Ulcerative colitis is a disease of incompletely understood etiology and is characterized by inflammation of the colonic mucosa. In its chronic form, inflammation extends into the muscle layer of the colon, with acute (symptomatic) manifestations, asymptomatic periods and relapses after months, years or even decades. Ulcerative colitis inflammation affects the rectum and extends in a retrograde manner, with extensive areas of superficial lesions. In more severe cases, the entire colon can be affected [¹,²].

The epidemiology of inflammatory bowel disease suggests that environmental factors such as personal hygiene, smoking and diet contribute to disease onset [³]. Pro-inflammatory markers including IL(interleukin)-6, IL-1 and TNF(tumor necrosis factor)-alpha are increased in colitis. These markers are also increased in obesity that have an inflammatory component [⁴,⁵]. Obesity is a multifactorial disease involving endocrine factors, genetics and behavior and directly contributes to systemic inflammation. Several studies have established a relationship between weight gain and levels of inflammatory proteins such as TNF, IL-1, IL-6 and leptin [⁴,⁵]. Moreover, studies have suggested that adipokines secreted by adipose tissue (TNFα, IL-1, IL-6, leptin, resistin and adiponectin) are closely associated with inflammatory bowel diseases such as ulcerative colitis [^6-13]. In addition, some studies have shown increased pro-inflammatory cytokines in the intestine of obese animals as well as in the adipose tissue of animals with colitis [⁹,¹²]. Nonetheless, to the best of our knowledge, inflammatory profile in animals with both diseases was not determined.

The objective of this study was to elucidate the relationship between obesity induced by a hypercaloric, high-fat diet (HFD) and chronic ulcerative colitis induced by intermittent administration of dextran sodium sulfate (DSS).

As expected, caloric intake was higher and water intake was lower in groups receiving the HFD (Table 1). During DSS administration, both caloric intake and water were reduced in the colitis groups. As a result, weight loss occurred, although body weights returned to normal in the weeks following treatment (data not shown). Colitis was confirmed by rectal bleeding, beginning on the third day after DSS introduction and persisting until one day after its removal. Although caloric intake was similar in both HFD groups, total weight gain was higher only in the HFD group without colitis.

Lipid profiles (triglycerides, total cholesterol, and LDL and HDL cholesterol) were not affected by diet or colitis (see additional file 1 - Table S1). The glucose tolerance test was altered in both HFD groups. Fasting glycemia. insulinemia, the homeostatic model assessment (HOMA) index and insulin sensitivity were similar among groups (see additional file 1 - Table 1). Despite alterations in glucose tolerance, no changes in expression of serine/threonine protein kinase (AKT/PKS) and glucose transporter type 4 (GLUT4), which are involved in the cascade of insulin signaling and glucose uptake, were detected in adipose tissue (Table 1).

The percentages of blood lymphocytes and monocytes were higher in animals with colitis that received a high-fat diet compared to others (see additional file 2 - Table S2). Other leukocyte subtypes remained unchanged.

Our study shows that HFD prolongs and aggravates the inflammatory manifestations of chronic ulcerative colitis, thereby increasing the pro-inflammatory status of adipose tissue. In our experiment, the analyses were performed 3 weeks after the second cycle of DSS (8^th experimental weeks) to permit the evaluation of this relationship out of the acute phase of ulcerative colitis in which metabolic abnormalities are more intense. In the same manner, we obtained a moderate grade of obesity due to the HFD, as seen by the significantly increased weight gain, adiposity and adipocyte area in the HFD group.

As previously described [¹⁴], animals provided with HFD consumed less liquid than those fed the standard chow diet. It could be assumed that DSS intake was also reduced in the colitis+HFD group. However, reduced DSS intake was not reflected in the intensity of the disease, as histopathological scores were even more intense in the colitis + HFD group compared to the colitis group.

Colon analyses showed mild inflammation in the colitis group, which became more severe when associated with HFD. Immune dysfunctions have been described both in obesity [¹⁵,¹⁶] and ulcerative colitis [¹⁷]. It is well-known that HFD leads to increased intestinal permeability by several mechanisms, including changes in the expression of tight junction proteins, such as occludins, ZO1 and claudin [¹⁸,¹⁹]. Thus, we hypothesized that chronic HFD consumption compromises the integrity of the intestinal barrier by disrupting the balance between injury and regeneration of the intestinal epithelium [¹⁶,²⁰] and exaggerating the mucosal immune response as result of increased pathogens and antigen entry from the intestinal lumen (including LPS), thereby enhancing the inflammation of ulcerative colitis [²⁰]. TLR4 plays an important role in the intestinal inflammatory response because it is activated either by lipopolysaccharide (LPS) produced by colonic bacteria or saturated fatty acids (FAs), both of which were present in the Colitis + HFD animals. The activation of TLR4 in colon cells could induce the expression and release of pro-inflammatory cytokines [²¹], as seen in the present study, via activation of NF(nuclear factor)-κB [²²], thereby increasing intestinal permeability in ulcerative colitis [¹⁷,²³]. As a result, luminal antigens may gain access to the lamina propria and trigger naive T cell differentiation, activation and production of inflammatory cytokines. Macrophages in the lamina propria are stimulated by these cytokines and secrete TNFα and IL-6, which enhances local inflammation [²⁴]. Our study supports this sequence of events, as we found increased colonic TLR4 expression, leukocyte (macrophages, lymphocytes and neutrophils) infiltration and inflammatory cytokine release.

