The Role And Potential Mechanism Of Tollip In Progress Of Active Ulcerative Colitis

Background and AimThe innate immune responses play an equally significant,even more primary character compared with adaptive immune responses in IBD initiation and progression.The host’s natural immunity is started through TLRs pathway to clear the invaded pathogens by recognizing the microbial conserved construction.So,Initiation of TLR signaling is tightly regulated because prolonged and excessive activation of TLRs can lead to uncontrolled inflammation detrimental to the host.The Toll-interacting protein(Tollip)is a critical negative regulator of Toll-like receptor(TLR)-mediated innate immune responses.Some studies revealed that low expression of TLRs and high expression of Tollip existed in the intestinal epithelial cells,which is helpful to maintain tolerance to bacteria products.As well,recent studies have revealed that the reduced expression of Tollip can lead to higher expression of TLRs and improve the susceptibility of TLRs,which can initiate the TLRs mediated innate immunity responses and promote the inflammation occurred in the intestine.Herein,we suspected that the downregulated Tollip expression or upregulated TLRs may provide a necessary background for the initiation and progression of IBD.There was one published paper which indicated Tollip-/-mice are more susceptible to chronic and acute colitis by breeding Tollip-/-mice on IL10-/background.This animal study is performed by breeding Tollip-/-mice on IL10-/background,and also,they did not explore the potential mechanism.While,there is still no data showing the role and potential mechanism of Tollip in the progress on DSS-induced acute UC by breeding Tollip-/-mice on wild type background.So,we thought using Tollip-/-mice is more direct and proper to explore the role and potential mechanism of Tollip in progress of UC.As we all known,neutrophils,the most abundant circulating leukocyte,act as the first line of innate immune defense.Activated neutrophils migrate to the site of inflammation and control microbes by phagocytosis,secretion of antimicrobials and formation of neutrophil extracellular traps(NET).A.Bhattacharya showed,for the first time,that autophagy mediates degranulation from neutrophils by regulating fusion of granules with phagolysosomes and that autophagy deficiency leads to global reduction in neutrophil-mediated inflammation and autoimmunity.A previous study suggested Tollip may be involved in autophagy.So.the potential influence of Tollip on neutrophil immunological activity still remains unknown.So.This study aimed to explore the role and potential mechanism of Tollip in progress of UC through animal study,neutrophil purify and culture,chemotaxis.as well as,collection samples of peripheral blood and intestine tissue,hopefully provide one more target for treatment of UC.Materials and methodsInduction of acute colitisWild type(WT)C57BL/6 mice were purchased from the Charles River laboratory.Tollip-/-mice on C57BL/6 background were provided by Dr.Jürg Tschopp in the University of Lausanne at Switzerland.All mice were housed under specific pathogen-free conditions,bred and maintained in the animal facility at Virginia Tech with the approved Animal Care and Use Committee protocol.Male mice with body weight:25±1g and 7-9 weeks of age were used in all experiments.WT or Tollip-/-mice were randomly separated to DSS group and control group:Mice of DSS group were given 3%(w/v)DSS(MW 47.000;MP Biomedicals)in drinking water ad libitum for 6 days followed by normal drinking for another 6 days:control group were given regular drinking in the whole experimental process.Mice were sacrificed at day 6 and day 12 during the experiment.TDisease activityThe disease activity of colitis was performed by clinical score as shown in Table.1.Weight loss,rectal bleeding and stool consistency were assessed,then averaged every day during the whole animal experimental process.In addition,colon lengths were measured for each animal at the completion of each study.PathologyColonic tissues were embedded in paraffin and stained with H&E.Histological severity of acute colitis in DSS-treated mice was determined using combined scores as reported:each sample was graded from 0 to 5 according to the score shown in Table.2.All histopathological