IFN-γ And IFN-λ Regulate Burkholderia Pseudomallei Infection Via Vitamin D-mediated Antimicrobial Peptide Production Pathway

Objective:As a pathogen,Burkholderia pseudomallei（B.p）causes human melioidosis.Pulmonary melioidosis is one of the most common clinical B.p infection type.Pulmonary melioidosis shows high case fatality rate and leads to great harm to patients.Currently,no treatment has shown effectiveness for pulmonary melioidosis prevention and therapy.To explore a effective way for pulmonary melioidosis prevention and treatment,we attempt to understand the roles of IFN-γand IFN-λin regulation of HCC827 resistance against B.p,which may provide a new theoretical basis for the pathogenesis of pulmonary melioidosis.Furthermore,to find a new way for pulmonary melioidosis intervention,we evaluate the effect of IFN-γand IFN-λin regulating antimicrobial ability via vitamin D-mediated antimicrobial peptide production pathway.Methods:（1）To observe the vitamin D-mediated resistance enhancement of HCC827 to B.p,we added 1,25（OH）₂D₃to HCC827 cell culture system.The levels of Cathelicidin antimicrobial peptide（CAMP）andβ-Defensin 4（DEFB4）were measured using Western Blot and ELISA.The gene expression levels of CAMP and DEFB4 were quantified by RT-q PCR.The B.p resistance level of HCC827 was assessed by count of CFU.CAMP and DEFB4 knocked-down HCC827 cell lines were used as the positive controls.（2）HCC827 cells were treated with exogenous IFN-γto evaluate the expression levels of vitamin D receptor（VDR）,CAMP and DEFB4.When HCC827 cells were infected with B.p,exogenous IFN-γand IFN-γneutralizing antibodies were added,respectively.The expression levels of vitamin D receptor（VDR）,CAMP and DEFB4 were observed by RT-q PCR,Western Blot and ELISA assay.The B.p resistance level of HCC827 cells was assessed by count of CFU.Meanwhile,exogenous 1,25（OH）₂D₃was added in the absence of IFN-γsignal to observe the expression levels of related genes and the B.p resistance level of HCC827 cells.（3）The knocked-down strains of IFN-λspecific receptor IFNLR1 in HCC827 cells were constructed to compare the expression of VDR,CAMP and DEFB4 genes in wild and IFNLR1 knocked-down strains of HCC827 cells.The differences in the expression levels of VDR,CAMP and DEFB4 were observed by RT-q PCR,Western Blot and ELISA assays when B.p infected wild strains and IFNLR1^-/-strains.The B.p resistance level of HCC827 cells was assessed by count of CFU.In the meantime,the expression levels of the above-mentioned relevant genes and the resistance of cells against B.p infection when exogenous 1,25（OH）₂D₃was added to wild and IFNLR1 knocked-down strains of HCC827 cells were evaluated.Results:（1）After exogenous 1,25（OH）₂D₃was added to HCC827 cells,the expression levels of CAMP and DEFB4 increased significantly,the CFU count was significantly decreased and the resistance of cells against B.p infection was also remarkably strengthened.However,after knockdown of CAMP and DEFB4 genes in HCC827 cells,the expression level of antimicrobial peptides decreased significantly after B.p infection compared with that in wild strain cells.（2）When HCC827 cells were infected with B.p,exogenous IFN-γand IFN-γneutralizing antibodies were added,respectively.It was found that under exogenous IFN-γstimulation,the expression levels of VDR,CAMP and DEFB4 in the vitamin D-mediated antimicrobial peptide production pathway increased significantly,the CFU count was significantly decreased and the resistance of cells against B.p infection was also significantly enhanced.The expression levels of VDR,CAMP and DEFB4 decreased significantly and the CFU count was significantly increased by incubation with IFN-γ-neutralizing antibodies in the culture system in advance,followed by infection of HCC827 cells with B.p.However,after exogenous 1,25（OH）₂D₃was added,the expression levels of VDR,CAMP and DEFB4 increased significantly,the CFU count was significantly decreased and the resistance of HCC827 cells against B.p infection was also remarkably strengthened.（3）The knocked-down strains of IFN-λspecific receptor IFNLR1 in HCC827 cells were constructed.After the wild and IFNLR1knocked-down strains of HCC827 cells were infected with B.p,the expression levels of VDR,CAMP and DEFB4 in the vitamin D-mediated antimicrobial peptide production pathway in IFNLR1 knocked-down strain increased significantly,the CFU count was significantly decreased and the resistance of IFNLR1 knocked-down strain against B.p increased significantly.Conclusions:（1）1,25（OH）₂D₃improves the expression levels of CAMP and DEFB4 in HCC827 cells and enhances the resistance of the cells against B.p infection.（2）IFN-γpromotes the expression of the key gene,VDR,in vitamin D against B.p infection pathway,inducing the up-regulated expression of CAMP and DEFB4 and strengthening the resistance of HCC827 cells against intracellular infection of B.p.（3）IFN-λinhibits the expression of the VDR in vitamin D-mediated resistance of B.p infection.IFN-λreduces the expression of CAMP and DEFB4,which results in feeble resistance of HCC827 cells against intracellular infection of B.p.