| Background and objectivesIL-22belongs to the IL-10superfamily and has both proinflammatory and anti-inflammatory properties depending on the milieu in which it is expressed. Cigarette smoke (CS) exposure is an accepted risk factor for the development and progression of obstructive airway diseases,such as COPD and asthma,and its harmful effect continues even after smoking cessation,i.e.the persistent inflammatory responses still exist.Although there are a large number of studies about the functions of IL-22in the non-infectious inflammatory pulmonary dieases,such as in the model of bleomycin-induced pulmonary fibrosis,which showed protective function,the role of IL-22in the CS-induced pulmonary inflammation remains unclear Therefore, in our study,we explored the effect of IL-22on pulmonary inflammation after CS exposure using a mouse model.Meanwhile,we observed the cytokine/chemokine profiles which are associated with pulmonary inflammation after smoking cessation(SC).Methods· Male C57B1/6mice (n=6per group) were divided into five groups randomly: control group;CS group; IL-22group;IL-22+CS group and SC group. The CS group was exposed to the smoke of five cigarettes, four times a day with a30min smoke-free interval for3consecutive days,and received a single intraperitoneal injection of isopyknic saline half an hour before the CS exposure; The control group was exposed to filtered air,except CS exposure,other conditions as the CS group;The IL-22group was exposed to filterd air and received a single intraperitoneal injection of rhIL-22(at a dose of100μug/kg body weight) per day, other conditions as the control group;The CS+IL-22group received a single intraperitoneal injection of rhIL-22(at a dose of100μg/kg body weight) half an hour before the CS exposure, other conditions as the CS group;The SC group received the same conditions as the CS group in the first three days,at the beginning of the forth day,mice were exposed to the filtered air,and sacrificed after1week. ·To detect the tissue damping(G),elastance(H) and airway resistance (Rn) of mice by the Flexivent.·To evaluate the lung morphology by HE and Masson staining.·To count the inflammatory cells and classification in BALF.·To analyze the CXCR3ligands,MMPs,IL-17A and TGF-β1in lung tissue at mRNA and protein levels by Real-time PCR and immunohistochemical methods.·To determine the concentrations of CXCR3ligands,IL-22,IL-6,IL-8,TNF-α and TGF-β1in BALF by ELISA.ResultsPart I The effect of IL-22on the pulmonary inflammation in mice induced by cigarette smoke exposure and associated mechanism·Lung function The Rn was significantly increased in the CS,IL-22and IL-22+CS groups when compared with the control group(0.67±0.08cmH2O.s/ml,0.68±0,08cmH2O.s/ml,0.78±0.10cmH2Os/ml vs0.52±0.03cmh2O.s/ml respectively,and P values were less than0.01,0.01and0.001respectively);While the G and H were significantly decreased(G:7.60±0.64cmH2O/ml,6.78±0.89cmH20/ml and5.59±0.57cmH20/ml vs9.31±1.89cmH20/ml,P values were less than0.05,0.01and0.001;H:32.10±2.59cmH2O/ml,28.52±2.22cmH2O/ml and25.66±2.54cmH2O/ml vs46.20±8.11cmH20/ml,P values were all less than0.001).Compared with the CS group,the IL-22+CS group was significantly augmented in the Rn (P<0.05),while decreased in the G and H(P values were less than0.01and0.05).·Lung morphology We observed epithelial damage,inflammatory cell infiltration,squamous cell metaplasia,and collogen deposition in the CS and IL-22+CS groups.According to scoring standard,the histopathological scores in the CS,IL-22and IL-22+CS groups were higher than that of in the control group(4.83±1.47,2.67±0.52and7.00±0.89vs1.33±0.52respectively, and P values were less than0.001,0.05and0.001respectively).Compared with the CS group,the scores in the IL-22+CS group were significantly increased(P<0.01).·Inflammatory cells in BALF Inflammatory cells,including the macroph-ages,neutrophils and lymphocytes,were significantly increased in in the CS,IL-22and IL-22+CS groups when compared with the control group (16.75±0.88*104/ml,13.08±0.82*104/ml and22.04±1.34*104/ml vs6.25±0.97*104/ml respectively,and all P values were less than0.001).Meanwhile, compared between the CS and IL-22+CS groups,the inflammatory cells were further increased in the latter group (P<0.001).