The Role And Mechanism Of Cbl-b/c-Cbl In Regulation Of Inflammation In Alveolar Macrophages

As we all known,E3 ubiquitin ligase plays an important role in innate immunity and adaptive immune regulation.Cbl protein family is a kind of E3 ubiquitin ligase with RING-finger domain.They are highly conservative in many species.All of the Cbl family of proteins mainly constituted by the tyrosine kinase binding domain(PTB),the structure of the RING finger domain(RF),a proline-rich region(PR)and a connecting area.There are three main members in mammals: c-Cbl,Cbl-b and Cbl-c,especially c-Cbl and Cbl-b are principally expressed in the hematopoietic system.So far a lot of evidence has been proved that the two molecules play an important role in lymphocyte development and activation,furthermore signaling pathways that associated with Cbl also play an enormous role in cell proliferation,differentiation and cell morphology.Macrophages are the most malleable immune cells in the hematopoietic system,and they exist in all tissues with extremely strong functional diversity.They play important roles in body development,self-stabilization,damage repair and immune response.Through the experiment of bone marrow chimeric research,the scientists found that the recruiting ability was enhaced in Cbl-b deficency peritoneal macrophages after injection of thioglycolic acid salt compared with WT peritoneal macrophages;Studies have also shown that ablation of Cbl-b can lead to infiltration and activation of macrophages in adipose tissue.Moreover,the c-Cbl deleted macrophages can form dense colonies,and their proliferation rate is faster than WT macrophages clones at the certain CSF-1 concentration.The migration of macrophages is regulated by Vav1 activation,and Tomoki Abe's group have demonstrated that ablation of Cbl-b can promote the phosphorylation of Vav1 in mouse peritoneal macrophages.These studies suggest that Cbl-b and c-Cbl may play a role in the regulation of macrophage inflammation.In order to explore whether the Cbl-b and c-Cbl play a role in regulating macrophage function,we established Cbl-b and c-Cbl conditional double knockout mouse model(dKO).Interestingly,two weeks old dKO mice began to grow slowly,and all dKO mice died around five weeks.H&E staining showed that the lung structure was fateful damaged in dKO mice,accompanied by a large number of inflammatory cell invasion.Sirius red staining indicated dKO mice had idiopathic pulmonary fibrosis like symptom.Furthermore,FACS results demonstrated that those inflammation cells were mainly activated alveolar macrophages,and this alveolar macrophages showed a reduction of apoptosis,produced more inflammation related cytokines,such as IL-1?,TGF-? and IL-4,but lower expression of IL-12,IL-6 and IL-10.To further explore how Cbl-b and c-Cbl regulate the inflammation of alveolar macrophages and finally destroyed the lung,firstly,bone marrow(BM)cells from WT,Cbl-b KO,c-Cbl KO and dKO mice were cultured with GM-CSF and M-CSF in vitro,we found BM from dKO mice could develop more colonies than BM from WT,Cbl-b KO,and c-Cbl KO mice under stimulating with M-CSF,and the proliferation rate of BMDM is faster than the WT group.However,the number of colonies and proliferation of dKO BM were similar to BM from WT,Cbl-b KO,and c-Cbl KO mice under stimulating with GM-CSF alone.These suggested that Cbl-b/c-Cbl may function on the M-CSF signaling pathway,thus,affects the differentiation and development of macrophages.Therefore,we detected the expression of M-CSFR in BMDM and alveolar macrophages respectively,and we found the expression of M-CSFR has significantly increased in c-Cbl KO macrophages and dKO macrophage.And in vitro studies demonstrated that c-Cbl could directly inhibited the protein expression level of M-CSFR.Also,c-Cbl could down-regulated the m RNA expression of M-CSFR by ubiquitin modification of PU.1 which are transcriptional factor of M-CSFR.Moreover,Co-IP and mass spectrometry analysis showed that c-Cbl and Cbl-b both could interacted with tyrosine kinase Lyn,and inhibited the protein level of Lyn through ubiquitin modification.Western-blot also showed that Lyn was obviously higher expression in dKO alveolar macrophages as well as in dKO BMDM.Meanwhile,addition of Lyn inhibitor in BMDM culture system,could reverse the cytokine producing profiles to some extent.All these suggested that c-Cbl and Cbl-b together regulated the inflammation of macrophages by modifying the ubiquitin of Lyn.Further experiment indicated that Lyn may regulating the inflammation of macrophage by inhibiting the expression of NF-kB.On conclusion,c-Cbl inhibits the differentiation and proliferation of macrophages by directly down-regulating the protein expression of M-CSFR and ubiquitin modification of PU.1 to further inhibit the m RNA expression of M-CSFR.And c-Cbl and Cbl-b both regulate the inflammation of macrophage through ubiquintination of Lyn,all together,finally results in Lung destroyed and idiopathic pulmonary fibrosis like symptom in c-Cbl and Cbl-b deficiency mice.These results enrich the molecular mechanism of macrophage differentiation and development,and provide new clues for the treatment of autoimmune diseases caused by the abnormal increase of macrophages.