulate the expression of its receptor (Fig. 1C). After IL-22 treatment, we did not observe altered expression of lipid metabolism- or inflammation-associated genes in the WAT (Supplementary Fig. 1A). We did, however, observe significantly elevated expression of Pnpla2 and Lipe in the liver of mice treated with IL-22.Fc (Fig. 1D). In addition, we observed increased staining by Oil Red O (ORO), a dye that specifically stains lipid droplets, of HepG2 cells that were treated with 4% lipid mixture (LM) and 80 ng IL-22.Fc, as opposed to pure LM-treated groups (Fig. 1E). Altogether, this suggests that IL-22 increases hepatic lipid metabolism. Upon analyzing the effects of IL-22 in the WAT and liver, we evaluated how IL-22.Fc treatment regulated intestinal function. As expected, we observed that the expression of IL-22 inducible antimicrobial peptide genes, Reg3g and Reg3b, were upregulated in the ileum (Fig. 1F). Opposite to what we observed in the liver, the expression of Pnpla2 and Lipe were significantly downregulated in the ileum after IL-22.Fc treatment (Fig. 1G). While we did not detect a difference in the expression of peroxisome proliferator-activated receptor genes, Ppara and Pparg (key transcription factors that aid peroxisomal fatty acid metabolism), in the liver after IL-22.Fc treatment, we observed that IL-22.Fc treatment resulted in decreased expression of these genes in the ileum (Fig. 1D, G). This decrease in Ppara coincides with previous studies that display that Pnpla2 activates transcription of Ppara and its target genes^18,19. To examine the ex vivo effects of IL-22, small intestinal organoids from wildtype (C57BL/6) mice were cultured in the presence or absence of 4% LM and 5 ng rIL-22 and were stained with ORO. We observed decreased ORO staining in the LM and rIL-22-treated organoids as opposed to the pure LM-treated groups (Fig. 1H). This impaired lipid accumulation in rIL-22-treated organoids correlates with the reduced expression of ileal lipid metabolism genes after IL-22.Fc treatment and implies that IL-22 acts to downregulate intestinal lipid metabolism. To assess whether rIL-22 acts directly on intestinal epithelial cells to mediate the expression of these genes, we stimulated C57BL6/J mice small intestinal organoids with rIL-22 (Fig. 1I). Expression of Pnpla2, Lipe, Ppara, and Pparg were all reduced after rIL-22 stimulation which supports this conclusion.

Our data indicate that systemic IL-22 induces opposing metabolic effects depending on its tissue-specific target. However, the tissue-specific effects of IL-22RA1 signaling remain unclear.

Deficiency of intestinal epithelium-specific IL-22RA1 signaling mediates the onset of systemic metabolic disorders

We next examined how IL-22RA1 signaling to specific tissues regulates metabolism. Since the intestines regulate the absorption of nutrients, communicate with other metabolic tissues, and highly express Il22ra1 (Fig. 1C), we assessed the intestinal epithelium-specific role of IL-22RA1 signaling in mediating systemic metabolism. To do so, we used Il22ra1^fl/fl;Villin-cre mice. Knockdown of Il22ra1 in Il22ra1^fl/fl;Villin-cre+ mice was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) (Fig. 2A). No difference was observed in weight gain or systemic glucose clearance between control diet-fed Il22ra1^fl/fl;Villin-cre+ and cre- mice (Fig. 2B; Supplementary Fig. 2A).

**Fig. 2: Systemic metabolism (but not weight gain) is regulated by intestinal-epithelium- and liver-specific IL-22RA1 signaling.**

To assess the effects of intestinal IL-22RA1 signaling in mediating the onset of diet-induced obesity metabolic disorders, we placed Il22ra1^fl/fl;Villin-cre mice on a long-term (16 weeks) HFD. The HFD used contains approximately 29% greater fat than the control diet, and HFD-fed mice receive 60% of their dietary calories from fat (Supplementary Table 1). After HFD, Il22ra1^fl/fl;Villin-cre mice displayed greater weight gain than control diet-fed mice, but no difference in weight was observed between HFD-fed cre- and cre+ mice (Fig. 2B). However, upon conducting glucose tolerance tests (GTTs), Il22ra1^fl/fl;Villin-cre+ mice displayed impaired glucose clearance when compared to their littermate controls (Fig. 2C), indicating that intestinal-specific IL-22RA1 signaling is important for increasing the clearance of systemic glucose. No differences in insulin resistance or serum c-peptide and insulin levels were detected between HFD-fed Il22ra1^fl/fl;Villin-cre mice to account for their differences in glucose clearance (Supplementary Fig. 2B, C).

To assess whether intestinal IL-22RA1 signaling mediates extra-intestinal metabolism, we first examined the liver of Il22ra1^fl/fl;Villin-cre mice. Il22ra1^fl/fl;Villin-cre mice fed a control diet displayed similar hepatic histology (Supplementary Fig. 2D). However, HFD-fed Il22ra1^fl/fl;Villin-cre+ mice displayed increased hepatocyte ballooning and lipid droplet deposition (Fig. 2D, E). Lipid deposition was quantified via hepatic ORO staining (Fig. 2E [right]). RT-PCR analysis of key glucose (Foxo1, G6PC) and lipid (Acc, Ppara) metabolism genes revealed decreased transcripts of Acc and Ppara in Il22ra1^fl/fl;Villin-cre+ mice after HFD but not control diet (Fig. 2F). No changes were observed in the expression of additional metabolic genes after HFD feeding (Supplementary Fig. 2E). Correlating with these findings, gas chromatography analysis of liver tissues from HFD-fed Il22ra1^fl/fl;Villin-cre+ mice also displayed elevated levels of total ceramides (significant) and cholesterol (Fig. 2G). However, significant effects were not detected when analyzing individual groups of lipids (Supplementary Fig. 2F).

Next, we assessed if the absence of intestinal IL-22RA1 signaling affects WAT functions. While control diet-fed Il22ra1^fl/fl;Villin-cre mice displayed similar WAT histology (Supplementary Fig. 3A), WAT from HFD-fed Il22ra1^fl/fl;Villin-cre+ mice displayed significantly greater inflammation (i.e., accumulation of crown-like structures) than their littermate controls (Fig. 2H, I). Macrophage accumulation was confirmed via immunofluorescence microscopy (Supplementary Fig. 3B, C). IL-22 also has been associated with elevated adipose inflammation in patients with obesity and after HFD^15,20,21. No difference in epididymal WAT fat pad mass was observed between HFD-fed Il22ra1^fl/fl;Villin-cre- and cre+ mice (Supplementary Fig. 3D). RT-PCR analysis of lipid catabolizing (Acox1 and Lipe) and synthesizing (Fasn) genes revealed that Il22ra1^fl/fl;Villin-cre+ mice displayed greater transcript levels of Acox1 after HFD but not control diet (Fig. 2J). A trend for elevated Lipe and decreased Fasn expression was present (Fig. 2J). We further examined changes in WAT lipid metabolism by gas chromatography. WAT from Il22ra1^fl/fl;Villin-cre+ mice displayed lower levels of diacylglycerides (significant), phospholipids, and ceramides (Fig. 2K). However, significant effects were not detected when analyzing individual groups of lipids (Supplementary Fig. 3E). No difference in WAT triglyceride levels was observed (Supplementary Fig. 3F).

We next assessed whether altered levels of systemic IL-22 between Il22ra1^fl/fl;Villin-cre- and cre+ mice may mediate the onset of these HFD-induced phenotypes. However, no differences in ileal Il22 expression as well as liver-specific or serum-specific IL-22 levels were detected (Supplementary Fig. 3G–I).

Altogether, our data suggest that intestinal IL-22RA1 signaling plays an important role in regulating extra-intestinal gene expression and lipid metabolism and accumulation.

Deficiency of liver-specific IL-22RA1 signaling impairs systemic glucose metabolism

To determine whether impaired systemic glucose and lipid metabolism are primary consequences of intestinal IL-22RA1 signaling, we assessed the liver-specific role of IL-22RA1 signaling. We generated Il22ra1^fl/fl;Albumin-cre mice and confirmed Il22ra1 knockdown by RT-PCR (Fig. 3A). No differences in weight gain were observed between Il22ra1^fl/fl;Albumin-cre- and cre+ mice after feeding either a control diet or HFD, although increased weight gain was observed after HFD (Fig. 3B). However, we observed that Il22ra1^fl/fl;Albumin-cre+ mice displayed significantly impaired glucose clearance compared to their cre- counterparts after HFD but not control diet (Fig. 3C; Supplementary Fig. 4A). Thus, in addition to intestinal signaling, hepatic IL-22RA1 signaling regulates systemic glucose metabolism.

**Fig. 3: Liver-specific IL-22RA1 signaling regulates systemic glucose metabolism.**

No major differences in hepatic histology or lipid deposition were observed between Il22ra1^fl/fl;Albumin-cre mice after HFD (Fig. 3D, E). Except for G6pc, no significant changes were observed in glucose or lipid metabolism genes that were downregulated in Il22ra1^fl/fl;Villin-cre+ mice (Figs. 2F and 3F; Supplementary Fig. 4B). Notably, no difference in Ppara expression was observed (Fig. 3F). This coincides with our data from Fig. 1D and additionally suggests that signaling outside the liver (including the intestine) is necessary for mediating hepatic expression of Ppara. To further assess changes in lipid metabolism, we performed gas chromatography analysis of liver tissues from HFD-fed Il22ra1^fl/fl;Albumin-cre mice. Unlike Il22ra1^fl/fl;Villin-cre mice, Il22ra1^fl/fl;Albumin-cre+ mice displayed significantly decreased levels of triglycerides but similar levels of ceramides and cholesterol (Figs. 2H and 3G; Supplementary Fig. 4C). Upon examining WAT histology, epididymal adiposity, and gene expression (Lipe, Acox1, Fasn), no differences were observed between Il22ra1^fl/fl;Albumin-cre mice (Fig. 3H–J). Altogether, this data further highlights the tissue-specific role that IL-22RA1 signaling induces in regulating metabolism.

