The Role And Potential Mechanism Of IRAK-M During Progression Of Inflammatory Bowel Disease And Inflammation Driven Colorectal Cancer

Background and AimCrohn's disease(CD)and ulcerative colitis(UC)represent the clinical manifestations of inflammatory bowel disease(IBD).In humans,there is a strong association in patients afflicted with IBD to further develop colitis associated cancer(CAC).Gastrointestinal(GI)immune homeostasis is,in part,maintained by pattern recognition receptors(PRRs);including,but not limited to the Toll-like receptors(TLRs).In healthy individuals,these TLR signaling cascades are responsible for rapid activation of innate host defenses and further activation of the adaptive immune response.However,uncontrolled activation of TLR signaling promotes chronic inflammation and is associated with a variety of autoimmune diseases,including IBD.The TLRs that utilize the MyD88 dependent pathway have been reported to signal through a family of Interleukin Receptor Associated Kinases(IRAKs),which contain four known family members being:IRAK-1,IRAK-2,IRAK-M,and IRAK-4.IRAK-1,IRAK-2,and IRAK-4 have been described to play a role in the transduction of downstream signaling from MyD88;conversely,IRAK-M is rather unique and has been shown to be a negative regulator of TLR signaling.Prior studies have evaluated IRAK-M in models of experimental colitis and colitis associated tumorigenesis utilizing IRAK-M-/-mice and shown that these animals are highly sensitive to both inflammation and neoplasia.However,contrary to these prior observations,more recent studies have revealed that IRAK-M-/-mice are actually protected from inflammation driven tumorigenesis.IRAK-M was found to support colorectal cancer progression through the reduction of antimicrobial defenses and the stabilization of STAT-3.Due to these conflicting reports,we sought to better elucidate the contribution of IRAK-M during IBD and colitis associated tumorigenesis.Materials and methodsIn order to assess acute experimental colitis,age(8-10 weeks)and weight(25-30 g)matched WT and IRAK-M-/-mice were given either 3 or 5%DSS dissolved in drinking water available ad libitum for 6 days as previously described.On day 6,mice were withdrawn from DSS and given regular drinking water ad libitum until euthanasia was performed on day 12,In order to evaluate colitis associated colorectal tumorigenesis,age(8-10 weeks)and weight(25-30 g)matched WT and IRAK-M-/-mice were intraperitoneally injected with AOM at a dose of lOmg/kg body weight.A week after AOM injection,the mice were given three cycles of 2%DSS for 5 days followed by 14 days of normal drinking water.While subjected to DSS,body weight,stool consistency,bleeding was measured as part of clinical.After the last water cycle,mice were sacrificed and tissues were harvested for further analysis.WT and IRAK-M-/-mice(8-10 weeks)were sacrificed and bone marrow cells were harvested.Bone marrow neutrophils were purified using 65%percoll gradient and the purity was about 85%-90%confirmed by Ly6G+CD11b+ staining.Purified BM neutrophils were cultured in complete RPMI medium with G-CSF(lOng/ml)or GM-CSF(1ng/ml),5%C02?37? for overnight.On one hand,neutrophils were harvested and were further used to coculture with T cell to analysis neutrophil immunosuppression on T cell proliferation and activation.On the other hand,cultured neutrophils were used to do chemotaxis,Western Blotting,Flow cytometry,and miRNA detection.Results1.IRAK-M-/-mice are protected in DSS induced acute colitis modelWe found that IRAK-M-/-mice are protected in DSS induced acute colitis model.Clinically,the IRAK-M-/-mice demonstrate significant improvements in weight change and clinical parameters associated with disease progression compared to the wild-type(WT)animals.Consistent with our clinical observations,IRAK-M-/-mice also displayed significantly increased colon length and less severe tissue damage as evident by histopathology evaluation of H&E stained colon sections compared to the WT counterparts.Interestingly,when viewing the histopathology of the colon from IRAK-M-/-mice we observed localized and highly structured areas of lymphoid cells throughout the colon.These structures were identified and confirmed to be expanded areas gut associated lymphoid tissue(GALT)by a board certified veterinary pathologist(T.L.).Further,we observed increased levels of IL-6 from IRAK-M-/-colon organ culture supernatant following DSS treatment,which