Obesity has reached epidemic proportions, but little is known about its influence on the intestinal immune system. Here we show that the gut immune system is altered during high-fat diet (HFD) feeding and is a functional regulator of obesity-related insulin resistance (IR) that can be exploited therapeutically. Obesity induces a chronic phenotypic pro-inflammatory shift in bowel lamina propria immune cell populations. Reduction of the gut immune system, using beta7 integrin-deficient mice (Beta7 null ), decreases HFD-induced IR. Treatment of wild-type HFD C57BL/6 mice with the local gut anti-inflammatory, 5-aminosalicylic acid (5-ASA), reverses bowel inflammation and improves metabolic parameters. These beneficial effects are dependent on adaptive and gut immunity and are associated with reduced gut permeability and endotoxemia, decreased visceral adipose tissue inflammation, and improved antigen-specific tolerance to luminal antigens. Thus, the mucosal immune system affects multiple pathways associated with systemic IR and represents a novel therapeutic target in this disease.

Here, we show that the gut immune system is an important modulator of IR. Diet-induced obesity (DIO) is accompanied by a low-grade pro-inflammatory shift in lamina propria (LP) immune cell polarity, consistent with changes previously described in response to an intestinal barrier defect (). Targeting these changes may lead to new classes of potentially effective, minimal-side-effect therapies for IR.

In IR and T2D, treatment with systemic anti-inflammatory therapies such as salicylates and IL-1β antagonists has shown some efficacy in clinical trials (), and systemic targeting of T and B cells has shown effects in rodent models (). However, many systemic immune modulators carry potential serious side effects; thus, the development of locally active, well-tolerated, and efficient therapies is a principal goal of IR therapy research. One locally active, gut-specific anti-inflammatory agent is mesalamine (5-ASA), a first-line maintenance therapy for inflammatory bowel disease (IBD) (). 5-ASA is a salicylic acid derivative with anti-inflammatory properties that acts locally in the gut with minimal systemic absorption and side effects. As IBD is characterized by increased intestinal inflammation and altered permeability (), we hypothesized that 5-ASA might have beneficial effects in obesity-related IR and may help elucidate roles of gut immune cells in this disease.

TINSAL-T2D (Targeting Inflammation Using Salsalate in Type 2 Diabetes) Study Team The effects of salsalate on glycemic control in patients with type 2 diabetes: a randomized trial.

Dysbiosis is believed to cause low-grade inflammation both systemically, through enhanced leakage of bacterial products such as lipopolysaccharides (LPS), and locally in the small bowel and colon (). Systemically, some of these bacterial products, including gut-derived antigens, are thought to accumulate and potentiate inflammation in the VAT (). Thus, manipulation of the gut barrier to reduce LPS leakage (e.g., with IL-22) improves insulin sensitivity (). In the bowel, increased tumor necrosis factor alpha (TNF-α) and NF-κB activation occurs in the ileum, while IL-1β and IL-12p40 levels are elevated in colons of HFD-fed mice (). However, the local effects of HFD on most immune cell populations in the gut and their function in IR remain unclear.

Propensity to high-fat diet-induced obesity in rats is associated with changes in the gut microbiota and gut inflammation.

In addition to VAT, recent evidence has pointed to the bowel as a key site that becomes altered in obesity-related IR (). These alterations include changes in the gastrointestinal flora, known as dysbiosis, which can impact body fat, systemic inflammation, and IR (). Under normal physiological conditions, dysbiosis is kept in check through maintenance of an intact intestinal barrier, characterized by increased mucus, transforming growth factor (TGF)-β, interleukin (IL)-10, IL-22, and luminal secretion of IgA ().

Obesity and its associated metabolic abnormalities, including type 2 diabetes (T2D) and its precursor, insulin resistance (IR), have become global diseases that carry considerable morbidity and mortality (). Obesity-related IR can arise through multiple pathways, but chronic inflammation in visceral adipose tissue (VAT) has become a prominent pathological mechanism (). Cells of both the innate and adaptive immune system residing in VAT have been shown to play a key role in IR. More specifically, M1 macrophages, interferon (IFN) γ-secreting Th1 T cells, CD8+ T cells, and B cells promote IR, in part, through secretion of pro-inflammatory cytokines (). In contrast, Foxp3+ regulatory T cells (Tregs), eosinophils, Th2 T cells, and type 2 innate lymphoid cells (ILC2) are associated with protection from IR through local control of VAT inflammation ().

While permeability-related gut-derived endotoxin alone may contribute to VAT inflammation and potentially IR (), it is thought that this trigger works alongside other gut-associated antigens to activate antigen-specific T cells in VAT, thereby influencing glucose homeostasis (). Thus, to further understand how a gut-specific anti-inflammatory agent may contribute to reduced inflammation in VAT, we examined the effects of 5-ASA on oral immune tolerance to a gut-derived antigen. NCD-, HFD-, or HFD 5-ASA-fed C57BL/6 mice were administered oral ovalbumin (OVA) antigen for 1 week prior to immunization with OVA-CFA. Interestingly, HFD 5-ASA-fed mice showed a stronger oral tolerance response to OVA antigen systemically, as reflected by an increased OVA-specific IgG1/IgG2c ratio (indicative of reduced Th1 inflammatory responses), and a nearly 3-fold increase in OVA-specific IgA ( Figure 6 D). Moreover, draining lymph nodes in mice fed HFD 5-ASA demonstrated a reduction in OVA-specific T cell-derived IL-2 and IFNγ, which is also consistent with the improved oral tolerance and reduced antigen-specific inflammation to gut antigen ( Figure 6 E). Finally, 5-ASA treatment induced a nearly 4-fold increase in antigen-specific Tregs to OVA in VAT as measured using OVA/I-Atetramers ( Figure 6 F). Collectively, the data suggest that reducing low-grade inflammation in the gut during HFD feeding can impact multiple pathways associated with IR, including gut barrier function, tolerance to gut-derived antigen, and antigen-specific immunity to gut-derived antigen in VAT. Taken together, these results suggest that anti-inflammatory targeting of gut immune cells is a novel approach to treat obesity-related IR.

