Obesidad, hiperleptinemia e inflamación intestinalestudios en modelos animales

Resumen

1. INTRODUCTION Overweight and obesity are defined as abnormal or excessive fat accumulation that may impair health. WHO describes obesity as one of the most blatantly visible, yet most neglected, public-health problems that threatens to overwhelm both more and less developed countries. Overall, about 13% of the world’s adult population (11% of men and 15% of women) were obese in 2014, and 39% of adults aged 18 years and over (38% of men and 40% of women) were overweight. In the last decades, obesity and autoimmune diseases, including inflammatory bowel disease (IBD), have experienced a dramatic increase in Western countries, following the same outbreak. The involvement of environmental factors is strongly suspected to explain this rise. Since the exciting discovery of the secretory properties of adipose tissue, the relationship between obesity and autoimmunity and the understanding of the underlying mechanisms have become of major interest. Indeed, the fat tissue has been found to produce a wide variety of “adipokines”, involved in the regulation of numerous physiological functions, including the immune response. Inflammatory Bowel Disease (IBD) is a chronic relapsing inflammatory disorder of the gastrointestinal tract that encompasses two idiopathic and major inflammatory diseases: Crohn's disease (CD) and ulcerative colitis. Both forms of IBD significantly impair quality of life, and require prolonged medical and/or surgical interventions. What makes it particularly challenging is its still unknown cause, its unpredictable presentations and symptoms, the less than optimal treatments, and a rise in its incidence and prevalence in many areas of the world. Obesity has previously been considered to be uncommon in IBD. As the prevalence of obesity has increased worldwide, this epidemic has also influenced the IBD patients population. Moreover, mesenteric adipose depots, so-called “creeping fat” have long been recognized as hallmarks of CD, its extent being correlated with the severity of intestinal inflammation. Due to the pro-inflammatory state induced by the adipose tissue, it is necessary to clarify its impact on disease progression. Studies on this topic are unfortunately rare. Recently, increased attention has been paid to the link between gut microbial composition, intestinal permeability and obesity. Gut microbiota is a source of endotoxins whose increase in plasma is related to obesity and insulin resistance through increased intestinal permeability in animal models. The proper functioning of the intestinal barrier is essential to avoid the excessive translocation of inflammatory molecules (eg, LPS) to the circulation. It is reported that microbiota can influence the integrity of intestinal epithelium and the tonus of mucosal inflammation, which, in turn, can affect intestinal permeability properties that are used to evaluate dysfunctions of the gastrointestinal barrier. Leptin, a molecule that is critical in the regulation of energy balance, body weight, and reproductive function, is a strong regulator of T cell function. As part of its immune-modulating function, it is able to increase activation and proliferation of monocytes and T-cells and mediates homeostasis in a variety of immune cells. This is one of many examples of redundancy and of the overlapping roles of molecules within the neuroendocrine and immune systems. Leptin is part of the helical cytokine family along with interleukin (IL-) 6, IL-12, and IL-15, its receptor (LepR) belonging to the group of class I cytokine receptors, which includes gp-130, the common signal transducing component for the IL-6 related family of cytokines. The long form of the receptor (LepRb) contains all canonical signaling sites, whereas the short forms lack much of the intracellular domain and capacity to signal. LepRb is expressed in numerous cell types, including neurons, intestinal epithelial cells (IECs), and immune cells, such as macrophages, T cells, natural killer cells and polymorphonuclear cells. Leptin is expressed particularly in adipose tissue and to a lesser extent in other tissues such as muscle, stomach, and placenta. Recent reports have shown that leptin secreted by the gastric mucosa is not fully degraded by proteolysis and can reach the intestine in an active form able to control the expression of sodium/glucose and peptide transporters on intestinal epithelial cells and also may stimulate gut mucosal cell proliferation. Therefore, it may be speculated that leptin displays a dual nature: as a growth factor for the intestine, involved in the absorption of carbohydrates and proteins and cell proliferation on the one hand, and as a mediator of the intestinal inflammation induced by T lymphocytes on the other. Thus, further research into the cellular targets of leptin and the downstream effector functions mediated through LepR are critical in understanding how leptin functions outside of the neuroendocrine axis and affects host’ susceptibility to disease. 2. AIMS Based on all the above, we proposed 3 main objectives in this doctoral thesis: 1. To assess the burden of obesity in the development of acute experimental colitis in the Zucker rat model, which is characterized by its leptin resistance. 2. To describe the role of leptin in intestinal inflammation in mice with a non-altered leptin signaling. 3. To set up a more suitable approach to evaluate protein loading control in Western blotting as alternative to housekeeping protein when pathological conditions such as obesity or inflammation can disrupt the normal amount of housekeeping reference. 