is consistent with the increased GALT.While increased IL-6 is typically associated with detrimental inflammation,our histopathology assessments revealed that the inflammation was highly localized to these areas of GALT,with minimal damage to the epithelial cell barrier.Attenuation of experimental colitis in the IRAK-M-/-Mice is associated with enhanced neutrophil and T-Cell Responses.Under basal conditions,we found the neutrophil count was significantly higher in the blood from naive IRAK-M-/-mice compared to WT animals.Interestingly,following exposure to DSS,the number of neutrophils significantly increased in WT mice;however,the number of neutrophils in the IRAK-M-/-mice maintained elevated levels with or without exposure to DSS.We further measured the levels of CXCR2 and CD14 from both naive animals and mice in the experimental colitis model.Under both naive and DSS treated conditions,we observed increased levels of CXCR2 and CD 14 from IRAK-M-/-Ly6G+/CD11b+leukocytes,suggesting IRAK-M-/-neutrophils are primed prior to tissue insult,which likely improves the efficiency in recruitment to a site of infection,such as the colon when bacterial translocation occurs.Further,we also observed increased numbers of CD4+,CD8+ T-cells and monocytes isolated from the spleen of IRAK-M-/-mice following exposure to DSS.Monocytes isolated from the spleen of IRAK-M-/-mice following DSS exposure displayed increased cell surface expression of IAE and decreased expression of CD62L.2.IRAK-M-/-mice are resistant to AOM+DSS induced colitis associated colorectal cancer progressionIRAK-M supports AOM+DSS induced colitis associated colorectal cancer progression.At the endpoint of AOM/DSS treatment,compared to WT mice,the IRAK-M-/-mice showed both decreased tumor numbers and reduced tumor size.In particular,the average numbers of "micro"(<2mm diameter)and "macro"(?2mm diameter)polyps in WT mice(12±1.57,6 ±0.73)were about 3-4 times higher than that of IRAK-M-/-mice(3.875±0.581,1.5±0.681).Moreover,IRAK-M-/-mice displayed much lower disease scores and reduced weight loss during the course of AOM/DSS treatment,suggesting attenuation of disease progression compared to the WT counterparts.Histologically,compared to IRAK-M-/-mice,WT mice present a significant extent of inflammation throughout of mucosa,alterations of epithelia structure,loss of crypts and enhanced inflammatory cell infiltration.Likewise,Ki67 immunostaining of colon tissue showed that IRAK-M-/-mice displayed a significant reduction in proliferating cell number,compared with WT ones.Attenuation of AOM/DSS induced tumorigenesis in the IRAK-M-/-Mice is associated with enhanced neutrophil and T-Cell Responses.The percentages of neutrophils within spleen were similar among naive WT(2.3±0.2779)and IRAK-M-/-(3.495±0.4523)mice.While,after challenge of AOM/DSS,the percentages of neutrophils significantly increased in WT(13.08±2.113)mice as compared with IRAK-M-/-(5.318±0.7514)mice.Meanwhile,in naive mice,IRAK-M-/-mice had significantly higher amount of CD4 T cells and similar amount of CD8 T cells compared to WT mice.Whereas,at the end of the DSS cycle,IRAK-M-/-mice had significantly higher amount of both CD4 and CD8 T cells with more CD 107a expression,a classical indicator of T cell activation,as compared to WT mice within the spleen.Splenic neutrophils from IRAK-M-/-mice expresscd significantly higher CD80 and lower PD-L1 than WT mice at the end of AOM-DSS cycle,suggesting IRAK-M-/-neutrophils have more potential to remove immunosuppression on T cell and improve T cell anti-tumor response through interaction with T cells.Correlated with elevated T cell populations,we observed IRAK-M-/-mice expressed higher levels of TNF-a,IFN-r,IL-12 which are several cytokines involved in anti-tumor response induced by activation of TLR-family members following AOM-DSS challenge.Despite elevated T cell populations and enhanced T-cell promoting neutrophils in IRAK-M-/-mice,we observed reduced circulating inflammatory cytokine IL-1? and increased anti-inflammatory mediator TGF-? in IRAK-M-/-mice challenged with AOM-DSS as compared to WT mice.IRAK-M mediates the immunosuppression of neutrophil on T cell proliferation and activation via PD-L1/CD80/CD40.We observed GM-CSF primed neutrophils showed typical immunosuppressive phenotype,as reflected with reduced T cell proliferation with the addition of neutrophils.However,compared with WT neutrophils,IRAK-M-/-neutrophils had significantly