Modulation of the IL-33/IL-13 Axis in Obesity by IL-13Rα2

IL-13Rα2 expression in adipose tissue is elevated in mouse models of obesity

IL-13Rα2 is expressed at low or undetectable levels in the absence of tissue inflammation, but is induced in the presence of IL-4 or IL-13, in combination with TNF-α. Levels may be further enhanced by IL-17 or other inflammatory cytokines (14, 35). Obesity is characterized by a state of systemic low-grade inflammation, which is marked by elevated levels of circulating TNF-α (36). In a model of diet-induced obesity, IL-13Rα2 mRNA expression levels in adipose tissue from mice fed an HFD for 13 wk were significantly increased compared with levels in adipose tissue from mice fed a normal chow diet during the same time period (Fig. 1A). Expression of IL-13Rα1 and common γ-chain (IL-2Rγ) was also elevated, whereas expression of IL-4Rα was not increased in adipose tissue of obese mice. As has been reported previously (36), TNF-α mRNA expression was also elevated in adipose tissue from obese mice. Similar increases in adipose tissue IL-13Rα2 expression were seen in leptin-deficient ob/ob mice, a genetic model of obesity (data not shown). Serum concentrations of sIL-13Rα2 were also higher under conditions of obesity (Fig. 1B). In accordance with the elevated IL-13Rα2, which inactivates and depletes IL-13 in tissue (15, 37), adipose tissue levels of IL-13 cytokine were reduced in mice fed an HFD (Fig. 1C).

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FIGURE 1.

IL-13Rα2 expression is induced with HFD, associated with depletion of tissue IL-13. C57BL/6 mice were fed normal chow or a 60% HFD for 13 wk. (A) Gene expression analysis in scWAT, normalized to housekeeping genes. (B) sIL-13Rα2 in mouse serum. (C) IL-13 concentration in adipose tissue extract. (D) IL-13Rα2 protein concentration in extracts of eWAT or scWAT tissue of mice fed chow or HFD. scWAT and eWAT were collected from the same animals. The dashed line represents the limit of assay detection. (E) il13rα2 transcripts in human adipose tissue. VAT denotes the omental adipose depot, corresponding to visceral adipose tissue. Data represent three independent experiments for (A–C) (n = 6–8 mice per group), two independent experiments for (D) (n = 11 mice per group), and one experiment for (E) (n = 7 and 12 subjects for the VAT and scWAT groups, respectively). *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0005.

We compared IL-13Rα2 expression in epididymal WAT (eWAT), a depot of VAT associated with insulin resistance and dyslipidemia, and in scWAT, a storage depot associated with adipokine secretion, enhanced lipid oxidation, and insulin action (38). In mouse adipose tissue, IL-13Rα2 protein concentration was low in both eWAT and scWAT of mice on a chow diet, and was significantly elevated in eWAT but not scWAT of mice fed an HFD (Fig. 1D). Human adipose tissue was also examined for il13ra2 gene expression. Subjects providing omental fat and scWAT for this analysis had average ages of 31.9 ± 3.5 and 48.6 ± 8.4 y, respectively, and all were female. Average BMI of the subjects providing omental fat and those providing scWAT was 41.9 ± 11.0 (n = 6) and 33.3 ± 3.6 (n = 7), respectively. The difference in BMI was not statistically significant, and with an average BMI >30, each group was considered obese. Expression of il13rα2 was found in both omental WAT (VAT) and in scWAT (Fig. 1E). Given that the subjects providing VAT differed in age and trended toward a higher BMI than those providing scWAT, and that gene expression values could not be directly compared between VAT and scWAT, these findings demonstrate expression of il13rα2 in human adipose tissue, but do not allow firm conclusions to be drawn concerning the relative expression of il13rα2 in VAT compared with scWAT.