Because HFD 5-ASA reduces IFNγ expression compared to control HFD and is associated with improvements in intestinal permeability, we next assessed the role of IFNγ in intestinal permeability during HFD feeding. HFD-fed IFNγ-deficient mice showed improved intestinal barrier function as reflected in reduced plasma FD4 levels ( Figure 6 C, left). IFNγ was also able to reduce ZO-1 tight junction gene expression in intestinal epithelial cells, suggesting one possible mechanism for its ability to influence gut permeability ( Figure 6 C, right). Thus, the shifts in HFD bowel immune cells to IFNγ-producing immune cells likely impact metabolic function at the level of intestinal permeability. To further corroborate this notion, we assessed bowel permeability in Beta7mice, which showed reduced numbers of intestinal immune cells that most prominently affected IFNγ-expressing T cells. Beta7mice showed improved/reduced intestinal permeability as measured by FD4 assay and reduced anti-LPS IgG ( Figures 6 A and 6B, left).

Since both DIO and intestinal inflammation are associated with impairment of the gut epithelial barrier, which can trigger systemic endotoxemia and IR (), we next investigated the effects of 5-ASA on intestinal permeability and on serum and VAT endotoxin levels. 5-ASA treatment induced significant improvements in intestinal epithelial barrier permeability, as measured by fluorescence FD4 assay ( Figure 6 A), which is consistent with the reduced gut immune cell inflammatory shift and overall improvement in glucose homeostasis previously observed. Moreover, IgG responses to LPS were markedly diminished in 5-ASA-treated mice ( Figure 6 B, left) accompanied by reduced levels of serum endotoxin ( Figure 6 B, middle). VAT endotoxin levels also trended lower in 5-ASA-treated mice, though this result did not reach significance ( Figure 6 B, right). Taken together, these results show effects of 5-ASA on reducing HFD-induced gut leakage to endotoxins.

(F) OVA tetramer-stained Treg cells from VAT of age-matched oral or non-oral challenged HFD WT and HFD 5-ASA WT mice 2 weeks after immunization with OVA-CFA ( ∗ p = 0.03; n = 2 experiments, 6 pooled mice). Data in bar graphs represent mean ± SEM.

(E) OVA-specific recall IL-2 and IFNγ responses in age-matched HFD WT and HFD 5-ASA WT mice from axillary lymph nodes, 2 weeks after immunization with OVA-CFA ( ∗ p = 0.01 for IL-2, ∗ p = 0.006 for IFNγ; n = 4–5 mice in duplicates).

(D) Ratio of OVA-specific IgG1/IgG2c (left) and OVA-specific IgA (right) in age-matched NCD WT, HFD WT, and HFD 5-ASA WT mice 2 weeks after immunization with OVA-CFA ( ∗ p = 0.03 for IgG1/IgG2c, ∗ p = 0.03 for oral NCD versus oral HFD IgA, ∗ p = 0.02 for oral HFD versus oral HFD 5-ASA IgA; n = 4–6 mice).

(C) Left: plasma FD4 concentrations, following oral gavage, of age-matched IFNγ null mice after 10 weeks of HFD ( ∗ p = 0.01, n = 4 mice). Right: ZO-1 mRNA expression relative to housekeeping gene expression in MODE-K intestinal cells treated with indicated amounts of IFNγ in vitro ( ∗ p = 0.006, n = 3 in each treatment).

(B) Far left: serum anti-LPS IgG levels of age-matched NCD WT, HFD WT, HFD 5-ASA WT, and HFD Beta7 null mice after 14 weeks HFD ( ∗ p ≤ 0.03, n = 5–8). Middle and far right: serum endotoxin levels (middle) and VAT endotoxin levels (right) of age-matched NCD WT, HFD WT, and HFD 5-ASA WT after 14 weeks HFD ( ∗ p < 0.05 for serum endotoxin, p = 0.19 for VAT endotoxin; n = 3–4 for serum endotoxin, n = 5 for VAT endotoxin).

(A) Plasma FD4 concentration of age-matched NCD WT, HFD WT, HFD 5-ASA WT, and HFD Beta7 null mice after 12–16 weeks of diet following oral gavage as a measure of intestinal permeability ( ∗ p = 0.02 for NCD Control versus HFD Control, ∗ p = 0.04 for HFD Control versus HFD 5-ASA, and ∗ p = 0.04 for HFD Control versus HFD Beta7 null ; n = 10 for NCD Control, n = 10 for HFD Control, n = 8 for HFD 5-ASA, and n = 6 for HFD Beta7 null mice).

Since knock-out studies linked potential effects of 5-ASA on glucose metabolism to the gut immune system, and 5-ASA has been reported to possess PPARγ agonist properties (), we next determined if 5-ASA could be directly influencing intestinal immune cell function in HFD through targeting PPARγ. As the effects of 5-ASA were more robust with small bowel T cells than colonic T cells, we focused our studies on small bowel T cells. Indeed, we observed higher PPARγ gene expression in small bowel T cells compared to total splenic T cells in both HFD and NCD-fed mice ( Figure 5 E; Figure S5 E). Mice fed HFD 5-ASA showed increased PPARγ functional activity in purified small bowel T cells compared to those fed with control HFD ( Figure 5 F). We next tested if 5-ASA can suppress IFNγ production in vitro. Indeed, similar to another PPARγ agonist, rosiglitazone, 5-ASA reduced IFNγ production by anti-CD3/CD28-activated small bowel but not splenic T cells ( Figure S5 F). Furthermore, loss of PPARγ in T cells (Lck-Cre PPARγ) abrogated the suppressive effects of 5-ASA, confirming that 5-ASA acts in a PPARγ-dependent manner ( Figure 5 G). In addition, 5-ASA indirectly reduced T cell IFNγ expression by modulating intestinal dendritic cell function, as shown by reduced IFNγ levels in antigen-specific co-culture systems using OT-II CD4+ T cells and 5-ASA-pre-treated small bowel but not splenic dendritic cells ( Figure 5 H).

