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

Abstract

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.

Keywords: Brucella suis; Omp25; macrophage; miRNA; tumor necrosis factor-α.

Figure 1

Deficiency of omp25 enhances B. suis-induced tumor necrosis factor (TNF)-α production in porcine alveolar macrophages (PAMs) and mouse RAW264.7 cells. (A,B) PAMs and RAW264.7 cells were infected with wild-type (WT) B. suis, Omp25-deficient mutant (Δomp25 B. suis), Omp31-deficient mutant (Δomp31 B. suis) or were uninfected (ctrl), and TNF-α secretion was measured at 24 h post-infection in culture supernatants by enzyme-linked immunosorbent assay (ELISA). (C,D) PAMs and RAW264.7 cells were infected with WT B. suis, Δomp25, or Δomp31 and cultured for 6 h, Q-PCR was used to measure TNF-α mRNAs levels. (E,F) PAMs and mouse RAW264.7 cells were infected with Δomp25, the complemented Δomp25 strain of B. suis (Δomp25-omp25 B. suis), Δomp31, or the complemented Δomp31 strain of B. suis (Δomp31-omp31 B. suis), followed by ELISA detection of TNF-α in culture supernatants. The results are mean ± SEM of three independent experiments. **P < 0.01 versus WT B. suis-infected cells. ^##P < 0.01 versus Δomp25 B. suis-infected cells.

Figure 2

Omp25, but not Omp31, inhibits LPS-induced tumor necrosis factor (TNF)-α production in porcine alveolar macrophages (PAMs) and mouse RAW264.7 cells. (A,B) Evaluation of the expression of Omp25 and Omp31 in PAMs and RAW264.7 cells infected with LV-Blank, lentivirus expressing Omp25 (LV-Omp25), or LV-Omp31. PAMs and RAW264.7 cells were, respectively, infected with 100 multiplicities of infection (MOIs) of lentivirus for 24 h, and then treated with or without LPS for 24 h. The expression of protein was detected by western blotting. (C,D) Omp25 inhibits LPS-induced TNF-α production in PAMs and RAW264.7 cells. Cells were infected and expression of TNF-α was detected by enzyme-linked immunosorbent assay in culture supernatants. (E) Omp25 decreases the levels of TNF-α mRNA in LPS-treated PAMs and RAW264.7 cells. Cells were, respectively, infected with 100 MOIs of lentivirus for 24 h and stimulated with LPS for 6 h, Q-PCR was used to measure the levels of TNF-α mRNA. Values are mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01 versus LV-Blank-infected cells in the same processing.

Figure 3

Omp25 inhibits the transcriptional expression of tumor necrosis factor (TNF)-α by suppressing NF-κB pathway activation. (A–D) Porcine alveolar macrophages (PAMs) and RAW264.7 cells were, respectively, transfected with pCI-neo, pCI-neo-Omp31, or pCI-neo-Omp25 along with TNF-α or NF-κB luciferase reporter plasmids for 24 h; cells were stimulated with or without LPS for another 24 h, and TNF-α promoter activities (A,B) and the relative transcriptional activities of NF-κB (C,D) were examined. (E,F) PAMs and RAW264.7 cells were infected with 100 multiplicities of infection of LV-Blank, lentivirus expressing Omp25 (LV-Omp25), or LV-Omp31 for 24 h and the expression levels of cytoplasmic p-IκB, IκB, or p65 and nucleoprotein p65 at 0, 0.5, 1, and 3 h following LPS stimulation were determined by western blotting. The results are mean ± SEM of three independent experiments. *P < 0.05 versus LV-Blank-infected cells; ^#P < 0.05, ^##P < 0.01 versus control (Ctrl) for same transfection.

Figure 4

Omp25 upregulates miR-130a-3p, -146a, -181a, -181b, or -301a-3p in porcine alveolar macrophages (PAMs) and miR-146a, -181a, -181b, -301a-3p, or -351-5p in mouse RAW264.7 cells. (A,B) Expression profiling of microRNAs in Omp25-expressing PAMs and mouse RAW264.7 cells. Quantitative polymerase chain reaction (Q-PCR) assay was used to measure the levels of 17 specific miRNAs normalized by RNU6B at 24 h following infection. (C–H) Q-PCR was used to measure the kinetics of miR-130a-3p, miR-146a, miR-181a, miR-181b, miR-301a-3p, and miR-155 expression in PAMs infected with LV-Blank or lentivirus expressing Omp25 (LV-Omp25). (I–N) Q-PCR was used to measure the kinetics of miR-146a, miR-181a, miR-181b, miR-301a-3p, miR-351-5p, and miR-155 expression in mouse RAW264.7 cells infected with LV-Blank or LV-Omp25. Results are mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01 versus LV-Blank-infected cells for same miRNAs or same time point.