IL-13 activity gene signature is enhanced in adipose tissue of il13rα2^−/− mice

As obesity-induced IL-13Rα2 expression was associated with reduced adipose tissue IL-13 concentrations, we asked whether IL-13Rα2 deficiency would enhance tissue IL-13 levels. Wt and il13rα2^−/− mice were subjected to HFD feeding for 13 wk. At the end of this time, concentrations of IL-13 in scWAT did not differ between wt and il13rα2^−/− mice (data not shown). Weight gain (Fig. 2A) and total fat mass (Fig. 2B) were similar between il13rα2^−/− mice and littermate controls. There were no significant differences in glucose tolerance or insulin sensitivity either on a chow diet (data not shown) or HFD (Fig. 2C, 2D). A small panel of genes was differentially expressed in adipose tissue of il13rα2^−/− mice compared with wt controls (Fig. 2E). Genes with elevated expression in il13rα2^−/− mice were known to be IL-13 responsive, including the chitinase family members Ym1 and Ym2 (39), the macrophage lectin Mgl2 (40), the eosinophil chemokine eotaxin 1 (Ccl11) (41), and the repair marker, amphiregulin (42).

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FIGURE 2.

HFD-induced metabolic changes do not differ between wt and il13rα2^−/− mice, despite adipose tissue IL-13 response profile. C57BL/6 mice were fed a 60% HFD for 13 wk. (A) Body weight; (B) % fat mass, determined by nuclear magnetic resonance; (C) glucose tolerance was assessed in fasted mice after injection of 1.5 g/kg glucose i.p.; (D) insulin tolerance was assessed in fasted mice after i.p. injection of 1 U/kg human insulin; (E) relative mRNA expression of representative genes in eWAT of il13rα2^−/− mice compared with wt, normalized to housekeeping genes. Graphs represent median, high, and low values from three independent experiments (n = 10–16 mice per group). *p < 0.05, **p < 0.01, ****p < 0.0001 by t test.

IL-33 promotes Th2 inflammation in chow-fed mice

HFD feeding was not sufficient to differentiate systemic metabolic responses in wt and il13rα2^−/− mice, as reflected in the body weight, percentage of fat mass, glucose tolerance, and insulin tolerance findings (Fig. 2A–D). With limiting IL-13 concentrations under resting conditions, lack of IL-13Rα2 may have little impact on metabolic endpoints. IL-33 is known to be a potent inducer of IL-13, IL-5, and other cytokines, both in vitro and in vivo (29). Therefore, we administered IL-33 to mice to elevate systemic IL-13 concentrations. Recombinant IL-33 was given by i.p. injection to wt and il13rα2^−/− mice, every other day for seven administrations, at doses of 0.0125 or 0.125 mg/kg. We observed dose-dependent modulation of a range of physiological and metabolic endpoints (Supplemental Fig. 1). Based on these observations, a dose of 0.125 mg/kg was chosen to induce responses of sufficient magnitude to allow interrogation of the role of IL-13Rα2.

IL-33 induced marked splenomegaly in both wt and il13rα2^−/− mice on chow or HFD (Fig. 3). After IL-33 administration on chow diet, IL-13 concentration was greatly elevated in serum and in tissues, including liver, eWAT, and scWAT (Fig. 3A). Compared to wt, il13rα2^−/− mice on chow diet had reduced concentrations of IL-13 in serum, but higher concentrations in liver, eWAT, and scWAT (Fig. 3A). These observations are consistent with findings that sIL-13Rα2 acts as a reservoir for the cytokine, such that mice lacking sIL-13Rα2 have a reduced IL-13 concentration in the blood (15). The cell-surface form of IL-13Rα2 acts to internalize and deplete IL-13, such that mice lacking IL-13Rα2 have elevated IL-13 concentration in tissues (15). IL-13 administration under comparable conditions failed to elevate tissue IL-13 levels (data not shown), suggesting that endogenous IL-13 production was required to sustain the responses.

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FIGURE 3.

IL-33 promotes tissue IL-13 elevation, splenomegaly, and adipose tissue inflammation in: (A) chow-fed; or (B) HFD-fed wt and il13rα2^−/− mice. Mice were administered PBS (□) or IL-33 (▪) i.p. at 0.125 mg/kg every other day for 2 wk. Serum and tissues were harvested 24 h after the last injection. Panels depict IL-13 concentration in serum or the indicated tissues, spleen weight, % CD45⁺ cells in the stromal vascular fraction (SVF) of scWAT or eWAT, % eosinophils (CD45⁺,CD11b⁺, SiglecF⁺) in scWAT or eWAT, % ILC2s (CD45⁺, Lin⁻, CD25⁺, ST2⁺) in the SVF of eWAT. Data represent mean ± SEM of n = 5–6 mice per group from two to three independent experiments. scWAT and eWAT were collected from the same animals. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, by t test, for comparisons between IL-33–treatment groups.