3. METHODS To carry out these objectives, we use a number of techniques, including tissue culture techniques, real time PCR, histology techniques, flow cytometry, Ussing chambers, Western Blot, ELISA as well as experimental models of colitis (TNBS, DSS) and obesity (Zucker rat). 4. RESULTS AND DISCUSSION The obese Zucker rat, which is commonly used as genetic model of obesity because of its leptin resistance, was selected to assess obesity consequences in the development of TNBS-induced acute model of colitis. Overall, obese Zucker rats seemed to be less inflamed than lean rats, showing lower levels of colonic myeloperoxidase (MPO) activity, inducible nitric oxide synthase (iNOS) and tumor necrosis factor alpha (TNFα), as well as downward trend in gamma interferon (IFNγ), IL-1β and IL-17A colonic expression and less phosphorylation of signal transducer and activator of transcription-(STAT-) 3. Surprisingly, the increase of forkhead box P3 (Foxp3) expression was not correlated with IL-10 expression levels in colonic tissue. Obese Zucker rats showed less pronounced weight loss but it must be taken into account that energy expenditure is reduced in this model. Actually, caloric intake was the same in lean and obese Zucker rats, and absolute weight loss was also identical (34 g). However, all of this was accompanied by an increase of colonic thickening and intestinal permeability. Besides, the overall appearance of obese rats was worse than that exhibited by the lean group. Therefore, although it may globally be considered that obese Zucker rats are relatively resistant to TNBS-induced colitis, it is equally true that the absence of leptin signaling appears to have some detrimental consequences. There was no change between colitic groups on cyclooxygenase-2 (COX-2), IL-10 and Reg3γ mRNA levels either on alkaline phosphatase (AP) activity, however, in vitro sensitivity of AP to the inhibitor levamisole was reduced in the obese group as well as TNAP mRNA expression in colonic tissue. Plasmatic leptin was reduced in the lean colitic group but not in obese rats. Mesenteric adipose tissue (MAT) of colitic rats showed a significantly decrease in leptin mRNA expression as well as adiponectin, resistin and ghrelin, indicating that MAT is involved in the inflammatory process, which is in line with the induction of TNFα and IL-1β (but not iNOS), assessed by RT-PCR. TNBS-induced colitis increased bacterial translocation from gut to liver and mesenteric lymph nodes (MLN), portal plasma levels of LPS and augmented intestinal permeability to FITC-dextran assessed in vitro. Data were always higher in obese rats, but differences never became significant. Altogether, these results suggest that mucosal barrier function (MBF) was impaired in obese Zucker rats. To describe the role of leptin in intestinal inflammation we carried out a second experiment giving pegylated-leptin to C57BL6/J mice. After a week, experimental DSS-induced colitic model was performed. Inflammatory response was attenuated in leptin-treated mice, showing less colonic thickness, histological damage, AP activity and fecal blood, recovery from anemia, lower IFN, IL-6 (not significant), IL-1, IL-27, Reg3, STAT3 activation, CD4+ IFN-+ T cells relative presence in MLN, etc. Besides, TNF, IFN-, IL-1, IL-17A, IL-12 p70 secretion was reduced in colonic explant cultures of leptin-treated mice. Zonula occludens 1, claudins 4 and 5 mRNA expression was induced by leptin both colitic and non-colitic mice; cyclin D1, Myc and proliferating cell nuclear antigen (PCNA) was increased in non colitic leptin-treated mice. Moreover, alcian blue staining showed a significant mucus production in leptin-treated colitic mice. Altogether, these data suggest that leptin treatment reinforces MBF, giving protection to DSS-induced colitis. Western blot analysis was widely used in this doctoral thesis. It is currently a routine practice to require a measurement of a housekeeping reference protein, including actin, glyceraldehyde-3-phosphate dehydrogenase, and β-tubulin, among others, in Western blots, as it is the rule in RNA blots. In some cases, such as obesity or inflammation, these proteins are indeed affected by the experimental conditions. In trying to improve this approach, we have tested a new technique, with the Stain-Free gels from Bio-Rad, against both Ponceau staining and housekeeping protein immunodetection under different conditions. Our results show that Stain-Free gels outperform Ponceau staining and that both are more consistent than housekeeping proteins as a loading control. 5. CONCLUSIONS 1. Stain-Free gels outperform Ponceau reversible staining, and both are more consistent than the immunodetection of housekeeping proteins as a loading control for Western blotting. 2. In the Zucker rat, a well-established model of genetic obesity featured by leptin resistance, the TNBS-induced inflammatory response is notably reduced, but this correlates with an enhanced epithelial damage. Besides, exogenous leptin administration to healthy mice has beneficial effects on the subsequently induced DSS model of colitis, due to an improved intestinal barrier function. 3. Leptin plays a complex role on the experimental colitis, given that its effects are both protective or harmful depending on the context. Our results are in good agreement with those published by other authors making use of animals with an impaired leptin signaling (ob/ob mice and db/db mice), rats treated with exogenous leptin, mice exposed to leptin antagonists, and others.