Intestinal IL-22RA1 signaling mediates extra-intestinal metabolism in a microbiota-dependent manner

We next assessed what intestinal factors may be regulated by IL-22 to mediate systemic metabolism and inflammation. Notably, the intestinal microbiota mediates key host functions including the absorption of nutrients and lipids^22,23. However, during obesity and after HFD, the microbiota undergoes a dysbiosis^24,25. Microbial dysbiosis has been associated with altered lipid absorption, metabolism, and adiposity^22,23,26,27. We and others have shown that IL-22 induces various antimicrobial proteins including Reg3 family proteins and Lyz1^8,11,28. Likewise, an interconnected nature exists between the intestinal microbiota and IL-22^29,30. Therefore, we evaluated if IL-22-mediated regulation of the microbiota affects systemic metabolism after HFD.

As expected, 16S rRNA sequencing revealed differences in alpha and beta diversity when comparing the genus-level microbial composition of cre- and cre+ mice before (day 0) and after (day 105) HFD; however, no differences were observed between the two genotypes in the same period (Supplementary Fig. 5A, B). We then assessed differences among specific bacterial genera. Thirteen genera were significantly differentially regulated in a two-factor (diet, genotype) analysis with the predominant effect of IL-22 deficiency (Fig. 4A). Notably, among these microbes, we detected the genus Oscillibacter (Fig. 4A, B), which has been previously reported to worsen glucose metabolism³¹. Furthermore, while Il22ra1^fl/fl;Villin-cre+ mice displayed only a trend of increased levels of Oscillibacter before HFD (Supplementary Fig. 5C, D), cre+ mice displayed a significantly greater abundance after HFD (Fig. 4B; Supplementary Fig. 5D). During obesity, Oscillibacter stimulates Mmp12 expression by WAT-associated macrophages which results in impaired glucose metabolism³¹. Reduction of this microbe in the gut is associated with decreased signatures of insulin-resistance-associated macrophages in the WAT and improved systemic glucose metabolism³¹. To verify if this mechanism may be operating in our system, we evaluated Mmp12 expression in epididymal WAT and found that Il22ra1^fl/fl;Villin-cre+ mice displayed elevated transcripts of Mmp12 as well as MMP12 immunofluorescence staining after HFD (Fig. 4C, D). These results indicate a potential contribution of Oscillibacter for driving WAT inflammation and, in turn, dysregulation of glucose metabolism in Il22ra1^fl/fl;Villin-cre+ mice.

**Fig. 4: Intestinal IL-22RA1-mediated alterations in microbiota composition regulate systemic metabolism and inflammation.**

To further assess whether differences in microbiota composition mediate extra-intestinal metabolic and inflammatory responses after HFD, we treated Il22ra1^fl/fl;Villin-cre mice with broad-spectrum antibiotics during their last 4 weeks of HFD to deplete their microbiota. After HFD and antibiotics treatment, we did not observe any differences in weight gain between cre- and cre+ mice (Fig. 4E). In addition, Il22ra1^fl/fl;Villin-cre- and cre+ mice displayed similar clearance of glucose from their blood (Fig. 4F). This is notable since non-antibiotics-treated Il22ra1^fl/fl;Villin-cre+ mice displayed impaired clearance of blood glucose when compared to cre- mice (Fig. 2C).

To additionally verify if microbiota-induced changes influence systemic glucose metabolism, we gavaged cecal content homogenate collected from Il22ra1^fl/fl;Villin-cre mice after 16 weeks of HFD into control diet-fed germ-free mice. We observed that mice colonized with Il22ra1^fl/fl;Villin-cre+ cecal contents displayed significantly impaired glucose clearance 30 min after glucose injection (Supplementary Fig. 5E). We also cohoused Il22ra1^fl/fl;Villin-cre- and cre+ mice over a 16 week HFD period. Unlike our antibiotics and microbiota transplantation data, cohoused Il22ra1^fl/fl;Villin-cre- and cre+ mice displayed similar trends in glucose metabolism as non-cohoused mice (Figs. 2C and 4F; Supplementary Fig. 5F). This indicates that the microbiota is indeed one of the key factors that regulate systemic metabolism, but other factors, such as tissue-specific IL-22RA1 signaling or HFD consumption, play additional roles.

To further assess the importance of IL-22 mediation of microbiota composition in regulating the onset of HFD-induced disorders, we analyzed the liver and WAT of antibiotics-treated mice. Respectively, H&E and ORO staining of liver tissues revealed that Il22ra1^fl/fl;Villin-cre- and cre+ mice displayed similar histology and lipid accumulation after antibiotics treatment (Fig. 4G; Supplementary Fig. 5G). While RT-PCR analysis revealed impaired expression of Foxo1, G6PC, Acc, and Ppara from liver tissues of non-antibiotics-treated Il22ra1^fl/fl;Villin-cre+ mice, no notable differences in the expression of glucose and lipid metabolism genes were observed among mice after antibiotics treatment (Fig. 2F and 4H). Additionally, H&E analysis of WAT post antibiotics revealed that Il22ra1^fl/fl;Villin-cre- and cre+ mice displayed similar histology (Fig. 4I). While greater trends for Acox1 and Lipe transcripts were detected via RT-PCR in non-antibiotics-treated Il22ra1^fl/fl;Villin-cre+ mice, this difference was ablated after antibiotics treatment (Figs. 2J and 4J).

Our data indicate that intestinal IL-22RA1 signaling is important for maintaining a microbiota that aids systemic metabolism. Furthermore, we identify an association between impaired intestinal IL-22RA1 signaling in elevating Oscillibacter levels and increasing levels of WAT-associated Mmp12-expressing macrophages.

WAT-specific IL-22RA1 signaling does not protect against HFD-driven metabolic disorders

WAT is important for regulating lipid and glucose metabolism^32,33. Furthermore, IL-22 decreases fat pad mass and regulates the expression of lipid metabolism genes⁴. Our observations that epididymal WAT of Il22ra1^fl/fl;Villin-cre+ display elevated macrophage accumulation and Mmp12 expression also suggest the importance of IL-22 in mediating WAT-specific functions. Therefore, we investigated the WAT-specific role of IL-22RA1 signaling in mediating systemic metabolism by generating and validating Il22ra1^fl/fl;Adiponectin-cre+ and cre- mice (Fig. 5A).

**Fig. 5: WAT-specific IL-22RA1 signaling does not ameliorate HFD-induced metabolic disorders.**

We did not observe any differences in weight gain or systemic glucose clearance between Il22ra1^fl/fl;Adiponectin-cre- and cre+ mice after placement on long-term HFD (Fig. 5B, C). Likewise, no notable differences in hepatic morphology, lipid deposition, and metabolic transcript levels were observed between Il22ra1^fl/fl;Adiponectin-cre- and cre+ mice (Fig. 5D–F). These mice also displayed no differences in WAT morphology, epididymal fat pad weight, and metabolic transcript levels (Fig. 5G–I; Supplementary Fig. 6A). Similar transcript levels of Il22 were also detected (Supplementary Fig. 6B). These results indicate that WAT-specific IL-22RA1 signaling may not play a critical role in directly regulating HFD-associated metabolic disorders.

IL-22RA1 signaling directly regulates intestinal lipid metabolism

While we showed that intestinal IL-22RA1 signaling regulates extra-intestinal metabolism after HFD (Fig. 2), we next assessed how IL-22RA1 signaling affects intestinal metabolic pathways. To do so, we performed RNA sequencing on ileal tissues of HFD-fed Il22ra1^fl/fl;Villin-cre mice (Fig. 6A). Many genes associated with lipid storage and metabolism were upregulated in the absence of IL-22RA1 signaling (Fig. 6A [left]). Furthermore, many peroxisome-associated genes, some of which are also important for lipid metabolism, were upregulated in Il22ra1^fl/fl;Villin-cre+ mice (Fig. 6A [right]). This is notable since peroxisomes are key organelles for coordinating lipid metabolism. Specifically, we noticed elevated expression of Ppara and Pparg (Fig. 6A [right]). To determine whether these phenotypes were diet-induced, we performed RT-PCR analysis of Ppara and Lipe on ileal tissues from Il22ra1^fl/fl;Villin-cre-/+ mice fed either an HFD or chow diet. We observed significant upregulation of Ppara and Lipe transcripts in Il22ra1^fl/fl;Villin-cre+ mice after HFD but not chow diet (Fig. 6B). This suggests that IL-22RA1 signaling is important for regulating intestinal lipid metabolism pathways after HFD.