Figure 5

Upregulation of miR-130a-3p, miR-146a, miR-181a, miR-181b, miR-301a-3p, and miR-351-5p blocks LPS-stimulated TNF-α production. (A–D) Porcine alveolar macrophages (PAMs) and mouse RAW264.7 cells were transfected with mimics control, or indicated miRNA mimics, and cells were treated with LPS for 24 h, and the levels of TNF-α protein and mRNA were detected by enzyme-linked immunosorbent assay and quantitative polymerase chain reaction, respectively. (E–H) HEK-293 cells were transfected with wild-type or mutated TNF-α 3′ untranslated region (UTR) firefly luciferase reporter plasmids along with pTK-Renilla luciferase plasmids, mimics control or different miR-mimics for 48 h, and luciferase activity was measured. (I,J) PAMs and RAW264.7 cells were transfected mimics control, or indicated miR-mimics; cells were stimulated with LPS for 24 h, and the levels of TRAF6, IRAK1 and IRAK2 were determined by western blotting. The results are mean ± SEM of three independent experiments. ^#P < 0.05, ^##P < 0.01 versus cells transfected mimics control (Ctrl).

Figure 6

Omp25-induced miR-146a and miR-351-5p inhibit the transcriptional expression of tumor necrosis factor (TNF)-α by targeting to TRAF6 and IRAK1. (A,B) Porcine alveolar macrophages (PAMs) and mouse RAW264.7 cells were infected with LV-Blank and lentivirus expressing Omp25 (LV-Omp25), and western blotting was used to determine the expressions of TRAF6, IRAK1, and IRAK2 at 0, 24, and 48 h. (C,D) PAMs and mouse RAW264.7 cells were transfected with anti-miRNA control or indicated anti-miRNAs; then, cells were infected with LV-Blank or LV-Omp25 for 24 h, following LPS stimulation for another 1 h, and cells were lysed and examined for TRAF6, IRAK1, IRAK2, p-IκB, and IκB by western blotting. (E,F) Cells were transfected with anti-miRNA control or indicated anti-miRNAs and then infected with LV-Blank or LV-Omp25 for 24 h; following LPS treatment for 6 h, quantitative polymerase chain reaction was used to measure the level of TNF-α mRNA. The results are mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01 versus LV-Blank-infected cells; ^##P < 0.01 versus LV-Omp25-infected cells with anti-control (Anti-Ctrl).

Figure 7

miR-146a, miR-181a, and miR-301a-3p participate in the regulation of tumor necrosis factor (TNF)-α in both porcine alveolar macrophages (PAMs) and mouse RAW264.7 cells, whereas miR-130a-3p and miR-351-5p differentially regulate TNF-α expression in porcine and murine cells. (A,B) PAMs and mouse RAW264.7 cells were transfected with anti-control, or indicated anti-miRNA, or anti-miRNAs mix (4 miRNA inhibitors); then, cells were infected with LV-Blank or lentivirus expressing Omp25 (LV-Omp25) for 24 h, and the levels of TNF-α were measured by enzyme-linked immunosorbent assay. The results are mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01 versus LV-Blank-infected cells; ^#P < 0.05, ^##P < 0.01 versus LV-Omp25-infected cells with anti-control (Anti-Ctrl); ^&&P < 0.01 versus LV-Omp25-infected cells with anti-mix (Anti-mix).

Figure 8

Deficiency of Omp25 decreases B. suis-induced miR-130a-3p, miR-146a, miR-181a, miR-301a-3p, or miR-351-5p whereas inhibition of these miRNAs upregulates tumor necrosis factor (TNF)-α and promotes the intracellular clearance of wild-type (WT). B. suis. (A–D) Porcine alveolar macrophages (PAMs) were infected with WT B. suis or Δomp25 B. suis for 0, 6, 12, and 24 h, and quantitative polymerase chain reaction (Q-PCR) was used to analyze the levels of indicated miRNAs. (E) PAMs were transfected anti-control or anti-miRNAs mix; cells were infected with WT B. suis for 24 h, and TNF-α production was measured by enzyme-linked immunosorbent assay (ELISA). (F–I) Mouse RAW264.7 cells were infected with WT B. suis or Δomp25 B. suis for 24 h, and cells were harvested to examine the expression of indicated miRNAs by Q-PCR at 0, 6, 12, and 24 h. (J) Mouse RAW264.7 cells were treated as in (E) and followed by ELISA measurement of TNF-α in culture supernatants. (K) Cells were transfected with anti-miRNA control or anti-miRNAs mix and infected with WT B. suis for 24 h; following stimulation with LPS for another 24 h, TNF-α production was measured by ELISA. (L) Cells were transfected anti-miRNA control or anti-miRNAs mix; cells were infected with WT B. suis for 48 h and the numbers of viable intracellular bacteria were determined as described in Section “Materials and Methods.” The results are mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01 versus WT B. suis-infected cells at same time point; ^##P < 0.01 versus Anti-Ctrl.

Figure 9

Model for Brucella Omp25 modulation of tumor necrosis factor (TNF)-α suppression in porcine and murine macrophages. Brucella Omp25 inhibits LPS-induced TNF-α at the transcriptional levels via miR-146a (in both porcine and murine macrophages), or miR-351-5p (in murine macrophages) by targeting TRAF6 or IRAK1 to inhibit the activation of NF-κB signaling pathway. At the posttranscriptional levels, Brucella Omp25 inhibits LPS-induced TNF-α via upregulating miR-181a and miR-301a-3p (in both porcine and murine macrophages), or miR-130a-3p (in porcine macrophages), or miR-351-5p (in murine macrophages).