To determine if the effects of 5-ASA were mediated through anti-inflammatory actions that require adaptive immune cells rather than direct effects on gut epithelium, we treated 6-week-old Rag1mice with HFD 5-ASA. Preventative treatment of Rag1mice with HFD 5-ASA had no effect on body weight, glucose tolerance, or IR ( Figures 5 A and 5B ), suggesting that the beneficial effects of 5-ASA required components of the adaptive immune system. To further pinpoint the location of 5-ASA action on glucose tolerance, we fed Beta7mice a HFD with 5-ASA. Interestingly, similar to the Rag1mice, treatment with 5-ASA had no major effects on glucose tolerance and IR ( Figures 5 C and 5D). Thus, the beneficial metabolic effects of 5-ASA require an “intact” gut immune system.

(H) Levels of secreted IFNγ cytokine from OT-II T cells stimulated with 5-ASA-treated (0.1 or 1.0 mM) or untreated splenic (left) or small bowel (right) dendritic cells presenting the indicated concentrations of OVA 323-339 peptide ( ∗ p ≤ 0.03, n = 3 samples, 3 spleens; n = 2 samples, 4 pooled bowels).

(G) Levels of secreted IFNγ cytokine from small bowel (SB) T cells (left) or splenic (SP) T cells (right) from HFD-fed WT mice compared to HFD-fed Lck-Cre + PPARγ fl/fl mice treated with the indicated doses of 5-ASA in vitro ( ∗ p ≤ 0.02 at all doses of 5-ASA for small bowel T cells, n = 3 mice).

(A and B) Body weights (A), GTT (B, left), and ITT (B, right) of Rag1 null mice after 8 weeks of HFD or HFD 5-ASA (1,500 mg/kg/day) (n = 4–5 mice).

Consistent with a dominant anti-inflammatory effect in the gut, 5-ASA treatment showed an overall reversal of the local pro-inflammatory immune shift in both the colon ( Figure 4 A) and small bowel ( Figure 4 B), characterized by a reduction in Th1 cells, IFNγ-secreting CD8+ T cells, and IL-17-secreting γδ T cells. There was also an increase in Tregs in the small bowel ( Figure 4 B). Interestingly, associated with the anti-inflammatory changes in the bowel, 5-ASA also reversed local VAT inflammation by reducing percentages of Th1 cells, IFNγ-secreting CD8+ T cells ( Figure 4 C), and M1 inflammatory macrophages ( Figure 4 D) in VAT while increasing Tregs ( Figure 4 C, third from left). Significant anti-inflammatory effects on immune cell populations were not seen in the bowels or VATs of NCD 5-ASA-treated mice compared to untreated NCD mice, suggesting that an increased inflammatory environment was needed to elicit significant differences in immune cell populations ( Figures S5 A–S5D). In line with the anti-inflammatory changes in gut immune populations seen with HFD 5-ASA-fed mice, HFD 5-ASA treatment was also associated with shifts in gut bacteria that are typically seen with administration of the drug, including increased bacterial diversity, increased Firmicutes, and increased Clostridiales ( Figures S6 A–S6E) ().

(C and D) Flow cytometriy analysis of T cell (C) and M1 macrophage subset (D) in VAT of mice after 16 weeks of HFD or HFD 5-ASA (1,500 mg/kg/day) ( ∗ p = 0.03 for Th1, ∗ p = 0.02 for CD8, ∗ p = 0.03 for Treg, ∗ p = 0.01 for macrophages, n = 2 experiments, 8 mice). Data in bar graphs represent mean ± SEM.

(A and B) Intracellular staining of cytokines and Foxp3 in LP T cell populations in the colon (A) or small bowel (B) of mice after 16 weeks of HFD or HFD 5-ASA (1,500 mg/kg/day) ( ∗ p = 0.001 for Th1, ∗ p = 0.01 for CD8, ∗ p = 0.045 for γδ T cell IL-17, n = 2–3 experiments, 9 mice for colon; ∗ p = 0.01 for Th1, ∗ p = 0.02 for CD8 IFNγ, ∗ p = 0.005 for Treg, ∗ p = 0.03 for γδ T cell IFNγ, ∗ p < 0.0001 for γδ T cell IL-17, n = 2–4 experiments, 6–8 mice for small bowel).

To begin understanding the mechanisms by which 5-ASA can exert effects on glucose homeostasis, we examined the effects of 5-ASA on systemic and local immune function during HFD feeding. 5-ASA treatment showed no effects on immune cell populations in the spleen ( Figure S4 A), on stimulated spleen immune cell cytokine secretion, or on circulating immune cell polarity in the blood ( Figures S4 B and S4C). Similarly, serum levels of cytokines in mice treated with 5-ASA were mostly unchanged, though we did identify a significant but small increase in RANTES and a reduction in TNF-α ( Figure S4 D). Consistent with little systemic effects on immune cell function, we could identify only traces of 5-ASA compound in the serum of mice, including mice treated with high-dose 5-ASA for 12 weeks, by use of high-performance liquid chromatography (HPLC) with an internal 4-ASA standard ( Figure S4 E, left). Instead, 5-ASA was concentrated in the colon and small bowel (approximately 20× enriched compared to serum, given the density of tissue), and importantly, 5-ASA was undetectable in VAT ( Figures S4 E, right, and S4F). Consistently, as mentioned previously, 5-ASA did not alter expression of adipogenesis-related genes in VAT or SAT (refer back to Figure S3 D). The results are in agreement with previous literature demonstrating poor systemic absorption of 5-ASA upon oral administration () and highlight the relative specificity of our gut anti-inflammatory therapy.