evaluations were done in a blinded fashion by an independent pathologist.Colon tissue culture and proinflammatory mediator assessmentsColons were washed with PBS containing penicillin/streptomycin followed the distal-most 3 cm were isolated and further cut intol-2cm sections.Colon sections were covered with RPMI media,including 1%FBSand 1%penicillin/streptomycin,and incubated overnight.Cell-free supernatants were harvested for further ELISA assay.ELISA of CytokinesCollected plasma and the supernatants of colon tissue culture were used for the measurement of TNF-α,IL1-β.IL-6 and IL-10 levels by enzyme-linked immunosorbent assay(ELISA)using ELISA Kit from eBioscience respectively.Myeloperoxidase(MPO)levels of colon culture and neutrophils culture medium were assessed using mouse MPO Elisa Kit from R&D system.Chemotaxis AssaysChemotaxis of neutrophils was measured with 48-well micro-chambers and polycarbonate filters(5μm poresize)(NeuroProbe.Cabin John,MD).The results were expressed as the Mean± SEM of the chemotaxis index(CI),representing the fold increase in the number of migrated cells in response to chemo-attractants over spontaneous cell migration(to control medium)as previously mentioned.Purification and culture for bone marrow-derived neutrophilNeutrophils from bone marrow were isolated from the tibias and femurs of WT and Tollip-/-mice,and cultured with completed RPMI(10%FBS,1%penicillin/streptomycin.1%Glycine)including long/ml GM-CSFin untreated tissue culture dishes for overnight.For purification.62.5%percoll was used to get neutrophil pellet.Cells were suspended in PBS for further experiment.ImmunoblottingCells were washed with PBS and harvested in a l×SDS lysis buffer containing protease inhibitor cocktail and subjected to SDS-PAGE.The protein bands were transferred to an immunoblot PVDF membrane(BioRad).Western analyses were performed with specified antibodies as shown in reagents above.Measurement for neutrophil extracellular traps(NET)by FACSNeutrophils were harvested from bone marrow,cultured,and treated with LPS overnight.For assay of NET formation by FACS as reported.Cells were fixed and permeabilized(BD PhosflowTM buffer,BD Biosciences.San Jose,CA),then labeled with anti-citrullinated Histone H4 antibody(Millipore,Billerica,MA),anti-Ly6G and anti-CD11b antibodies(BioLegend.SanDiego,CA).The samples were then analyzed by FACSCanto Ⅱ(BD Biosciences).The data were processed by Flow Jo(Tree Star,Ashland,OR).StatisticsResults were expressed as Mean ± SEM.Statistical significance between groups were determined using one-way analysis of variance(ANOVA)with Tukey’s multiple comparison test.Differencesbetween two groups were evaluated using an unpaired two-tailedStudent’s t test.P values<or=0.05 were considered statistically significant.GraphPad Prism statistical software was used for analysis.ResultsTollip deficiency increased colonic mucosal damage in DSS induced acute colitis Since Tollip deficiency dose not interfere with gut mucosal homeostasis under steady-state condition,we at first examined whether Tollip contributes to colonic mucosal inflammation.We found that in acute model of colitis,Tollip-deficient mice displayed a quick and significant decrease in weight loss,as well as a higher mortality compared with WT.Tollip-deficient mice presented significant clinical features associated with gastrointestinal disease,which was in agreement with the weight change and mortality data.Also,the colons of Tollip-deficient mice were significantly shorter than those of their WT littermates.Histological analysis revealed that the HAI of colon in Tollip-deficient mice is low at day 6.and high at day 12 as compared to WT mice.Taken together,these data indicated a stronger protective role of Tollip in acute ulcerative colitis and Tollip deficiency increases the susceptibility to DSS-induced injury.Tollip deficiency reduced recruitment of neutrophils and increased migration of monocytes to inflamed colon mucosa in DSS induced acute colitisThe phenomenon that Tollip deficiency increases the susceptibility to DSS-induced injury promoted us to investigate the role of Tollip in host defense to microbiota in the large intestine.In Tollip-deficient mice,the number of neutrophils in the damaged colon mucosa was dramatically decreased at days 6 and 12 as compared to WT mice with up-regulation