The ESX-1 Virulence Factors Downregulate miR-147-3p in Mycobacterium marinum-Infected Macrophages

Abstract

As important virulence factors of Mycobacterium tuberculosis, EsxA and EsxB not only play a role in phagosome rupture and M. tuberculosis cytosolic translocation but also function as modulators of host immune responses by modulating numerous microRNAs (miRNAs). Recently, we have found that mycobacterial infection downregulated miR-148a-3p (now termed miR-148) in macrophages in an ESX-1-dependent manner. The upregulation of miR-148 reduced mycobacterial intracellular survival. Here, we investigated miR-147-3p (now termed miR-147), a negative regulator of inflammatory cytokines (e.g., interleukin-6 [IL-6] and IL-10), in mycobacterial infection. We infected murine RAW264.7 macrophages with Mycobacterium marinum, a surrogate model organism for M. tuberculosis, and found that the esxBA-knockout strain (M. marinum ΔesxBA) upregulated miR-147 to a level that was significantly higher than that induced by the M. marinum wild-type (WT) strain or by the M. marinum ΔesxBA complemented strain, M. marinum ΔesxBA/pesxBA, suggesting that the ESX-1 system (potentially EsxBA and/or other codependently secreted factors) is the negative regulator of miR-147. miR-147 was also downregulated by directly incubating the macrophages with the purified recombinant EsxA or EsxB protein or the EsxBA heterodimer, which further confirms the role of the EsxBA proteins in the downregulation of miR-147. The upregulation of miR-147 inhibited the production of IL-6 and IL-10 and significantly reduced M. marinum intracellular survival. Interestingly, inhibitors of either miR-147 or miR-148 reciprocally compromised the effects of the mimics of their counterparts on M. marinum intracellular survival. This suggests that miR-147 and miR-148 share converged downstream pathways in response to mycobacterial infection, which was supported by data indicating that miR-147 upregulation inhibits the Toll-like receptor 4/NF-κB pathway.

Keywords: CFP-10; ESAT-6; EsxA; EsxB; Mycobacterium marinum; Mycobacterium tuberculosis; miR-147; miR-147-3p.

FIG 1

M. marinum ΔesxBA induced in infected macrophages elevated levels of miR-147 expression that were significantly higher than those induced by M. marinum (Mm). RAW264.7 cells were infected by M. marinum, M. marinum ΔesxBA, and M. marinum ΔesxBA/pesxBA at an MOI of 10. At 6 hpi and 12 hpi, the cell lysates were collected and subjected to qRT-PCR for quantification of the mRNA levels of miR-147 (A), IL-6 (B), and IL-10 (C). The mRNA levels in noninfected cells were set as the controls (for which the value was set equal to 1). The values on the y axes are the fold increase in mRNA levels relative to those in uninfected cells. The data were calculated from at least three independent experiments with at least three technical repeats in each experiment. The one-way ANOVA method was used to determine statistical significance. The Holm-Sidak test was used for multiple comparisons. P values of <0.05 indicate that the differences were statistically significant. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 2

The recombinant proteins EsxA and EsxB and the EsxBA heterodimer downregulated the expression of miR-147. RAW264.7 cells were incubated with 5 μg/ml of the purified proteins EsxA (A) and EsxB (B) and 10 μg/ml of the EsxBA heterodimer (C). Meanwhile, RAW264.7 cells were incubated with 5 μg/ml of bovine serum albumin (BSA) for comparison (D). At 3, 6, 9, and 12 h of incubation, the mRNA level of miR-147 was determined by qRT-PCR. The uninfected cells were used as controls (for which the value was set equal to 1). The values on the y axes are the fold increase in mRNA levels relative to those for uninfected cells. The data were calculated from at least three independent experiments with at least three technical repeats in each experiment. The one-way ANOVA method was used to determine statistical significance. The Holm-Sidak test was used for multiple comparisons. P values of <0.05 indicate that the differences were statistically significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 3