We next assessed whether 5-ASA could be used to treat established obesity-associated IR. C57BL/6 mice on HFD for 8 weeks, with established metabolic disease, were switched onto a HFD with 5-ASA for 8 additional weeks and compared to mice on only HFD from the beginning. Similar to the preventative protocol, 5-ASA did not change body weight ( Figure 3 F), but did produce significant improvements in glucose tolerance and insulin tolerance ( Figure 3 G). To assess whether the beneficial metabolic effects of 5-ASA require a HFD-induced milieu, we placed 6-week-old C57BL/6 mice on either NCD or NCD with 5-ASA (1,500 mg/kg/day). After 12 weeks of treatment, there was little or no difference in body weight, fasting glucose, glucose tolerance, or IR ( Figure S3 I). These results suggest that the use of 5-ASA has specific therapeutic effects on glucose homeostasis in the setting of DIO.

(F and G) Body weights (F), GTT (G, left), and ITT (G, right) after 8 weeks of HFD or HFD 5-ASA (1,500 mg/kg/day) in mice switched over from 8 weeks of HFD ( ∗ p < 0.05, n = 5 mice, GTT was performed with an i.p. glucose challenge at a dose of 1.0 g/kg). Data in bar graphs represent mean ± SEM.

(E) Left: insulin signaling in VAT, liver, and muscle of NCD, HFD, and HFD 5-ASA-fed mice injected with (+) or without (−) insulin and immunoblotted for pAkt and total Akt proteins (image shown is representative of 1 of 3 experiments with similar results, n = 3–4 mice). Right: fold change of pAkt/Akt protein ratios in mice fed HFD 5-ASA relative to HFD-fed controls ( ∗ p ≤ 0.01, n = 4 mice for VAT, 3–4 mice for liver and muscle).

(C and D) Fasting glucose (C, left), fasting insulin (C, right), glucose tolerance test (GTT, D, left), and insulin tolerance test (ITT, D, right) of mice after 14 weeks of HFD or HFD 5-ASA (1,500 mg/kg/day) ( ∗ p = 0.001 for glucose, n = 10 mice, ∗ p = 0.02 for insulin, n = 8–9 mice, ∗ p < 0.05 for tolerance testing, n = 10 mice for GTT, n = 13–15 mice for ITT).

(B) Relative fat cell diameter of mice (left) or number of VAT “crown-like structures” (CLS) (right) per 100× low power field, after 14 weeks of HFD or HFD 5-ASA (1,500 mg/kg/day) (n = 3).

(A) Left: body weights of HFD and HFD 5-ASA (1,500 mg/kg/day)-fed C57BL/6 mice over time, starting at 6 weeks of age (n = 10). Right: VAT weights of mice, after 14 weeks of HFD or HFD 5-ASA (1,500 mg/kg/day) (n = 10).

In terms of metabolic parameters, there were no differences in weight gain during 12 weeks of HFD feeding between wild-type (WT) and Beta7mice ( Figure 2 B). Interestingly, Beta7mice demonstrated improved fasting glucose, glucose tolerance (using glucose tolerance test, GTT), and insulin sensitivity (using insulin tolerance test, ITT) compared to WT mice after 12 weeks of HFD ( Figure 2 C). These mice also showed similar food intake, oxygen consumption, and carbon dioxide production ( Figure 2 D). Histological analysis of bowels in HFD-fed Beta7mice did not show signs of active colitis ( Figure S2 C). Interestingly, HFD-fed Beta7mice presented no difference in adipocyte size, but showed a marked reduction in crown-like structures (CLS) in the VAT, along with reduced liver steatosis ( Figure 2 E; Figure S2 D). Consistent with the reduced CLS, HFD Beta7mice had overall less VAT immune cell infiltrates ( Figures S2 E–S2J). This change was likely not due to the beta7 integrin deficiency imparting an intrinsic defect on T cells to home to VAT, as Beta7T cells were equally capable at trafficking and engrafting to VAT upon transfer as their WT counterparts ( Figure S2 K). When Beta7mice were fed a NCD, we saw little differences in body weight ( Figure S2 L), fasting glucose, or insulin tolerance, but some mild improvements in glucose tolerance ( Figure S2 M), suggesting that functional glucose modulation by the gut immune system is more pronounced in the setting of HFD but may also be relevant to a lesser degree under normal physiological conditions such as a NCD. Collectively, these results demonstrate that changes to the makeup of the gut immune system may have ramifications in the development of obesity-associated IR.

We next determined if the gut immune system as a whole could contribute to the development of obesity-associated IR. We placed beta7 integrin-deficient C57BL/6 mice (Beta7mice) on either normal chow diet (NCD) or HFD for 12 weeks and then assessed metabolic parameters. Beta7 pairs with alpha4 integrin on leukocytes to form the mucosal addressin molecule LPAM-1, and mice deficient in beta7 show hypoplasia of gut lymphoid tissue due to reduced homing of leukocytes to colon and small bowel (). Consistently, we observed a reduction mainly in the absolute numbers but not proportions of most immune cells, especially in IFNγ-producing T cell subsets in the LP of colons and small bowels of Beta7mice after 12 weeks of HFD ( Figure 2 A; Figure S2 A). There were no differences in the relative proportions of these subsets in the spleen ( Figure S2 B), suggesting that the lack of beta7 integrin does not attenuate systemic immunity.

(E) Relative fat cell diameter (left) of mice, or number of VAT “crown-like structures” (CLS) per 100× low power field (right), after 12 weeks of HFD ( ∗ p < 0.0001, fields counted from n = 3 mice). Data in bar graphs represent mean ± SEM.