of Fpr2 expression.However,the frequency of neutrophils in the blood and MPO levels in the total colon,were increased at days 6 and 12 in Tollip-deficient mice significantly as compared to WT mice,suggesting that the Tollip-/-neutrophils were retention of the blood and derma.and unable to migrate the damaged colon mucosa.Interestingly,the frequency of blood monocytes(CD11b+Ly6C+Ly6G-cells)was increased at days 6 after fed with DSS in Tollip-/-mice significantly as compared to WT mice although the frequency of na(?)ve blood Tollip-/-monocytes was low significantly as compared to WT micewith up-regulation of CCR5 expression.The macrophages(MOMA-2+cells)persisted in the damaged colon mucosa in Tollip-/-mice at day 12 after DSS treatment,indicating that Tollip deficiency delays the recovery from acute colitis.Tollip deficiency increased systemic inflammatory responses in DSS induced acute colitisWe further investigated the systemic and local inflammatory responses and found that the levels of plasma TNF-α,IL-1β at day 6 and 12,IL-6 at day 12,IL-10 at day 6 were increased in Tollip-deficient mice significantly as compared to WT mice.The high levels of TNF-α,IL-1β and IL-10 were still detected in the supernatants of colon tissue culture in Tollip-/-mice significantly as compared to WT mice.Histological analysis revealed that the most of hepatocytes displayed degeneration or necrosis and a few inflammatory cells infiltrating into the location of damaged hepatocytes in the liver of Tollip-/-mice.In contrast,in WT mice the most of hepatocytes were intact,infiltrated inflammatory cells aggregated in a mass were often observed.These data suggested that Tollip deficiency reduced host defense to gut microbiota invasion.Tollip deficiency reduced Fpr2 expression in neutrophils via down-regulation of LPS-induced signalingSince the recruitment of neutrophils to the damaged colon mucosa sites plays a key role for the development of inflammatory process and Fpr2 deficiency fails to aggregation for neutrophils in the damaged tissue sites,here we focused the influence of Tollip deficiency on the function of neutrophils.Bacterial product LPS up-regulation of Fpr2 expression 4,we investigated the influence of Tollip deficiency on LPS induced signaling in neutrophils and found that LPS increased neutrophil migration in response to Fpr2 ligand fMLF with up-regulation of Fpr2 expression in WT mice.In contrast.LPS induced low cell migration in response to fMLF and Fpr2 expression in Tollip-/-neutrophils significantly as compared to WT neutrophils.It was associated with low levels of p-CREB,p-AKT(S-473)and LAMP-2a expression induced by LPS in Tollip-/-neutrophils.Tollip deficiency reduced the capacity of neutrophils to kill bacteria via down-regulation of Neutrophil Extracellular Traps(NETs)We further found that Tollip deficiency diminished the formation of NETs by neutrophils induced by LPS and reduced the capacity of bacterial killing significantly as compared to WT mice.Put together.Tollip plays an important role to sustain the function of neutrophils in defense to gut microbiota invasion during acute colitis.ConclusionTollip-/-mice display serious rectal bleeding.quicker weight loss,higher mortality,and shorter colon length significantly as compared to WT mice.Histological analysis showed that histological activity index(HAI)was decreased at day 6 and increased at day 12 in Tollip-/-mice significantly as compared to WT mice.Further studies showed that bacterial colonies in blood and PMN infiltration in the liver,the levels of plasma-derived and colon-derived IL-6.IL-10,IL-1β and TNFα were significantly increased as compared to WT mice.The proportion of neutrophils in blood was increased,meanwhile reduced in colon tissues significantly as compared to WT mice.The mechanism studies showed that Tollip deficiency resulted in down-regulation of Fpr2 expression,thereby reduced the recruitment of neutrophils to bacteria-derived Fpr2 ligand fMLF via down-regulation of p-CREB,p-AKT,and p-MSK1 activity.Overall,Tollip plays a key role in the defense to flora-derived damageon the DSS-induced acute colitis.Tollip deficiency results in reduced recruitment of neutrophils to the colon via down-regulation of Fpr2 expression,thereby increased bacteremia,invasion of liver,and systemic inflammatory responses.