Molecular Pathogenesis

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

Xiaoshu Zuo, Lin Wang, Yanqing Bao, Jianjun Sun
Sabine Ehrt, Editor
DOI: 10.1128/IAI.00088-20

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.

INTRODUCTION

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains a major threat to public health. M. tuberculosis infects one-third of the world’s population and kills more than 1 million people each year, which ranks it as the leading cause of death as a single infectious agent (1, 2). Both innate and adaptive immune responses are required for host control of M. tuberculosis infection (3, 4). After the aerosolized droplets containing M. tuberculosis are inhaled into the lung, M. tuberculosis is internalized into the phagosomes of alveolar macrophages through phagocytosis. The ability of M. tuberculosis to penetrate the phagosome membrane and translocate into the cytosol is dependent on the ESX-1-secreted virulence factors EsxA, or the 6-kDa early-secreted antigenic target (ESAT-6), and EsxB, or the 10-kDa culture filtrate protein (CFP-10), presumably through the membrane-lytic activity of EsxA (5, 6). Several studies suggest that EsxA membrane-permeabilizing activity is the major determinant of the virulence phenotypes of mycobacterial species (7–10). Not only is EsxA recognized as a virulence factor essential for M. tuberculosis pathogenesis, but it is also found to be an immune modulator by inhibiting macrophage activation and promoting an inflammatory response (11).

Studies of the antimycobacterial responses in macrophages isolated from patients with active or latent TB as well as healthy individuals have revealed significantly altered gene expression profiles (12–14). These alterations might be regulated by microRNAs (miRNAs), a group of small noncoding RNAs that have been identified to be important regulators of gene expression at the posttranscriptional level (15, 16). For instance, miRNAs have been found to control the expression of multiple protein-encoding genes in the immune system and have significant influences on pathogenesis, diagnosis, and the treatment of infectious diseases (17–21). Some miRNAs have been reported to be involved in the regulation of host immunity during M. tuberculosis infection (22, 23). In vitro and clinical studies have found that miR-29 was overexpressed in mycobacterial infections, which downregulated gamma interferon (15, 24, 25). miR-223 and miR-424 were highly expressed in peripheral blood mononuclear cells from patients with active TB (12). Interestingly, some of the miRNAs are up- or downregulated in an EsxA-dependent manner. M. tuberculosis infection (EsxA antigen exposure) of murine macrophages upregulated miR-155, which inhibited the expression of two innate immunomodulators, interleukin-6 (IL-6) and cyclooxygenase 2 (26). M. tuberculosis strain H37Rv induced miR-155 expression in cultured murine macrophages in an EsxA-dependent manner (27). M. tuberculosis infection downregulated miR-let7f in an EsxA-dependent manner (28). Most recently, we have presented evidence that M. marinum downregulated miR-148a-3p (now termed miR-148) in macrophages in an EsxA-dependent manner (29). Interestingly, it has been reported that miR-147-3p (now termed miR-147) is highly induced in TB patients (15). The upregulation of miR-147 inhibited the expression of proinflammatory cytokines (such as tumor necrosis factor alpha [TNF-α] and IL-6), indicating that miR-147 has potent anti-inflammatory properties and functions as a negative regulator of Toll-like receptor (TLR)/NF-κB-mediated proinflammatory cytokines (30). Therefore, we hypothesize that miR-147 is involved in mycobacterial infection by acting as a negative regulator of the inflammatory response. Here, we found that a Mycobacterium marinum strain with a deletion of esxBA (M. marinum ΔesxBA) upregulated miR-147 to a level that was significantly higher than that for wild-type (WT) M. marinum, suggesting that the Esx-1 system is the negative regulator of miR-147. Moreover, the overexpression of miR-147 reduced the production of inflammatory cytokines (such as IL-6 and IL-10) and inhibited mycobacterial intracellular survival.

