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.

Circoviruses are the smallest DNA viruses known to infect mammalian and avian species. Although circoviruses are known to be associated with a range of clinical diseases, the details of circovirus DNA release still remain unknown. Here, we identified p32 as a key regulator for porcine circoviral nuclear egress. Upon porcine circovirus type 2 (PCV2) infection, p32 was recruited into the nucleus by the viral capsid (Cap) protein; simultaneously, protein kinase C isoform δ (PKC-δ) was phosphorylated at threonine 505 by phospholipase C (PLC)-mediated signaling at the early stage of infection, which was further amplified by Jun N-terminal protein kinase (JNK) and extracellular signal-regulated kinase (ERK) signaling at the late infection phase. p32 functioned as an adaptor to recruit phosphorylated PKC-δ and Cap to the nuclear membrane to phosphorylate lamin A/C, resulting in a rearrangement of nuclear lamina and thus facilitating viral nuclear egress. Consistent with these findings, knockout (KO) of p32 in PCV2-infected cells markedly reduced the phosphorylation of PKC-δ and impeded the recruitment of p-PKC-δ and Cap to the nuclear membrane, hence abolishing the phosphorylation of lamin A/C and the rearrangement of nuclear lamina. As a result, p32 depletion profoundly impaired the production of cell-free viruses during PCV2 infection. We further identified the N-terminal 24RRR26 of Cap to be crucial for binding to p32, and mutation of these three arginine residues significantly weakened the replication and pathogenesis of PCV2 in vivo. In summary, our findings highlight a critical role of p32 in the activation and recruitment of PKC-δ to phosphorylate lamin A/C and facilitate porcine circoviral nuclear egress, and they certainly help understanding of the mechanism of PCV2 replication. IMPORTANCE Circovirus infections are highly prevalent in mammalian and avian species. Circoviral capsid protein is the only structural protein of the virion that plays an essential role in viral assembly. However, the machinery of circovirus nuclear egress is currently unknown. In this work, we identified p32 as a key regulator of porcine circovirus type 2 (PCV2) nuclear egress that forms a complex with the viral capsid (Cap) protein to enhance protein kinase C isoform δ (PKC-δ) activity; this resulted in a recruitment of phosphorylated PKC-δ to the nuclear membrane, which further phosphorylates lamin A/C to promote the rearrangement of nuclear lamina and facilitate viral nuclear egress. Notably, we found that the N-terminal 24RRR26 of Cap, a highly conserved motif among circovirus species, was required for interacting with p32, and that mutation of this motif markedly impeded PCV2 nuclear egress. These data indicate that p32 is a critical regulator of PCV2 nuclear egress and reveal the importance of this finding in circovirus replication.

Porcine circovirus (PCV) type 2 (PCV2), an immunosuppression pathogen, is often found to increase the risk of other pathogenic infections. Yet the relative immune mechanisms determining the susceptibility of PCV2-infected animals remain unclear. In this study, we confirmed that PCV2 infection suppressed IL-12p40 expression and host Th1 immune response, leading to a weakened pathogenic clearance upon porcine reproductive respiratory syndrome virus (PRRSV) or infection. PCV2 infection suppressed pathogens, LPS/IFN-γ, or LPS/R848-induced IL-12p40 expression in porcine alveolar macrophages. PCV2 capsid (Cap) was the major component to suppress IL-12p40 induction by LPS/IFN-γ, LPS/R848, PRRSV, or Either wild-type PCV2 or mutants PCV2-replicase 1 and PCV type 1-Cap2, which contained PCV2 Cap, significantly decreased IL-12p40 levels and increased the replication of PRRSV and in the lung tissues relative to mock or PCV type 1 infection. gC1qR, a Cap binding protein, was not involved in IL-12p40 induction but mediated the inhibitory effect of PCV2 Cap on IL-12p40 induction. PCV2 also activated PI3K/Akt1 and p38 MAPK signalings to inhibit IL-12p40 expression via inhibition of NF-κB p65 binding to promoter and upregulation of miR-23a and miR-29b. Knockdown of Akt1 and p38 MAPK downregulated miR-23a and miR-29b and increased IL-12p40 expression. Inhibition of miR-23a and miR-29b attenuated the inhibitory effect of PCV2 on IL-12p40 induction, resulting in an increased IL-12p40 expression and Th1 cell population and reduced susceptibility to PRRSV or Taken together, these results demonstrate that PCV2 infection suppresses IL-12p40 expression to lower host Th1 immunity to increase the risk of other pathogenic infection via gC1qR-mediated PI3K/Akt1 and p38 MAPK signaling activation.

Interleukin (IL)-10, as a key anti-inflammatory cytokine, increases during porcine circovirus type 2 (PCV2) infection, but the role of IL-10 in the process remains to be defined. In the present study, using an IL-10 deficient mice model, we found that IL-10 deficiency prevented the reduction of splenic lymphocytes (CD45+ cells) induced by PCV2 and promoted CD4+ and CD8+ T cell infiltration in lungs through inducting more T cell chemokines (CCL3, CXCL9, and CXCL10). Simultaneously, PCV2 infection induced a significant increase of pro-inflammatory cytokines and PCV2-specific antibodies in IL-10 deficient mice than in wild-type mice, resulting in a lower viral load in lung and a milder lung lesion in IL-10 deficient mice relative to wild-type mice. Moreover, the amounts of pulmonary CD4+ and CD8+ T cells were all inversely correlated with the lung lesions, as well as the viral load of PCV2. These results demonstrate that PCV2 infection employs IL-10 to block the transfer of T cells to the lungs of mice, and IL-10 attenuates the production of pro-inflammatory cytokines and PCV2-specific antibodies. The lack of T cell infiltration, pro-inflammatory cytokines, and PCV2-specific antibodies promote PCV2 replication, leading to a more severe lung lesion in mice.

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