Cat L促进猪肺炎支原体感染后SIgA免疫应答的作用机制

Mycoplasmal pneumonia of swine is one of the important global epidemic diseases with high morbidity, which causes huge economic losses, since it often results in chronic and long-term inflammation. And, it is impossible to clear the pathogenic bacteria -M.hyopneumoniae(Mhp) in lung completely, since few antibiotics can penetrate the respiratory mucosal layer to reach the mucosal surface where the pathogen stays. Immunization with various vaccines didn't provide ideal protections against Mhp infections, although they may significantly reduce lesions caused by challenge with Mhp.According to the reports published recently, SIgA induced by mucosal immune responds play an important role in providing protections against Mhp infections. Moreover, we also found that Cat L associates with SIgA responds and the protection in mice infected by Mhp with the aid of gene kock-out techniques. In order to prove that Cat L can also promote the secretion of SIgA in airway and provide protection for pigs, and uncover the concrete mechanisms, we plan to set up a model of mycoplasmal pneumonia in swine, and try to find the relationships between SIgA and Cat L. At the same time, we plan to set up an induction model of SIgA in vitro, so we can find whether Cat L gene-silencing disturb the secretion of SIgA by B lymphocytes. If the above tests show positive results, We will go further study and try to find if Cat L can promote the phagocytosic function by the inhibition of the activity of Cat L,and the antigeic peptide loading ability of the MHC II molecular of antigen presentation cells(APCs) by the digestion of MHC-DR3-αβIi complex with recombinant Cat L, by which we may interpret the concrete mechanism about how Cat L promotes the responds of SIgA in pigs following infection with Mhp. Finally, we would like to try to increase the protection rates for the pigs infected by Mhp with the use of recombinant Cat L, so as we can find a more effective method for the control of the infections of Mhp.

猪肺炎支原体(Mhp)是猪的重要病原之一，流行广泛，感染率高，而抗生素难以到达呼吸道粘膜表面对其进行清除。有研究表明，Mhp感染后粘膜免疫分泌的SIgA为猪提供了主要的抗感染保护。我们发现，CatL基因敲除小鼠感染Mhp后SIgA分泌量明显降低，发病率显著增加。由此我们设想，猪体内CatL也能促进呼吸道粘膜SIgA免疫应答并为猪提供抵抗Mhp感染的保护作用。为此，本项目拟通过建立猪感染Mhp模型来研究CatL与SIgA、Mhp菌数和保护力的相关性，通过建立SIgA诱导细胞模型来研究CatL基因沉默对B细胞分泌SIgA的影响，通过抑制CatL活性来检测其对抗原递呈细胞吞噬能力的影响，以及重组CatL消化活性对MHC II分子荷肽能力的影响，以期证明CatL能够促进Mhp感染后SIgA免疫应答并揭示其具体机制。最后，拟用重组CatL提升对Mhp感染猪的保护力，期望找到控制Mhp感染的有效途径。

猪肺炎支原体（Mhp）是猪的重要病原之一，流行广泛，感染率高，而抗生素难以到达呼吸道黏膜表面对其进行清除。有研究表明，Mhp感染后粘膜免疫分泌的SIgA为猪提供了主要的抗感染保护。我们利用Mhp活疫苗RM48株对猪进行气管内和鼻腔注射攻毒，成功建立了Mhp感染猪的模型。用ELISA方法检测外周血血清及鼻拭子中IgA的含量明显上升，免疫组化检测鼻黏膜，气管，肺门淋巴结，肺脏组织中 IgA+ B 细胞显著增多，提示IgA在Mhp感染中提供了重要的免疫保护作用，免疫组化方法分别检测Mhp感染猪的肺脏和肺门淋巴结中 IgA+ B 细胞与cd11c+DC，发现两种细胞可以共定位，提示cd11c+DC在Mhp感染引起IgA型粘膜免疫中发挥着重要的作用，分别分离小鼠骨髓，脾脏与肺脏来源的cd11c+DC以及肺脏来源的cd64+巨噬细胞（LMφ）与脾脏来源的cd19+ B细胞分别共培养，用Mhp全菌裂解液分别刺激四种细胞模型，发现Mhp全菌裂解液能使骨髓与肺脏来源的cd11c+DC+cd19+ B细胞分泌IgA，其中肺脏来源的cd11c+DC（LDC）+cd19+ B细胞分泌IgA的量最多，而Mhp全菌裂解液刺激脾脏来源的cd11c+DC+cd19+ B细胞模型及肺脏来源的cd64+ LMφ +cd19+ B细胞模型分泌IgA的量较少，LDC+cd19+ B细胞模型随着DC的比例与Mhp全菌裂解液浓度的增加，IgA的量逐渐增加。分别用TLR2，TLR4，TLR7/9，TLR8的抑制剂作用于LDC+cd19+ B细胞模型，发现在抑制了TLR2，TLR4后Mhp全菌裂解液不能使细胞模型产生IgA，而TLR7/9，TLR8的抑制剂则对Mhp全菌裂解液刺激细胞模型产生IgA无明显影响，说明Mhp全菌裂解液通过TLR2，TLR4刺激LDC+cd19+ B细胞模型产生IgA。我们的研究表明，DC在Mhp感染猪引起IgA型粘膜免疫中发挥着重要的作用，Mhp全菌裂解液可以通过TLR2，TLR4刺激小鼠LDC+cd19+ B细胞模型产生IgA。