(D) Food intake (left), metabolic cage analysis including oxygen consumption (left middle), carbon dioxide production (right, middle), and respiratory exchange ratio (RER) (right) of HFD WT and Beta7 null mice (n = 7 for food intake, n = 7 WT and n = 6 Beta7 null mice for metabolic cage analysis).

(A) Absolute cell counts, including CD45+ (top far left), CD3+ (top middle left), CD3+ CD4+ or CD3+ CD8+ (top far right), CD4+ subsets (bottom far left), IFNγ+ CD8+ (bottom middle), and γδ+ T cell subsets (bottom far right) from colon and small bowel (SB) LP after 12 weeks of HFD in WT and Beta7 null (Beta7 null ) mice. Entire colons were processed, or the distal 10 cm of SB (jejunum + ileum). ∗ p = 0.0008 for CD45 colon, ∗ p < 0.0001 for CD45 SB; ∗ p = 0.045 for CD3 colon, ∗ p = 0.0004 for CD3 SB; ∗ p = 0.009 for CD4 colon, ∗ p = 0.0006 for CD4 SB, ∗ p < 0.0001 for CD8 SB; ∗ p = 0.02 for γδ SB; ∗ p = 0.0008 for CD4 IFNγ colon; ∗ p = 0.008 for CD4 IFNγ SB; ∗ p = 0.03 CD8 IFNγ SB; ∗ p = 0.001 for γδ IL-17 colon, n = 4 experiments, 8–11 mice.

Collectively, these results demonstrate a reduction in gut Tregs and a pro-inflammatory shift in some adaptive and innate T cell populations in the gut of HFD-fed mice, with a similar observation in our specific cohort of obese humans. Interestingly, this inflammatory shift was not associated with any apparent histological changes of chronic or active inflammation on H&E-stained sections of obese human ( Figure S1 F, top) or HFD-fed mouse colons ( Figure S1 F, bottom).

To determine if humans showed similar changes in gut immune populations during obesity, we correlated patient BMI with relative numbers of pro-inflammatory T-bet+ (Th1, ILC1 []) T cells, anti-inflammatory Foxp3+ (Treg) T cells, and CD8+ T cells in the LP of colon and ileum resection specimens. Table S1 summarizes relevant clinical parameters of patients included in the study. Obese patients showed increases in colon and small bowel T-bet+ cells and CD8+ cells and a reduction in Tregs ( Figures 1 E and 1F).

To examine the effects of DIO on gut immunity, we first investigated if adaptive immune cell populations in the colon and small bowel LP are altered by HFD feeding in C57BL/6 mice at 3 or 12–16 weeks of HFD. After 3 weeks of HFD, changes in the proportions of bowel immune populations began in the colon, including a reduced percentage of Tregs and an increase in IL-17-producing γδ T cells ( Figures S1 A and S1B). However, after 12–16 weeks of diet, HFD induced a pro-inflammatory shift in immune cells in both the colon and small bowel. In colonic immune cells, there was an increase in the proportion and/or absolute number of IFNγ-producing Th1 T cells and CD8+ T cells and reduction in the proportion of CD4+ Foxp3+ Tregs ( Figure 1 A; Figure S1 C). In the small bowel of HFD mice, there was an increase in the frequencies and/or numbers of Th1 CD4+ T cells and IFNγ-producing CD8+ T cells and a decrease in the proportion and absolute number of CD4+ Foxp3+ Tregs ( Figure 1 B; Figure S1 D). We next assessed the effects of 12–16 weeks of HFD feeding on γδ T cells and innate lymphoid cell populations in the bowel. HFD increased the frequency and/or number of IL-17-producing but not IFNγ-producing γδ T cells in the colon and small bowel ( Figures 1 C and 1D; Figures S1 C and S1D). Furthermore, there was an increase in total cell numbers of innate lymphoid cells in the colon, though the relative proportion of NKp46+ CD4− cells was reduced in HFD-fed mice ( Figure S1 E).

(F) Representative images of double staining for T-bet (red, examples indicated by blue arrows) and Foxp3 (brown, examples indicated by black arrows) (top and third row) and CD8+ stain (brown) (second and last row) in lean (left) versus obese (right) colons (top four quadrants) and ileums (bottom four quadrants).

(E) T-bet (far left), Foxp3 (middle), and CD8+ (far right) staining in colon and ileum of human subjects with lean or obese BMI ( ∗ p = 0.04, n = 7 for colon, ∗ p = 0.02, n = 3–4 for ileum for T-bet; ∗ p = 0.005, n = 7 for colon, ∗ p = 0.047, n = 3–4 for ileum for Foxp3; ∗ p = 0.03, n = 7 for colon, ∗ p = 0.006, n = 3–4 for ileum for CD8+).

(C and D) Intracellular cytokine staining in γδ T cells from colon (C) or small bowel (D) LP after 12–16 weeks of HFD ( ∗ p = 0.02 for colon, ∗ p = 0.001 for small bowel; n = 4 experiments, 10 mice for colon and n = 4–5 experiments, 10 mice for small bowel).

(A and B) Intracellular cytokine staining in T cells from colon (A) or small bowel (B) LP after 12–16 weeks of HFD ( ∗ p = 0.005 for Th1, ∗ p = 0.02 for CD8, ∗ p = 0.02 for Tregs, n = 3–4 experiments, 8–10 mice for colon; ∗ p = 0.03 for Th1, ∗ p = 0.046 for CD8, ∗ p = 0.002 for Tregs, n = 5–6 experiments, 10–12 mice for small bowel).

Discussion

Ding et al., 2010 Ding S.

Chi M.M.

Scull B.P.

Rigby R.

Schwerbrock N.M.

Magness S.

Jobin C.