In mice on an HFD, the magnitude of IL-13 induction in response to IL-33 in eWAT and scWAT was reduced compared with that of mice on a chow diet (Fig. 3). In eWAT, the IL-13 concentration was significantly higher in IL-33–treated il13ra2^−/− mice compared with wt mice on chow diet, but significantly lower in il13ra2^−/− mice compared with wt mice on an HFD. Trends toward similar findings were seen in scWAT (Fig. 3), showing a strong effect of diet on adipose tissue IL-13 regulation in response to IL-33. In mice on a chow diet, IL-33 also increased adipose tissue eosinophils, ILC2s, and total CD45⁺ leukocytes in both wt and il13rα2^−/− mice (Fig. 3A). On an HFD, IL-33 induction of leukocytes and eosinophils was maintained in wt mice, but attenuated in il13rα2^−/− mice (Fig. 3B), emphasizing that effects of IL-13Rα2 deficiency became apparent under the stress of an HFD.

IL-33–induced metabolic changes are IL-13 dependent

Administration of IL-33 at 0.125 mg/kg every other day for 2 wk did not produce appreciable weight change in mice on a chow diet, but caused weight loss in obese mice (Fig. 4A), consistent with previous reports (29, 43). Il13rα2^−/− mice showed a greater degree of weight loss than wt mice (Fig. 4A). There was no effect of IL-33 on body weight in il13^−/− mice on an HFD, confirming IL-13 dependence of the effect (Fig. 4A). Similarly, fasting blood glucose concentrations were reduced in HFD-fed wt mice administered IL-33, and reduced to a greater extent in il13rα2^−/− mice, but were not reduced in il13^−/− mice (Fig. 4B). Under conditions of HFD, IL-33 also significantly decreased fat mass (Fig. 4C) and adipocyte size (data not shown). In the course of these studies, we noted that the wt and il13rα2^−/− mice administered IL-33 on chow diet exhibited mild perianal irritation and diarrhea, which was relatively more pronounced in the il13rα2^−/− animals (Fig. 4E). The perianal irritation and diarrhea were both increased in wt mice fed an HFD compared with chow diet, and heightened further in HFD-fed il13rα2^−/− animals (Fig. 4E, 4F). This may have contributed to the markedly reduced food consumption observed in the IL-33–treated animals on an HFD (Fig. 4D). Mice that were deficient in IL-13 were protected from the perianal irritation and diarrhea (Fig. 4E), and maintained food consumption (Fig. 4D) and body weight (Fig. 4A) on HFD.

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FIGURE 4.

Differential effects of IL-33 administration in chow versus HFD for il13rα2^−/− (α2KO), il13^−/− (13KO), and wt mice. Mice on chow diet were administered IL-33 i.p. at 0.125 mg/kg every other day for 2 wk. (A) Body weight; (B) fasting blood glucose; (C) % fat mass; (D) food consumption; and (E) perianal irritation/diarrhea score were assessed 24 h after the final injection of IL-33; (F) perianal irritation in IL-33–treated il13rα2^−/− mice fed an HFD. Data represent the mean ± SEM of three independent experiments, with 6–12 mice per group for the chow groups, and 10–26 mice per group for the HFD groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 for the indicated comparisons, by t test.

Elevations in serum concentrations of liver enzymes alanine aminotransferase and aspartate aminotransferase, but not alkaline phosphatase, were seen in mice fed an HFD as compared with those on a chow diet (Fig. 5A–C). Concentrations of these markers were reduced to variable degrees in mice administered IL-33 (Fig. 5A–C). Levels of alkaline phosphatase and aspartate aminotransferase were lower in the il13^−/− mice compared with il13rα2^−/− animals, suggesting an influence of IL-13 on liver function. Serum levels of insulin, low-density lipoprotein, HDL, triglycerides, and cholesterol were all significantly reduced in HFD-fed mice administered IL-33 (Fig. 5D–H). There was a trend toward greater reduction in serum lipids in il13rα2^−/− animals, and attenuated reduction in il13^−/− mice, producing a significant difference between lipid levels in il13rα2^−/− and il13^−/− animals (Fig. 5E–H). Taken together, these data implicate IL-13 in mediating IL-33-induced protection from HFD-induced changes in liver enzymes, insulin, and serum lipids.

