Wound healing in FAT-1 mice : involvement of the cholinergic anti-inflammatory pathway

Orientador: Hosana Gomes RodriguesDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências AplicadasResumo: A inflamacao e a primeira fase do processo de cicatrizacao de feridas e esta pode ser dividida em resposta inflamatoria inicial e tardia. A resposta inflamatoria tardia e essencial ao controle de mediadores pro-inflamatorios e resolucao da inflamacao, sendo determinante ao inicio da proxima fase de formacao de tecido ou proliferacao. Acidos graxos poli-insaturados das familias 'omega'-6 e 'omega'-3, modulam o perfil inflamatorio, sendo cada vez mais estudados no processo de reparo tecidual. Assim, buscando compreender o papel destes acidos graxos na fase inflamatoria tardia do processo de cicatrizacao de feridas, utilizamos como objeto de estudo, camundongos transgenicos denominados FAT-1, capazes de produzir endogenamente, acidos graxos 'omega'-3 a partir de acidos graxos 'omega'-6. Para verificar a composicao lipidica destes camundongos, avaliamos por cromatografia gasosa, o plasma e o tecido cicatricial, 3 dias apos a inducao da ferida. No plasma, os camundongos transgenicos reduziram a concentracao do acido graxo araquidonico (AA) e aumentaram a concentracao dos acidos graxos alfa-linolenico (ALA), eicosapentaenoico (EPA) e docosahexaenoico (DHA), reduzindo a proporcao 'omega'-6/'omega'-3 em 32,9%, quando comparados ao grupo wild type (WT). Ja no tecido cicatricial, estes camundongos apresentaram reducao da porcentagem de AA nas classes lipidicas teciduais de fosfatidilcolina (PC) e fosfatidiletalonamina (PE) e, aumento dos acidos graxos docosapentaenoico (DPA) e DHA nas classes lipidicas teciduais de PC, PE e de triacilglicerol (TAG). Com isto, os camundongos transgenicos obtiveram uma reducao da proporcao 'omega'-6/'omega'-3, equivalente a 56,4% em PC, 55,1% em PE e 39,1% em TAG, em relacao ao grupo WT. Em analise do fechamento da ferida pelo programa Image J, os camundongos FAT-1 mantiveram a area da ferida maior desde o 1¿ dia apos a inducao, sendo que no ultimo dia de analise (14¿ dia), a area da ferida era duas vezes maior que a do grupo WT (p<0.01). Na analise da atividade da mieloperoxidase (MPO) por espectrofotometria, os camundongos transgenicos exibiram aumento da atividade no tecido cicatricial de 3 dias, quando comparados ao grupo WT (p<0.05). Considerando este mesmo tempo de ferida para todas as demais analises, observamos atraves de PCR em tempo real, que os camundongos FAT-1 aumentaram a expressao genica dos marcadores celulares CD206 e CD280 (p<0.05), representativos de macrofagos M2. Contudo, pelo metodo de ELISA, verificou-se que estes camundongos aumentaram a producao dos mediadores: IL-6 (p<0.05), CXCL1 (p<0.05), CXCL2 (p<0.01), TIMP-1 (p<0.01) e VEGF (p<0.05) e reduziram IL-10 (p<0.05), indicando uma modulacao da resposta inflamatoria tardia. Buscando investigar se a via anti-inflamatoria colinergica estaria influenciando esta resposta, analisamos a producao de acetilcolina (ACh) e colina livre pelo metodo colorimetrico, a atividade da acetilcolinesterase (AChE) por espectrofotometria e a expressao das proteinas 'alfa'7nAChR, STAT3 e pSTAT3, por western blottting. Os camundongos FAT-1, aumentaram a expressao proteica da 'alfa'7nAChR (p<0.001) e reduziram pSTAT3 (p<0.05), exibindo um comprometimento da propagacao do sinal anti-inflamatorio, no ambiente intracelular. Para analisar o impacto desta inflamacao tardia exacerbada, analisamos por PCR array, a expressao de genes presentes em todo o processo de cicatrizacao de feridas. No grupo FAT-1, genes essenciais a formacao e manutencao da matriz extracelular (MEC) foram negativamente modulados. Assim, concluimos que o nao controle da resposta inflamatoria tardia pelos camundongos FAT-1, pode ter prejudicado as funcoes da MEC e contribuido para o atraso no reparo tecidualAbstract: Inflammation is the first stage of the wound healing process and can be divided into initial and late inflammatory response. The late inflammatory response is essential for the control of pro-inflammatory mediators and resolution of inflammation, and is decisive at the beginning of the next phase of tissue formation or proliferation. Polyunsaturated fatty acids of 'omega'-6 and'omega'-3 families, modulate the inflammatory profile, being increasingly studied in the tissue repair process. Thus, to understand the role of these fatty acids in the late inflammatory phase of the wound healing process, we used FAT-1 transgenic mice capable of endogenously producing 'omega'-3 fatty acids from 'omega'-6. To verify the lipid composition of these mice, we evaluated by gas chromatography, plasma and scar tissue 3 days after wound induction. In the plasma, transgenic mice reduced arachidonic acid (AA) concentration and increased the concentration of alpha-linolenic acid (ALA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) fatty acids, reducing the ù-6/ù-3 ratio in 32,9%, when compared to the wild type (WT) group. In the scar tissue, these mice had a reduction in the percentage of AA in the lipidic tissue classes of phosphatidylcholine (PC) and phosphatidylalonamine (PE) and, increase in the fatty acids docosapentaenoic acid (DPA) and DHA in the lipidic tissue classes of PC, PE and triacylglycerol (TAG). Thus, the transgenic mice obtained a reduction of 'omega'-6/'omega'-3 ratio, equivalent to 56,4% in PC, 55,1% in PE and 39,1% in TAG, in relation to the WT group. In the analysis of wound closure by the Image J program, FAT-1 mice maintained the major wound area from the 1st day after induction, and on the last day of analysis (14th day), the area of the wound was two times greater than that of the WT group (p<0.01). In the analysis of myeloperoxidase activity (MPO) by spectrophotometry, the transgenic mice showed increased activity in the cicatricial tissue of 3 days, when compared to the WT group (p<0.05). Considering this same time of wound for all other analyzes, we observed through PCR real time, that FAT-1 mice increased the gene expression of CD206 and CD280 cell markers (p<0.05), representative of M2 macrophages. However, by the ELISA method, these mice were found to increase the production of mediators: IL-6 (p<0.05), CXCL1 (p<0.05), CXCL2 (p<0.01), TIMP-1 (p<0.01) and VEGF (p<0.05) and reduced IL-10 (p<0.05), indicating a modulation of the late inflammatory response. In order, to investigate whether the cholinergic anti-inflammatory pathway was influencing this response, we analyzed the production of acetylcholine (ACh) and free choline by colorimetric method, acetylcholinesterase activity (AChE) by spectrophotometry and expression of 'alpha'7nAChR, STAT3 and pSTAT3 proteins, by western blotting. FAT-1 mice increased the protein expression of 'alpha'7nAChR (p<0.001) and reduced pSTAT3 (p<0.05), exhibiting impairment of the propagation of the anti-inflammatory signal, in the intracellular environment. To analyze the impact of this late inflammation exacerbated, we analyzed by PCR array, the expression of genes present throughout the wound healing process. In FAT-1 group, genes essential for the formation and maintenance of the extracellular matrix (ECM) were negatively modulated. Thus, we concluded that the non-control of the late inflammatory response by the FAT-1 mice, may have impaired the ECM functions and contributed to delayed tissue repairMestradoNutriçãoMestra em Ciências da Nutrição e do Esporte e Metabolismo2016/02021-3FAPES