Lund P.K. High-fat diet: bacteria interactions promote intestinal inflammation which precedes and correlates with obesity and insulin resistance in mouse. Wang et al., 2013 Wang N.

Wang H.

Yao H.

Wei Q.

Mao X.M.

Jiang T.

Xiang J.

Dila N. Expression and activity of the TLR4/NF-κB signaling pathway in mouse intestine following administration of a short-term high-fat diet. Kim et al., 2012 Kim K.A.

Gu W.

Lee I.A.

Joh E.H.

Kim D.H. High fat diet-induced gut microbiota exacerbates inflammation and obesity in mice via the TLR4 signaling pathway. Li et al., 2008 Li H.

Lelliott C.

Håkansson P.

Ploj K.

Tuneld A.

Verolin-Johansson M.

Benthem L.

Carlsson B.

Storlien L.

Michaëlsson E. Intestinal, adipose, and liver inflammation in diet-induced obese mice. Wang et al., 2014 Wang X.

Ota N.

Manzanillo P.

Kates L.

Zavala-Solorio J.

Eidenschenk C.

Zhang J.

Lesch J.

Lee W.P.

Ross J.

et al. Interleukin-22 alleviates metabolic disorders and restores mucosal immunity in diabetes. We have identified the gut immune system as an active orchestrator and therapeutic target in obesity-related IR. Previous work has shown that HFD increases ileal TNF-α mRNA, induces expression of TLR4 and NF-κB in small bowels (), and increases IL-1β, IL-12p40, NF-κB, and TLR4 in colons of DIO mice (). Consistently, we show that DIO promotes a pro-inflammatory shift in gut immune cell populations, characterized by reduced LP Foxp3+ Treg cells, increased IFNγ-producing Th1 and CD8+ T cells, as well as increased IL-17-producing γδ T cells. Similar to the changes in mice, altered ratios of T-bet+ cells—Foxp3+ Treg cells, as well as changes in CD8+ T cells—were found in both small and large bowels of obese humans, though these studies involved the use of negative margin specimens from patients with tumors and thus need more rigorous follow-up in additional cohorts of patients, including bariatric patients. A recent report has also demonstrated reduction in IL-22 in the gut of obese mice post-immune challenge (). Consistently, we saw reduced percentages of NKp46+ CD4− ILCs, which are important producers of IL-22. Moreover, the pro-inflammatory shift in immune cell populations observed in the gut was not associated with obvious inflammatory histological changes, and so we classify this pro-inflammatory shift as a sub-histological change, or “low-grade subclinical inflammation.”

null mice, which have marked hypoplasia of the gut lymphoid system. We noted improved metabolic parameters in the Beta7null mice despite similar body weights. These mice showed reduced immune cell infiltrates in the gut during HFD, including reductions in IFNγ-producing CD4+ and CD8+ T cells, consistent with a potential pathogenic role for some intestinal immune cells in DIO. However, additional work is needed to rule out whether other off-target effects of this molecule, such as potential traffic to other tissues, exist in the setting of DIO that might also contribute to the phenotype. Furthermore, Beta7null mice are susceptible to bacterial overgrowth ( Wagner et al., 1996 Wagner N.

Löhler J.

Kunkel E.J.

Ley K.

Leung E.

Krissansen G.

Rajewsky K.

Müller W. Critical role for beta7 integrins in formation of the gut-associated lymphoid tissue. Upadhyay et al., 2012 Upadhyay V.

Poroyko V.

Kim T.J.

Devkota S.

Fu S.

Liu D.

Tumanov A.V.

Koroleva E.P.

Deng L.

Nagler C.

et al. Lymphotoxin regulates commensal responses to enable diet-induced obesity. Upadhyay et al., 2012 Upadhyay V.

Poroyko V.

Kim T.J.

Devkota S.

Fu S.

Liu D.

Tumanov A.V.

Koroleva E.P.

Deng L.

Nagler C.

et al. Lymphotoxin regulates commensal responses to enable diet-induced obesity. Brown et al., 2013 Brown E.M.

Sadarangani M.

Finlay B.B. The role of the immune system in governing host-microbe interactions in the intestine. Pastorelli et al., 2013 Pastorelli L.

De Salvo C.

Mercado J.R.

Vecchi M.

Pizarro T.T. Central role of the gut epithelial barrier in the pathogenesis of chronic intestinal inflammation: lessons learned from animal models and human genetics. Kahles et al., 2014 Kahles F.

Meyer C.

Möllmann J.

Diebold S.

Findeisen H.M.

Lebherz C.

Trautwein C.

Koch A.

Tacke F.

Marx N.

Lehrke M. GLP-1 secretion is increased by inflammatory stimuli in an IL-6-dependent manner, leading to hyperinsulinemia and blood glucose lowering. Caesar et al., 2012 Caesar R.

Reigstad C.S.

Bäckhed H.K.

Reinhardt C.

Ketonen M.

Lundén G.O.

Cani P.D.

Bäckhed F. Gut-derived lipopolysaccharide augments adipose macrophage accumulation but is not essential for impaired glucose or insulin tolerance in mice. Wang et al., 2010 Wang Y.

Li J.

Tang L.

Wang Y.

Charnigo R.

de Villiers W.

