Viruses use diverse strategies to impair the antiviral immunity of host in order to promote infection and pathogenesis. Herein, we found that PCV2 infection promotes the infection of DNA viruses through inhibiting IFN-β induction in vivo and in vitro. In the early phase of infection, PCV2 promotes the phosphorylation of cGAS at S278 via activation of PI3K/Akt signaling, which directly silences the catalytic activity of cGAS. Subsequently, phosphorylation of cGAS at S278 can facilitate the K48-linked poly-ubiquitination of cGAS at K389, which can been served as a signal for recognizing by the ubiquitin-binding domain of histone deacetylase 6 (HDAC6), to promote the translocation of K48-ubiquitinated-cGAS from cytosol to autolysosome depending on the deacetylase activity of HDAC6, thereby eventually resulting in a markedly increased cGAS degradation in PCV2 infection-induced autophagic cells relative to Earle’s Balanced Salt Solution (EBSS)-induced autophagic cells (a typical starving autophagy). Importantly, we found that PCV2 Cap and its binding protein gC1qR act as predominant regulators to promote porcine cGAS phosphorylation and HDAC6 activation through mediating PI3K/AKT signaling and PKCδ signaling activation. Based on this finding, gC1qR-binding activity deficient PCV2 mutant (PCV2RmA) indeed shows a weakened inhibitory effect on IFN-β induction and a weaker boost effect for other DNA viruses infection compared to wild-type PCV2. Collectively, our findings illuminate a systematic regulation mechanism by which porcine circovirus counteracts the cGAS-STING signaling pathway to inhibit the type I interferon induction and promote DNA virus infection, and identify gC1qR as an important regulator for the immunosuppression induced by PCV2.

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-α) expression in different mammalian macrophages remains unclear. In this study, we investigated the potential mechanisms by which Omp25 regulates TNF-α expression and found that Omp25-deficient mutant of B. suis exhibited an enhanced TNF-α expression compared with wild-type (WT) B. suis, whereas ectopic expression of Omp25 suppressed LPS-induced TNF-α 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-α transcription by targeting TRAF6 and IRAK1 at the 3′ untranslated region (UTR), resulting in inhibition of NF-kB pathway, while upregulation of miR-130a-3p, -181a, or -301a-3p correlated temporally with decreased TNF-α 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-α 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-α production and decreased intracellular bacteria in both cells. Taken together, our results demonstrate that Brucella downregulates TNF-α to promote intracellular survival via Omp25 regulation of different microRNAs in porcine and murine macrophages.

Brucella spp. infection results in compromised Type1 (Th1) cellular immune response. Several reports have described an immunomodulatory function for Brucella major outer membrane protein Omp25. However, the mechanism by which Omp25 modulates macrophage dysfunction has not been defined. Herein, we report that Omp25-deficient mutant of B. suis exhibited an enhanced ability to induce interleukin (IL)-12 whereas ectopic expression of Omp25 protein inhibited TLR agonists-induced IL-12 p70 production through suppression of both IL-12 p40 and p35 subunit expression in THP-1 cells. In addition, Omp25 significantly up-regulated miR-155, -23b and -21-5p, as well as the immunomodulator molecule programmed death-1 (PD-1) in monocyte/macrophages. The up-regulation of miR-155 and -23b correlated temporally with decreased TAB2 levels, IκB phosphorylation and IL-12 p40 levels by targeting TAB2 and il12B 3’UTR, respectively, while miR-21-5p increase directly led to the reduction of LPS/R848-induced IL-12 p35 protein by targeting il12A 3’UTR. Consistent with this finding, reduction of miR-155 and -23b attenuated the inhibitory effects of Omp25 on LPS/R848-induced IL-12 p40 expression at both transcriptional and post-transcriptional levels, while reduction of miR-21-5p attenuated the inhibitory effects of Omp25 on LPS/R848-induced IL-12 p35 expression at the post-transcriptional level, together significantly enhanced IL-12 p70 production upon LPS/R848 stimulation. We also found that blocking PD-1 signaling decreased the expression of miR-155, -23b and -21-5p induced by Omp25 and enhanced IL-12 production in monocyte/macrophages. Altogether, these data demonstrate that Brucella Omp25 induces miR-155, -23b and -21-5p to negatively regulate IL-12 production at both transcriptional and post-transcriptional levels via regulation of PD-1 signaling, which provides an entirely new mechanism underlying monocyte/macrophages dysfunction during Brucella spp infection.

Recent studies have demonstrated that ovarian granular cells (OGCs) pyroptosis is present in the ovaries of polycystic ovary syndrome (PCOS) mice and that NLRP3 activation destroys follicular functions. Metformin has been shown to protect against PCOS by reducing insulin resistance in women, whereas its role in OGC pyroptosis is unknown. This study aimed to investigate the impact of metformin on OGC pyroptosis and the underlying mechanisms. The results showed that treating a human granulosa-like tumor cell line (KGN) with metformin significantly decreased LPS-induced expression of miR-670-3p, NOX2, NLRP3, ASC, cleaved caspase-1, and GSDMD-N. Cellular caspase-1 activity; ROS production; oxidative stress; and the secretion of IL-1β, IL-6, IL-18, and TNF-α were also diminished. These effects were amplified by adding N-acetyl-L-cysteine (NAC), a pharmacological inhibitor of ROS. In contrast, metformin’s anti-pyroptosis and anti-inflammatory effects were robustly ameliorated by NOX2 overexpression in KGN cells. Moreover, bioinformatic analyses, RT-PCR, and Western blotting showed that miR-670-3p could directly bind to the NOX2 (encoded by the CYBB gene in humans) 3’UTR and decrease NOX2 expression. Metformin-induced suppression of NOX2 expression, ROS production, oxidative stress, and pyroptosis was significantly alleviated by transfection with the miR-670-3p inhibitor. These findings suggest that metformin inhibits KGN cell pyroptosis via the miR-670-3p/NOX2/ROS pathway.