Involvement of GPR43 Receptor in Effect of Lacticaseibacillus rhamnosus on Murine Steroid Resistant Chronic Obstructive Pulmonary Disease: Relevance to Pro-Inflammatory Mediators and Oxidative Stress in Human Macrophages.

BACKGROUND: Cytokine storm and oxidative stress are present in chronic obstructive pulmonary disease (COPD). Individuals with COPD present high levels of NF-κB-associated cytokines and pro-oxidant agents as well as low levels of Nrf2-associated antioxidants. This condition creates a steroid-resistant inflammatory microenvironment. Lacticaseibacillus rhamnosus (Lr) is a known anti-cytokine in lung diseases; however, the effect of Lr on lung inflammation and oxidative stress in steroid-resistant COPD mice remains unknown. OBJECTIVE: Thus, we investigated the Lr effect on lung inflammation and oxidative stress in mice and macrophages exposed to cigarette smoke extract (CSE) and unresponsive to steroids. METHODS: Mice and macrophages received dexamethasone or GLPG-094 (a GPR43 inhibitor), and only the macrophages received butyrate (but), all treatments being given before CSE. Lung inflammation was evaluated from the leukocyte population, airway remodeling, cytokines, and NF-κB. Oxidative stress disturbance was measured from ROS, 8-isoprostane, NADPH oxidase, TBARS, SOD, catalase, HO-1, and Nrf2. RESULTS: Lr attenuated cellularity, mucus, collagen, cytokines, ROS, 8-isoprostane, NADPH oxidase, and TBARS. Otherwise, SOD, catalase, HO-1, and Nrf2 were upregulated in Lr-treated COPD mice. Anti-cytokine and antioxidant effects of butyrate also occurred in CSE-exposed macrophages. GLPG-094 rendered Lr and butyrate less effective. CONCLUSIONS: Lr attenuates lung inflammation and oxidative stress in COPD mice, suggesting the presence of a GPR43 receptor-dependent mechanism also found in macrophages.

GPR43 stimulation on TCRαß+ intraepithelial colonic lymphocytes inhibits the recruitment of encephalitogenic T-cells into the central nervous system and attenuates the development of autoimmunity.

INTRODUCTION: Gut microbiota plays a critical role in the regulation of immune homeostasis. Accordingly, several autoimmune disorders have been associated with dysbiosis in the gut microbiota. Notably, the dysbiosis associated with central nervous system (CNS) autoimmunity involves a substantial reduction of bacteria belonging to Clostridia clusters IV and XIVa, which constitute major producers of short-chain fatty acids (SCFAs). Here we addressed the role of the surface receptor-mediated effects of SCFAs on mucosal T-cells in the development of CNS autoimmunity. METHODS: To induce CNS autoimmunity, we used the mouse model of experimental autoimmune encephalomyelitis (EAE) induced by immunization with the myelin oligodendrocyte glycoprotein (MOG)-derived peptide (MOG35-55 peptide). To address the effects of GPR43 stimulation on colonic TCRαß+ T-cells upon CNS autoimmunity, mucosal lymphocytes were isolated and stimulated with a selective GPR43 agonist ex vivo and then transferred into congenic mice undergoing EAE. Several subsets of lymphocytes infiltrating the CNS or those present in the gut epithelium and gut lamina propria were analysed by flow cytometry. In vitro migration assays were conducted with mucosal T-cells using transwells. RESULTS: Our results show a sharp and selective reduction of intestinal propionate at the peak of EAE development, accompanied by increased IFN-γ and decreased IL-22 in the colonic mucosa. Further analyses indicated that GPR43 was the primary SCFAs receptor expressed on T-cells, which was downregulated on colonic TCRαß+ T-cells upon CNS autoimmunity. The pharmacologic stimulation of GPR43 increased the anti-inflammatory function and reduced the pro-inflammatory features in several TCRαß+ T-cell subsets in the colonic mucosa upon EAE development. Furthermore, GPR43 stimulation induced the arrest of CNS-autoreactive T-cells in the colonic lamina propria, thus avoiding their infiltration into the CNS and dampening the disease development. Mechanistic analyses revealed that GPR43-stimulation on mucosal TCRαß+ T-cells inhibits their CXCR3-mediated migration towards CXCL11, which is released from the CNS upon neuroinflammation. CONCLUSIONS: These findings provide a novel mechanism involved in the gut-brain axis by which bacterial-derived products secreted in the gut mucosa might control the CNS tropism of autoreactive T-cells. Moreover, this study shows GPR43 expressed on T-cells as a promising therapeutic target for CNS autoimmunity.

GPR43 regulates sodium butyrate-induced angiogenesis and matrix remodeling.