Eckhardt E. T-lymphocyte responses to intestinally absorbed antigens can contribute to adipose tissue inflammation and glucose intolerance during high fat feeding. We next investigated if the gut immune system as a whole could exert systemic effects on glucose homeostasis. In this model, we used Beta7mice, which have marked hypoplasia of the gut lymphoid system. We noted improved metabolic parameters in the Beta7mice despite similar body weights. These mice showed reduced immune cell infiltrates in the gut during HFD, including reductions in IFNγ-producing CD4+ and CD8+ T cells, consistent with a potential pathogenic role for some intestinal immune cells in DIO. However, additional work is needed to rule out whether other off-target effects of this molecule, such as potential traffic to other tissues, exist in the setting of DIO that might also contribute to the phenotype. Furthermore, Beta7mice are susceptible to bacterial overgrowth (), which can cause changes in the microbiome and contribute to the observed phenotype. This phenotype may be similar to lymphotoxin-deficient mice that show hypoplasia of Peyer’s patches and improved glucose tolerance due to altered gut colonization of segmented filamentous bacteria (SFB) and reduced energy-harvesting bacteria (). Nonetheless, taking together the phenotypic data of both models, it appears that active gut inflammation contributes to downstream pathways, ultimately leading to obesity or related IR. Potential pathways include modulation of the gut flora with effects on energy-harvesting bacteria (), bile acid and short-chain fatty acid release (), modulation of the gut epithelial barrier (), control of gut hormone release such as GLP-1 leading to hyperinsulinemia (), and/or a role in dictating inflammatory responses to gut-derived antigen and endotoxin ().

null mice, we observed overall improvements in gut barrier function, characterized by reduced FD4 and anti-LPS response. These findings are potentially linked to reduced infiltrates of IFNγ-producing cells in the bowel, as IFNγ has direct pathological effects on disrupting barrier function ( Beaurepaire et al., 2009 Beaurepaire C.

Smyth D.

McKay D.M. Interferon-gamma regulation of intestinal epithelial permeability. null mice have improved barrier function compared to HFD WT mice, implicating local intestinal IFNγ production as one critical pathogenic mediator of intestinal permeability in DIO. In HFD Beta7mice, we observed overall improvements in gut barrier function, characterized by reduced FD4 and anti-LPS response. These findings are potentially linked to reduced infiltrates of IFNγ-producing cells in the bowel, as IFNγ has direct pathological effects on disrupting barrier function (). Consistently, HFD IFNγmice have improved barrier function compared to HFD WT mice, implicating local intestinal IFNγ production as one critical pathogenic mediator of intestinal permeability in DIO.

Di Paolo et al., 1996 Di Paolo M.C.

Merrett M.N.

Crotty B.

Jewell D.P. 5-Aminosalicylic acid inhibits the impaired epithelial barrier function induced by gamma interferon. Liu et al., 2009 Liu X.C.

Mei Q.

Xu J.M.

Hu J. Balsalazine decreases intestinal mucosal permeability of dextran sulfate sodium-induced colitis in mice. Barreau et al., 2010 Barreau F.

Madre C.

Meinzer U.

Berrebi D.

Dussaillant M.

Merlin F.

Eckmann L.

Karin M.

Sterkers G.

Bonacorsi S.

et al. Nod2 regulates the host response towards microflora by modulating T cell function and epithelial permeability in mouse Peyer’s patches. Beaurepaire et al., 2009 Beaurepaire C.

Smyth D.

McKay D.M. Interferon-gamma regulation of intestinal epithelial permeability. Rousseaux et al., 2005 Rousseaux C.

Lefebvre B.

Dubuquoy L.

Lefebvre P.

Romano O.

Auwerx J.

Metzger D.

Wahli W.

Desvergne B.

Naccari G.C.

et al. Intestinal antiinflammatory effect of 5-aminosalicylic acid is dependent on peroxisome proliferator-activated receptor-gamma. Cipolletta et al., 2012 Cipolletta D.

Feuerer M.

Li A.

Kamei N.

Lee J.

Shoelson S.E.

Benoist C.

Mathis D. PPAR-γ is a major driver of the accumulation and phenotype of adipose tissue Treg cells. null mice and Beta7null mice, the lack of changes in adipogenesis gene expression in VAT and SAT, and the lack of detectable compound in VAT of HFD 5-ASA-fed mice. Thus, intestinal immune cell PPARγ may be another potential target of action for immune modulatory drugs with PPARγ agonistic effects. We further show that inhibition of low-grade gut inflammation with a gut-specific anti-inflammatory agent, 5-ASA, during HFD feeding can alter systemic glucose metabolism. Treatment with gut anti-inflammatory agents, including 5-ASA and Balsalazide, has beneficial effects on the intactness of the gut epithelial barrier in IBD models (). We observed similar beneficial effects of 5-ASA on gut barrier function during HFD feeding, which are linked to reduced levels of inflammatory cytokines, such as TNF-α and IFNγ, that can directly worsen barrier leakage of gut bacteria (). Accordingly, similar alterations in intestinal IFNγ-producing cells contribute to gut barrier defects in the setting of DIO. In addition to its well-known role as a COX-2 inhibitor, 5-ASA has PPARγ agonistic effects, which also contribute to our observed anti-inflammatory phenotype (). We noted increased PPARγ activity from bowel T cells of HFD 5-ASA-fed mice and that PPARγ contributes to 5-ASA inhibitory effects on IFNγ production by intestinal T cells in vitro. Interestingly, PPARγ induction in T cells can also bolster Treg function and numbers in other tissues, including VAT (). However, systemic effects of PPARγ agonism in fat or liver are unlikely in our study due to minimal metabolic effects seen in HFD 5-ASA-fed Rag1mice and Beta7mice, the lack of changes in adipogenesis gene expression in VAT and SAT, and the lack of detectable compound in VAT of HFD 5-ASA-fed mice. Thus, intestinal immune cell PPARγ may be another potential target of action for immune modulatory drugs with PPARγ agonistic effects.

Andrews et al., 2011 Andrews C.N.

Griffiths T.A.

Kaufman J.

Vergnolle N.

Surette M.G.

Rioux K.P. Mesalazine (5-aminosalicylic acid) alters faecal bacterial profiles, but not mucosal proteolytic activity in diarrhoea-predominant irritable bowel syndrome. Andrews et al., 2011 Andrews C.N.

Griffiths T.A.

Kaufman J.

Vergnolle N.