·The mRNA expressions of chemokines or cytokines in lung tissue Compared with the control group,the CS,IL-22and IL-22+CS groups were significantly increased in the following chemokines:CXCL9(P values were less than0.01,0.001and0.01respectively);CXCL10(P values were less than0.05,0.05and0.001respectively);While the CXCL11was obviously increased only in the IL-22+CS group(P<0.01),compared with the CS group,the IL-22+CS group was significantly increased in CXCL10and CXCL11mRNA expression(all P values were less than0.01);In the expressions of TGF-β1and IL-17A mRNA,there were significantly increased in the CS and IL-22+CS groups when compared with the control group(TGF-β1:.P values were less than0.01and0.001respectively;IL-17A:all P values were less than0.001), the expression of TGF-β1mRNA in the IL-22+CS group was further increased even when compared with the CS group(P<0.05).· The protein expressions of chemokines or cytokines in lung tissue Compared with the control group,the CS and IL-22+CS groups were significantly increased in the following chemokines or cytokines: CXCL9(3.17±0.41and3.50±0.55vs1.17±0.41respectively,and all P values were less than0.001);CXCL10(2.17±0.41and2.83±0.75vs1.17±0.41resp-ectively,and P values were less than0.01and0.001respectively);CXCL11(3.17±0.41and3.17±0.41vs1.50±0.55respectively,and all P values were less than0.001),compared with the CS group,the IL-22+CS group was significantly increased in CXCL10(P<0.05);TGF-β1(2.67±0.52and3.67±0.52vs1.17±0.41respectively,and all P values were less than0.001),and the IL-22+CS group was increased significantly even when compared with the CS group(.P<0.01).In the expression of IL-17A,the CS and IL-22+CS groups increased significantly when compared with the control group(3.17±0.41and3.33±0.52vs1.67±0.52respectively,and all P values were less than0.001),while no significant difference between those two groups(P<0.05).·The concentrations of chemokines or cytokines in BALF Compared with the control group, the CS and IL-22+CS groups were significantly increased in the following chemokines or cytokines:CXCL9(75.13±8.63pg/ml and85.04±8.32pg/ml vs66.33±5.40pg/ml respectively,and P values were less than0.05and0.001respectively);CXCL10(143.86±5.43pg/ml and152.90±9.34pg/ml vs104.15±4.86pg/ml respectively,and all P values were less than0.001);CXCL11(53.67±5.26pg/ml and56.34±3.47pg/ml vs44.47±4.15pg/ml respectively,and P values were less than0.01and0.001respectively),in addition,the CXCL9and CXCL10in the IL-22+CS group were significantly increased when compared with the CS group(all P values were less than0.05);IL-8(77.05±4.14pg/ml and92.41±5.22pg/ml vs58.39±7.79pg/ml respectively,and all P values were less than0.001),and the IL-22+CS was increased significantly even when compared with the CS group (P<0.001);TGF-β1(173.63±4.53pg/ml and187.29±3.38pg/ml vs127.06±5.32pg/ml,and all P values were less than0.001),and the IL-22+CS group was significantly increased even compared with the CS group(P<0.01);IL-6(109.78±6.69pg/ml and112.91±9.67pg/ml vs87.74±6.96pg/ml respectively,and all P values were less than0.001);TNF-a (407.83±20.69pg/ml and423.54±43.74pg/ml vs343.78±25.95pg/ml,and P values were less than0.05and0.01respectively),while no significant difference between those two groups;IL-22(91.52±5.09pg/ml and95.45±3.75pg/ml vs74.53±0.73pg/ml respectively,and all P values were less than0.001),and the IL-22+CS group was significantly increased even compared with the CS group(P<0.05).Part II The pulmonary inflammatory changes in mice after smoking cessation and the role of CXCR3ligands· Lung fucntion The Rn was significantly increased in the CS and SC groups when compared with the control group(0.67±0.08cmH2O.s/ml and0.65±0.12cmH20.s/ml vs0.52±0.03cmH2O.s/ml respectively,and P values were less than0.01and0.05respectively);While the G and H were significantly decreased(G:7.60±0.64cmH2O/ml and6.09±0.99cmH2O/m cmH2O/ml vs9.31±1.89cmH2O/ml,P values were less than0.05and0.001;H:32.10±2.59cmH2O/ml and25.59±5.17cmH20/ml vs46.20±8.11cmH20/ml,P values were all less than0.001).Compared with the CS group,the SC group was significantly decreased in the G and H(P values were all less than0.05). while no significant difference in the Rn (P>0.05).·Lung morphology We observed epithelial damage,inflammatory cell infiltration,squamous cell metaplasia,and collogen deposition in the CS and SC groups.According to scoring standard,the histopathological scores in the CS and SC groups were higher than that of in the control group(4.83±1.47and4.17±0.75vs1.33±0.52respectively,and all P values were less than0.001),there were no significant difference between those two groups (.P>0.05).