Project

MODULATION OF EPITHELIAL AUTOPHAGY BY MACROPHAGES: RELEVANCE FORWOUND HEALING IN CROHN ́S DISEASE

Updates
0 new
0
Recommendations
0 new
0
Followers
0 new
1
Reads
0 new
12

Project log

Pedro Salvador Escribano
added 3 research items
Background:IntestinalfibrosisisacommoncomplicationofIBD.Fibrosisisaconsequenceoflocalchronicinflammationandischaracterizedbyanexcessiveextracellularmatrixdepositionandlossofnormalfunction.Residentmacrophagesplayakeyroleinmaintainingintestinalhomeostasisaswellasininjuryrepair,andtheirphenotypeevolvesduringthephasesofinflammation,remissionandwoundhealing.Stat6isthechieftranscriptionfactorinvolvedinmacrophagepolarizationtowardstheanti-inflammatoryphenotypeinducedbyIL4treatment,andwehavepreviouslyreportedthatStat6(-/-)micedevelopincreasedmucosaldamageandfibrosisinamodelofTNBS-colitis.OurpresentaimistoanalysetherelevanceofmacrophagesontheincreasedsusceptibilityofStat6-KOmice. Methods:Stat6(-/-)micereceivedincreasingdosesofTNBS(0.5,0.5,0.75,0.75,1and1mg,intrarectally)onceaweek.TwodaysaftereachTNBSadministration,theyreceivedasuspensionofIL4-treatedperitonealmacrophages(Mf,2x106cells)obtainedfromwildtype(WT)orStat6(-/-)donors.AscontrolgroupweusedStat6(-/-)TNBS-treatedmicethathadnotreceivedmacrophageadministration.Sevendaysafterthelastdose,micewheresacrificed,andtheexpressionofseveralmarkersoffibrosis(Tgf-β,E-Cadherin,Vimentin,Col1a1,α-Sma,Mmp2,Fsp-1andTimp-1)andthatofCD16,asamarkerofM2c-polaryzedmacrophages,wereanalysedbyqPCRincolonictissue. Results:TheweeklyinjectionofIL4-treatedMfinducedareductioninmortalitythatreachedstatisticalsignificancewhenthoseMfwereobtainedfromWTmice(p=0.037),butnotwhenMfcamefromStat6(-/-)donors(p=0.149)(Figure1).ThefibroticstateinTNBS-treatedStat6(-/-)micewasdemonstratedbytheincreasedexpressionofmostofthemarkersanalysed(Vimentin,α-Sma,Mmp2,Timp1,Col1a1)(ECCO2016).MicereceivingWT/IL4-treatedMfshowedareducedexpressionofTNBS-inducedfibroticmarkerswhencomparedtothatobservedinmicereceivingStat6(-/-)/IL4-treatedMf(Figure2).ThelevelsofsomeofthesemarkerscorrelatedwiththeexpressionoftheM2cmarkerCD16inSTAT6-KOmicebutnotinWTanimals(Figure3A).ThiscorrelationbetweenCD16andfibroticmarkerswasalsopresentinmicereceivingmacrophagesfromStat6(-/-)donorsbutnotinmicetreatedwithmacrophagesobtainedfromWTmice(Figure3B). Conclusion:Exogenous administration of macrophages with a Stat6-dependent phenotype exerts a protective effect and reverses the increased susceptibility of Stat6 (-/-) mice to TNBS-induced colitis and fibrosis, which seems related with the presence of M2c macrophages.
Background and pourpose: A defective autophagy is involved in the pathogenesis of inflammatory disorders such as IBD. Cross talk interactions between autophagy and inflammation have been reported and we analyse the effects of autophagy stimulators on murine colitis. Experimental approach: Mice were treated with intrarectal administration of TNBS (3.5 mg/20 g BW) and body weight was measured every day and histological damage score analysed two or four days after treatment. Some mice received trehalose (3% in drinking water three weeks before TNBS administration) or a daily administration of rapamycin (1.25 mg/kg, i.p.), betanin (1g/kg, i.p.) or betanin + 3MA (10mg/kg, i.p.). Mucosal protein levels of p-mTOR, p62, LC3, BCL10, NFκB, IκBα and p-IκBα were determined by WB and mRNA expression of TNFα, IL1β, IL6, IL10, COX2, CCR7, CD11c, iNOS and CD86 by qRT-PCR. Key results: An impaired autophagy associated with body weight loss and intestinal damage was detected in the mucosa of TNBS-treated mice. Administration of trehalose, rapamycin or betanin prevented the impaired autophagic flux induced by TNBS and decreased mucosal protein levels of BCL10, p-IκBα and NFκBp65 and the expression of pro-inflammatory cytokines and M1 macrophage markers. Blockade of the autophagosome formation by treatment of mice with 3MA, prevented the reduction in protein levels of p62, BCL10, p-IκBα and NFκBp65 induced by betanin in TNBS-treated mice and weakened the protective effects of betanin on murine colitis. Conclusions and implications: Our results demonstrate that pharmacological stimulation of mucosal autophagy reduces intestinal inflammation and ameliorates murine colitis.
Intestinal fibrosis, which is caused by excessive extracellular matrix deposition, is a common complication of inflammatory bowel disease. Macrophages assume a wide spectrum of different functional phenotypes (M1, M2a, M2b, and M2c) that differ in the expression of surface proteins, transcription factors, and cytokine production. It is believed that macrophages contribute to all phases of repair, inflammation, proliferation, and remodelling through their progression from M1 to M2c. We have demonstrated that STAT6 (-/-) mice exhibit an impaired M2a polarisation and delayed wound healing in an acute model of colitis. We have hypothesised that these animals are more susceptible to fibrosis development in response to chronic bowel damage.
Pedro Salvador Escribano
added 5 research items