Surette M.G.

Rioux K.P. Mesalazine (5-aminosalicylic acid) alters faecal bacterial profiles, but not mucosal proteolytic activity in diarrhoea-predominant irritable bowel syndrome. Sartor, 2010 Sartor R.B. Genetics and environmental interactions shape the intestinal microbiome to promote inflammatory bowel disease versus mucosal homeostasis. Sartor, 2010 Sartor R.B. Genetics and environmental interactions shape the intestinal microbiome to promote inflammatory bowel disease versus mucosal homeostasis. Sartor, 2010 Sartor R.B. Genetics and environmental interactions shape the intestinal microbiome to promote inflammatory bowel disease versus mucosal homeostasis. Consistent with other reports (), we noted that 5-ASA could elicit changes in gut bacteria, including increased bacterial diversity, and increased abundances of Firmicutes, Clostridiales, and Ruminococcaceae. While these changes could reflect primary effects of the drug, they could also be secondary to reduced inflammation (). Reduced bacterial diversity and decreased abundance of certain Clostridial groups and Ruminococcaceae have been linked to increased inflammation in IBD (). Ruminococcaceae are prominent producers of short-chain fatty acids, including butyrate, which have protective activity in the intestine (). Thus, an interesting future direction will be to tease out specific effects of 5-ASA-associated microbial influences on improving metabolic syndrome.

Reagan-Shaw et al., 2008 Reagan-Shaw S.

Nihal M.

Ahmad N. Dose translation from animal to human studies revisited. Burger and Travis, 2011 Burger D.

Travis S. Conventional medical management of inflammatory bowel disease. We were able to obtain beneficial effects on glucose tolerance using a 5-ASA dose range of 150 mg/kg/day up to 1,500 mg/kg/day in mice, which using body surface area calculations () equates to approximate equivalent doses of 730 mg/day up to 7 g/day in a 60 kg human. Typical daily maintenance dosing of 5-ASA for mild to moderate IBD varies but often ranges between 1.5 and 4.8 g/day (). Thus, our work highlights novel uses of such drugs in obesity-related IR, which may require dosing not unlike existing protocols for IBD, though controlled human clinical studies are needed to better validate the effects and safety of different formulations of 5-ASA on IR.

null HFD-fed mice, with no effect on the inflammatory status of systemic hematolymphoid organs such as the spleen, suggest a possible linked circuit between adipose tissue and bowel inflammation. Other studies have also suggested that bowel inflammation may directly contribute to VAT inflammation ( Li et al., 2008 Li H.

Lelliott C.

Håkansson P.

Ploj K.

Tuneld A.

Verolin-Johansson M.

Benthem L.

Carlsson B.

Storlien L.

Michaëlsson E. Intestinal, adipose, and liver inflammation in diet-induced obese mice. Teixeira et al., 2011 Teixeira L.G.

Leonel A.J.

Aguilar E.C.

Batista N.V.

Alves A.C.

Coimbra C.C.

Ferreira A.V.

de Faria A.M.

Cara D.C.

Alvarez Leite J.I. The combination of high-fat diet-induced obesity and chronic ulcerative colitis reciprocally exacerbates adipose tissue and colon inflammation. Teixeira et al., 2011 Teixeira L.G.

Leonel A.J.

Aguilar E.C.

Batista N.V.

Alves A.C.

Coimbra C.C.

Ferreira A.V.

de Faria A.M.

Cara D.C.

Alvarez Leite J.I. The combination of high-fat diet-induced obesity and chronic ulcerative colitis reciprocally exacerbates adipose tissue and colon inflammation. Improvements in systemic glucose tolerance with 5-ASA treatment were found to be dependent on adaptive and gut immune systems. The observed direct effect of 5-ASA in vitro on purified intestinal dendritic cells modulating antigen-specific T cell responses and IFNγ production highlights potential crosstalks between intestinal adaptive and innate immune cells in mediating the effects of 5-ASA. The improvements in intestinal and VAT inflammation in 5-ASA-treated or Beta7HFD-fed mice, with no effect on the inflammatory status of systemic hematolymphoid organs such as the spleen, suggest a possible linked circuit between adipose tissue and bowel inflammation. Other studies have also suggested that bowel inflammation may directly contribute to VAT inflammation (). For instance, induction of colitis during HFD leads to marked increases in VAT macrophages, lymphocytes, and neutrophils (). Such results raise the possibility of downstream trafficking between immune cells of the bowel and VAT or the possibility that tolerance to leaked gut-soluble antigens in VAT is dependent on mechanisms governed by the gut immune system. Additional studies are needed to determine whether bowel immune cells routinely traffic to VAT and whether trafficking of gut-derived anti-inflammatory immune cells (or reduced trafficking of gut inflammatory cells) to VAT represents another mechanism of action of 5-ASA.

Wang et al., 2010 Wang Y.

Li J.

Tang L.

Wang Y.

Charnigo R.

de Villiers W.

Eckhardt E. T-lymphocyte responses to intestinally absorbed antigens can contribute to adipose tissue inflammation and glucose intolerance during high fat feeding. Another contributing role of the gut immune system during HFD may be in dictating downstream systemic inflammation to soluble gut-derived antigens, including in metabolic tissues like VAT, where inflammation directly impacts systemic disease. Improved oral tolerance may also manifest as reduced inflammatory responses, including IgG against gut-derived endotoxin. Oral tolerance to gut-derived antigens has been previously linked to reduced inflammation in VAT and improvements in IR, though the mechanisms were unknown (). We show that aberrant handling of gut antigen is likely due to the gut inflammatory environment during HFD, which is reversible with gut anti-inflammatory medication. HFD-induced low-grade gut inflammation may be a key trigger for antigen-specific T cell responses in VAT, linking the inflammatory phenotype we describe in the bowel to downstream responses in VAT.