Exploring The Potential Of CXCR1/2 Antagonist G31P In Ameliorating Ulcerative Colitis

Introduction:Inflammatory bowel disease(IBD)is a generic term which encompasses all chronic inflammatory diseases of the gastrointestinal canal.It remains a major health challenge due to its incurable nature,difficult to treat and limited options for treatment and management Its global scope in terms of incidence and prevalence keeps expanding.Crohn' s disease(CD)and ulcerative colitis(UC)are the principal subtypes of IBD,which are similar symptomatically but also exhibit significantly precise divergence.CD is aggressive and very diverse in pathogenesis.It is trans-mucosal and can affect the entirety of the gastrointestinal tract,even though it predominantly involves the small intestines.Symptomatically,CD is characterized by abdominal discomfort and pain,feverishness,and marked bloody-mucoid diarrhea.UC is symptomatically similar to CD but marked by bloody diarrhea,and anatomically restricted to the colon and rectal regions of the gastrointestinal tract Affected persons may frequently suffer tenesmus and rectal urgency.Currently,the armamentarium available for the treatment and management of IBD covers amino-salicylates,anti-diarrheal agents,antibiotics,corticosteroids,immune-regulators,and biologic agents such as anti-TNF-a and anti-?4?7 integrin.Other studies have reported probiotics as potential therapeutic agents in the maintenance of gut integrity,crucial for the management and prevention of IBD.However,most of the treatment regimens prescribed for IBD have serious adverse effects.Immune-suppressors such as corticosteroids compromise immunity and make patients more susceptible to infection.Their long term use may lead to osteoporosis and enhance susceptibility to fracture.The biologic agents have a strong link with lymphoma,while anti-diarrhea and antibiotics potentiate gut microbiome dysbiosis.The complications associated with the present therapeutic agents,coupled with their inability to offer complete cure,demand the need for alternative therapies for IBD.CXCL8 is one of the earliest identified and sequenced chemokines.CXCL8 was found to induce the production of neutrophil activating factor(NAF),capable of stimulating exocytosis,and producing H202 and superoxide,via its receptors.Its structure was first elucidated in 1991.It consists of 72 amino acid peptides with three anti-parallel beta strand,and an alpha helix consisting of C-terminal residue.Its disulfide bonds are located at Cys7-Cys34 and Cys9-Cys50,giving the protein its stability.CXCL8 is classified under CXC chemokine because it Cys7 and Cys9 are separated by Gln8(glutamine).Additionally,CXCL8 exist as a dimer,stabilized by hydrogen bond.Even though CXCL8 monomer exhibit stronger affinity for CXCRl,both CXCL8 monomer,and dimer exhibit similar affinity towards CXCR2.It has been identified that mutation in the first fifteen amino acids to alanine results in Glu-Leu-Arg(ELR)motif.This change or mutation results in less affinity of CXCL8 to its cognate receptors in a competitive binding assay and is considered a clue to anti-IL8 therapy.CXCL8 secretion is almost ubiquitous and constitutive in many cells including monocytes,macrophages,neutrophils,fibroblast,endothelial,and epithelial.In un-insulted cells or tissues,CXCL8 is not or negligibly detected.However,following cellular or tissue assault,its secretion is elevated and easily detectable.It can be induced by other cytokines including IL-1,IL-6,CXCL8 itself,CXCL12,and TNF.Also,environmental stress,infection,and reactive oxygen species(ROS)modulate CXCL8 activation and secretion.Additionally,CXCL8 activation is mediated by transcription factors NF-k?,and activator protein 1(AP-1).Structurally,human recombinant CXCL8 antagonist is similar to CXCL8,however,it K11 and G31 positions have been mutated.The K11 position originally lysine(K),has been replaced with arginine(R),while the G31 position glycine(G),is now proline(P).These purposeful