·Inflammatory cells in BALF After CS exposure,the inflammatory cells,including the macrophages,neutrophils and lymphocytes,were signific-antly increased in in the CS group when compared with the control group(16.75±0.88*104/ml vs6.25±0.97*104/ml,P<0.001).After smoking cessation,the inflammatory cells in the SC group decreased significantly when compared with the CS group(11.54±0.68vs16.75±0.88*104/ml*104/ml,P<0.001),but it still increased significantly when compared with the control group.·The mRNA expressions of chemokines or cytokines in lung tissue Compared with the control group,the CS and SC groups were significantly increased in the following chemokines:CXCL9(P values were less than0.01and0.001respectively);CXCL10(P values were less than0.05and0.001respectively),in addition,the SC group was significantly increased in the CXCL9and CXCL10mRNA expressions when compared with the CS group(P values were less than0.05and0.01). Compared with the control group,the CS and SC groups were significantly increased in the following MMPs mRNA:MMP9CP values were less than0.001and0.05respectively); MMP12(P values were less than0.01and0.001respectively);While the MMP2mRNA was significantly increased only in the CS group(P<0.05),in addtion,the MMP12in the SC group increased significantly even when compared with the CS group(P<0.001);In the expressions of TGF-|31mRNA,there. was significantly increased only in the CS group when compared with the control group(P<0.01).·The protein expressions of chemokines or cytokines in lung tissue Compared with the control group,the CS and SC groups were significantly increased in the following chemokines or cytokines:CXCL9(3.17±0.41and2.67±0.52vs1.17±0.41respectively,and all P values were less than0.001); CXCL10(2.17±0.41and2.00±0.63vs1.17±0.41respectively,and P values were less than0.01and0.05respectively);CXCL11(3.17±0.41and2.83±0.41vs1.50±0.55respectively,and all P values were less than0.001); TGF-β1(2.67±0.52and2.17±0.75vs1.17±0.41respectively,and P values were less than0.001and0.01respectively);MMP2(3.17±0.41and2.33±0.52vs1.17±0.41respectively,and P values were less than0.001);MMP9(3.17±0.41and2.50±0.55vs1.50±0.55respectively,and P values were less than0.001and0.01respectively);MMP12(2.83±0.41and2.67±0.52vs1.17±0.41respectively,and P values were less than0.001).Among those chemo-kines or cytokines,only MMP2and MMP9were increased significantly in the CS group when compared with the SC group(P values were less than0.01and0.05respectively).·The concentrations of chemokines or cytokines in BALF Compared with the control group, the CS and SC groups were significantly increased in the following chemokines or cytokines:CXCL9(75.13±8.63pg/ml and79.45±8.72pg/ml vs66.33±5.40pg/ml respectively,and P values were less than0.05and0.01respectively);CXCL10(143.86±5.43pg/ml and132.99±6.65pg/ml vs104.15±4.86pg/ml respectively,and all P values were less than0.001);CXCL11(53.67±5.26pg/ml and53.82±7.34pg/ml vs44.47±4.15pg/ml respectively,and all P values were less than0.01),in addition,the CXCL10in the CS group was significantly increased when compared with the SC group (P<0.01);IL-8(77.05±4.14pg/ml and77.48±6.64pg/ml vs58.38±7.79pg/ml respectively,and all P values were less than0.001);TGF-β1(173.63±4.53pg/ml and161.21±8.63pg/ml vs127.06±5.32pg/ml,and all P values were less than0.001).The following cytokines were only significantly increased in the CS group:IL-6(109.78±6.96pg/ml vs87.74±6.96pg/ml,P<0.001);TNF-a(407.83±20.69pg/ml vs343.78±25.95pg/ml,P<0.05). ConclusionsPart I The effect of IL-22on the pulmonary inflammation in mice induce by cigarette smoke exposure and associated mechanismIL-22exacerbates the aforementioned inflammatory responses after CS exposure,probably associated with some certain chemokines or cytokines, such as CXCR3ligands and IL-17A.Part II The pulmonary inflammatory changes in mice after smoking cessation and the role of CXCR3ligandsCS exposure triggers inflammatory cell flux and accumulation in the lung parenchyma and BALF.As a consequence,the inflammatary cytokines increase dramatically.Among the cytokines/chemokines accumulated after CS,the CXCR3ligands may play a critical role in mediating the persistent inflammatory responses after smoking cessation. |
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