Role Of Monocyte/macrophage In Ulcerative Colitis Exacerbated By Symptoms Of Anxiety/depression

Objective:Studies demonstrated that the prevalence of anxiety,depression in inflammatory bowel disease(IBD)was evidently higher than the normal population,and anxiety/depression caused a bad effect on IBD.However,studies on psychological disorders in ulcerative colitis in China are few.This paper intends to study the prevalence of ulcerative colitis(UC)patients with symptoms of anxiety/depression,and analyze the impact of symptoms of anxiety/depression on ulcerative colitis.Meanwhile,figure out the role of monocyte and macrophage in ulcerative colitis patients exacerbated by symptoms of anxiety/depression,and the impact of macrophage colony stimulating factor(M-CSF)on the monocyte/macrophage.Materials and Methods:(1)Ulcerative colitis inpatients were included in this cross-sectional study from March 2019 to January 2021 in West China Hospital,Sichuan University.Patients were divided into two groups based on the Hospital Anxiety and Depression Scale(HADS),UC patients with symptoms of anxiety/depression and UC patients without symptoms of anxiety/depression.The prevalence of symptoms of anxiety/depression in UC was calculated and the difference of clinical features,inflammatory cytokines,intestinal barrier and quality of life between the two groups were compared;In addition,risk factors of symptoms of anxiety/depression in ulcerative colitis were analyzed.(2)Peripheral blood samples were collected for the analysis of monocyte immunophenotype,phagocytic function and CD4+T cell differentiation by flow cytometry.Intestinal biopsies were collected for detecting the number of intestinal macrophages by immunofluorescence.The immunophenotype and phagocytosis of intestinal macrophages were tested by flow cytometry.Transmission electron microscopy was used to observe the ultrastructure of intestinal macrophages,and RTPCR for expression of intestinal inflammatory cytokines.The levels of macrophage colony-stimulating factor(M-CSF),granulocyte-macrophage colony-stimulating factor(GM-CSF)and monocyte chemotactic protein(MCP)in each group were compared by Luminex.(3)THP-1 monocyte lines were stimulated with different concentrations of MCSF,and cell viability was detected by CCK-8 assay.Phagocytosis and expression of CD115 were tested by flow cytometry.PMA induced THP-1 monocytes to differentiate into macrophages,and flow cytometry was used to find the change of immunophenotypes and phagocytosis after M-CSF stimulation.The expression of inflammatory cytokines was analyzed by RT-PCR and WB was used for the expression of Akt,Erk1/2,p38 and NF-κB.Results:Part 1: Impact of symptoms of anxiety/depression on ulcerative colitis patients(1)A number of 139 UC patients were included and were evaluated based on HADS.The prevalence of UC patients with symptom of anxiety was 37.41%(51/139),and 32.37%(45/139)for symptom of depression in UC patients.UC patients with symptom of anxiety or depression,or with symptoms of anxiety and depression were defined as UC patients with symptoms of anxiety/depression,including 15 cases with only symptom of anxiety,8 cases with only symptom of depression,and 37 cases with symptoms of anxiety and depression,so the percentage of UC patients with symptoms of anxiety/depression was 43.17%(60/139).(2)Patients were divided into two groups,UC patients with symptoms of anxiety/depression and without symptoms of anxiety/depression.UC patients with symptoms of anxiety/depression were significantly older than UC patients without[48(33,55)vs.32(24,51),P = 0.0012],and the percentage of low education level(primary school and junior school graduation or even below)was evidently higher in UC patients with symptoms of anxiety/depression(43.33% vs.26.58%,P =0.0164).In addition,Mayo score,platelet count,erythrocyte sedimentation rate,and endoscopic score were significantly higher than UC patients without symptoms of anxiety/depression(P < 0.05),and UC patients with symptoms of anxiety/depression more frequently used steroid(58.33% vs.29.11%,P = 0.0005).There were no significant differences in the ratio of male to female,BMI or disease duration between the two groups(P = 0.5114,0.7696 and 0.0969,respectively).(3)The plasm levels of proinflammatory cytokines,including IL-1β、IL-6 and TNF-α were significantly higher in UC patients with symptoms of anxiety/depression(P < 0.05),while anti-inflammatory cytokine TGF-β1 was evidently lower in UC patients with symptoms of anxiety/depression(P < 0.05).(4)Histological score(Geboes score)was significantly higher in UC patients with symptoms of anxiety/depression[5.1(4.2,5.2)vs.4.1(3.1,4.2),P < 0.0001].(5)The intestinal epithelial villus were shorter,more sparse,partial loss and irregular arrangement,in addition,the tight junction was loose,and the levels of claudin and occludin were lower in UC patients with symptoms of anxiety/depression(P < 0.05).(6)IBDQ score was significantly lower(P < 0.05)and scores of FSS,PSQI and COMPASS-31 were evidently higher(P < 0.05)in UC patients with symptoms of anxiety/depression.