**Fig. 6: Intestinal IL-22RA1 signaling downregulates the expression of lipid metabolism but not ISC- or progenitor cell-associated markers after HFD.**

The importance of intestinal IL-22RA1 in mediating lipid metabolism may explain why Il22ra1^fl/fl;Albumin-cre+ mice but not Il22ra1^fl/fl;Villin-cre mice displayed significantly decreased levels of triglycerides. It is possible that IL-22-mediated inhibition of intestinal lipid metabolism enhances liver lipid accumulation. We show that systemic IL-22 regulates the expression of Pnpla2, a triglyceride hydrolyzer, in an opposing manner in the small intestine (reduced) and liver (increased; Fig. 1D, G). It has been shown that intestinal Pnpla2 (ATGL) deficiency has no impact on plasma and liver triglyceride accumulation, but the small intestinal tissues of these mice possess an increased accumulation of triglycerides¹⁸. Furthermore, crosstalk between the liver and intestines can occur via circulation, thereby opening the possibility that liver-specific IL-22RA1 signaling may release soluble mediators that influence intestinal metabolism. To test this, we placed Il22ra1^fl/fl;Villin-cre and Il22ra1^fl/fl;Albumin-cre mice on 6 weeks of HFD and injected them with 80 µg IL-22.Fc. In addition, HFD-fed control mice were intraperitoneally injected with either 0 or 80 µg IL-22.Fc. Control mice injected with IL-22.Fc lost significantly greater weight than those treated with pure PBS (Supplementary Fig. 7A). In addition, we observed that Il22ra1^fl/fl;Villin-cre+ mice were injected with IL-22.Fc lost significantly less weight when compared to their cre- counterparts (Supplementary Fig. 7B); however no significant difference in weight loss was observed between treated Il22ra1^fl/fl;Albumin-cre mice (Supplementary Fig. 7C). This indicates that intestinal IL-22RA1 signaling may partially mediate weight loss under conditions with elevated systemic IL-22. However, it is also possible that weight gain relies on IL-22RA1 signaling to tissues besides the intestines, liver, and WAT and/or coordinated IL-22RA1 signaling to different tissue types. Next, we examined the tissue-specific effects of IL-22RA1 signaling on the expression of intestinal lipid metabolism genes. Ileal expression of Ppara, Lipe, and Pnpla2 were downregulated by intestinal-specific but not liver-specific IL-22RA1 signaling (Supplementary Fig. 7D), highlighting the importance of tissue-specific IL-22RA1 signaling in mediating intestinal metabolism.

IL-22 exerts its effects by activating downstream transcription factors including Stat3³⁴. We observed Stat3 binding sites upstream of the start site for Ppara (Supplementary Fig. 7E), indicating that its expression may be regulated in a Stat3-dependent manner. We observed a modest reduction in Ppara expression after treatment of organoids with S3I-201 (Stat3 inhibitor) and a significant reduction after treatment with rIL-22 (Supplementary Fig. 7F). However, treatment of S3I-201 did not reverse or enhance the inhibitory effects of rIL-22 on the expression of Ppara, indicating that IL-22 may not be responsible for mediating Ppara transcription in a Stat3-dependent manner (Supplementary Fig. 7F).

While we show that IL-22 mediates the expression of intestinal lipid metabolism genes (Fig. 6A, B), it is unclear whether IL-22 is solely responsible for promoting these effects. IL-22 has also been shown to induce the production of epithelial IL-18 to provide protection against parasitic and bacterial infections^34,35. Therefore, we evaluated the role of IL-18 in mediating the expression of intestinal lipid metabolism genes. Whereas IL-22RA1 deficiency solely reflects a lack of IL-20/IL-22/IL-24-mediated effects, IL-22 supplementation induces additional proteins/pathways such as IL-18. Upon intraperitoneal injection, we reproduced data indicating that IL-22 results in elevated ileal Il18 transcript levels (Fig. 6C). We did not observe any significant difference in Il18r1 transcripts after injection (Fig. 6C). Furthermore, we did not observe significant differences in Il18 or Il18r1 transcripts in the liver or WAT after IL-22.Fc injection (Supplementary Fig. 7G), indicating that intestinal IL-18 responses are more strongly regulated by IL-22. Upon stimulating C57BL/6 small intestinal organoids with rIL-18, we observed significantly downregulated expression of Lipe and Pnpla2 while Ppara showed a trend in the same direction (Fig. 6D). This implies that IL-22 may mediate some of its metabolic effects in conjunction with or by inducing IL-18, possibly explaining why systemic IL-22.Fc injection opposingly mediated gut and liver expression of Lipe and Pnpla2. These results may partially explain why we observed impaired hepatic functions in HFD-fed Il22ra1^fl/fl;Villin-cre+ mice, but prior studies show that systemic IL-22 treatment mediates cytoprotective effects in the liver.

IL-22RA1 signaling is not critical for regulating Lgr5⁺ ISC and progenitor cell-associated genes and epithelial proliferation during HFD

Thus far, we observed that IL-22RA1 signaling regulates the expression of intestinal lipid metabolism genes after HFD (Fig. 6A, B). Next, we examined how specific intestinal cell types may be affected by IL-22RA1 signaling to mediate these HFD-driven effects. HFD enhances crypt regeneration, reduces the dependence of Lgr5⁺ ISCs on Paneth cell-derived growth factors, and enhances PPARδ/α-mediated programs that induce ISC and progenitor cell proliferation^36,37. Unlike Lgr5⁺ ISCs and progenitor cells, which display increased functionality after HFD, Paneth cells display impaired number and function^36,38. To assess the effects of HFD on Lgr5⁺ ISCs (Lgr5⁺ high), progenitor cells (Lgr5⁺ low), and Paneth cells, we analyzed publicly available RNA-seq data (GSE67324) from small intestinal sorted cells of Lgr5-EGFP-IRES-CreERT2 mice fed a long-term (9–14 months) chow diet or HFD (Supplementary Fig. 8A)³⁶. We assessed transcript levels of lipid storage and metabolism genes as well as peroxisome-associated genes based on our data from Fig. 6A. As expected, HFD resulted in upregulated function of many genes associated with lipid metabolism. Compared to Lgr5⁺ ISCs and Paneth cells, progenitor cells generally displayed higher overall expression of many lipid metabolism- and peroxisome-associated genes including Ppara, Ppard, and Pparg (Supplementary Fig. 8A). However, we did observe that the expression of several of these genes (including Ppara and Ppard) was upregulated in Paneth cells after HFD. Furthermore, expression of IL-22-associated genes (Il10rb and Il22ra1) was highest in Paneth cells (Supplementary Fig. 8A), which is consistent with our prior work⁸. We also observed that Paneth cell transcripts of Il22ra1 increased after HFD, while no striking difference in expression was observed in progenitor cells and a modest decrease in expression was observed in ISCs (Supplementary Fig. 8A). Our analysis suggests that HFD regulates the transcriptional profile of lipid metabolism- and peroxisome-associated genes in Paneth cells and that IL-22RA1 signaling to Lgr5⁺ ISCs, progenitor cells, and/or Paneth cells may regulate intestinal metabolism.

We next investigated whether IL-22 regulates Lgr5⁺ ISC function after exposure to fatty acids ex vivo. Ileal organoids were grown from C57BL/6 mice over 6 days during which they were treated with or without 10 ng/mL rIL-22 or 30 μM palmitic acid (PA), a fatty acid found in HFD. Fewer numbers of organoids were observed after treatment with rIL-22 (Supplementary Fig. 8B), which has previously been reported³⁹. RT-PCR analysis revealed no difference in Lgr5 or Olfm4 transcripts after stimulation with pure media, IL-22, PA, or PA + rIL-22 (Supplementary Fig. 8C). This suggests that rIL-22 stimulation either in the absence or presence of PA may not affect the ex vivo presence of Lgr5⁺ ISCs. In line with these observations, we did not detect any differences in transcripts of progenitor cell- (Dll1, Dll4, Prom1, Zpf652) or Lgr5⁺ ISC- (Lgr5, Olfm4, Sox9) associated genes in the ileum of HFD-fed Il22ra1^fl/fl;Villin-cre mice (Supplementary Fig. 6D, E). No difference in ISC number was observed after staining ileal tissues from the control diet or HFD-fed Il22ra1^fl/fl;Villin-cre mice for OLFM4 (Supplementary Fig. 8F). Likewise, no striking difference in cellular proliferation was observed upon staining ileal tissues for PCNA and examining Mki67 transcript levels (Supplementary Fig. 8G). This suggests that IL-22RA1 signaling does not mediate epithelial proliferation, Lgr5⁺ cell stemness, or induction of progenitor cells in vivo during HFD. However, it is possible that IL-22RA1 signaling regulates other functions of Lgr5⁺ ISCs and progenitor cells after HFD or that IL-22RA1 signaling to other enterocytes such as Paneth cells plays an important role in regulating HFD-driven changes in metabolism.

Intestinal IL-22RA1 signaling upregulates Paneth cell number and function to mediate systemic glucose metabolism during HFD

Although transcript levels of some Paneth cell lipid metabolism genes were upregulated after HFD (Supplementary Fig. 8A), the primary role of Paneth cells is to regulate the intestinal microbiota by producing shared antimicrobial peptides, including Reg3 proteins, and unique proteins such as Lyz1, α-defensins, and MMP7. Ileal tissues from HFD-fed Il22ra1^fl/fl;Villin-cre+ mice displayed decreased expression of Paneth cell-associated antimicrobial genes after HFD (Fig. 7A). In addition, HFD-fed cre+ mice displayed decreased numbers of LYZ1+ cells (Fig. 7B, C). This indicates that intestinal IL-22RA1 signaling mediates Paneth cell antimicrobial programs and numbers after HFD.

**Fig. 7: Paneth cell-specific IL-22RA1 signaling mediates their antimicrobial functions and systemic fasting glucose metabolism after HFD.**

Since Paneth cell-related programs are not maintained in Il22ra1^fl/fl;Villin-cre+ mice after HFD (Fig. 7A–C), it is possible that IL-22RA1 signaling is important in mitigating the downregulatory effects that HFD has on the Paneth cell differentiation/transcriptional program of Lgr5⁺ ISCs and progenitor cells. To study this, we placed non-tamoxifen injected Lgr5 reporter mice (see methods) on HFD and control diet, intraperitoneally injected them with 0 or 80 μg IL-22.Fc, and sorted single Lgr5-GFP low (progenitor) and high (Lgr5⁺ ISCs) cells to assess their expression of Paneth cell-associated genes. The following gating strategy was used (Supplementary Fig. 9A). We determined the purity of Paneth cells (CD24+, UEA+) isolated from all sorted cells. Our sorted CD24+, UEA+ population was enriched for Paneth cells (Lyz1) upon RT-PCR analysis (Supplementary Fig. 9B). Fairly higher (but not significant) Paneth cell numbers were detected from mice injected with 80 μg IL-22.Fc (Supplementary Fig. 9A, C). We observed that IL-22.Fc treatment significantly upregulated the expression of Mmp7 in Lgr5⁺ ISCs after HFD (Fig. 7D). However, IL-22.Fc treatment did not significantly upregulate the expression of Mmp7 in progenitor cells or the expression of Lyz1 in Lgr5⁺ ISCs or progenitor cells (Fig. 7D). Altogether, this suggests that IL-22 may not facilitate the differentiation of Lgr5+ ISCs or progenitor cells into Paneth cells.

