Macrophage-inducible C-type Lectin Signaling Pathway Contributes To The Pathogenesis Of Lung Fibrosis

BackgroundIdiopathic pulmonary fibrosis(IPF)is a devastating disease with an unknown aetiology.The exact etiology of IPF is unknown and may be diverse.Macrophage-induced C-type lectin receptor(Mincle)is a pattern recognition receptor(PPR)on the surface of innate immune cells.Recent studies showed that macrophages polarisation and danger-associated molecular patterns play critical roles in the process of lung fibrosis.ObjectiveTo determine the expression of Mincle and its ligand recognition in lung tissue of subjects with lung fibrosis,and the role in mediating specific signaling pathways in the pathogenesis of lung fibrosisMethods1.The expressions of Mincle in human IPF biopsies,monocyte and BAL,as well as in lung of mice treated with Bleomycin(BLM)were examined.Meanwhile,the expression of Mincle in MHS treated with BLM in vitro was also investigated.2.The expression of spliceosome-associated protein 130(SAP130)in human IPF lung tissue,blood and BAL,as well as in lung of mice treated with BLM were determined.We also determine whether Mincle in fibrotic lung tissue can recognize and bind the endogenous ligand SAP130 using immunofluorescence multi-label technique.3.Genetic ablation,or Mincle activation using TDB and recombinant SAP130 were employed to ascertain the functional significance of Mincle in lung fibrosis.4.The effect of Mincle knockout on the polarization of M2 macrophages was investigated in the BLM-induced lung fibrosis model.The effects of Mincle +/+ alveolar macrophages on lung fibrosis was further conducted by using alveolar macrophages elimination and adoptive experiments.5.RNA microarray analysis was performed to identify differential expression of genes.We also further defined the roles of Syk/CARD9 through Syk inhibitor and CARD9 KO mice in BLM-induced lung fibrosis.Results1.Increased expressions of Mincle in lungs of IPF patients and BLM-injured mice were observed.Mincle expression was increased in MHS cells stimulated by BLM.2.Increased expressions of SAP130 in lungs of IPF patients and BLM-injured mice were observed.The PPR Mincle on the macrophage membrane in the fibrotic lung tissue of IPF patients and mouse treated with BLM can recognize the endogenous ligand SAP130.3.Mincle deficiency alleviated the severity of lung fibrosis induced by BLM.Stimulating Mincle signing pathway by using recombinant SAP130 and TDB exacerbated BLM induced fibrosis in mice.4.Mincle deficiency reduced the M2 polarization.Adoptive transfer of Mincle-expressing alveolar macrophages promoted lung fibrosis in BLM-induced Mincle KO mice.5.Consistent with our microarray data,BLM-treated Mincle KO mice expressed lower levels of CARD9 compared with WT controls.Mincle deficiency reduced the M2 polarization by regulating the Syk/Card9 signaling pathway,and alleviated BLM-induced lung fibrosis in mice.ConclusionMincle recognized SAP130 and mediates the Syk/Card9 signaling pathway to polarize of lung M2 macrophages and thus affected lung fibrosis.Mincle/Syk/Card9 signaling pathway may act a crucial role in the pathogenesis of lung fibrosis,and provide a new,clinically significant therapeutic target.