Brucella Downregulates Tumor Necrosis Factor-α to Promote Intracellular Survival via Omp25 Regulation of Different MicroRNAs in Porcine and Murine Macrophages

Brucella spp. impedes the production of pro-inflammatory cytokines by its outer membrane protein Omp25 in order to promote survival and immune evasion. However, how Omp25 regulates tumor necrosis factor (TNF-alpha) expression in different mammalian macrophages remains unclear. In this study, we investigated the potential mechanisms by which Omp25 regulates TNF-alpha expression and found that Omp25-deficient mutant of B. suis exhibited an enhanced TNF-alpha expression compared with wild-type (WT) B. suis, whereas ectopic expression of Omp25 suppressed LPS-induced TNF-alpha production at both protein and mRNA levels in porcine alveolar macrophages (PAMs) and murine macrophage RAW264.7 cells. We observed that Omp25 protein as well as WT B. suis upregulated miR-146a, -181a, -181b, and -301a-3p and downregulated TRAF6 and IRAK1 in both PAMs and RAW264.7 cells, but separately upregulates miR-130a-3p in PAMs and miR-351-5p in RAW264.7. The upregulation of miR-146a or miR-351-5p attenuated TNF-alpha transcription by targeting TRAF6 and IRAK1 at the 3' untranslated region (UTR), resulting in inhibition of NF-kappa B pathway, while upregulation of miR-130a-3p, -181a, or -301a-3p correlated temporally with decreased TNF-alpha by targeting its 3' UTR in PAMs or RAW264.7 cells. In contrast, inhibition of miR-130a-3p, -146a, -181a, and -301a-3p attenuated the inhibitory effects of Omp25 on LPS-induced TNF-alpha in PAMs, while inhibition of miR-146a, -181a, -301a-3p, and -351-5p attenuated the inhibitory effects of Omp25 in RAW264.7, resulting in an increased TNF-alpha production and decreased intracellular bacteria in both cells. Taken together, our results demonstrate that Brucella downregulates TNF-alpha to promote intracellular survival via Omp25 regulation of different microRNAs in porcine and murine macrophages.