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FIGURE 5.

Effects of IL-33 administration on serum metabolic markers in HFD-fed il13rα2^−/− (α2KO), il13^−/− (13KO), and wt mice after 13 wk of HFD. (A) Alanine aminotransferase (ALT; units per milliliter); (B) alkaline phosphatase (ALP; units per liter); (C) aspartate aminotransferase (AST; units per liter); (D) insulin (picograms per milliliter); (E) low density lipoprotein (LDL; milligrams per deciliter); (F) HDL (milligrams per deciliter); (G) TG (triglyceride; milligrams per deciliter); and (H) cholesterol (milligrams per deciliter). The dashed line indicates concentration in untreated wt mice on chow diet. Data represent the mean ± SEM of three independent experiments, with 5–19 mice per group for the HFD groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 for the indicated comparisons, by t test.

IL-33–induced gene expression changes are modulated by IL-13Rα2

Following IL-33 administration, increased expression of numerous genes was noted in adipose tissue, including those encoding cytokines, cytokine receptors, and metabolic markers (Fig. 6, Supplemental Fig. 2). Whether these changes signified altered expression per cell, or reflected altered cellular composition of the adipose tissue, could not be determined. In eWAT, the expression of genes encoding Th2 cytokines IL-13, IL-4, and IL-5 was increased 30–50-fold following administration of IL-33 to wt mice on an HFD, and was reduced in eWAT of il13rα2^−/− mice (Fig. 6A), consistent with the relative concentrations of IL-13 cytokine in eWAT (Fig. 3B). In contrast to the effect of IL-13Rα2 deficiency, expression of IL-5 was elevated in eWAT of il13^−/− mice, suggesting that IL-13 normally functions to limit IL-5 elevation (Fig. 6A). Among other Th2-associated cytokines, IL-25 expression was not induced in eWAT. IL-33 expression was mildly increased following IL-33 administration, but the response did not differ between wt, il13rα2^−/−, and il13^−/− mice (Fig. 6A).

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FIGURE 6.

Gene expression changes in eWAT of il13rα2^−/− or il13^−/− mice with matched wt controls, administered 0.125 mg/kg IL-33 every other day for 2 wk. Expression of each gene was determined relative to housekeeping controls. The dashed line represents the average expression level in all PBS-treated groups (il13rα2^−/− mice and wt controls, il13^−/− mice and wt controls), normalized to a value of one. Genes were grouped into those encoding: (A) cytokines; (B) cytokine receptors; (C) cell differentiation markers; (D) IL-13 response markers. Graphs represent median, high, and low values from two to three independent experiments, with 5–10 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with matched wt, by t test.

Among cytokine receptor genes, expression of il13rα2 was greatly reduced in eWAT of il13^−/− mice, below the baseline level seen in the absence of IL-33 (dashed line, Fig. 6B), suggesting that an active inhibitory pathway exists to block IL-13Rα2 expression in mice lacking IL-13. The IL-13Rα1 and IL-4Rα signaling chains were not regulated by IL-33. The IL-33–associated expression of both IL-2Rγ and IL-1RL1 (ST2) was reduced in il13rα2^−/− mice, but elevated in il13^−/− mice (Fig. 6B). Among markers of cell differentiation, il13rα2^−/− mice also displayed reduced expression of genes encoding the Th2 transcription factor, Gata3, the costimulatory molecule, PD1, the ILC2 transcription factor, Rorα, the Treg marker, Foxp3, and the Th17 transcription factor, Rorc. For all but Rorc, the transcripts were elevated in il13^−/− mice (Fig. 6C). Collectively, these gene expression changes in mice lacking IL-13Rα2 displayed an inverse profile to those seen in mice lacking IL-13. A similar gene expression profile was seen in scWAT (Supplemental Fig. 2). Overall, the reduced expression of cellular differentiation genes in adipose tissue of il13rα2^−/− mice reflected the reduced level of CD45⁺ leukocyte infiltration in the stromal vascular fraction of IL-33–treated il13rα2^−/− mice compared with wt on an HFD (Fig. 3B). In contrast to the elevated expression of lymphocyte differentiation markers in eWAT of il13^−/− mice given IL-33, expression of most IL-13 response markers was reduced in il13^−/− animals (Fig. 6D, Supplemental Fig. 2), and did not differ between wt and il13rα2^−/− mice.