Butyrate is a short-chain fatty acid (SCFA) derived from microbiota and is involved in a range of cell processes in a concentration-dependent manner. Low concentrations of sodium butyrate (NaBu) were shown to be proangiogenic. However, the mechanisms associated with these effects are not yet fully known. Here, we investigated the contribution of the SCFA receptor GPR43 in the proangiogenic effects of local treatment with NaBu and its effects on matrix remodeling using the sponge-induced fibrovascular tissue model in mice lacking the Gpr43 gene (Gpr43-KO) and the wild-type (WT) mice. We demonstrated that NaBu (0.2 mM intraimplant) treatment enhanced the neovascularization process, blood flow, and VEGF levels in a GPR43-dependent manner in the implants. Moreover, NaBu was able to modulate matrix remodeling aspects of the granulation tissue such as proteoglycan production, collagen deposition, and α-smooth muscle actin (α-SMA) expression in vivo, besides increasing transforming growth factor (TGF)-ß1 levels in the fibrovascular tissue, in a GPR43-dependent manner. Interestingly, NaBu directly stimulated L929 murine fibroblast migration and TGF-ß1 and collagen production in vitro. GPR43 was found to be expressed in human dermal fibroblasts, myofibroblasts, and endothelial cells. Overall, our findings evidence that the metabolite-sensing receptor GPR43 contributes to the effects of low dose of NaBu in inducing angiogenesis and matrix remodeling during granulation tissue formation. These data provide important insights for the proposition of new therapeutic approaches based on NaBu, beyond the highly explored intestinal, anti-inflammatory, and anticancer purposes, as a local treatment to improve tissue repair, particularly, by modulating granulation tissue components.NEW & NOTEWORTHY Our data show the contribution of the metabolite-sensing receptor GPR43 in the effects of low dose of sodium butyrate (NaBu) on stimulating angiogenesis and extracellular matrix remodeling in a model of granulation tissue formation in mice. We also show that human dermal fibroblasts, myofibroblasts, and endothelial cells express the receptor GPR43. These data provide important insights for the use of NaBu in local therapeutic approaches applicable to tissue repair in sites other than the intestine.

The Metabolic Sensor GPR43 Receptor Plays a Role in the Control of Klebsiella pneumoniae Infection in the Lung.

Pneumonia is one of the leading causes of death and mortality worldwide. The inflammatory responses that follow respiratory infections are protective leading to pathogen clearance but can also be deleterious if unregulated. The microbiota is known to be an important protective barrier against infections, mediating both direct inhibitory effects against the potential pathogen and also regulating the immune responses contributing to a proper clearance of the pathogen and return to homeostasis. GPR43 is one receptor for acetate, a microbiota metabolite shown to induce and to regulate important immune functions. Here, we addressed the role of GPR43 signaling during pulmonary bacterial infections. We have shown for the first time that the absence of GPR43 leads to increased susceptibility to Klebsiella pneumoniae infection, which was associated to both uncontrolled proliferation of bacteria and to increased inflammatory response. Mechanistically, we showed that GPR43 expression especially in neutrophils and alveolar macrophages is important for bacterial phagocytosis and killing. In addition, treatment with the GPR43 ligand, acetate, is protective during bacterial lung infection. This was associated to reduction in the number of bacteria in the airways and to the control of the inflammatory responses. Altogether, GPR43 plays an important role in the "gut-lung axis" as a sensor of the host gut microbiota activity through acetate binding promoting a proper immune response in the lungs.

Characterization of free fatty acid receptors expression in an obesity rat model with high sucrose diet.

INTRODUCTION/AIMS: In recent years, it has been shown that free fatty acids receptors (FFAR) of whose function in the cell surface plays a significant role in the regulation of cell function and nutrition as well are activated by various endogenous ligands, but mainly by fatty acids. Within FFAR of our interest are GPR 41, 43 and 120. The functions of these receptors are varied and dependent on the tissue where they are. The activation and signaling of these receptors, FFAR, are involved in many physiological processes, and currently the target of many drugs in metabolic disorders like obesity, diabetes and atherosclerosis. MATERIAL AND METHODS: Obesity was induced with hypercaloric diet (HD) in male Wistar rats for 20 weeks (n = 10). At the end, adipose tissue (abdominal and subcutaneous) was taken to perform assays for relative quantification mRNA expression by end-point RT-PCR and protein level expression by Western blot. RESULTS: These present data have shown for the first time that total mRNA isolation and protein expression from both adipose tissues (abdominal and subcutaneous) of rat in obesity condition yield significative statistical difference among the control versus obese groups, showing that the diet high in carbohydrates modifies the total presence of mRNA and protein level expression of the receptors GPR41, 43 and 120. CONCLUSIONS: Further comparative methods are in process to clarify whether or not the obesity changes the functional receptors in these two tissues for new pharmacological approaches.