| Objective:Ulcerative colitis(UC)is a chronic idiopathic inflammatory bowel disease(IBD)that affects the large intestine and exhibits a relapsing and remitting course.This disease is a complex and considerable public health problem.Its incidence and prevalence continue to increase worldwide.What is worse,approximately 15%of the patients do not respond to treatment with immunosuppressant and biological drugs;in such cases,colectomy is necessary.However,the pathogenesis of UC remains unclear.Several pathogenic factors are involved in the pathogenesis of UC,such as dysregulated immune responses,genetic predisposition,epithelial barrier defects,and environmental factors.Dysregulated immune responses,such as the increased activation of peripheral leukocytes(particularly monocytes);infiltration of monocytes/macrophages into the colonic mucosa;and increased expression of pro-inflammatory factors,such as tumor necrosis factor(TNF)-?,interleukin(IL)-6,and IL-1?,play a pivotal role in the occurrence and development of UC.Furthermore,in a mathematical model of colonic inflammation,it was confirmed that macrophages constitute an important target for interventional strategies.Most medicines prescribed for UC therapy could interfere with the activation of monocytes/macrophages,although without the exhibition of specificity.Thus,the data suggest that monocytes/macrophages play a pivotal role in the regulation of the immune response during the progression of UC and serve as an important therapeutic target.The CD30 ligand(CD30L,CD153,gene name:TNFSF8)is a membrane-associated glycoprotein belonging to the TNF superfamily(TNFSF).CD30L is mainly expressed by activated T cells,monocytes/macrophages,B cells,and dendritic cells.In our previous work,Cd30l or Cd30 gene knockout mice were used to investigate the expression of CD30L and CD30 on intestinal mucosal adaptive immune cells and the effect on the development of intestinal inflammation in mice with IBD.It proved that CD30L/CD30signal played an important proinflammatory role in the formation mechanism of IBD intestinal inflammatory lesions.However,there are few reports on the mechanism of CD30L in the innate immune cells for IBD.Therefore,in order to deeply explore the influence of CD30L on innate immunity,mainly monocytes/macrophages,which in turn affects the occurrence and development of IBD.In the present study,peripheral blood samples of patients with UC were collected to investigate the expression and function of CD30L in peripheral blood mononuclear cells(PBMCs)and the relationship between CD30L-positive monocytes and UC.Meanwhile,we used Cd30l gene knockout mice to establish enteritis model,so as to further study the effect of Cd30l gene deletion on the role of monocytes in the occurrence and development of mouse enteritis.It will provide a reference theoretical basis for further in-depth study of the mechanism of CD30L on monocyte subsets in the occurrence and development of IBD.Methods:1.Patients with UC1.1 Select diagnosed patients with UC(n=35):Diagnosis of UC was based on clinical symptoms,laboratory tests,colonoscopy and pathology,and exclude other system diseases.All patients presented with the active phase of the disease.The disease activity of UC was classified as mild(3–5),moderate(6–10),and severe(11–12)according to the Mayo score of Rutgeerts[1]et al.Sex-and age-matched healthy polpys were defined for patients with polyps(n=35).1.2 Peripheral blood mononuclear cell extraction:the peripheral blood mononuclear cells were isolated using Lymphoprep according to the manufacturer's instructions from patients with UC and control group after EDTA-anticoagulated treatment.1.3 GEO dataset was used to analyze the expression levels of TNFSF8 in the colon and peripheral blood mononuclear cells in UC and normal control group.1.4 The expression levels of CD30L protein in peripheral blood mononuclear cells of UC and control group was detected by Western Blotting.1.5 CD30L+monocytes and CD30L+T lymphocytes,respectively,as percentages of the whole population of monocytes and T lymphocytes in peripheral blood,were measured using flow cytometry in patients with UC and normal controls.Meanwhile,the cell number of CD30L+monocytes was also analyzed.1.6 The mRNA levels of a few important pro-inflammatory mediators,including TNFA,IL1B and IFNG,chemokine CCL2,vascular endothelial growth factor-A(VEGFA),and two common anti-inflammatory mediators,IL10 and ARG1,were examined in sorted monocytes from PBMCs by RT-q PCR in patients with UC and controls.Furthermore,plasma levels of TNF-?and IL-1?secreted from monocytes were also measured by Elisa in patients with UC and controls.1.7 The percentage of CD30L+classical monocytes(CLM),intermediate monocytes(INM),and non-classical monocytes(NCM),as well as the number of CD30L+CLM was also measured using flow cytometry in patients with UC and normal controls.1.8 Correlation analysis was used to analyze the relationship between the percentage of CD30L+CLM and Mayo score.2.Animal model2.1 Establish C57BL/6J mice CONTROL and DSS animal models:dynamically compare and observe the changes of weight,DAI and intestinal length during the formation of DSS enteritis in mice.At the same time,the histopathological changes of mice intestinal tract were detected by HE staining technology and the lesions of intestinal mucosa in