The mechanisms of increased intestinal permeability related to obesity are not yet fully understood [²⁵,²⁶]. The persistently high levels of inflammatory cytokines produced by inflamed adipose tissue can cause an imbalance of the intestinal barrier function by altering tight junction proteins [²⁵]. These proteins regulate the selective transport of ions, solutes and peptides from the lumen of the intestine to the bloodstream. Thus, the association between diet-induced obesity and chronic ulcerative colitis enhances transport of pathogens or their derivatives (such as LPS) that were previously restricted by the intestinal barrier to the systemic circulation resulting in inflammatory pathways activation [²³,²⁷] in peripheral organ adipose tissue. Interestingly, the changes triggered by chronic colitis were more significant in visceral adipose tissue than in closely related immune organs, such as the spleen and lymph nodes, highlighting the influence of intestinal inflammation on organs that are not traditionally associated with immune responses, such as adipose tissue.

In addition to adipocytes, adipose tissue is composed of stromal vascular cells, monocytes and lymphocytes, which are responsible for immunological balance [²⁸]. Adipose tissue expansion triggers immunological imbalance [¹⁵,²²] by increasing expression and also activation of TLR-4, which leads to NFκB activation and, consequently, production of pro-inflammatory cytokines [²²]. Moreover, the influx of Th1 lymphocytes and macrophages further increases pro-inflammatory cytokine and chemokine production, attracting and activating more macrophages to perpetuate the inflammation [¹⁵,²²,²⁸]. In our study, the increase in adipose tissue inflammation seen in the Colitis + HFD group was confirmed by the increase in CLS, activated macrophages, lymphocytes and neutrophils and the overexpression of adhesion molecules, TNFα, IL-6, MCP1/CCL2 and TLR4.

This pro-inflammatory scenario was confirmed in vivo by the increased rolling and adhesion of leukocytes in adipose tissue microvasculature.

Although the colitis + HFD group presented lower adiposity and adipocyte area, the number of CLS-forming macrophages was higher compared to that of other groups. CLS represents the conclusion of the relatively long life of adipocytes and is related to the macrophage response to changes in adipose tissue that lead to adipocyte death or apoptosis [²⁹]. In addition to the CLS, the increase in activated CD4+ T cells in colitis + HFD mice leads to increased MCP-1/CCL2 release in this tissue [¹⁵], which induces migration and activation of macrophages that originate from the CLS and enhance inflammation [²⁹].

Although it is a classical inflammatory marker, we observed an increase in serum resistin in the colitis group that was not observed in the Colitis + HFD group. Increased serum resistin in patients with ulcerative colitis has been previously described [⁷,⁸,¹³] and is an early marker of inflammation [⁸]. Resistin is also produced by peripheral blood mononuclear cells and macrophages [³⁰]. Therefore, serum levels were not correlated to resistin expression in epididymal adipose tissue. The absence of increased resistin in the HFD group disagrees with previous literature [³¹]. This discrepancy may be due to the duration of the present experiment and the moderate obesity observed, which were not sufficient to induce changes in this parameter.

Although circulating levels of adiponectin were similar among groups, its expression in adipose tissue was reduced in all groups compared to the control group. This fact has been previously described in animals fed HFD [³²], but its pathological significance in inflammatory bowel disease is still controversial. Human studies have shown both increased [⁷,³³] and decreased [¹³,³⁴] serum adiponectin levels in patients with ulcerative colitis. Studies in mice are even more controversial. Two studies using adiponectin knockout mice with DSS-induced ulcerative colitis showed opposite results. One found a protective effect of adiponectin, as the knockout animals displayed a more severe ulcerative colitis [¹⁰], while the other study showed a protective effect of adiponectin deletion against the development of ulcerative colitis [⁶]. Pro-inflammatory factors may decrease the expression of adiponectin [³⁵], which would explain its decreased expression in visceral adipose tissue. Moreover, subcutaneous fat, rather than visceral fat (as measured in this study), is the main site of adiponectin production.

Leptin is produced in adipose tissue in response to fat mass and controls appetite and energy expenditure by hypothalamic pathways [³⁶]. Leptin also modulates several immune and inflammatory responses, such as monocyte and T lymphocyte activation, phagocytosis and TNFa, IL-6, and IFN-y production by peripheral blood mononuclear cells [^36-38]. In our study, leptin expression was increased in adipose tissue in both HFD groups compared to the colitis group. However, the higher circulating leptin concentration was seen only in the HFD group and not the Colitis + HFD group. This may be due to increased leptin/Ob-R receptor binding because the receptor is overexpressed in inflamed colons of colitis animals [³⁹,⁴⁰]. Therefore, we believe that leptin/receptor interactions in the colons of the colitis + HDF animals are partially responsible for maintaining local colonic inflammation.