mutations brought about conformational and functional change,antagonizing CXCL8 production in a competitive manner via CXCR1/2.This newly engineered chemokine,CXCL8 antagonist,CXCL8(3-72)K11R/G31P(abbreviated as G31P),acts as neutrophils inhibitor.CXCL8 is one of the members of the CXC chemokine family which recruits neutrophil to inflamed sites.The activation of CXCL8 pathway starts with the binding of CXCL8 to the G protein-coupled receptors(GPCRs)CXCR1(with relatively high affinity),and CXCR2 and other CXC chemokine receptors with less affinity.The role of CXCR1/2 as therapeutic targets have been underscored in previous studies,and G3 1P have been proven to be efficacious in many solid cancers including breast,liver,lung,ovarian,and prostate tumors.However,few studies have reported the potential application of CXCR1/2-G31P interaction in non-tumor diseases.Given the neutrophil infiltration inhibitory property of G3 1P and reportedly fewer side effects,it was thought appropriate to investigate its potency in IBD,particularly UC.Therefore,the purpose of this study was to investigate the therapeutic effect of CXCL8 antagonist,G31P,against IBD,focusing on UC.Method:The techniques employed included ex vivo and in vivo studies.The ex vivo investigations covered conducting functional and molecular assays on THP-1 cells.Inflammation was induced by application of LPS,and intervened with either 20 or 30?g/ml G31P,informed by CCK-8 outcome.In the in vivo phase,twenty-eight(28)C57/BL/6J male mice of age eight weeks and weighing between 22-26 grams were randomized into four groups,seven per group according to the treatment options;Control,dextran sodium sulfate(DSS),DSS+G31P,and DSS+G31P+Lactobacillus acidophilus(DSS+G31P+LACT)groups.G31P administration was via subcutaneous injection at a dose of 0.5mg/kg body weight every other day,using 0.9%sterile normal saline as diluent.The DSS+G31P+LACT group was given daily dose of 106 CFU/mL L acidophilus in a total volume 200 ?1 of normal saline via oral gavage,in addition to the DSS and G31P.The dosage of G31P administered was informed by previous reports.Aside the observational studies,functional and molecular techniques employed in this study included proliferation assay by CCK-8,transwell migration assay,ELISA,PCR,western blot,hematoxylin and eosin staining,immunohistochemistry staining,and flow cytometry.Results:The CCK-8 assay revealed G31P confers significant inhibition to THP-1 cells proliferation at concentrations 20?g/ml and 30?g/ml after 48h incubation,and co-treated with LPS(1?g/ml)exhibited significant inhibition at 30?g/ml after 48h incubation.LPS stimulation marginally elevated CXCR1 mRNA expression and significant increased CXCR2 mRNA,but both were mitigated by G31P.Both transcription and translation levels of CXCL8(IL-8)were significantly down-regulated.The transcriptional activity of selected inflammatory cytokines where measured by RT-PCR.LPS associated elevation of IL-1?,IL-6,and TNF-? mRNA were significantly down-regulated by G31P in concentration dependent manner.However,IL-10 mRNA expression remained relatively unchanged compared with Control and LPS induced groups.Also,LPS induced elevation of inflammatory associated enzymes including COX-2,MMP-2,and MMP-9 were reverted by G31P.The treatment also restricted the migration of the monocyte-derived macrophage.To further probe how G31P affects cytokines expression,inflammatory associate transcription factors including c-Fos,c-Jun,SP-1,and NF-k? were detected by RT-PCR.While G31P treatment significantly retards mRNA expression of c-Fos and NF-k?,marginal elevation of c-Jun mRNA was observed.Also,SP-1 mRNA was seen to increase appreciably by G31P treatment in concentration dependent mannner.The in vivo treatment of colitis model with G31P showed protection against DSS induce colitis demonstrated by improved disease activity index.The histopathological examination established severe damage in DSS group,with significant defamation of colon integrity