| Part Ⅰ Fusobacterium nucleatum was positively correlated with clinical activity in inflammatory bowel diseaseObjectiveTo observe the abundance of F.nucleatum in colon tissues of IBD and to explore the correlation between F.nucleatum and clinical activity.Methods1.Formalin-fixed,paraffin-embedded IBD intestinal tissues(60 UC and 41 CD)were obtained from the pathology department.Detecting the abundance of F.nucleatum by FISH in colon tissues of IBD patients.2.The diversity and differences of gut microbiome in intestinal tissues of IBD patients were identified by 16S rDNA high-throughput sequencing technology and assessed whether F.nucleatum was associated with clinical activity.Results1.F.nucleatum is enriched in inflammatory bowel disease colonic tissues.2.We defined a high abundance of F.nucleatum as>20 visualized F.nucleatum per 200× magnification field on average.F.nucleatum was detectable by FISH in 53.5%of colonic tissues from IBD patients.We observed that F.nucleatum was an independent predictor of clinical activity in IBD.In particular,the majority of F.nucleatum-high cases seemed to be refractory phenotypes or with surgical history.There were no significant differences between the F.nucleatum-high group and F.nucleatum-low group with respect to age,location,behavior or perianal disease in UC and CD because of the low statistical power due to the subgroup analysis.3.We compared sequencing data obtained via 16S rDNA sequencing of 6 inflamed tissues from patients with active UC and 5 inflamed tissues from patients with remitted UC.We found that Fusobacterium were enriched in AUC tissues.ConclusionF.nucleatum is enriched in inflammatory bowel disease colonic tissues and correlated with clinical activity.Part Ⅱ The role of F.nucleatum in the pathogenesis of inflammatory bowel disease and intestinal macrophage polarization.ObjectiveTo explore the role of F.nucleatum in the pathogensis of IBD and intestinal macrophage polarization.Methods1.Detecting the abundance of F.nucleatum by fluorescence in situ hybridization(FISH)in colon tissues of IBD patients.We equally dichotomized the cases with detectable F.nucleatum into low versus high and immunofluorescent staining was used to detect the infiltration of immune cells and the distribution of macrophages in inflamed colon tissues.2.we isolated a strain of F.nucleatum from IBD tissues.C57BL/6 mice were randomly divided into 4 groups:PBS group,F.nucleatum group,DSS group and F.nucleatum+DSS group.Mice were pre-treated with F.nucleatum before administration of 3%DSS for 7 days.The clinical signs of colitis,including body weight,stool consistency and rectal bleeding,were monitored daily,and the length and the histology of the colons were examined.3.Immunofluorescent staining was used to detect the distribution of macrophages in colon tissues,flow cytometry was used to detect the number and polarization of macrophages in colonic lamina propria cells,RT-PCR was used to detect the levels of IL-1β、iNOS、TNF-α、IL-10、Arg-1 and CD206 in colon tissues.Results1.We observed a higher number of CD68+macrophages and CD83+dendritic cells in F.nucleatum-high IBD than in F.nucleatum-low IBD,especially macrophages(P<0.001).2.F.nucleatum did not show any signs of weight recovery and had higher clinical scores compared to the PBS group(P>0.05).Upon DSS administration,the F.nucleatum+DSS group exhibited more severe colitis symptoms,including rapid weight loss(P<0.001)and higher disease activity index(P<0.001).And we found distinctly shorter colons in the F.nucleatum+DSS group than in the DSS group(P<0.01).Consistent with these observations,the histological assessment of colons revealed more severe disease,disruption of mucosal structures,and increased inflammatory cell infiltration and revealed a significantly higher histological score(P<0.01)in the F.nucleatum+DSS group.3.The densities of M1 macrophages were significantly higher than those of M2 macrophages in F.nucleatum-high IBD tissues,and the densities of M1 macrophages were increased in F.nucleatum-high IBD tissues compared with F.nucleatum-low IBD tissues(P<0.05).4.CD11c+macrophages had accumulated in the colonic lamina propria in mice treated with F.nucleatum following DSS exposure(P<0.001).5.Immunofluorescence results showed that CD86+cells were highest in the F.nucleatum-fed DSS mice group compared to other groups(P<0.01).Colonic lamina propria cells macrophages were sorted from the inflamed colon,and the resultant macrophage subtypes were analyzed.The percentage of M1(CD11b+CD11c+)macrophages was significantly increased in DSS mice pretreated with F.nucleatum compared with that of mice without F.nucleatum infection(P<0.01),while the percentage of M2(F4/80+CD206+)populations reduced(P<0.05).Conclusion1.F.nucleatum selectively promotes macrophages infiltration in IBD2.F.nucleatum infection exacerbated intestinal inflammation in DSS-induced colitis.3.F.nucleatum promotes the skewing of