The miR-147 mimic and inhibitor up- and downregulated endogenous miR-147 expression and inflammatory factors, respectively. RAW264.7 cells were transiently transfected with the miR-147 mimic (final concentrations, 50 and 100 nM) (A) or inhibitor (final concentrations, 100 and 150 nM) (B) for 24 h. The cells were harvested, and the mRNA level of miR-147 was measured by qRT-PCR. The cells with mock transfection were set as controls. The values on the y axes are the fold increase in mRNA levels relative to those for mock-transfected cells. The data were calculated from at least three independent experiments with at least three technical repeats in each experiment. The one-way ANOVA method was used to determine statistical significance. The Holm-Sidak test was used for multiple comparisons. P values of <0.05 indicate that the differences were statistically significant. ****, P < 0.0001.

FIG 4

Effects of the miR-147 mimic and inhibitor on M. marinum intracellular survival in M. marinum-infected cells. RAW264.7 cells were transfected with the miR-147 mimic (50 nM) or miR-147 inhibitor (100 nM) for 24 h, which was followed by infection with M. marinum, M. marinum ΔesxBA, and M. marinum ΔesxBA/pesxBA at an MOI of 10 for another 12 h (A to C) and 24 h (D). (A) The cells were harvested, and the mRNA level of miR-147 was measured by qRT-PCR. (B and C) The mRNA levels of IL-6 (B) and IL-10 (C) were measured by qRT-PCR. (D) M. marinum, M. marinum ΔesxBA, and M. marinum ΔesxBA/pesxBA intracellular survival was determined by a CFU assay. The mRNA levels in the noninfected cells were set as controls (for which the value was set equal to 1). The values on the y axes are the fold increase in mRNA levels relative to those for uninfected cells. The data were calculated from at least three independent experiments with at least three technical repeats in each experiment. The one-way ANOVA method was used to determine statistical significance. The Holm-Sidak test was used for multiple comparisons. The significance of the difference relative to the results for the WT strain are indicated. P values of <0.05 indicate that the differences were statistically significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 5

Effects of cotransfection of the mimics and inhibitors of miR-147 and miR-148 on M. marinum survival in M. marinum-infected cells. RAW264.7 cells were transfected with the miR-147/miR-148 mimic (50 nM) or the miR-147/miR-148 inhibitor (100 nM) in the indicated combinations for 24 h, which was followed by infection with M. marinum at an MOI of 10 for 24 h. (A) The expression levels of miR-147 and miR-148 in the indicated transfection combinations were measured by qRT-PCR. (B) The effects of the indicated cotransfection combinations on M. marinum intracellular survival were measured by CFU assays. The cells with mock transfection were set as a control (for which the value was set equal to 1). The values on the y axes are the fold increase in mRNA levels relative to those in mock-transfected cells. The data were calculated from three independent experiments with at least three technical repeats in each experiment. The one-way ANOVA method was used to determine statistical significance. The Holm-Sidak test was used for multiple comparisons. P values of <0.05 indicate that the differences were statistically significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 6

miR-147 negatively regulated TLR4/NF-κB expression in M. marinum-infected cells. RAW264.7 cells were transfected with the miR-147 mimic (50 nM) or miR-147 inhibitor (100 nM) for 24 h, which was followed by infection with M. marinum at an MOI of 10 for 12 h. (A and B) The expression of TLR4 was detected by Western blotting (A), and the relative expression was calculated (B). (C and D) The expression of NF-κB (p-NF-κB) was detected by Western blotting (C), and the relative expression of p-NF-κB was calculated (D). β-Actin was used as an internal control. The data were calculated from three independent experiments with at least three technical repeats in each experiment. The one-way ANOVA method was used to determine statistical significance. The Holm-Sidak test was used for multiple comparisons. P values of <0.05 indicate that the differences were statistically significant. *, P < 0.05.