DSS treated mice were detected by mice colonscopy.2.2 Flow Cytometry was used to detect the changes after enteritis induction in peripheral blood of mice:the percentage and number of CLM and NCM;the percentage and MFI of CD30L in CLM and NCM;also,the percentage and MFI of CCR2 in CLM were detected;the percentage of CD30L+CCR2+CLM and the MFI change of CCR2 in CD30L+CLM.2.3 Flow Cytometry was used to detect the percentage and number of pro-inflammatory monocytes in the lamina propria of intestinal mucosa in CONTROL and DSS animal models;the percentage,number and MFI of CD30L and CCR2 in proinflammatory monocytes in enteritis model were detected;and the percentage and number of CD30L+CCR2+proinflammatory monocytes were detected;also,the MFI of CCR2 in CD30L positive proinflammatory monocytes were measured.2.4 Establishment of DSS animal model by WT and Cd30l-/-mice:dynamic comparative observation of the changes of body weight,DAI and intestinal tube length in mice with Cd30l gene deletion during the process of enteritis formation.Meanwhile,the pathological changes of intestinal tissue was detected by HE staining in mice,and the lesions of intestinal mucosa after DSS enteritis in WT and Cd30l-/-mice were measured by mouse endoscope.2.5 During the formation of DSS enteritis,the percentage and number of CLM and NCM in peripheral blood were detected using flow cytometry in WT and Cd30l-/-mice.The percentage and MFI expression of chemokine receptor CCR2 in CLM after enteritis induction were detected.2.6 During the formation of DSS enteritis,the percentage and number of pro-inflammatory monocytes in the lamina propria of intestinal mucosa were detected using flow cytometry in WT and Cd30l-/-mice,as well as the percentage,number and MFI expression of chemokine receptor CCR2 in proinflammatory monocytes.2.7 The pro-inflammatory monocytes in the lamina propria of intestinal mucosa were sorted using flow cytometry sorting technology,and the mRNA expression changes of chemokine Ccl2 and its receptor Ccr2,pro-inflammatory cytokines Ifng?Tnfa?Il1b and Il6 were detected by Real time-q PCR in WT and Cd30l-/-mice.In addition,the effector cytokines levels of IFN-?,TNF-?,IL-1?and IL-6 secreted by the pro-inflammatory monocytes after cultured in vitro were measured by ELISA in WT and Cd30l-/-mice.Results:1.Patients with UC1.1 Through the analysis of two GEO datasets:the expression of TNFSF8 in the inflammatory sites of the descending colon,sigmoid colon and PBMCs of patients with UC was significantly higher than that in healthy individuals.To verify the findings obtained from the analysis of the GEO datasets,we extracted PBMCs from patients with UC and controls.The results showed that the expression of CD30L increased in the PBMCs obtained from patients with UC.It suggests that CD30L may play a potential important role in the formation of lesions in patients with ulcerative colitis.1.2 PBMCs were extracted from UC and controls.The percentage and number of CD30L+monocytes increased in patients with UC.However,the percentage of CD30L+T lymphocytes was comparable between patients with UC and those with polyps.These results indicate the importance of expression of CD30L in the monocytes of patients with UC.1.3 Peripheral blood monocytes were sorted in UC patients and the control group.Relative to the control group,the mRNA expression levels of pro-inflammatory factor TNFA?IL1B and IFNG,chemokine CCL2,vascular endothelial growth factor-A(VEGFA)were significantly upregulated in the UC group;however,the mRNA expression levels of anti-inflammatory factors,IL10 and ARG1 were comparable.Furthermore,plasma levels of IL-1?and TNF-?increased in patients with UC.These results indicated that a major subpopulation of circulating monocytes showed pro-inflammatory properties in patients with UC.1.4 PBMCs were extracted from UC patients and controls.The percentage and number of CD30L+CLM were higher in patients with UC than in those with polyps using flow cytometry.Regarding the correlation between CD30L+CLM and UC severity,we found that the Mayo score was significantly positively associated with the percentage of CD30L in CLM of patients with UC.Above all,these results suggested that the percentage of CD30L+CLM was positively correlated with the severity of UC.From the above results,we conclude that the pro-inflammatory CLM express high levels of CD30L in the peripheral blood of patients with UC.In order to explore the important role of CD30L-positive CLM in IBD,we used Cd30l gene knockout mice to induce IBD model for further in-depth research.2.Animal model2.1 First,we use WT mice to establish a DSS enteritis model.Compared with the control group,the weight loss rate and DAI score were significantly increased,and the colon length was remarkably shortening in DSS enteritis model.Meanwhile,the pathological histology showed diffuse loss of goblet cells,more mucosal ulcer,structural disorder of crypt,submucosal edema,epithelial hyperplasia in the intestinal mucosa,and the infiltration of immune cells(such as polymorphonuclear leukocytes or plasma cells)in LP and submucosa of DSS enteritis mice.Also,thickening of the colon,changes of the vascular pattern,fibrin visible,granularity of the mucosal surface,and stool consistency in the intestinal mucosa were shown in DSS enteritis mice by endoscopic examination.It proved that