(7)Age,erythrocyte sedimentation rate,Mayo score,UCEIS score and Geboes score in ulcerative colitis were positively correlated with anxiety symptom score and depression symptom score of HADS,while the level of albumin was negatively correlated.(8)The level of IL-1β in UC patients was positively correlated with anxiety symptom score and depression symptom score of HADS,while TGF-β1 was negatively correlated.(9)IBDQ score in UC patients was negatively correlated with anxiety symptom score and depression symptom score of HADS,while FSS,PSQI and COMPASS-31 were positively correlated.(10)The risk factors of symptoms of anxiety/depression in UC patients were age and disease activity.Part 2: Role of monocyte/macrophage in ulcerative colitis exacerbated by symptoms of anxiety/depression(1)The percentages of intermediate monocyte(CD14++CD16+ monocytes)and nonclassical monocyte(CD14+CD16++ monocytes)were evidently higher in UC patients with symptoms of anxiety/depression[17.29(10.00,21.40)vs.8.73(6.26,11.50),P = 0.0004;7.46 ± 0.76 vs.4.18 ± 0.45,P = 0.0010].(2)Phagocytosis was significantly decreased in UC patients with symptoms of anxiety/depression compared to UC patients without symptoms of anxiety/depression(68.88 ± 2.39 vs.78.79 ± 1.78,P = 0.0027).(3)Monocytes from UC patients with symptoms of anxiety/depression inhibited CD4+ T cells from healthy volunteers polarized to Treg cells[1.37(0.68,2.34)vs.2.40(2.15,4.56),P = 0.0124],but induced CD4+ T cells to differentiate into Th1cells[4.52(3.05,6.37)vs.3.25(2.07,3.77),P = 0.0409].(4)The total number of intestinal mucosal macrophages was increased in UC patients contrasted to healthy controls(P < 0.05).UC patients with symptoms of anxiety/depression had a larger number of CD68+ cells in the intestine mucosal layer compared to UC patients without symptoms of anxiety/depression[43(38,49)vs.29(26,31),P < 0.0001].And the ratio of M1/M2 was evidently higher in UC patients with symptoms of anxiety/depression(2.15 ± 0.18 vs.1.31 ± 0.11,P = 0.0004).(5)Phagocytosis of intestinal lamina propria macrophages in UC patients with symptoms of anxiety/depression had a downward tendency,but the difference showed no statistical significance(29.10 ± 2.70 vs.36.71 ± 2.63,P = 0.1489).(6)The levels of proinflammatory cytokines,IL-6,TNF-α were significantly higher in the intestine of UC patients with symptoms of anxiety/depression(34.21 ±21.91 vs.8.29 ± 5.91,P = 0.0173;23.17 ± 16.94 vs.7.78 ± 6.58,P = 0.0303),and the level of TGF-β1 was lower compared to UC patients without symptoms of anxiety/depression(10.73 ± 9.04 vs.34.24 ± 16.11,P = 0.0173).(7)Levels of M-CSF in the plasm and intestine were significantly increased in UC patients with symptoms of anxiety/depression(P < 0.05).Part 3: Impact of M-CSF on monocyte/macrophage(1)When M-CSF stimulated THP-1 monocyte line for 12 hours,M-CSF with concentrations of 50ng/m L and 100ng/m L could maintain the cell viability(P = 0.0133,0.0094,respectively).M-CSF with a concentration of 100ng/m L could also save the cell viability(P = 0.0339)when stimulated for 24 hours.However,there was no significance among different groups with various concentrations of M-CSF stimulating for 48 hours(P = 0.6775).(2)Compared to no M-CSF stimulated group,phagocytosis in groups of M-CSF stimulation with concentrations of 20ng/m L and 50ng/m L was significantly impaired(P = 0.0441,0.0053,respectively),but there was no significant difference between the concentration of 100ng/m L and blank control(P = 0.6332).(3)In the inflammatory environment,THP-1 macrophages tended to polarize into M1 macrophages in the group of M-CSF and LPS stimulation contrasted to only LPS stimulation(P = 0.0325),and there was no significant difference in the proportion of M2 macrophages between the above two groups(P = 0.1465).(4)Compared to only LPS stimulation group,phagocytosis of THP-1macrophages was evidently inhibited in the group of M-CSF and LPS stimulation(P= 0.0295).(5)M-CSF not only significantly increased the expression of pro-inflammatory cytokine,TNF-α in THP-1 macrophages(P = 0.0300),but also anti-inflammatory cytokine,TGF-β1(P = 0.0088).However,the expressions of TGF-β1 and IL-1β in THP-1 macrophages did not differed significantly(P = 0.0997,P = 0.1666),and the expression of TNF-α was significantly increased(P = 0.0282)in the inflammatory environment.Meanwhile,the activations of Erk1/2 and p38 were significantly increased(P < 0.05).Conclusion:(1)Ulcerative colitis patients with symptoms of anxiety/depression were more severe.(2)Peripheral blood monocyte and intestinal macrophage evidently polarized to proinflammatory subtype in UC patients with symptoms of anxiety/depression,and phagocytosis of monocyte was inhibited,phagocytosis of intestinal macrophages also had a downward tendency.(3)M-CSF induced monocyte/macrophage to proinflammatory subtype,and impaired their phagocytosis,which may offer a new idea for the treatment of ulcerative colitis.