The expression of a panel of metabolism-related genes was examined in the adipose tissue of mice administered IL-33 compared with PBS-treated control animals (Supplemental Fig. 3A). In eWAT, expression of most genes in the panel was reduced following IL-33 treatment, in both wt and il13rα2^−/− mice. In all cases, expression was further reduced in mice lacking IL-13, suggesting that IL-33–induced IL-13 normally functions in eWAT to regulate expression of metabolic response genes (Supplemental Fig. 3A). Similarly, in scWAT, expression of most genes in the panel was reduced in response to IL-33 administration, and was not significantly affected by IL-13Rα2 deficiency. Unlike in eWAT, however, IL-13 deficiency did not exacerbate the IL-33–associated gene expression changes in scWAT (Supplemental Fig. 3B). These observations suggest that although IL-13 plays a key regulatory role in IL-33–induced metabolic changes in eWAT, additional regulatory pathways exist in scWAT, abrogating their IL-13 dependence.

Finally, we @examined expression of a panel of genes in the GI tract of wt and il13rα2^−/− mice to identify correlates of the metabolic changes seen with HFD feeding and IL-33 administration. The mice expressed IL-13Rα2 throughout the GI tract, progressively increasing from duodenum to jejunum to ileum to colon (Supplemental Fig. 4A). As has been reported previously (44), IL-13Rα2 expression in the gut was increased following IL-33 administration, and this was seen in the duodenum, jejunum, and ileum (Supplemental Fig. 4A). Because the greatest difference between expression of IL-13Rα2 in IL-33–treated mice compared with PBS-treated mice was seen in the jejunum, we focused on this region for analysis of type 2 markers that could differ in wt versus il13rα2^−/− mice. Neither IL-13Rα1 nor IL-4Rα expression was induced in the jejunum following IL-33 dosing (Supplemental Fig. 4B). Expression of IL-1RL1 (ST2) was increased with IL-33 administration, but was not modulated in the absence of IL-13Rα2 (Supplemental Fig. 4B). Several genes were induced with IL-33 in wt mice, and displayed significantly lower expression in mice lacking IL-13Rα2, including those encoding the M2/ILC2 marker Arg1, the Th2 transcription factor, Gata3, the costimulatory molecule, PD1, the Treg marker, Foxp3, the eosinophil chemokine, Ccl24, and the inflammatory marker, IL-12Rβ1 (Supplemental Fig. 4C). Additional markers were elevated throughout the GI tract with IL-33, including IL-5, IL-13, and Ym1, but the expression level did not differ between wt versus il13rα2^−/−mice (data not shown).

IL-33–induced extramedullary hematopoiesis and inflammation are modulated by IL-13Rα2

As previously reported (30, 45), IL-33 administration induced infiltration of inflammatory cells in multiple tissues, and resulted in splenomegaly (Fig. 7A, 7B), associated with microscopic evidence of disrupted splenic architecture and increased extramedullary hematopoiesis (EMH) (Fig. 7C, data not shown). Increased cellularity was also observed in bone marrow in IL-33–administered mice compared with PBS-treated controls, consistent with the splenic EMH (data not shown). Marginally elevated eosinophil numbers were found in the circulation, spleen, and peritoneal lavage of wt mice in response to IL-33 (Fig. 7D–F). In contrast, the peritoneal lavage of IL-33–treated wt and il13rα2^−/− mice was characterized by large numbers of vacuolated macrophages, comprising up to 70% of total leukocytes (Fig. 7G–I). Because they were found in il13rα2^−/− mice that displayed reduced body weight on an HFD, the macrophages were examined for lipid in the vacuoles by Oil Red O staining, and found to be negative (data not shown).

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FIGURE 7.