Background & aims: IBD is a chronic disorder of the gastrointestinal tract characterized by mucosal inflammation and epithelial damage. Biologic therapy has significantly improved the course of the disease but there are still a high percentage of patients that do not respond to current therapies. We aim to determine the effects of the flesh ethanolic extract of Hylocereus polyrhizus (EH) in a mice model of colitis induced by TNBS. Methods: Balb/c mice received TNBS (175 mg/kg, 100 μl, i.r.) and six and thirty hours later were administered with EH (1 g/kg, i.p.). Mice were weighted daily and after sacrificing (2 and 4 days after TNBS) we analyzed mucosal histology, myeloperoxidase activity (MPO), the expression of pro-inflammatory molecules (qPCR) and NF-κB and Iκβ-α protein levels. The chemical characterization of the EH was determined by LC-MS/MS. Results: The administration of EH to TNBS-treated mice prevented (P < 0.05) the loss of body weight and significantly reduced in the colon: a) histological damage score, b) MPO enzymatic activity c) the expression of pro-inflammatory molecules and d) Iκβ-α degradation and nuclear NF-κβ protein levels. The LC-MS analysis detected metabolites such as polyphenols and fatty acids. Conclusion: Systemic administration of the ethanolic extract of H. polyrhizus exerts an anti-inflammatory effect and prevents murine colitis induced by TNBS.
Background Vitamin D signaling modulates inflammation through the vitamin D receptor (VDR) which is a member of the nuclear receptor family of transcription factors. The presence of C instead of T in the single-nucleotide polymorphism (SNP) rs731236 in the VDR gene has been associated with a higher risk for Crohn's disease (CD). We analysed the relevance of the presence of risk allele C in the evolution of the disease. Methods DNA was extracted from blood samples from 99 patients diagnosed with CD and 72 healthy donors from the Hospital of Manises (Valencia) and the SNP was genotyped using PCR-RFLP. We collected clinical data for each patient, including the Montreal classification in several phenotypes. Also, peripheral blood mononuclear cells (PBMCs) from 16 CD patients with the TT or CC genotype were obtained and gene expression of some cytokines was quantified in these cells by real-time RT-PCR. Results The allelic frequency of the risk allele was higher in CD patients related to healthy controls (p=0.2881, Fisher's test) and it was significantly different when compared with patients showing a B3 phenotype (p=0.026, Fisher's test). In addition, CD patients homozygous for the risk allele C initiated with the disease at a lower age (Fig. 1; p=0.05, t-test CC vs TT), and exhibited a significant higher risk to have a B3-penetrating phenotype (Fig. 2; p=0.0018, Chi-square; p=0.0078, Fisher's test CC vs TT, OR=5.3) and to need surgery (Fig. 3; p=0.013, Chi-square; p=0.021, Fisher's test CC vs TT, OR=4.3). Finally, PBMCs from patients with the CC genotype showed a higher level of IL1 β (p=0.13, t-test), IL18 (p=0.05, t-test) and IFN γ (p=0.36, t-test) mRNA than patients with the TT genotype. Conclusion Our study indicates that homozygosity for the allele C in the SNP rs731236 in the VDR gene confers a higher risk to develop a B3-penetrating phenotype in CD patients, associated with an elevated expression of pro-inflammatory cytokines in PBMCs.
Background: A defective autophagy is involved in the pathogenesis of inflammatory disorders such as IBD. Cross talk interactions between autophagy and inflammation have been reported and we analyse the effects of autophagy stimulators on murine colitis. Methods: Mice were treated with intrarectal administration of TNBS (3.5 mg/20 mg mice) and body weight was measured every day (and expressed as a percentage of starting weight), and histological damage score analysed two or four days after treatment. Some mice received trehalose (3% in drinking water three weeks before TNBS administration) or a daily administration of rapamycin (1.25 mg/kg, i.p.), betanin (1g/kg, i.p.) or betanin + 3MA (10mg/kg, i.p.). Mucosal protein levels of p-mTOR, p62, LC3, BCL10, NF-κB, IκBα and p-IκBα were determined by WB and mRNA expression of TNFα, IL1β, IL6, IL10, COX2, CCR7, CD11c, iNOS and CD86 by qRT-PCR. Results: An impaired autophagy associated with body weight loss and intestinal damage was detected in the mucosa of TNBS-treated mice. Administration of trehalose, rapamycin or betanin prevented the impaired autophagic flux induced by TNBS and decreased the expression of pro-inflammatory cytokines and M1 macrophage markers (Fig. 1A) and mucosal protein levels of BCL10, p-IκBα and NF-κBp65. Blockade of the autophagosome formation by treatment of mice with 3MA prevented the reduction in both body weight loss (Fig. 1B) and protein levels of p62, BCL10, p-IκBα and NF-κBp65 (Fig 1C) induced by betanin in TNBS-treated mice and weakened the protective effects of betanin on murine colitis. View largeDownload slide Figure 1 View largeDownload slide Figure 1 Conclusions: Our results demonstrate that pharmacological stimulation of mucosal autophagy reduces intestinal inflammation and ameliorates murine colitis.