Expression Of Gene CARD9 In Leprosy And Mycobacterium Infected Macrophages

Background:Leprosy,also known as Hansen’s disease(HD),is a chronic granulomatous infectious disease caused by Mycobacterium leprae infection in susceptible individuals which mainly affects peripheral nerves and skin.It can cause irreversible impairment of nerve function and con-sequent chronic disabilities.Both innate and adaptive immunity play important roles in the recognizing and defending of Mycobacterium leprae.Due to different immune responses to Mycobacterium leprae,the clinical manifestation of leprosy is divided into five types.Mycobacterium leprae has not been successfully cultured in vitro by now,which limited our understanding of Mycobacterium leprae and the pathogenesis of this disease.Gene CARD9 is located on chromosome 9 and encodes caspase recruitment domain protein 9(CARD9).CARD9 protein is a member of the CARD family and is an important adapter protein that regulates intracellular signaling through protein-protein interactions.CARD9 is a cytoplasmic protein which is ubiquitously expressed in a variety of tissues,such as thymus,spleen,lung,bone marrow,especially in bone marrow-derived macrophages and dendritic cells.It can regulate the inflammatory response,integrate a variety of signals transducted by inherent immune receptor,and participate in the immune respnses against fungi,bacteria,viruses and other pathogenic microorganisms.Objectives:To analyze the differential expressions of CARD9 in skin and peripheral blood between leprosy patients and healthy controls.To analyze the differential expressions of genes associated with CARD9 pathway between leprosy patients and healthy controls.And to verify the correlation between gene CARD9 and leprosy in mouse macrophages.methods:1.Using transcriptome sequencing(RNA-Seq)technique to measure and compare the expression of CARD9 mRNA in skin lesions of 27 cases(19 multibacillary leprosy,8 paucibacillary leprosy)and normal skin of 18 healthy volunteers.The expressions of CARD9 mRNA in 11 patients’ skin lesions and 5 normal skin tissues of healthy controls were detected by qPCR.The expression of CARD9 mRNA was detected by qPCR in peripheral blood of 10 patients and 11 normal controls.2.Using transcriptome sequencing(RNA-Seq)technique to measure and compare the expression of genes associated with CARD9 pathway in skin lesions of 27 cases(19 multibacillary leprosy,8 paucibacillary leprosy).3.The expression level of Card9 mRNA in mouse RAW264.7 cells infected by different concentrations of Mycobacterium marina was measured by qPCR.Result:1.The expression of CARD9 gene in the lesion tissue was significantly higher than that in the normal control group,and the difference was statistically significant(RNA-Seq:p<7.92E-5,qPCR:p<0.007(P<0.022).No significant difference was observed in the expression of CARD9 gene in peripheral blood(p<0.124).2.There were significant differences in the expression of 13 genes associated with CARD9 between leprosy patients and normal controls(p<0.05).But most of the genes showed no significant difference between multibacillary and paucibacillary leprosy.3.The mRNA expression level of Card9 in mouse marcrophageswas significantly increased after stimulation with Mycobacterium marina for 12h.Conclusion:The mRNA level of CARD9 was significantly higher in skin lesions of leprosy than that in normal skins of healthy controls.This result was also validated in the mouse macrophages infection experiments.CARD9 showed some association with leprosy.