Using primary organoid culture, we evaluated whether rIL-22 and PA treatment alter the expression of Paneth cell antimicrobial genes from C57BL/6 mice-derived ileal organoids. As expected, we observed a significant increase in Lyz1 expression in the rIL-22 treated group when compared to the control group (Fig. 7E [left]). While no difference in Lyz1 expression was noted between the control or PA-treated groups, we observed that the rIL-22-treated group possessed significantly greater Lyz1 expression than the PA-treated group. While PA + rIL-22 treatment resulted in decreased Lyz1 expression than the group treated only with rIL-22, this decrease was not as significantly low as the group treated only with PA (Fig. 7E [left]). Altogether, this indicates that while IL-22 induces Paneth cell antimicrobial gene expression, fatty acids (such as PA) can act to dampen this regulation. To assess whether this downregulation is a general phenomenon or is more specific to Paneth cells, we also examined the expression of Reg3g. As expected, transcript levels of Reg3g were elevated in the presence of rIL-22 (Fig. 7E [right]). Treatment with PA + rIL-22, however, still resulted in elevated expression of Reg3g comparable to that of the group treated only with rIL-22 (Fig. 7E [right]). This indicates that PA does not indeterminately reduce antimicrobial gene expression of enterocytes and that Paneth cell transcriptional functions may be more specifically regulated by the presence of fatty acids.

To determine whether Paneth cell number directly mediates systemic metabolism, we generated diphtheria toxin-depleted Paneth cell knockout (Defa6-cre+, ROSA26^DTA) and littermate control (Defa6-cre-, ROSA26^DTA) mice. Validation of knockout was confirmed (Supplementary Fig. 9D). Upon performing GTTs, we observed that Paneth cell-specific knockout mice fed a chow diet displayed impaired systemic glucose metabolism (Fig. 7F). To further elucidate the role of IL-22RA1 signaling to Paneth cells, we generated Paneth cell-specific Il22ra1 knockout (Il22ra1^fl/fl;Defa6-cre+) and littermate control mice. These mice were previously validated⁸. Il22ra1^fl/fl;Defa6-cre- and cre+ mice displayed similar numbers of LYZ1+ cells after long-term control diet and HFD (Fig. 7G, H). We next assessed if Paneth cell-specific IL-22RA1 signaling is important for regulating systemic metabolism. As expected, Il22ra1^fl/fl;Defa6-cre- and cre+ mice displayed similar weight gain when placed either on control diet or HFD (Fig. 7I; Supplementary Fig. 9E). Upon conducting GTTs, we did not observe any differences in glucose clearance or fasting glucose levels between control diet-fed Il22ra1^fl/fl;Defa6-cre mice (Supplementary Fig. 9F, G). After HFD, Il22ra1^fl/fl;Defa6-cre+ mice displayed significantly greater levels of blood glucose directly after fasting (Fig. 7J). However, glucose was similarly cleared between groups within 30 min after injection (Supplementary Fig. 9H). No differences in hepatic or WAT gene expression were observed in these mice after HFD (Supplementary Fig. 9I, J). Overall, our data suggest that IL-22 may act on Paneth cells to regulate their function and, in part, maintain a baseline glucose metabolism during HFD.

Discussion

In this study, we elucidate the tissue-specific role of IL-22RA1 signaling in mediating the onset of HFD-driven metabolic disorders. We observed that IL-22RA1 signaling differentially mediates the metabolic functions of intestinal, liver, and adipose tissues. Furthermore, our work highlights a key role of intestinal IL-22RA1 signaling in regulating intestinal and extra-intestinal metabolism along with the underlying immune and microbial mechanisms that shape these outcomes.

Our study indicates that IL-22-mediated regulation of the intestinal microbiota mediates systemic metabolism during HFD. Furthermore, we observed that the absence of intestinal IL-22RA1 signaling during HFD is associated with a microbial dysbiosis marked by an increased abundance of Oscillibacter which promotes the accumulation of metabolically damaging macrophages in the WAT³¹. This coincided with our observation of elevated macrophage infiltration and Mmp12 levels in the WAT of Il22ra1^fl/fl;Villin-cre+ mice, thereby supporting a protective role of intestinal IL-22RA1 signaling in mediating the microbiota to limit systemic WAT inflammation. Mechanistically, we also show that IL-22 may elicit its effects by activating IL-18. IL-22 protects against parasitic and bacterial infections by inducing epithelial IL-18^34,35. IL-18 also protects against obesity and its associated systemic metabolic disorders^40,41. While studies indicate that IL-18 can act on various sites, including skeletal muscle, adipose tissue, and the brain, to mediate metabolism and food intake, the direct metabolic role of intestinal IL-18 remained unclear⁴². Our study shows that IL-22 can also induce IL-18 to upregulate the expression of intestinal epithelial metabolic genes.

In addition, our study highlights a key regulatory role of Paneth cell-specific IL-22RA1 signaling. Prior studies illustrate the key role of IL-22 in mediating the differentiation of Paneth cells as well as their antimicrobial functions to regulate the intestinal microbiota and protect against infection^8,34,43. However, we did not observe any differences in LYZ1⁺ cell number in Il22ra1^fl/fl;Defa6-cre+ and littermate control mice. One reason for this could be the difference in age between these mice. The previously published study by our group used 6–8-week-old mice, whereas the present study uses mice fed a long-term diet (~23 weeks old at euthanasia)⁸. With an increase in age, there could be a compensatory mechanism by which Paneth cell numbers are maintained. Therefore, the role of age-related changes in cytokine levels and their effect on Paneth cell numbers should be investigated. Our study further explores the regulatory role of IL-22 on Paneth cells and provides evidence that Paneth cell-specific IL-22RA1 signaling is important for mediating baseline systemic glucose metabolism post-HFD. It should be noted that IL-22RA1 signaling may directly mediate glucose metabolism by Paneth cells since Paneth cells are glycolytic cell types and IL-22 has been shown to upregulate glycolysis^4,44,45,46. Furthermore, IL-22RA1 signaling to other enterocytes likely plays a critical role in facilitating systemic glucose metabolism. It is possible that intestinal IL-22RA1 signaling regulates systemic metabolism by mediating the production of certain metabolic hormones by enteroendocrine cells. For instance, IL-22 regulates the production of peptide tyrosine tyrosine (PYY), a hormone important for increasing insulin secretion and glucose metabolism^4,47,48. While PYY is produced by enteroendocrine cells, it has also been shown to be produced by Paneth cells^48,49. Therefore, IL-22RA1 signaling may directly induce PYY produced from these cells to regulate systemic glucose metabolism.

It should be noted that we analyzed publicly available RNA-sequencing data from the small intestine (Supplementary Fig. 8A) and, therefore, discrepancies between different facilities and experimental protocols (diet duration/composition, housing conditions, microbiota composition) may exist to influence sequencing results. In addition, while our study evaluated the role of IL-22RA1 signaling to WAT, we did not assess how IL-22 may specifically mediate the function of brown adipose tissue, a key mediator of energy expenditure and lipid metabolism. Regarding further examination of intestinal lipid and peroxisome pathways that may be mediated by IL-22RA1 signaling, it should be noted that the WAT and liver also express these genes and associated pathways. Therefore, blockade experiments to study the effects of a given pathway in Il22ra1^fl/fl;Villin-cre-/+ mice will also impact liver/WAT functions. An ideal approach would be to use intestinal epithelium-specific knockouts of a suspected IL-22 target such as Ppara. Lastly, how tissue-specific IL-22RA1 signaling regulates weight gain remains an open question.

Overall, the crosstalk between immune and metabolic functions in the intestinal epithelium observed herein has a potential relevance for human physiology. Indeed, similar regulatory interactions have been observed in patients with common variable immunodeficiency, HIV enteropathy, and celiac disease^50,51,52. Future studies would benefit from studying how IL-22RA1 signaling to additional metabolic organs, such as the pancreas, mediates obesity-associated disorders. Finally, our results elucidate how IL-22 or IL-22RA1 can be used to develop future tissue-specific treatments for obesity-associated metabolic disorders.

Methods

Mice

C57BL/6J (WT), Albumin-cre (C57BL/6J background), Villin-Cre (C57BL/6J background), Adiponectin-Cre (C57BL/6J background), and ROSA-DTA (C57BL/6J background) mice were purchased from The Jackson Laboratory. Germ-free C57BL/6Tac mice (male, 6-week-old) were ordered from Taconic Biosciences. Il22^−/− mice received from Dr. Jay Kolls, Tulane University. WT and Il22^−/− mice cohoused for 2 weeks and then separated for the entire duration of HFD. Lgr5-EGFP-creERT2 mice (C57BL/6 background) were provided by Dr. Vincent W. Yang, Stony Brook University. Defa6-Cre mice (C57BL/6 background) were provided by Dr. Richard Blumberg, Brigham and Women’s Hospital, Harvard. Generation and characterization of IL-22-Floxed (Il22ra^fl/fl) mice were performed as described⁵³. Il22ra1^fl/fl mice were bred with Albumin-cre, Villin-cre, Adiponectin-cre, Lgr5-EGFP-creERT2, or Defa6-cre mice to generate entire liver-, intestinal epithelium-, WAT-, Lgr5⁺ ISC-, or Paneth cell-specific IL-22RA1 knockout and littermate control mice. Defa6-cre mice were bred with ROSA-DTA mice to generate Paneth cell-specific knockout and littermate control mice. For all HFD and control diet experiments, we placed 6-week-old male mice on 16 weeks of HFD (Bio-Serv, S3282) or control diet (Bio-Serv, F4031). Littermate mice were used in all experiments and mice were separated based on cre status before placement on HFD, chow diet (PicoLab, Rodent Diet 5053), or control diet. For cohousing experiments, mice were not separated based on cre status after weaning. Mice were housed at Stony Brook University, Stony Brook, NY under specific pathogen-free conditions. For all ex vivo culture experiments, male and female mice were at least 6 weeks old when used. All mice were housed under a 12/12 light/dark cycle, 64–79 °F, and 30–70% humidity. For all experiments, mice were euthanized with CO₂. Animal studies were conducted with the approval and under all relevant ethical regulations of Stony Brook University’s Institutional Animal Care and Use Committee (IACUC: 968871).

