CARD9 Protect Lung Cancer Development By Regulating The Recruitment And Function Of Myeloid-derived Suppressor Cells

Caspase recruitment domain-containing protein 9?CARD9?is a central adaptor protein and highly expressed in myeloid cells.Previous studies revealed that CARD9 played a crucial role in macrophages polarization in a liver metastasis of colon carcinoma.Nevertheless,the effect of CARD9 in lung cancer is still unclear.Here,using a mouse lewis lung cancer model,we found the tumor burden of CARD9^-/-mice was much heavier than that in wild-type?WT?mice.More myeloid-derived suppressor cells?MDSCs?was accumulated and less cytotoxicity T lymphocyte were found in tumor tissues of CARD9^-/-mice,compared with WT mice.Depleting MDSCs using anti-Grl antibody can significantly decrease tumor burden in CARD9^-/-mice.Furthermore,the expression of indoleamine 2,3-dioxygenase?IDO?,an intracellular enzyme,was up-regulated in MDSCs from CARD9^-/-mice.The high expression of IDO was resulted from the activation of noncanonical NF-?B pathway in MDSCs from CARD9^-/-mice,including increased NIK protein level,phosphorylation of p100 in cytoplasm,and RelB-p52 nuclear translation.Inhibiting NIK in CARD9^-/--derived MDSCs decreased IDO expression.Taken together,all results showed a CARD9-NF-?B-IDO pathway in MDSCs which promote lung cancer development.Our findings provide insights into understanding the important role and mechanism of CARD9-MDSCs in lung carcinoma.1.CARD9 affects the development of Lewis Lung Cancer and has something to do with MDSCsCARD9 is an adaptor protein of innate immunity which is related to many diseases.Selected fungi,bacteria and viruses could activate CARD9 signaling which regulates innate immune responses and affects adaptive immunity.Previous studies revealed that CARD9 played a crucial role in macrophages polarization in a liver metastasis of colon carcinoma.CARD9 also has something to do with MDSCs accumulation in Candida albicans infection.Therefore,CARD9 may play an important role in lung cancer.To figure out the role of CARD9 in lung cancer,lewis lung cancer mice model was established on wild-type?WT?and CARD9-knock out(CARD9^-/-)mice.All results suggested CARD9^-/-tumor-bearing mice had heavier tumor burden and much bigger tumor tissue.The Flow cytometric analysis also revealed more MDSCs accumulated and a decreased percentage of cytotoxicity T lymphocyte in CARD9^-/-tumor-bearing mice.The anti-Gr-1 antibody treatment significantly reduced the percentage and number of MDSCs in tumor site and increased the frequency of CD8+T cells in tumor microenvironment.So depletion of MDSCs could relieve the tumor burden in CARD9^-/-mice.All in all,CARD9 had an impact on lung cancer,which was related to MDSCs.2.CARD9 activates noncanonical NF-?B pathway in MDSCs to suppress expression of IDOTo explain how CARD9 affect the accumulation and function of MDSCs,bone marrow cells were acquired from WT and CARD9^-/-mice and were differentiated into MDSCs.Then BM-MDSCs were stimulated with supernatants derived from Lewis Lung cancer?LLC?cells.We found LLC supernatants increased the percentage of MDSCs in CARD9^-/-mice,the noncanonical NF-?B-IDO pathway was obviously activated in CARD9^-/-mice-derived MDSCs.The expression of NIK?P-P100?RelB?P52 and IDO were totally up-regulated.MDSC suppressive assay implied an enhanced inhibitory effect of MDSCs on T cells in CARD9^-/-mice.However,the function of WT and CARD9^-/--derived MDSCs showed no difference without LLC stimulation.Therefore CARD9 could interfere with lung cancer development.In conclusion,CARD9 activates noncanonical NF-?B pathway in MDSCs to suppress IDO which could affect the immune suppressive function of MDSCs,promoting the development of lung cancer.3.CARD9 is negative correlated with the expression of MDSCs-related genes in lung carcinoma patientsIn order to figure out the relationship between CARD9 and MDSCs in lung cancer,48 patients with lung cancers were recruited in our clinical study and lung tumor tissues were acquired.Then we detected the expression of MDSC-related genes in tumor tissue.Quantitative analysis showed that CARD9 was highly expressed in tumor tissues,especially increased in paracancerous tissues.Furthermore,the relationship between CARD9 and tumor volume was significant.We also found negative correlations between CARD9 expressions and MDSCs relative genes?iNOS,Arg-1,IDO?.The results were consistent with these in mice.