the mouse IBD model was successfully established.2.2 The peripheral blood of the mice was extracted.Compared with the normal group,the percentage and number of CLM in the peripheral blood increased significantly after DSS enteritis in WT mice;also,the expression levels of CD30L in CLM were significantly up-regulated,suggesting that CD30L may play a potentially important role in the CLM in peripheral blood in the formation of DSS lesions.We also found that the expression levels of CCR2 were up-regulated in CLM,especially in CD30L-positive CLM.It implied that CD30L may promote the expression of CCR2 on the CLM and the homing of CLM to inflammatory regions of intestinal mucosa in colitis.2.3 We studied the intestinal tract issue in mice.Compared with the normal group,the expression levels of CD30L in the pro-inflammatory monocytes in the lamina propria of the intestinal mucosa increased significantly after induced DSS enteritis in WT mice.It indicated that under inflammation,CD30L played a role in promoting the differentiation of CLM in the peripheral blood to the pro-inflammatory monocytes in the intestinal tract.In addition,we also found that,the expression leves of CCR2 in pro-inflammatory monocytes were up-regulated,especially in CD30L-positive pro-inflammatory monocytes.It is suggested that in the formation of DSS lesions,CD30L plays a role in promoting the homing process of CLM in peripheral blood to the intestinal mucosa under the action of CCR2.2.4 Cd30l gene was knockouted.During DSS enteritis,compared with WT,Cd30l-/-mice had a lower weight loss rate and DAI score,with a significant increase in intestinal tube length.Histopathological analysis showed that the more diffuse loss of goblet cells,serious mucosal ulcers,crypts structural disorders,submucosal edema,epithelial hyperplasia in the intestinal mucosa,and immune cells(such as polymorphonuclear leukocytes or plasma cells)infiltration in the lamina propria/submucosal in WT mice.The more thickening of the colon,changes of the vascular pattern,fibrin visible,granularity of the mucosal surface,and stool consistency in the intestinal mucosa were shown in WT enteritis mice by endoscopic examination.The results showed that the deletion of Cd30l gene leads to a significant decreased susceptibility and enhanced tolerance to DSS enteritis,suggesting that CD30L plays an important role in promoting inflammation in the formation of DSS enteritis.2.5 The deletion of Cd30l gene resulted in a significant decrease in the percentage and number of CLM in peripheral blood.Also,in CLM,the expression levels of CCR2decreased remarkbly.We further explore the changes of pro-inflammatory monocytes in the intestinal tract differentiated from the classical peripheral blood monocytes by Cd30l gene deletion,it found that the deletion of Cd30l gene leads to a significant decrease in the percentage and number of pro-inflammatory monocytes in the lamina propria of the intestinal mucosa.Compared with WT mice,the expression levels of CCR2 in the pro-inflammatory monocytes in the lamina propria of the intestinal mucosa were significantly decreased after induced DSS enteritis in Cd30l-/-mice.Again,it is suggested that,in the formation of DSS lesions,Cd30l gene deletion will inhibit the differentiation of CLM in peripheral blood into the pro-inflammatory monocytes in the intestinal tract.Also,the expression levels of CCR2 on the CLM were reduced,thereby weakening the CLM homing to the inflammatory tissues in the intestine.2.6 Finally,we sorted the pro-inflammatory monocytes in the intestinal tissue of IBD mice.The deletion of Cd30l gene resulted in a significant down-regulation of the mRNA gene expression levels of pro-inflammatory factors Ifng?Tnfa?Il1b and Il6.Meanwhile,the mRNA gene expression levels of the chemokine Ccl2 and its receptor Ccr2 were also significantly down-regulated.We also cultured the sorted pro-inflammatory monocytes from the IBD mice in vitro to detect the changes in the expression levels of inflammatory factors in the supernatant.The results showed that the expression levels of inflammatory factors decreased after the Cd30l gene was deleted.It implies that the deletion of Cd30l gene can reduce the pro-inflammatory effect of monocytes on mice by reducing the pro-inflammatory factors secreted by pro-inflammatory monocytes,thereby reducing the pathological formation of enteritis;CD30L may rely on the CCL2-CCR2 axis to promote the recruitment of circulating immune CLM to the intestine.Conclusion:1.The expression of CD30L in circulating classical monocytes increased in patients with UC and was positively related to the disease severity of UC.2.Cd30l gene can promote the differentiation of CLM in PBMCs into the pro-inflammatory monocytes in the intestinal tract,and upregulate the secretion of pro-inflammatory factors by the pro-inflammatory monocytes,also increasing the sensitivity to DSS enteritis.It plays a pro-inflammatory role in the formation of inflammatory lesions.3.Cd30l gene facilitates the homing of classical monocytes in peripheral blood to the intestinal inflammatory tissues by up-regulating the expression levels of intestinal homing receptor CCR2,so as to participate in the formation of intestinal inflammatory lesions and promote the occurrence and development of IBD. |
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