Cell lines

HepG2 cells, a human hepatoma cell line, were obtained from Dr. Patrick Hearing at Stony Brook University, Stony Brook, NY. Conditioned organoid media was prepared from cultured L-WRN cells as described⁵⁴.

Animal treatment

IL-22.Fc (F652; Evive Biotech, China) was reconstituted in sterile 1x PBS. Either 0 ug (pure PBS) or 80 μg IL-22.Fc was intraperitoneally injected in chow diet-fed wild-type mice. Mice were euthanized 24 h post injection and tissues were harvested for relevant analysis.

Wildtype, Il22^−/−, Il22ra1^fl/fl;Albumin-cre, Il22ra1^fl/fl;Villin-cre, Il22ra1^fl/fl;Adiponectin-cre, and Il22ra1^fl/fl;Defa6-cre mice (male, 6 weeks old) were placed on 16 weeks of HFD. A control diet was used wherever indicated. Mice were weighed weekly, feces were collected before (day 0) and after (day 115) HFD treatment and GTTs were conducted after 16 weeks of treatment. To conduct GTTs, mice were weighed and then fasted for 5 h in clean cages. After this period, an initial blood glucose reading (0 min) was recorded by obtaining a blood sample from the tail. A Contour next glucose monitor and test strips (Bayer) were used to obtain blood glucose readings. Mice were then intraperitoneally injected with a body weight-specific volume (equivalent to half of the body weight) of glucose (200 mg/mL, MP Biomedicals) as previously published. Blood glucose readings were obtained every 30 min over a 2 h period. GTTs for Defa6-cre-/+, ROSA26^DTA mice were conducted on male and female chow diet-fed mice (10-16 weeks old). Insulin tolerance tests were conducted using a modified protocol described for GTTs. Mice were fasted for 5 h in a clean cage and then intraperitoneally injected with 0.75 IU/kg body weight insulin (Lilly). Glucose readings were obtained at 0 (fasting), 15, 30, 45, 60, and 90 min post-injection.

Mice were treated with antibiotics using methods similar to those previously described⁵⁵. Briefly, drinking water was substituted with an antibiotics cocktail containing vancomycin (0.5 g/L), ampicillin (0.5 g/L), neomycin (1 g/L), and metronidazole (1 g/L) on week 12 of HFD. Mice received antibiotics-supplemented drinking water for 4 weeks. For cecal content transfer experiments, 6-week-old male germ-free C57BL/6NTac mice were purchased from Taconic Biosciences. Cecal contents from either Il22ra1^fl/fl;Villin-cre- or cre+ mice fed 16 weeks of HFD were pooled and homogenized. Upon arrival, germ-free mice were housed in freshly autoclaved cages, fed a sterilized control diet, and gavaged with 200 μL 0.1 g/mL cecal contents (day 0). Contents were additionally given on days 2 and 4. GTTs were conducted on day 7.

Lgr5-EGFP-IRES-CreERT2, Il22ra1^fl/fl;Villin-cre-/+, and Il22ra1^fl/fl;Albumin-cre-/+ mice were placed on HFD for 6 weeks. Lgr5-EGFP-IRES-CreERT2 mice were intraperitoneally injected with either 0 or 80 μg IL-22.Fc every other day starting on week 5 (4 injections total). Il22ra1^fl/fl;Villin-cre-/+ and Il22ra1^fl/fl;Albumin-cre-/+ mice were intraperitoneally injected with 80 μg IL-22.Fc every other day starting on week 5 (4 injections total). Body weight was recorded at the indicated time points. Mice were euthanized and tissues were harvested for transcriptional analyses.

Reverse transcriptase-polymerase chain reaction (RT-PCR)

Total RNA was extracted from the liver, WAT, and terminal ileum tissues using Trizol-based isolation. Total RNA was isolated from organoids using an RNeasy Mini Kit (QIAGEN). Following the manufacturer’s instructions, cDNA was synthesized using Bio-Rad iScript kits. RT-PCR assays were performed by mixing 5 μL of SsoAdvanced™ Universal Probes Supermix (Bio-Rad) or SsoAdvanced™ Universal SYBR Green Supermix (Bio-Rad), 4.5 μL of cDNA, and 0.5 μL of primers/probes per well. Expression of genes of interest was calculated relative to Hprt or Gapdh. Fold change (derived from ΔΔC_T analysis) is calculated for individual litters. Control groups for organoid experiments are set to a baseline expression of 1.0 to clearly assess the relative fold change (derived from ΔΔC_T analysis) of each treatment on a biological sample. Refer to Supplementary Table 2 for the full list of primers.

Histopathology

For H&E staining, 5 μm paraffin-embedded tissue sections were cut, deparaffinized using xylene (2 washes), and rehydrated through a descending ethanol gradient (100%, 95%, 70%, 2x pure dH2O). Hematoxylin (VWR) was added to all slides for 1 min. Slides were then washed in running tap water for approximately 15 min, incubated in dH₂O for 30 s, treated with 95% ethanol for 1 min, and stained with eosin (VWR) for 1 min. Staining was briefly evaluated under a microscope and slides were transferred to 95% and 100% ethanol for 2 min each followed by xylene for 2 min. Slides were mounted with toluene and images were acquired. Crypt/villus length was quantified using ImageJ.

For alcian blue staining, tissue sections were prepared as described above. Sections were covered with 3% acetic acid for 3 min. Acetic acid was removed and sections were stained with alcian blue for 30 min. Sections were washed with 3% acetic acid for 15 s. Slides were rinsed in running tap water for 10 min. Sections were stained with Nuclear Fast Red solution (Electron Microscopy Sciences) for 6 min. Slides were rinsed in running tap water for 2 min. Sections were dehydrated with an ascending ethanol wash (70%, 95%, and 100%) followed by xylene for 2 min each. Slides were mounted with toluene and images were acquired.

ORO tissue staining was performed as previously described⁵⁵. Briefly, liver tissues were harvested and frozen in an OCT compound medium (Sakura). Cryosections (5 μm) were cut. Before staining, tissues were dried at room temperature for 10 min. A 3:2 ratio solution of ORO Isopropanol Solution (Electron Microscopy Sciences): dH₂O was applied to the slides for 5 min. Slides were washed in running tap water for 30 min. Slides were dried and mounted with glycerol/gelatin-based medium. Images were acquired. Percent area coverage of ORO staining per image was quantified using ImageJ.

Scoring of H&E-stained WAT was performed by Dr. Kenneth R. Shroyer. A scale from 0–4 was used to subjectively score the degree of inflammation (i.e., the presence of crown-like structures). Scale: 0, no or minimal inflammation, 1 indicates low inflammation; 2, moderate inflammation; 3, high inflammation; and 4, severe inflammation.

Culture of murine intestinal organoids

The murine organoid culture was performed similarly to what we previously described⁵⁶. Briefly, small intestinal ileal tissues were harvested from C57BL/6 mice. Peyer’s patches and connective tissues were removed. Intestinal tissues were flushed with ice-cold 1x PBS, cut longitudinally, and quickly washed in 1x PBS. Tissues were cut into approximately 1 cm pieces and washed in cold 1x PBS until the liquid was predominantly clear of debris (usually 3–4 washes). To isolate intestinal crypts, tissues were incubated at 4 °C on an orbital shaker for 30 min in 1x PBS containing 3.75 mM EDTA. Tissues were washed once in ice-cold 1x PBS to remove residual EDTA. Fresh ice-cold 1x PBS was added and the tissues were shaken to promote the release of crypts into the supernatant. The supernatant was passed into a new tube over a 70 μm cell strainer. To collect more crypts, this step was repeated two more times with gradually stronger shaking. Crypts were then pelleted down at 200 x g and each sample’s overall number was calculated.

For plating in a 24-well plate, crypts were resuspended in Matrigel Matrix (Corning) at a concentration of 80–100 crypts per 30 μL Matrigel. Plated Matrigel domes were kept at 37 °C, 5% CO2 for 10 min until they fully polymerized. Domes were overlaid with 500 μL of Organoid Growth Medium (1:1 mixture of L-WRN and DMEM/F12). The following media supplements were added: 1x penicillin-streptomycin-glutamine (Invitrogen), 1x N2 (Invitrogen), 1x B27 (Invitrogen), 50 ng/mL EGF (R&D system), 1 mM N-acetylcysteine (Sigma-Aldrich), 10 nM Gastrin-Leu15 (Sigma-Aldrich), 500 nM A83-01 (Torics), 10 μM Y-27632 (Sigma-Aldrich), and 10 μM of CHIR99021 (Tocris). The media was changed every 2 days. Y-27632 and CHIR99021 were not added to the media after day 2.