Butyrate Protects Mice from Clostridium difficile-Induced Colitis through an HIF-1-Dependent Mechanism

Antibiotic-induced dysbiosis is a key factor predisposing intestinal infection by Clostridium difficile. Here, we show that interventions that restore butyrate intestinal levels mitigate clinical and pathological features of C. difficile-induced colitis. Butyrate has no effect on C. difficile colonization or toxin production. However, it attenuates intestinal inflammation and improves intestinal barrier function in infected mice, as shown by reduced intestinal epithelial permeability and bacterial translocation, effects associated with the increased expression of components of intestinal epithelial cell tight junctions. Activation of the transcription factor HIF-1 in intestinal epithelial cells exerts a protective effect in C. difficile-induced colitis, and it is required for butyrate effects. We conclude that butyrate protects intestinal epithelial cells from damage caused by C. difficile toxins via the stabilization of HIF-1, mitigating local inflammatory response and systemic consequences of the infection

Wound Healing and Omega-6 Fatty Acids: From Inflammation to Repair

Wound healing is an evolutionarily conserved process that is essential for species survival. Wound healing involves a series of biochemical and cellular events that are tightly controlled, divided into 3 concomitant and overlapping phases: inflammation, proliferation, and remodelling. Poor wound healing or a chronic wound represents a silent epidemic that affects billions of people worldwide. Considering the involvement of immune cells in its resolution, recent studies are focused on investigating the roles of immune nutrients such as amino acids, minerals, and fatty acids on wound healing. Among the fatty acids, much attention has been given to omega-6 (ω-6) fatty acids since they can modulate cell migration and proliferation, phagocytic capacity, and production of inflammatory mediators. The present review summarizes current knowledge about the role of ω-6 fatty acids in the wound healing context

