Rab GTPases Involve In Enhanced Bacterial Clearance By BLP-tolerized Macrophages

AIM: BLP as the most abundant protein in the outer membrane of both Gram-positive and Gram-negative bacteria is mainly released by the growing or cracked bacteria.BLP can also induced tolerance to a second BLP challenge, which is called BLP tolerance.Since the enhanced bacterial clearance effect of BLP-tolerized body(or cell) is closely associated with phagosome maturation, it will be helpful to explore the change of phagosome maturation relate protein, to know whether they are involved in the enhanced bacterial clearance, to clarify the relevant signaling molecules and pathways that control these changes, and most importantly to let us understand the underlying mechanisms of the protective regulatory role of BLP tolerance.Rab GTPases paly a important role in phagosome maturation.So we want to explore whether Rab GTPases are involved inthe enhanced bacterial clearance by BLP-tolerized macrophages(bonemarrow-derived macrophages, BMDMs). METHODS: Femur and tibia of SPF,18-20 g,male mice were separated and bone marrow was rushed out, then it was cultured with L929-conditioned medium until bone marrow-derived macrophages differentiate into a homogenous population of mature BMDMs after 7 days which later divides in Na?ve and BLP-tolerized BMDMS.Next, we compared engulfment of bacteria and intracellular bacterial killing ability of Na?ve BMDMs with BLP-tolerized BMDMs by Flow Cytometer.Real time PCR was used to compare the m RNA levels of Rab5 a, Rab5 b, Rab7, Rab9, Rab9 b, Rab11 a, Rab11 b, Rab12, Rab14, Rab20, Rab32, Rab34 between the naive BMDMs and the BLP-tolerized BMDMs.The up-regulation of Rab7 and Rab20 which were further confirmed in BLP-tolerized BMDMs infected with E. coli by quantitative PCR and Western blotting. Following down regulating the expression of Rab7 and Rab20 separately using RNA interference(RNAi), the bacteria uptake and intracellular bacterial killing of BLP-tolerized BMDMs treated with E. coli were detected. RESULTS:BLP-tolerized BMDMs(25.7±1.7%)engulfment of bacteria was more than Na?ve BMDMs(13.8±1.7%)on 15 min, BLP-tolerized BMDMs(75.1±0.1%)engulfment of bacteria was more than Na?ve BMDMs(39.8±5.4%)on 30min;The intracellular bacterial killing ability of BLP-tolerized BMDMs was 1.7 fold higher to Na?ve BMDMs on 30 min,which was 1.6 fold higher on 60min; m RNA levelsof Rab7 was significantly increased in BLP-tolerized BMDMs, which was 1.4 fold higher to the naive BMDMs, and m RNA level and protein expression in BLP-tolerized BMDMs stimulated with heat-killed E.coli were further confirmed to be up regulating;m RNA levelsof Rab20 was significantly increased in BLP-tolerized BMDMs, which was 2 fold higher to the naive BMDMs, and m RNA level and protein expression in BLP-tolerized BMDMs stimulated with heat-killed E.coli were further confirmed to be up regulating. Down-regulating the expression of Rab7 had no effect on BLP-tolerized BMDMs engulfment of bacteria, but the intracellular bacterial killing ability was significantly attenuated.The same to Rab20.CONCLUSION: Firstly, compared with Naive BMDMs, the bacterial engulfment and intracellular killing were both enhanced in BLP-tolerized BMDMs andthe expression of phagosome maturation related molecules such as Rab7 and Rab20 were up-regulated in BLP-tolerized BMDMs; Secondly, Rab7 and Rab20 involved in the enhanced bactericidal effect of BLP-tolerized BMDMs.