For Figs. 1I and 6D, organoids were either treated with pure media, rIL-22 (10 ng; R&D Systems), or rIL-18 (100 ng; R&D Systems) starting on day 2. On day 5, organoids were lysed in RLT buffer (QIAGEN) containing 1% β-mercaptoethanol for RNA isolation. For Fig. 7E and Supplementary Fig. 8B, C, organoids were either treated with pure media, rIL-22 (10 ng/mL; R&D Systems), PA (30 μM; Sigma), or rIL-22 and PA starting on day 0. On day 6, brightfield images were taken and organoids were lysed in RLT buffer (QIAGEN) containing 1% β-mercaptoethanol for RNA isolation.

Single-cell isolation and sorting

Il22ra1^fl/fl;Lgr5-EGFP-creERT2+ or Il17ra^fl/fl;Lgr5-EGFP-creERT2+ mice were placed on control diet or HFD for 1 week. These mice were not injected with tamoxifen and, therefore, were useful for specifically sorting Lgr5⁺ ISC (Lgr5-GFP^high) and progenitor (Lgr5-GFP^low) cells based on GFP expression. Mice were injected with IL-22.Fc or PBS 72 h and 24 h prior to being euthanized. Small intestinal crypts were isolated as discussed in the prior section one day after the last IL-22 injection. Once crypts were isolated, they were incubated in DMEM/F12 (Gibco) containing 20% FBS for 15 min at 4 °C. Crypts were dissociated into single cells upon incubation with TrypLE Express (Invitrogen) containing 10 μM Y-27632 (Sigma-Aldrich) and 10 μg/mL DNase I (Roche) for 5 min at 37 °C after which an equal volume DMEM/F12 was added. Cells were pipetted to further promote dissociation. Cells were washed with 1x PBS and pelleted by centrifugation at 4 °C at 200 xg for 5 min. Cells were stained with Aqua Dead Cell Stain Kit (1:500; Invitrogen; Ref: L34957) for 30 min to exclude dead cells, anti-CD45:APC-eFluor780 (1:200; Invitrogen; Ref: 47–0451) to exclude CD45+ cells, and anti-CD24:PE-Cyanine7 (1:200; Invitrogen; Ref: 25–0242) and UEA1:DyLight649 (1:200; Vector Laboratories; Ref: DL-1068-1) to sort Paneth cells. All antibodies were validated by their manufacturer and/or our lab. Cells were processed for fluorescence-activated cell sorting (FACS) sorting of Lgr5-GFP^hi and Lgr5-GFP^low cells using a BD FACSAria. Cells were sorted into RLT buffer (QIAGEN) containing 1% β-mercaptoethanol for RNA isolation.

Immunofluorescence (tissues and organoids)

Liver, adipose, and intestinal tissues were harvested and fixed in 10% formalin for 24 h. Tissues were processed and embedded in paraffin blocks. Sections (5 μm) were cut. Sections were deparaffinized and rehydrated using xylene (2 washes) and a descending ethanol gradient (100%, 95%, 70%, 2x pure dH2O). Antigen retrieval was performed by incubating tissues in citric acid buffer and placing them in a steamer for 40 min. Tissues were cooled to room temperature, encircled with an IHC PAP pen (ENZO Life Sciences), and permeabilized (10 min) with 1x PBS containing 0.1% Triton X-100 (Sigma-Aldrich). Tissues were washed 3x (5 min) with 1x PBS containing 0.01% Tween 20 (VWR). Tissues were blocked in 1x PBS containing 5% bovine serum albumin for 30 min at 37 °C. Tissues were incubated overnight at 4 °C in 1x PBS containing 5% bovine serum albumin and primary antibodies against LYZ1-FITC (1:200; Dako; Ref: F0372), OLFM4 (1:200; Cell Signaling Technology; Ref: 39141S), PCNA (1:200; Santa Cruz Biotechnology; Ref: Sc-56), anti-F4/80-FITC (1:50; Miltenyi Biotec; Ref: 130-117-509), MMP12 (1:50; Abcam; Ref: ab231109). Slides were washed 3x with 1x PBS. Tissues were incubated with anti-rabbit IgG Fab2 Alexa Fluor® 488 (1:200; Cell Signaling Technology; Ref: 4412). All antibodies were validated by their manufacturer and/or our lab. DAPI hard stain mounting media (Vector® Laboratories) was applied to visualize cell nuclei. Images were acquired using an Olympus CKX41 microscope.

For ORO staining, organoids were cultured in the presence or absence of 5 ng rIL-22 starting on day 2. After 24 h (day 3), organoids were treated with 4% LM. Organoids were dissociated from the Matrigel with cell recovery media (Corning). Organoids were placed on an orbital shaker at 4 °C for 5 min. Organoids were washed in DMEM, 10% FBS, spun down at 180 xg, and fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 30 min. Organoids were briefly washed with 1x PBS and then 60% isopropanol. ORO solution (similar to what we used for tissue staining) was added for 20 minutes. The solution was carefully removed, leaving organoids undisturbed at the bottom of the tube. Organoids were washed in 1x PBS. Pipette tips were coated with 5% bovine serum albumin to prevent organoids from sticking. Organoids were transferred to a slide and were encircled by an IHC PAP pen. DAPI hard stain mounting media (Vector® Laboratories) was applied to visualize cell nuclei. Images were acquired using an Olympus CKX41 microscope.

Cell line assays

HepG2 cells were grown until 70% confluency in a T-75 flask, dissociated into single cells, washed, and plated (200 μL) at equal concentrations in a 96-well plate. At 60–70% confluency, cells were stimulated with or without 80 ng IL-22.Fc. After 24 h, cells were treated with 4% LM. After 48 h, cells were washed and fixed with 4% paraformaldehyde. Cells were washed with 60% isopropanol and stained with ORO (similar to what we previously used) for 10 min. Cells were washed with dH₂O until the supernatant was clear (approximately 2x). ORO stain was extracted from cells using 250 μL 100% isopropanol. Extracted dye (200 μL) was plated in a clean well and its OD of 490 nm was measured.

Gas chromatography

The Folch-Lees method of lipid extraction was used⁵⁷. Extracts were filtered. Lipids were isolated in chloroform and separated via thin-layer chromatography. Separation was performed using Silica Gel 60 A plates developed in petroleum ether, ethyl ether, and acetic acid (80:20:1). Separation was visualized with rhodamine 6G. As performed by Morrison and Smith, phospholipids, diglycerides, triglycerides, and cholesteryl esters were isolated and methylated using BF3/methanol⁵⁸. Methylated fatty acids were extracted and analyzed by gas chromatographic analyses using an Agilent 7890 A gas chromatograph with flame ionization detectors and capillary column (SP2380, 0.25 mm×30 m, 0.25 µm film, Supelco, Bellefonte, PA). Helium was used as a carrier gas and oven temperature was set from 160–230 °C at 4 °C/min. To determine fatty acid methyl esters, sample and standard retention times were compared. Standards with odd-chain fatty acids were used to quantify sample lipids. The following standards were used: dipentadecanoyl phosphatidylcholine (C15:0), diheptadecanoin (C17:0), trieicosenoin (C20:1), and cholesteryl eicosenoate (C20:1).

16S rRNA microbial community analysis

Microbial community analysis was performed as previously described⁸. Feces were collected from mice before (day 0) and after (day 105) HFD treatment. Fecal DNA was isolated using a QiAMP stool DNA extraction kit (QIAGEN). Analysis utilizing PCR amplification of the V4 region of 16 S rRNA followed by sequencing on an Illumina MiSeq was performed by MR DNA (Shallowater, TX, USA). Identification of gut microbial amplicon sequence variants (ASVs) followed by chimera removal was performed with DADA2 (v1.21). Taxonomy assignment was performed using the Silva Project’s version 138.1 release and formatted for use with DADA2. The downstream and statistical analysis was carried out with Phyloseq (v1.34.0), quantile normalization, and differential abundance using BRB-ArrayTools (https://brb.nci.nih.gov/BRBArrayTools/) and alpha and beta diversity using MicrobiomeAnalyst⁵⁹. Raw sequencing data was deposited in the NCBI Gene Expression Omnibus.

RNA sequencing (RNA-seq)

RNA-seq analysis of RNA isolated from terminal ileum tissue was performed as we previously described⁵⁶. Ileal tissues were collected from Il22ra1^fl/fl;Villin-cre mice after 16 weeks of HFD. RNA was isolated via Trizol extraction methods and then purified for sequencing.

Small intestinal RNA-sequencing data previously deposited in the Gene Expression Omnibus (GEO) database (accession number: GSE67324) was analyzed. Methods for this sequencing were previously described³⁶. Small intestinal crypts were isolated from Lgr5-EGFP-IRES-CreERT2 mice fed an HFD or control diet for 9–14 months. Briefly, crypts were dissociated with TrypLe Express (Invitrogen) and stained with appropriate markers for sorting. ISCs were sorted as Lgr5-EGFP^highEpcam⁺CD24^low/−CD31⁻Ter119⁻CD45⁻7-AAD⁻. Lgr5-EGFP^low progenitor cells were sorted as EGFP^lowEpcam⁺CD24^low/−CD31⁻Ter119⁻CD45⁻7-AAD⁻. Paneth cells were sorted as CD24^highSidescatter^highLgr5-EGFP⁻Epcam⁺CD31⁻Ter119⁻CD45⁻7-AAD⁻. Viability dye 7-AAD (Life Technologies) was used to exclude dead cells³⁶.

Luminex multiplex assay

Serum was isolated from HFD-fed mice and analyzed via multiplex analysis (Luminex) using the Mouse Metabolic Hormone Panel (MilliporeSigma) for c-peptide and insulin and TH17 Panel (Millipore Sigma) for IL-22. Samples were run at the Stony Brook School of Medicine Genomics Core (Bio-Plex 200) and Vanderbilt University Hormone Assay and Analytical Services Core (MagPix).