Oral administration of EPA-rich oil impairs collagen reorganization due to elevated production of IL-10 during skin wound healing in mice

Wound healing is an essential process for organism survival. Some fatty acids have been described as modulators of wound healing. However, the role of omega-3 fatty acids is unclear. In the present work, we investigate the effects of oral administration of eicosapentaenoic acid (EPA)-rich oil on wound healing in mice. After 4 weeks of EPA-rich oil supplementation (2 g/kg of body weight), mice had increased serum concentrations of EPA (20:5ω-3) (6-fold) and docosahexaenoic acid (DHA; 22:6ω-3) (33%) in relation to control mice. Omega-3 fatty acids were also incorporated into skin in the EPA fed mice. The wound healing process was delayed at the 3rd and 7th days after wounding in mice that received EPA-rich oil when compared to control mice but there was no effect on the total time required for wound closure. Collagen reorganization, that impacts the quality of the wound tissue, was impaired after EPA-rich oil supplementation. These effects were associated with an increase of M2 macrophages (twice in relation to control animals) and interleukin-10 (IL-10) concentrations in tissue in the initial stages of wound healing. In the absence of IL-10 (IL-10−/− mice), wound closure and organization of collagen were normalized even when EPA was fed, supporting that the deleterious effects of EPA-rich oil supplementation were due to the excessive production of IL-10. In conclusion, oral administration of EPA-rich oil impairs the quality of wound healing without affecting the wound closure time likely due to an elevation of the anti-inflammatory cytokine IL-10

Docosahexaenoic acid slows inflammation resolution and impairs the quality of healed skin tissue

There is no consensus on the effects of omega-3 (ω-3) fatty acids (FA) on cutaneous repair. To solve this problem, we used 2 different approaches: 1) FAT-1 transgenic mice, capable of producing endogenous ω-3 FA; 2) wild-type (WT) mice orally supplemented with DHA-enriched fish oil. FAT-1 mice had higher systemic (serum) and local (skin tissue) ω-3 FA levels, mainly docosahexaenoic acid (DHA), in comparison to WT mice. FAT-1 mice had increased myeloperoxidase (MPO) activity and content of CXCL-1 and CXCL-2, and reduced IL-10 in the skin wound tissue three days after the wound induction. Inflammation was maintained by an elevated TNF-α concentration and presence of inflammatory cells and edema. Neutrophils and macrophages isolated from FAT-1 mice, also produced increased TNF-α and reduced IL-10 levels. In these mice, the wound closure was delayed, with a wound area 6-fold bigger in relation with WT group, on the last day of analysis (14 days post-wounding). This was associated with poor orientation of collagen fibers and structural aspects in repaired tissue. Similarly, DHA group had a delay during late inflammatory phase. This group had increased TNF-α content and CD45+F4/80+ cells at the 3rd day after skin wounding and increased concentrations of important metabolites derived from ω-3, like 18-HEPE and reduced concentrations of those from ω-6 FA. In conclusion, elevated DHA content, achieved in both FAT-1 and DHA groups, slowed inflammation resolution and impaired the quality of healed skin tissue

Microbiota-derived acetate protects against respiratory syncytial virus infection through a GPR43-type 1 interferon response

Severe respiratory syncytial virus (RSV) infection is a major cause of morbidity and mortality in infants <2 years-old. Here we describe that high-fiber diet protects mice from RSV infection. This effect was dependent on intestinal microbiota and production of acetate. Oral administration of acetate mediated interferon-beta (IFN-beta) response by increasing expression of interferon-stimulated genes in the lung. These effects were associated with reduction of viral load and pulmonary inflammation in RSV-infected mice. Type 1 IFN signaling via the IFN-1 receptor (IFNAR) was essential for acetate antiviral activity in pulmonary epithelial cell lines and for the acetate protective effect in RSV-infected mice. Activation of Gpr43 in pulmonary epithelial cells reduced virus-induced cytotoxicity and promoted antiviral effects through IFN-beta response. The effect of acetate on RSV infection was abolished in Gpr43(-/-) mice. Our findings reveal antiviral effects of acetate involving IFN-beta in lung epithelial cells and engagement of GPR43 and IFNAR10CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPES036270/2016sem informação17/2551-0001 380-