■类lioidosis,由革兰氏阴性细菌伯克霍尔德氏菌引起,是全球许多热带国家的地方病。临床表现变化很大,从无症状到致命的败血症,因此感染的结果可能取决于宿主的免疫反应。本研究的目的是首先,表征巨噬细胞对假单胞菌的免疫反应,其次,为了确定在存在靶向毒力因子的新型抑制剂的情况下免疫反应是否被修饰,巨噬细胞感染性增效剂(Mip)蛋白。我们假设抑制假单胞菌中的Mip将解除细菌的武装并导致宿主有益的免疫应答。
■在存在靶向Mip蛋白的小分子抑制剂的情况下,用假单胞菌K96243感染鼠巨噬细胞J774A.1细胞。在感染后4小时对感染的细胞进行RNA测序。在感染后24小时测量细胞培养上清液中分泌的细胞因子和乳糖脱氢酶。可行,也在感染后24小时计数巨噬细胞中的细胞内假单胞菌。
■通过RNA-seq对感染假球芽孢杆菌的巨噬细胞进行全局转录分析,证明了免疫相关基因的上调,特别是TNF信号通路中基因的显著富集。用Mip抑制剂治疗假单胞菌感染的巨噬细胞,与用DMSO处理的细胞相比,AN_CH_37导致il1b减少了5.3倍,抑制剂被溶解在其中。在感染AN_CH_37以及其他促炎细胞因子后24小时,观察到培养上清液中IL-1β水平的统计学显着降低,即IL-6和TNF-α。用AN_CH_37处理还降低了24小时后巨噬细胞中假球芽孢杆菌的存活,这伴随着由乳酸脱氢酶释放确定的假球芽孢杆菌诱导的细胞毒性的显著降低。
■这些数据强调了利用Mip抑制剂减少由巨噬细胞中的假单胞菌感染引起的潜在有害的促炎反应的潜力。这可能是重要的,因为过度刺激促炎反应可以导致免疫病理学,组织损伤和感染性休克。
UNASSIGNED: Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a disease endemic in many tropical countries globally. Clinical presentation is highly variable, ranging from asymptomatic to fatal septicemia, and thus the outcome of infection can depend on the host immune responses. The aims of this study were to firstly, characterize the macrophage immune response to B. pseudomallei and secondly, to determine whether the immune response was modified in the presence of novel inhibitors targeting the virulence factor, the macrophage infectivity potentiator (Mip) protein. We hypothesized that inhibition of Mip in B. pseudomallei would disarm the bacteria and result in a host beneficial immune response.
UNASSIGNED: Murine macrophage J774A.1 cells were infected with B. pseudomallei K96243 in the presence of small-molecule inhibitors targeting the Mip protein. RNA-sequencing was performed on infected cells four hours post-infection. Secreted cytokines and lactose dehydrogenase were measured in cell culture supernatants 24 hours post-infection. Viable, intracellular B. pseudomallei in macrophages were also enumerated 24 hours post-infection.
UNASSIGNED: Global transcriptional profiling of macrophages infected with B. pseudomallei by RNA-seq demonstrated upregulation of immune-associated genes, in particular a significant enrichment of genes in the TNF signaling pathway. Treatment of B. pseudomallei-infected macrophages with the Mip inhibitor, AN_CH_37 resulted in a 5.3-fold reduction of il1b when compared to cells treated with DMSO, which the inhibitors were solubilized in. A statistically significant reduction in IL-1β levels in culture supernatants was seen 24 hours post-infection with AN_CH_37, as well as other pro-inflammatory cytokines, namely IL-6 and TNF-α. Treatment with AN_CH_37 also reduced the survival of B. pseudomallei in macrophages after 24 hours which was accompanied by a significant reduction in B. pseudomallei-induced cytotoxicity as determined by lactate dehydrogenase release.
UNASSIGNED: These data highlight the potential to utilize Mip inhibitors in reducing potentially harmful pro-inflammatory responses resulting from B. pseudomallei infection in macrophages. This could be of significance since overstimulation of pro-inflammatory responses can result in immunopathology, tissue damage and septic shock.