Promoter analysis

The Eukaryotic Promoter Database was used to identify the transcriptional start site for murine Ppara. Sequence output was set to 2000 base pairs upstream of the transcriptional start site. The retrieved sequence was validated via NCBI. The upstream sequence was then analyzed using the JASPAR Core database to identify putative binding sites for murine Stat3.

Statistics and reproducibility

Statistics were conducted using GraphPad Version 7.05.237 software. The following tests were used as indicated in figure legends to determine p-values: Mann–Whitney test (two-tailed), Mann–Whitney test (one-sided), Unpaired t-test (one-tailed), 2-way ANOVA with Sidak multiple comparisons, and one-way ANOVA with Tukey multiple comparisons. For all tests, p < 0.05 was considered significant. Data are presented as mean ± standard error or standard deviation as indicated in the figure legends. At least 2 experimental replicates were performed whenever possible to verify reproducibility. Replicate number is indicated in the figure legends. Experimental sample size and replicates are indicated in the text. Data points were excluded if they were statistical outliers as determined by using GraphPad Prism (ROUT analysis, Q = 1%). Data acquisition and analysis were performed blind whenever possible.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The 16S rRNA microbial sequencing data are deposited in the NCBI BioProject database (accession code: PRJNA944165). The RNA-sequencing data from small intestinal tissues of HFD-fed Il22ra1^fl/fl;Villin-cre-/+ mice are deposited in the Sequence Read Archive (accession code: PRJNA1055251). The previously published small intestinal RNA-sequencing data from control or HFD-fed Lgr5-EGFP-IRES-CreERT2 mice are deposited in GEO (accession code: GSE67324). Relevant data underlying all non-sequencing-related findings are available in the Source Data file. Any additional information is available upon request to the corresponding author (Pawan Kumar, pawan.kumar@stonybrook.edu). Source data are provided in this paper.

References

Keir, M. E., Yi, T., Lu, T. T. & Ghilardi, N. The role of IL-22 in intestinal health and disease. J. Exp. Med. 217, e20192195 (2020).
Article PubMed PubMed Central Google Scholar
Parks, O. B., Pociask, D. A., Hodzic, Z., Kolls, J. K. & Good, M. Interleukin-22 signaling in the regulation of intestinal health and disease. Front. Cell Dev. Biol 3, 85 (2016).
Article PubMed PubMed Central Google Scholar
Sugimoto, K. et al. IL-22 ameliorates intestinal inflammation in a mouse model of ulcerative colitis. J. Clin. Investig. https://doi.org/10.1172/JCI33194 (2008).
Wang, X. et al. Interleukin-22 alleviates metabolic disorders and restores mucosal immunity in diabetes. Nature 514, 237–241 (2014).
Article ADS CAS PubMed Google Scholar
Zenewicz, L. A. et al. Innate and adaptive interleukin-22 protects mice from inflammatory bowel disease. Immunity 29, 947–957 (2008).
Article CAS PubMed PubMed Central Google Scholar
Salzman, N. H. et al. Enteric defensins are essential regulators of intestinal microbial ecology. Nat. Immunol. 11, 76–83 (2010).
Article CAS PubMed Google Scholar
Sato, T. et al. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469, 415–418 (2011).
Article ADS CAS PubMed Google Scholar
Gaudino, S. J. et al. IL-22 receptor signaling in Paneth cells is critical for their maturation, microbiota colonization, Th17-related immune responses, and anti-Salmonella immunity. Mucosal Immunol. https://doi.org/10.1038/s41385-020-00348-5 (2020).
Lindemans, C. A. et al. Interleukin-22 promotes intestinal-stem-cell-mediated epithelial regeneration. Nature 528, 560–564 (2015).
Article ADS CAS PubMed PubMed Central Google Scholar
Pickert, G. et al. STAT3 links IL-22 signaling in intestinal epithelial cells to mucosal wound healing. J. Exp. Med. 206, 1465–1472 (2009).
Article CAS PubMed PubMed Central Google Scholar
Zheng, Y. et al. Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens. Nat. Med. 14, 282–289 (2008).
Article CAS PubMed Google Scholar
NCD Risk Factor Collaboration (NCD-RisC). Rising rural body-mass index is the main driver of the global obesity epidemic in adults. Nature 569, 260–264 (2019).
Article ADS Google Scholar
Yang, L. et al. Amelioration of high fat diet induced liver lipogenesis and hepatic steatosis by interleukin-22. J. Hepatol. 53, 339–347 (2010).
Article CAS PubMed Google Scholar
Park, O. et al. Biologically active, high levels of interleukin-22 inhibit hepatic gluconeogenesis but do not affect obesity and its metabolic consequences. Cell Biosci. 5, 25 (2015).
Article PubMed PubMed Central Google Scholar
Dalmas, E. et al. T cell–derived IL-22 amplifies IL-1β–driven inflammation in human adipose tissue: relevance to obesity and type 2 diabetes. Diabetes 63, 1966–1977 (2014).
Article CAS PubMed Google Scholar
Ki, S. H. et al. Interleukin-22 treatment ameliorates alcoholic liver injury in a murine model of chronic-binge ethanol feeding: role of signal transducer and activator of transcription 3. Hepatology 52, 1291–1300 (2010).
Article CAS PubMed Google Scholar
Radaeva, S., Sun, R., Pan, H.-N., Hong, F. & Gao, B. Interleukin 22 (IL-22) plays a protective role in T cell-mediated murine hepatitis: IL-22 is a survival factor for hepatocytes via STAT3 activation. Hepatology 39, 1332–1342 (2004).
Article CAS PubMed Google Scholar
Obrowsky, S. et al. Adipose triglyceride lipase is a TG hydrolase of the small intestine and regulates intestinal PPARα signaling. J. Lipid Res. 54, 425–435 (2013).
Article CAS PubMed PubMed Central Google Scholar
Ong, K. T., Mashek, M. T., Bu, S. Y., Greenberg, A. S. & Mashek, D. G. Adipose triglyceride lipase is a major hepatic lipase that regulates triacylglycerol turnover and fatty acid signaling and partitioning. Hepatology 53, 116–126 (2011).
Article CAS PubMed Google Scholar
Kim, E.-Y. et al. Interleukin-22 induces the infiltration of visceral fat tissue by a discrete subset of duffy antigen receptor for chemokine-positive M2-like macrophages in response to a high fat diet. Cells 8, 1587 (2019).
Article CAS PubMed PubMed Central Google Scholar
Wang, Z. et al. High fat diet induces formation of spontaneous liposarcoma in mouse adipose tissue with overexpression of interleukin 22. PLoS ONE 6, e23737 (2011).
Article ADS CAS PubMed PubMed Central Google Scholar
Backhed, F. et al. The gut microbiota as an environmental factor that regulates fat storage. Proc. Natl Acad. Sci. USA 101, 15718–15723 (2004).
Article ADS PubMed PubMed Central Google Scholar
Martinez-Guryn, K. et al. Small intestine microbiota regulate host digestive and absorptive adaptive responses to dietary lipids. Cell Host Microbe 23, 458–469.e5 (2018).
Article CAS PubMed PubMed Central Google Scholar
Hildebrandt, M. A. et al. High-fat diet determines the composition of the murine gut microbiome independently of obesity. Gastroenterology 137, 1716–1724.e1–2. (2009).
Article CAS PubMed Google Scholar
Ley, R. E. et al. Obesity alters gut microbial ecology. Proc. Natl Acad. Sci. USA 102, 11070–11075 (2005).
Article ADS CAS PubMed PubMed Central Google Scholar
Ridaura, V. K. et al. Gut microbiota from twins discordant for obesity modulate metabolism in mice. Science 341, 1241214 (2013).
Article PubMed Google Scholar
Velagapudi, V. R. et al. The gut microbiota modulates host energy and lipid metabolism in mice. J. Lipid Res. 51, 1101–1112 (2010).
Article CAS PubMed PubMed Central Google Scholar
Ito, T. et al. IL-22 induces Reg3γ and inhibits allergic inflammation in house dust mite–induced asthma models. J. Exp. Med. 214, 3037–3050 (2017).
Article CAS PubMed PubMed Central Google Scholar
Hendrikx, T. et al. Bacteria engineered to produce IL-22 in intestine induce expression of REG3G to reduce ethanol-induced liver disease in mice. Gut 68, 1504–1515 (2019).
Article CAS PubMed Google Scholar
Yang, W. et al. Intestinal microbiota-derived short-chain fatty acids regulation of immune cell IL-22 production and gut immunity. Nat. Commun. 11, 4457 (2020).
Article ADS CAS PubMed PubMed Central Google Scholar
Li, Z. et al. Microbiota and adipocyte mitochondrial damage in type 2 diabetes are linked by Mmp12 + macrophages. J. Exp. Med. 219, e20220017 (2022).
Article CAS PubMed PubMed Central Google Scholar
Morigny, P., Boucher, J., Arner, P. & Langin, D. Lipid and glucose metabolism in white adipocytes: pathways, dysfunction and therapeutics. Nat. Rev. Endocrino.l 17, 276–295 (2021).
Article CAS Google Scholar
Rosen, E. D. & Spiegelman, B. M. Adipocytes as regulators of energy balance and glucose homeostasis. Nature 444, 847–853 (2006).
Article ADS CAS PubMed PubMed Central Google Scholar
Chiang, H.-Y. et al. IL-22 initiates an IL-18-dependent epithelial response circuit to enforce intestinal host defence. Nat. Commun. 13, 874 (2022).
Article ADS CAS PubMed PubMed Central Google Scholar
Muñoz, M. et al. Interleukin-22 induces interleukin-18 expression from epithelial cells during intestinal infection. Immunity 42, 321–331 (2015).
Article PubMed Google Scholar
Beyaz, S. et al. High-fat diet enhances stemness and tumorigenicity of intestinal progenitors. Nature 531, 53–58 (2016).
Article ADS CAS PubMed PubMed Central Google Scholar
Mana, M. D. et al. High-fat diet-activated fatty acid oxidation mediates intestinal stemness and tumorigenicity. Cell Rep. 35, 109212 (2021).
Article CAS PubMed PubMed Central Google Scholar
Liu, T.-C. et al. Western diet induces Paneth cell defects through microbiome alterations and farnesoid X receptor and type I interferon activation. Cell Host Microbe 29, 988–1001.e6 (2021).
Article CAS PubMed PubMed Central Google Scholar
Zha, J.-M. et al. Interleukin 22 expands transit-amplifying cells while depleting Lgr5+ stem cells via inhibition of Wnt and Notch signaling. Cell. Mol. Gastroenterol. Hepatol. 7, 255–274 (2019).
Article PubMed Google Scholar
Netea, M. G. et al. Deficiency of interleukin-18 in mice leads to hyperphagia, obesity and insulin resistance. Nat. Med. 12, 650–656 (2006).
Article CAS PubMed Google Scholar
Zorrilla, E. P. et al. Interleukin-18 controls energy homeostasis by suppressing appetite and feed efficiency. Proc. Natl Acad. Sci. USA 104, 11097–11102 (2007).
Article ADS CAS PubMed PubMed Central Google Scholar
Somm, E. & Jornayvaz, F. R. Interleukin-18 in metabolism: From mice physiology to human diseases. Front. Endocrinol. (Lausanne) 13, 971745 (2022).
Article PubMed Google Scholar
He, G.-W. et al. Optimized human intestinal organoid model reveals interleukin-22-dependency of paneth cell formation. Cell Stem Cell 29, 1333–1345.e6 (2022).
Article CAS PubMed PubMed Central Google Scholar
Liu, Y. et al. Interleukin-22 promotes aerobic glycolysis associated with tumor progression via targeting hexokinase-2 in human colon cancer cells. Oncotarget 8, 25372–25383 (2017).
Article PubMed PubMed Central Google Scholar
Rodríguez-Colman, M. J. et al. Interplay between metabolic identities in the intestinal crypt supports stem cell function. Nature 543, 424–427 (2017).
Article ADS PubMed Google Scholar
Trevejo-Nunez, G. et al. Interleukin-22 (IL-22) binding protein constrains IL-22 activity, host defense, and oxidative phosphorylation genes during Pneumococcal pneumonia. Infect. Immun. 87, e00550–19 (2019).
Article CAS PubMed PubMed Central Google Scholar
Ramracheya, R. D. et al. PYY-dependent restoration of impaired insulin and glucagon secretion in Type 2 diabetes following Roux-En-Y gastric bypass surgery. Cell Rep. 15, 944–950 (2016).
Article CAS PubMed PubMed Central Google Scholar
Guida, C. et al. PYY plays a key role in the resolution of diabetes following bariatric surgery in humans. EBioMedicine 40, 67–76 (2019).
Article PubMed PubMed Central Google Scholar
Pierre, J. F. et al. Peptide YY: a Paneth cell antimicrobial peptide that maintains Candida gut commensalism. Science 381, 502–508 (2023).
Article ADS CAS PubMed PubMed Central Google Scholar
Earley, Z. M. et al. GATA4 controls regionalization of tissue immunity and commensal-driven immunopathology. Immunity 56, 43–57.e10 (2023).
Article CAS PubMed PubMed Central Google Scholar
Shulzhenko, N. et al. Crosstalk between B lymphocytes, microbiota and the intestinal epithelium governs immunity versus metabolism in the gut. Nat. Med. 17, 1585–1593 (2011).
Article CAS PubMed PubMed Central Google Scholar
Shulzhenko, N. et al. CVID enteropathy is characterized by exceeding low mucosal IgA levels and interferon-driven inflammation possibly related to the presence of a pathobiont. Clin. Immunol. 197, 139–153 (2018).
Article CAS PubMed PubMed Central Google Scholar
Zheng, M. et al. Therapeutic role of interleukin 22 in experimental intra-abdominal Klebsiella pneumoniae infection in mice. Infect. Immun. 84, 782–789 (2016).
Article CAS PubMed PubMed Central Google Scholar
Miyoshi, H. & Stappenbeck, T. S. In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture. Nat. Protoc. 8, 2471–2482 (2013).
Article CAS PubMed PubMed Central Google Scholar
Gaudino, S. J. et al. Cutting edge: intestinal IL-17A receptor signaling specifically regulates high-fat diet–mediated, microbiota-driven metabolic disorders. J. Immunol. 207, 1959–1963 (2021).
Article CAS PubMed Google Scholar
Lin, X. et al. IL-17RA-signaling in Lgr5+ intestinal stem cells induces expression of transcription factor ATOH1 to promote secretory cell lineage commitment. Immunity 55, 237–253.e8 (2022).
Article CAS PubMed PubMed Central Google Scholar
Folch, J., Lees, M. & Sloane Stanley, G. H. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226, 497–509 (1957).
Article CAS PubMed Google Scholar
Morrison, W. R. & Smith, L. M. Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride-methanol. J. Lipid Res. 5, 600–608 (1964).
Article CAS PubMed Google Scholar
Chong, J., Liu, P., Zhou, G. & Xia, J. Using MicrobiomeAnalyst for comprehensive statistical, functional, and meta-analysis of microbiome data. Nat. Protoc. 15, 799–821 (2020).
Article CAS PubMed Google Scholar