less immunosuppressive effects on the proliferation of CD4 and CD8 T cells,as shown with increased CFSE negative CD4 and CD8 T cells.Likewise,T cells activation also was enhanced when co-cultured with IRAK-M-/-neutrophils compared with WT neutrophils.As evidence,the expressions of CD62Llow,CD40L on CD4 T cells and Granzyme B,CD107a,IFN-y on CD8 T cells were higher,while,the expressions of PD-1,Foxp3 on CD4 T cells and PD-1 on CD8 T cells were lower when co-cultured with IRAK-M-/-neutrophils compared with WT neutrophils.Consistent with in vivo study,we observed significantly elevated cell-surface level of CD80 and significantly reduced cell-surface level of PD-L1 on GM-CSF primed IRAK-M-/-neutrophils before or after co-culture with T cell.In the presence of anti-CD80 antibody during the co-culture,T cell proliferation co-cultured with IRAK-M-/-neutrophils were further blocked,as well as,lower CD40L,CD62Llow on CD4 T cells and lower CD 107a,CD62Llow on CD8 T cells were observed.In contrast,in the presence of anti-PD-L1 antibody,the suppression of WT neutrophils on T cell proliferation was partially released,as well as,lower PD-1 expressed on both CD4 T and CD8 T cells were observed.We observed that the decreased phosphorylation level of STAT1 and increased phosphorylation level of STAT5 in IRAK-M-/-neutrophils as compared to WT neutrophils,which were consisted with reduced PD-L1 expression and enhanced CD80 expression in IRAK-M-/-neutrophils as shown above.Further,we found that phosphorylation level of STAT3 was dramatically reduced in IRAK-M-/-neutrophils compared to WT neutrophils.Adoptive transfer of IRAK-M-/-neutrophils is sufficient to dampen colitis-associated tumor progression.We observed that mice receiving IRAK-M-/-neutrophils displayed much lower disease scores and reduced weight loss during the course of AOM/DSS treatment,indicating attenuation of disease progression compared to the mice receiving WT neutrophils.In addition,mice transferred with IRAK-M-/-neutrophils had a 2-fold reduction in both micro(5.0±0.8729)and macro(2.2±0.7348)polyps of colon,compared to that(9.8±0.3742;4.25±0.4787)in mice transferred with WT neutrophils.H&E staining revealed reduced inflammation and tumor load in mice transfused with IRAK-M-/-neutrophils.Ki67 immunostaining revealed that the colons from mice transfused with IRAK-M-/-neutrophils had reduced Ki67 positive cells,suggesting a significant reduction in proliferating cell number,compared to mice transfused with WT neutrophils.Mice transfused with IRAK-M-/-neutrophils had more splenic cell counts of CD8 T cells.Furthermore,CD4 and CD8 T cells in mice transfused with IRAK-M-/-neutrophils demonstrated significantly elevated activation status as reflected in the higher expression levels of CD62Llow and CD40L on CD4 T cells,as well as,higher expression levels of CD62Llow and CD 107a on CD8 T cells.In addition to activation,the T cells proliferation in mice transfused with IRAK-M-/-neutrophils also were significantly elevated as shown with higher Ki-67 expression in both CD4 and CD8 T cells.Correlated with elevated T cell population and activation,mice transfused with IRAK-M-/-neutrophils had higher circulating levels of anti-tumor cytokines,such as TNF-a,1L-12,and lower level of inflammatory cytokine IL-1? following AOM-DSS challenge.Conclusion1.Our data strongly suggests that IRAK-M functions to modulate inflammatory signaling pathways and is critical in maintaining immune system homeostasis in the gut.IRAK-M-/-mice are protected from experimental colitis due to efficient recruitment of neutrophils and T-cells following microbial translocation.These data provide a mechanism of acute colitis protection and lend support to the phenotype that GALT in IRAK-M-/-mice contributes to the overall protection observed in the experimental colitis model.2.Our current study reveals that,through IRAK-M deletion,neutrophils can be uniquely programed to serve as a highly effective anti-tumor immune modulator.Several lines of data support this novel conclusion.First,we observed that IRAK-M deficient mice have reduced colon tumor development when subjected to the AOM-DSS challenge.Second,we found that IRAK-M deficient neutrophils are re-programmed to be conducive for T cell proliferation,survival and activation.Third,we demonstrated that the transfusion of IRAK-M deficient neutrophils into WT mice is sufficient to alleviate AOM-DSS induced colon tumor formation.