| ObjectiveTo establish sarcoidosis granuloma C57BL/6mouse model by mycobacteriumtuberculosis superoxide dismutase A(SodA) polypeptide. Observing the variation ofTh17/Treg cells differentiation and relative cytokine levels in the lung of sarcoidosisgranuloma mouse model. Analyzing the relationship between Th17/Treg cellsdifferentiation and cytokines and further exploring the immunological pathogenesis ofsarcoidosis.Methods1. Establishment and identification of sarcoidosis granuloma C57BL/6mousemodel.Twenty-one female C57BL/6mice were randomly divided into3groups:At the1stday,the experimental group A were sensitized by subcutaneous injection of SodA(50μg)incorporated into incomplete freund’s adjuvant(IFA)(0.25ml). Group B were sensitized bysubcutaneous injection of PBS (0.25ml) incorporated into IFA(0.25ml).Group C weresensitized by subcutaneous injection of PBS0.5ml.On the14thdays,Group A werechallenged by tail vein injection of6000grains of sepharose4B beads(in0.5ml PBS)covalently coupled to50μg SodA.Group B were challenged by tail vein injection of6000grains of sepharose4B beads(in0.5ml PBS). Group C were challenged by tail veininjection of PBS0.5ml. All the mice were euthanized by cervical dislocation on the22ndday.Gross changes were observed in the lung and peripheral lymph nodes. Histologicand morphometric changes in lung tissue and lymph nodes were examined by HE stain tofind whether there were sarcoidosis granuloma formation. Immunohistochemistry ofgranuloma positive section was performed to identify the expression of macrophagesspecific antigen Mac-2and lymphocyte antigen CD4+T. Bronchoalveolar lavage was performed to examine the cell count percentage of macrophage, lymphocyte and neutrophilin bronchoalveolar lavage fluid (BALF).BALF CD4+/CD8+ratio was analyzed by flowcytometry. Typical cytokine of Th1such as IFN-γ,IL-12and cytokine of Th2such as IL–4in lung homogenate supernatant were measured using the flowcytomix system.2. Variation of Th17/Treg differentiation and relative cytokine levels in thelung of Sarcoidosis mouse model.Q-PCR was performed to examine mRNA expression of Th17specific transcriptionfactor:orphan nuclear receptor γ t(RORγt) and Treg specific transcriptionfactor:Forkhead/winged helix transcription factor（Foxp3) in mice lung.Western Blot wasperfomed to analyze the protein synthesis of RORγt and Foxp3. Th17/Treg relativecytokines such as TGF-β,IL-6and IL-17A in lung homogenate supernatant were measuredusing the flowcytomix system. The relationgship between Th17/Treg differentiation andthese cytokines was analyzed.Results1. The hilum pulmonis and peripheral lymph nodes(neck, armpit, groin) enlargedobviously in the mice of group A. No obvious anomalies were found in the pulmonary andperipheral lymph nodes in the mice of group B and C.2. Lymphocytes infiltration around bronchi and non-caseous necrotizing granulomawere found in the mice lung of group A by HE stain. The center of granuloma wascomposed of epithelioid cells and sometimes multinuclear giant cells were visible. Thegranuloma was surrounded by lymphocytes. Similar kind of quasi-circular granuloma werefound in mice lymph nodes of group A. Sepharose beads surrounded by a thin layer ofepithelioid cells of foreign body granuloma were found in the mice lung of group B andwithout sarcoidosis granuloma. There were no anomalies in the lymph nodes of group Band C and lung of the group C.3. Macrophages specific antigen Mac-2was positively expressed in granulomacenter and CD4was positively expressed at the circum by immunohistochemistry.4. The percentages of lymphocyte and neutrophil in BALF were significantlyhigher in group A than that of group B and C.(all P<0.01). The percentage of macrophagewas significantly lower in group A than that of group B and C(all P<0.01). The percentageof lymphocyte in group B is higher and macrophage percentage is lower than that of group C(all P<0.01).BALF-CD4+/CD8+T lymphocytes ratio in group A was significantly higherthan that of group B(P=0.0019)5. IL-12,IFN-γ levels in the lung homogenate supernatant in group A were bothsignificantly higher than that of group B and C.(all P<0.01).No statistical differences ofIL-12,IFN-γ levels were found between group B and C(P＞0.05). No statistical differencesof IL-4levels was found among three groups (P>0.05).6.Both of RORγt mRNA and protein synthesis in the lung of group A weresignificantly higher than in group B and C.(all P<0.05).No statistical differences of RORγtmRNA and protein synthesis were found between group B and C(P＞0.05).Foxp3mRNAexpression in the lung of group A were lower than in group B and C(all P<0.05) and nostatistical differences between group B and C. No statistical differences of Foxp3proteinsynthesis was found among three groups(all P>0.05).7.IL-6,IL-17A levels in the lung homogenate supernatant of group A were bothsignificantly higher than in group B and C.(all P<0.01).No statistical differences ofIL-6,IL-17A levels were found between group B and C(P＞0.05). No statistical differencesof TGF–β levels was found among three groups (P>0.05).8.RORγtmRNA expression in mice lung was positively correlated to IL-6andIL-17A level significantly (r=0.701,P=0.000, r=0.631,P=0.002).RORγt protein synthesislevel was positively correlated to IL-6and IL-17A levels significantly (r=0.705,P=0.000,r=0.546,P=0.010).Foxp3mRNA expression in the lung of mice was negatively correlatedto IL-6and IL-17A levels significantly (r=-0.444，P=0.044, r=-0.471,P=0.031).Conclusions1.Stimulation of SodA could induce the formation of lymph nodes and pulmonarynon-caseous necrotizing granuloma in C57BL/6mice,which was characterized by Th1cells reaction. The features of this granuloma were similar to human sarcoidosispathologically and immunologically.This model could be used as the animal model forstudying sarcoidosis immune mechanism.2. SodA could induce obvious Th17/Treg imbalance accompanied by upregulationof relative cytokines such as IL-6and IL-17A. SodA induced Th17/Treg imbalance ingranulouma animal model may be related to the formation of sarcoidosis granulouma. |
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