A previous study [²⁹] has reported increased leptin expression in the colons of mice with colon cancer that was secondary to DSS-induced ulcerative colitis. As in our work, the authors observed increased leptin expression in the fatty tissue of animals with colitis associated with HFD. These results agree with ours and reinforce the association between leptin and inflammation.

To the best of our knowledge, this is the first time that crosstalk between inflammatory components of ulcerative colitis and obesity has been shown to lead to mutual exacerbation. We believe that this relationship is supported by increased leptin uptake due to Ob-Rb overexpression induced by colitis. Leptin then activates immune cells by producing IL-6, IL-1p and chemokine (C-X-C motif) ligand 1 CXCL1[⁴¹] and supports intestinal inflammation caused by chronic ulcerative colitis. However, changes in intestinal permeability triggered by colitis (and aggravated by HFD) allow the greater input of LPS and fatty acids in the systemic circulation, which bind to TLR4 receptors on the surface of adipocytes and macrophages in the expanded adipose tissue, thereby activating NF-ĸB signaling and reinforcing the systemic inflammation.

In conclusion, HFD during chronic ulcerative colitis creates an environment of reciprocal activation and worsens inflammation in adipose tissue and colon.

The project was approved by the Ethics Committee for Animal Experimentation at the Federal University of Minas Gerais (CETEA/UFMG # 110/2010).

Six-week-old male C57BL/6 mice were group-housed in an environment with light cycles of 12 hours (7:00 to 19:00) and controlled temperature (20 to 24°C). All mice had free access to food and water for 8 weeks.

The animals were divided into the following groups: control, colitis, HFD and colitis + HFD. Animals from the control and colitis groups were fed a standard chow diet (Labina ^®, 7.8% energy as fat, 50.3% in the form of carbohydrates, 41.9% protein, caloric density of 2.18 kcal/g) for 8 weeks, while mice from the HFD and Colitis + HFD groups received a high-fat diet (HFD) to induce obesity [⁴²]. The composition of the HFD was 61% energy as fat, 24.5% carbohydrates, and 14.5% protein and had a caloric density of 5.21 kcal/g.

Chronic ulcerative colitis was induced in the colitis and colitis + HFD groups by replacing drinking water with a solution of 3% (w/v) dextran sulfate sodium (DSS, 36.000 to 50.000, MP Biomedicals) for 5 days as previously described [⁹,⁴³] at the 1^st and 4^th experimental weeks. The animals without induction of colitis (the control and HFD groups) received water throughout the experiment. At the end of the 8^th experimental week, all animals were euthanized under anesthesia after overnight fast. Blood, adipose tissue, spleens, lymph nodes and colons were removed for analysis.

AKT/PKS: serine/threonine protein kinase; BSA: bovine serum albumin; CD: cluster of differentiation; CLS: crown-like structure; CXCL1: chemokine (C-X-C motif) ligand 1; DSS: dextran sodium sulfate; EDTA/KF: Ethylenediaminetetraacetic acid/potassium fluoride; ELISA: Enzyme-Linked Immunoabsorbent Assay; GLUT4: glucose transporter type 4; HDL: high density lipoproteins; HFD: high-fat diet; HOMA: homeostatic model assessment; ICAM: inter-cellular adhesion molecule 1; IFN: interferon; IL: interleukin; IR: insulin resistance; Kcal/g: kilocalories per gram; LDL: low density lipoproteins; LPS: lipopolysacharide; MCP1/CCL2: monocyte chemotatic protein; RT-PCR: Reverse transcription polymerase chain reaction; NaCl: sodium chloride; NF-ĸB: nuclear factor ĸB; Ob-Rb: leptin receptor b; PMSF: phenylmethanesulfonylfluoride; TLR4: receptor like toll 4; TNF: tumor necrosis factor; VAT: visceral adipose tissue; VCAM: vascular cell adhesion protein

The authors declare that they have no competing interests.

LGT designed and conducted research, analyzed data and wrote the paper. AJL and ECA helped in conducting research. ACA and AMCF helped in flow cytometry and citokynes ELISA. NVB and DCCM carried out intravital microscopy and helped in histological analysis. CCC carried out the radioimmunoassay. AVMF carried out the adipokines ELISA. JIAL supervised the work and wrote the paper. All authors read and approved the final manuscript.

The authors are grateful to Maria Helena Alves de Oliveira who was responsible for the animal facility, and the sources of support: Pró-reitoria de Pesquisa of Universidade Federal de Minas Gerais, CNPq (National Counsel of Technological and Scientific Development), CAPES (Coordination of Personal Improvement of Higher Education), FAPEMIG (Foundation for Research Support of Minas Gerais).