Exacerbated splenomegaly, EMH, and eosinophilia in HFD-fed il13^−/− mice treated with IL-33. (A) Spleen size; (B) spleen weight; (C) EMH score; (D) % circulating eosinophils; (E) % eosinophils in spleen; (F) % eosinophils in peritoneal lavage; (G) % macrophages in peritoneal lavage; Wright’s Giemsa staining of peritoneal cells from IL-33–treated: (H) wt; (I) il13rα2^−/−; (J) il13^−/− mice. Data are representative of three independent experiments. (B–G) Data plotted as mean ± SEM of five to six mice per group. ****p < 0.0001 compared with matched wt, by t test.

In IL-13–deficient mice, eosinophilic inflammation was dramatically increased, with elevations of up to 40–50% total leukocytes in the circulation, spleen, peritoneal lavage (Fig. 7D–F, 7J), and bone marrow (data not shown). The increased eosinophils drove marked splenomegaly (Fig. 7A, 7B). Increased numbers of granulocyte precursors were observed in the bone marrow (data not shown), and were accompanied by a reduction in the relative percentage of vacuolated macrophages in the peritoneal lavage (Fig. 7G, 7J). The striking increase in eosinophil number seen in mice lacking IL-13 suggests that IL-13 acts to limit eosinophil development and/or infiltration in response to IL-33 administration.

IL-13–dependent GI toxicity develops in wt mice following IL-33 administration and is exacerbated in il13rα2^−/− mice under HFD conditions

As described above, diarrhea and perianal irritation were observed in mice administered IL-33, and were more severe in il13rα2^−/− mice compared with wt littermates (Fig. 4E, 4F). IL-13–deficient mice were protected from these GI effects, suggesting IL-13 dependence. Microscopic examination showed that IL-33 administration increased eosinophilic and mononuclear cell infiltrates in the jejunum and colorectum (Fig. 8A–D), with increased goblet cell size and increased size and secretory material within Paneth cells in the jejunum (Fig. 8E, 8K, 8L). In the pancreatic ducts, IL-33 induced epithelial cell hypertrophy with accumulation of eosinophilic material within epithelial cells (hyalinosis), periductal infiltration of inflammatory cells, and decreased zymogen content in the pancreas, which may have been related to the decreased food intake (Fig. 8F–J). Accumulation of similar eosinophilic material was also present within epithelial cells in the esophagus and stomach (data not shown). Although il13rα2^−/− mice had more severe diarrhea and perianal irritation (Fig. 4E, 4F), the microscopic changes throughout the GI tract were similar in severity and distribution between wt and il13rα2^−/− mice. Taken together, these findings show that IL-33 induced diarrhea and perianal irritation in the context of HFD, which was exacerbated in IL-13Rα2–deficient mice, associated with reduced food intake, but was not associated with an identifiable gene expression pattern or signature in the gut.

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FIGURE 8.

IL-33 increases mononuclear and eosinophilic infiltrates in the GI tract and pancreas, with perianal irritation. HFD-fed il13rα2^−/− and wt control mice were administered PBS or 0.125 mg/kg IL-33 every other day for 2 wk. (A) Mononuclear infiltrates in jejunum; (B) eosinophilic infiltrates in jejunum; (C) mononuclear infiltrates in colorectum; (D) eosinophilic infiltrates in colorectum; (E) goblet cells in jejunum; (F) epithelial hyalinosis/hypertrophy in pancreatic duct; (G) pancreatic periductal inflammatory infiltrates; (H) reduction in pancreatic zymogen content. For (A)–(H), data plotted are median of two to five mice per group. For (F), only one mouse was scored in the PBS-treated il13rα2^−/− group, representative of no detectable pathology in this group. Pancreatic ductal epithelium from: (I) wt mice administered PBS; or (J) il13rα2^−/− mice administered IL-33, showing increased eosinophilic material in ductal epithelial cells (*), decreased zymogen granules within acinar cells (+), and periductal inflammatory cell infiltrates (↑); jejunum from: (K) wt mice administered PBS; or (L) il13rα2^−/− mice administered IL-33 showing increased goblet cell numbers (*), increased inflammatory cell infiltrates in lamina propria (+), and increased size/secretory material in Paneth cells (↑).