marked with eroded crypts and goblet cells,and distorted colonic epithelial architecture.Comparatively,relatively improved colonic condition was observed in the DSS+G31P group(p<0.001)than DSS+G31P+LACT(p<0.05)when compared with the DSS group.Generally,the mRNA levels of TNF-?,INF-?,IL-1?,and IL-6 expressed by the colon tissues harvested from the mice were expectedly elevated in the DSS group,but were reduced significantly in both DSS+G31P and DSS+G31P+LACT groups.The expression levels of IL-10 mRNA were however not significantly different among all the groups compared with the Control group.In addition,serum samples were analyzed by ELISA technique for the expression of serum CXCL8.Though its up-regulation was reversed by G31P treatment,the reduction by DSS+G31P+LACT was more significant(p<0.001),suggesting synergism.Among the characteristics of inflammation is trafficking of immune cells to the affected site.Employing RT-PCR and IHC,the expression levels of F4/80,CD68,IL-17A,and IL-17F in the colon tissues were estimated.The DSS+G31P and DSS+G31P+LACT groups restricted the influx of macrophages in the colon,However,DSS+G31P group significantly reduced IL-17A expression,while that of DSS+G31P+LACT group showed elevated expression IL-17A.On the other hand,there were marginally elevated IL-17F expressions among all the groups,comparable to the Control group.The immuno-histochemistry(IHC)staining targeting CD68 as marker for macrophages,demonstrated significantly minimal infiltration of macrophages in the treatment groups compared with the DSS group.The effect of G31P on colon fibrosis were investigated by determining the expression levels of VEGF,TGF-P,and the gelatinases:MMP-2 and MMP-9.The mRNA expressions of VEGF and TGF-P were significantly reduced in both DSS+G31P and DSS+G31P+LACT groups when compared with the DSS group.Similarly,DSS+G31P and DSS+G31P+LACT groups,though insignificant,exhibited reduction in MMP-2 and MMP-9 levels,except MMP-9 mRNA which was significantly reduced by G31P+LACT treatment and comparable to the Control group.Expression of the tight junction proteins,E-cadherin and occludin as yardstick for colon tissue integrity were measured.The levels of these proteins were significantly reduced in the DSS group when compared with the Control group.However,there was significant restoration of E-cadherin in both DSS+G31P and DSS+G31P+LACT groups,while occludin expression was marginally increased when compared with the DSS group.Using immuno-blotting and RT-PCR,the effects of the treatments on AKT and ERK pathways were investigated.Activation of AKT1 and ERK1/2 observed in colitis model and in ex vivo study were reversed by G31P.This reduction had corresponding downstream restriction in the expression of HIFla and Egrl.Contrarily,flow cytometry analysis revealed that G31P increases ROS levels of IPS induced THP-1 monocytes in concentration-wise.Conclusion:This study has demonstrated for the first time that CXCL8 antagonist,G31P,has potent anti-inflammatory activity in vitro,and exhibits therapeutic capability of G31P and/or G31P+LACT against inflammatory bowel disease in vivo.The in vitro reports revealed that the CXCL8 antagonist,G31P,curbs activation of inflammatory cytokines IL-1?,IL-6,CXCL8,and TNF-a;enzymes associated with inflammation;COX-2,MMP2,and MMP-9,and variably influences inflammatory related transcription factors.The AP-1 transcription factor is suppressed by G31P via c-Fos inhibition,but not c-Jun.The in vivo investigations showed that G31P arrests macrophage migration and restricts their colonic infiltration in UC.Additionally,the treatment regiments(G31P and/or G31P+LACT)appreciably mitigates UC associated inflammation,ameliorates colonic fibrosis,and enhances tight junction proteins.G31P,but not the combination of G31P and LACT,significantly reduced T cell related cytokine,IL-17A expression.Mechanistically,G31P marshals its arsenal by inhibiting AKT1 and ERK1/2 signaling pathways,and up-surging ROS signaling.