macrophages towards the M1 subtype.Part Ⅲ The effects of CHOP-mediated endoplasmic reticulum stress in M1 macrophage polarization infected with F.nucleatumObjectiveTo investigate the role of CHOP-mediated endoplasmic reticulum stress in F.nucleatum induced M1 macrophage polarization.Methods1.RT-PCR was used to detect the levels of endoplasmic reticulum stress markers CHOP and Bip in CD11b+CD 11 c+M1 macrophages.2.This experiment established a model of THP-1-deverived macrophages incubated with F.nucleatum in vitro.interfering the expression of CHOP by endoplasmic reticulum stress inhibitors 4-PBA,and then using Western blot to detect endoplasmic reticulum stress related proteins such as CHOP,Bip,p-eIF2α,and immunofluorescence to detect related protein in endoplasmic stress.Immunofluorescent staining was used to analyze the correlation between CD86+CHOP+macrophages and M1 polarization in THP-1-deverived macrophages incubated with F.nucleatum.3.Bone marrow cells were isolated from C57/BL6 mouse in vitro.GM-CSF was induced to culture bone marrow-derived macrophages for 7 day,interfering the expression of CHOP by siRNA.F.nucleatum was added to stimulate their activation to M1 macrophages.Flow cytometry was uesed to detect the polarization level of macrophages in cultured cells(F4/80CD11c,F4/80CD206).RT-PCR was used to detect the levels of IL-1β、NOS、Arg-1 and CD206.4.C57BL/6 mice were randomly divided into 6 groups:PBS group,4-PBA group,F.nucleatum group,DSS group,F.nucleatum+DSS group and F.nucleatum+DSS+4PBA group.Mice were pre-treated with F.nucleatum and endoplasmic reticulum inhibitors 4-PBA before administration of 3%DSS for 7 days.The clinical signs of colitis,including body weight,stool consistency and rectal bleeding,were monitored daily,and the length and the histology of the colons were examined.5.Immunofluorescent staining was used to detect the distribution of macrophages in colon tissues,flow cytometry was used to detect the number and polarization of macrophages in colonic lamina propria cells,RT-PCR was used to detect the levels of IL-1β、IL-6、iNOS、TNF-α、IL-10 and Arg-1 in colon tissues.Results1.M1 and M2 macrophages in the colonic lamina propria were isolated from DSS-and F.nucleatum+DSS-treated mice,RT-PCR confirmed that F.nucleatum infection increased the expression of CHOP and Bip in M1 macrophages and decreased the expression of CHOP and Bip in M2 macrophages(P<0.05).2.The western blot results showed that CHOP,Bip and p-eIF2α were downregulated in THP-1-derived macrophages cocultured with F.nucleatum.These effects were not found in THP-1-derived macrophages incubated with E.coli.And 4-PBA markedly diminished F.nucleatum induced CHOP-mediated ER stress pathway activation in M1 macrophages.The frequency of CD86+CHOP+cells in F.nucleatum-challenged THP1-derived macrophages was significantly higher than that in THP-1-derived macrophages without infection and higher than that in cells pre-treated with 4-PBA before exposure to F.nucleatum(P<0.05).3.F.nucleatum promoted M1 macrophage polarization also measured by flow cytometry in BMDMs expressing siCHOP.The percentage of M1 and M2 macrophage were both significantly decreased in F.nucleatum-treated BMDMs expressing si CHOP(P<0.05).At protein level,F.nucleatum induced increase of CHOP was not detected in BMDMs expressing siCHOP,but in nontargeting siRNAs(NC)cells.Notably,increased CD86 were significantly prevented in F.nucleatum-treated BMDMs expressing siCHOP.At the mRNA level,F.nucleatum-induced inflammatory cytokines,including IL-1β,iNOS,Arg-1 and CD206 were remarkably decreased by siCHOP(P<0.05).4.Compared with mice treated with DSS and F.nucleatum+DSS,mice treated with F.nucleatum+DSS+4-PB A exhibited a slower decline in body weight(P<0.001),a lower DAI(P<0.001),and a lower histological score(P<0.001).F.nucleatum+DSSinduced colon shortening was mitigated in mice administered 4-PBA(P<0.001).H&E staining showed that the glandular structure of colonic tissues in F.nucleatum+DSS+4PBA group was more complete,and the infiltration of submucosal cells significantly decreased.5.Immunofluorescence results of CD86 and CD 206 further confirmed that CHOPmediated ER stress inhibition prevents F.nucleatum-mediated M1 polarization(P<0.0001).Similarly,upon 4-PBA inhibition,M1 markers such as IL-1β,iNOS and TNFα expression were also substantially decreased,while M2 markers such as IL-10,Arg1 and CD206 were increased in the inflamed tissues from F.nucleatum+DSS+4PBA-treated mice than tissues from F.nucleatum+DSS.Conclusion1.F.nucleatum promotes M1 polarization by up-regulating CHOP-mediated endoplasmic reticulum stress pathway.2.Endoplasmic reticulum stress inhibitors 4-PBA prevents F.nucleatum-induced M1 macrophages polarizarion.3.CHOP-mediated endoplasmic reticulum stress inhibition protected mice from DSSor F.nucleatum-induced colitis and downregulated M1 polarization in vivo. |
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