Download references

Acknowledgements

We would like to acknowledge the Flow Cytometry Core, Research Histology Core, and Genomics Core at Stony Brook University. We would like to acknowledge the RNA-sequencing core at Cold Spring Harbor Laboratory. We would like to acknowledge Vanderbilt University Medical Center’s Hormone Assay and Analytical Services Core. This work was supported by the NIHAI149257 to P.K., NRSA T32 training grant (T32AI007539) to S.J.G., and NIHDK088199 to R.B.. P.K. is supported by the Crohn’s and Colitis Foundation (476637), NIH R01 DK121798-01, and NIH R21 AI146696. We thank Jay Kolls (Tulane University), Jeremy McAleer (Marshall University), and Vishal Singh (Pennsylvania State University) for critically reading the manuscript and providing expert advice.

Author information

Authors and Affiliations

Department of Microbiology and Immunology, Renaissance School of Medicine, Stony Brook University, Stony Brook, NY, USA
Stephen J. Gaudino, Ankita Singh, Huakang Huang, Makheni Jean-Pierre, Cody Kempen, Tej Bahadur, Kiyoshi Shiomitsu & Pawan Kumar
College of Pharmacy, Oregon State University, Corvallis, OR, USA
Jyothi Padiadpu & Andrey Morgun
Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, 02115, USA
Richard Blumberg
Department of Pathology, Renaissance School of Medicine, Stony Brook University, Stony Brook, NY, USA
Kenneth R. Shroyer
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 11724, USA
Semir Beyaz
Carlson College of Veterinary Medicine, Oregon State University, Corvallis, OR, USA
Natalia Shulzhenko

Authors

Stephen J. Gaudino
View author publications
You can also search for this author in PubMed Google Scholar
Ankita Singh
View author publications
You can also search for this author in PubMed Google Scholar
Huakang Huang
View author publications
You can also search for this author in PubMed Google Scholar
Jyothi Padiadpu
View author publications
You can also search for this author in PubMed Google Scholar
Makheni Jean-Pierre
View author publications
You can also search for this author in PubMed Google Scholar
Cody Kempen
View author publications
You can also search for this author in PubMed Google Scholar
Tej Bahadur
View author publications
You can also search for this author in PubMed Google Scholar
Kiyoshi Shiomitsu
View author publications
You can also search for this author in PubMed Google Scholar
Richard Blumberg
View author publications
You can also search for this author in PubMed Google Scholar
Kenneth R. Shroyer
View author publications
You can also search for this author in PubMed Google Scholar
Semir Beyaz
View author publications
You can also search for this author in PubMed Google Scholar
Natalia Shulzhenko
View author publications
You can also search for this author in PubMed Google Scholar
Andrey Morgun
View author publications
You can also search for this author in PubMed Google Scholar
Pawan Kumar
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

P.K. conceptualized the idea. S.J.G., H.H., and P.K. designed the experiments. S.J.G. and P.K. wrote the manuscript. S.J.G. and H.H. weighed mice and performed GTTs. S.J.G. and M.J.-P. performed H&E staining and RNA isolation. K.S. assisted with RNA isolation. S.J.G. and A.S. performed flow cytometry and RT-PCR experiments. S.J.G., A.S., and C.K. performed immunofluorescence staining. S.J.G. performed ORO staining, alcian blue staining, ELISA, crypt and single-cell isolation, DSS experiments, and cell/organoid culture. T.B. assisted with organoid culture. R.B. provided mouse lines for generating Paneth cell-specific Il22ra1 knockouts. K.R.S. evaluated H&E slides of WAT for inflammation. S.B. provided small intestinal RNA-seq data from Lgr5-EGFP-IRES-CreERT2 mice. J.P., N.S., and A.M. analyzed and interpreted the 16S rRNA sequencing data. All authors discussed data and commented on the manuscript.

Corresponding author

Correspondence to Pawan Kumar.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Communications thanks Dechun Feng, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Reporting Summary

Peer Review File

Source data

Source Data

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Gaudino, S.J., Singh, A., Huang, H. et al. Intestinal IL-22RA1 signaling regulates intrinsic and systemic lipid and glucose metabolism to alleviate obesity-associated disorders. Nat Commun 15, 1597 (2024). https://doi.org/10.1038/s41467-024-45568-6

Download citation

Received: 28 April 2022
Accepted: 26 January 2024
Published: 21 February 2024
DOI: https://doi.org/10.1038/s41467-024-45568-6

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.