RESULTS

M. marinum ΔesxBA infection induced a significantly higher level of miR-147 expression than M. marinum WT infection in macrophages.Since miR-147 has been reported to enhance anti-TB immunity and acts as a negative regulator of the immune response against TB (31–33), here we tested how this miRNA responds to M. marinum infection in macrophages in the presence or absence of EsxBA. RAW264.7 cells were infected with M. marinum WT, M. marinum ΔesxBA, and M. marinum ΔesxBA/pesxBA (the M. marinum ΔesxBA strain complemented by esxB esxA) at a multiplicity of infection (MOI) of 10 for 6 and 12 h. The miR-147 transcription level at 6 and 12 h postinfection (hpi) was measured by quantitative reverse transcription-PCR (qRT-PCR) (Fig. 1A). At 6 hpi, the M. marinum WT, M. marinum ΔesxBA, and M. marinum ΔesxBA/pesxBA strains showed miR-147 levels similar to those for the noninfected control (for which the level was set equal to 1). At 12 hpi, M. marinum WT and M. marinum ΔesxBA/pesxBA induced an ∼4-fold increase in the miR-147 level, while M. marinum ΔesxBA induced an ∼8-fold increase (P < 0.01), indicating that the ESX-1 system plays a role in the downregulation of miR-147 at later times of infection. Both IL-6 and IL-10 have been shown to play important roles in M. tuberculosis infection. In response to M. tuberculosis infection, IL-6 is upregulated to enhance the killing of mycobacteria (34–36). IL-10 has also been reported to be an important regulator in response to M. tuberculosis infection not only in directing the adaptive immune responses but also in directing the innate immune responses, such as macrophage apoptosis (37–39). When miR-147 was upregulated in murine macrophages, the expression of proinflammatory cytokines, such as IL-6 and TNF-α, was attenuated (30). miR-147 was found to be negatively regulated in the IL-10-mediated anti-inflammatory response (40). Thus, we predict that the production of IL-6 and IL-10 is inversely correlated to miR-147 expression. Consistent with the findings of the above-mentioned studies, M. marinum WT or M. marinum ΔesxBA/pesxBA stimulated an ∼10-fold increase in IL-6 production compared with that for the noninfected control (for which the level was set equal to 1) at 6 hpi, while M. marinum ΔesxBA induced an ∼25-fold increase (P < 0.01) (Fig. 1B). At 12 hpi, however, M. marinum WT or M. marinum ΔesxBA/pesxBA induced an ∼25-fold increase in IL-6 production, while M. marinum ΔesxBA induced only an ∼2-fold increase (P < 0.0001) (Fig. 1B). Similarly, M. marinum WT or M. marinum ΔesxBA/pesxBA induced an ∼8-fold increase in IL-10 production compared to that for the noninfected control at 6 hpi, while M. marinum ΔesxBA induced an ∼12-fold increase (P < 0.01 and P < 0.001, respectively) (Fig. 1C). At 12 hpi, M. marinum WT or M. marinum ΔesxBA/pesxBA induced an ∼6-fold increase in IL-10 production, while M. marinum ΔesxBA induced an ∼2-fold increase in IL-10 production (P < 0.01 and P < 0.05, respectively) (Fig. 1C). Thus, it is clear that at 12 hpi, the production of IL-6 and IL-10 is inversely related to the expression of miR-147, which is negatively regulated by the ESX-1 system. This result is consistent with previous reports that M. tuberculosis induces potent IL-6 and IL-10 production in macrophages in an ESX-1-dependent manner (41, 42).

FIG 1
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.

The purified M. tuberculosis EsxA or EsxB protein or EsxBA heterodimer had differential effects on miR-147 expression in RAW264.7 cells.In order to dissect the effects of EsxA and EsxB on the expression of miR-147, the M. tuberculosis EsxA and EsxB proteins and the EsxBA heterodimer were purified from M. smegmatis and incubated with RAW264.7 cells. The miR-147 level in the EsxA-treated cells had no obvious change at 3 h from that in the control group (not treated with a protein) and the group treated with bovine serum albumin (BSA; an unrelated protein) (Fig. 2A); this was followed by an increase at 6 h and then a sharp decrease at 9 h and 12 h (P < 0.01 and P < 0.001, respectively) (Fig. 2A). Similarly, in the EsxB-treated cells, the miR-147 level was stable at 3 h but increased ∼2-fold at 6 h, followed by a significant decrease at 9 h and 12 h (P < 0.01) (Fig. 2B). Interestingly, the miR-147 level in the EsxBA heterodimer-treated cells was stable at 3 h but decreased from 6 to 12 h (P < 0.05, P < 0.01, and P < 0.01, respectively) (Fig. 2C). The cells treated with the unrelated protein BSA did not show a significant change in miR-147 levels over time (Fig. 2D). The data suggest that EsxA and EsxB upregulated miR-147 at the early time point of treatment (6 h) but inhibited miR-147 at the later time points of treatment (9 to 12 h). Interestingly, EsxBA, which is presumably the physiological form secreted through the ESX-1 system, inhibited miR-147 from 6 h to 12 h. Thus, these data are consistent with the data presented in Fig. 1.

FIG 2
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.

The mimic and the inhibitor of miR-147 up- and downregulated miR-147 expression, respectively.To modulate miR-147 expression, RAW264.7 cells were transiently transfected with an miR-147 mimic (final concentrations, 50 nM and 100 nM) or inhibitor (final concentrations, 100 nM and 150 nM) for 24 h. mmu-miR-147-3p is the miR base identifier, and mmu indicates that the miRNA is from the mouse (hsa refers to miRNA from human). miR-147 mimics are small, double-stranded RNAs that mimic endogenous precursor miR-147. One strand is identical to and effectively mimics mature miR-147. miR-147 mimics can overexpress endogenous miR-147 for enhanced performance in gain-of-function analysis. miR-147 inhibitors are single-stranded RNA-based oligonucleotides that are designed to bind and inhibit the activity of endogenous miR-147 when introduced into cells, which means that miR-147 inhibitors can inhibit the expression of endogenous miR-147. The mimics and inhibitors of miRNAs were designed and synthesized by Life Technologies (Invitrogen). The catalog numbers are included in Materials and Methods. As expected, the miR-147 mimic at concentrations of 50 nM and 100 nM upregulated miR-147 expression in a dose-dependent manner with ∼5,000- and ∼7,000-fold increases, respectively (P < 0.0001) (Fig. 3A), while the inhibitor downregulated miR-147 expression by ∼30% (P < 0.0001) (Fig. 3B).

FIG 3
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.

Up- and downregulation of miR-147 inversely regulated M. marinum intracellular survival.Next, we tested the effects of miR-147 on M. marinum intracellular survival. First, in the absence of the miR-147 mimic and inhibitor, M. marinum ΔesxBA induced a higher level of miR-147 expression than M. marinum WT and M. marinum ΔesxBA/pesxBA (P < 0.01) (Fig. 4A), which is consistent with the result in Fig. 1. In the presence of the miR-147 mimic, the miR-147 levels in cells infected with M. marinum WT, M. marinum ΔesxBA, and M. marinum ΔesxBA/pesxBA were all significantly increased (P < 0.0001) (Fig. 4A), and among these cells, the miR-147 level in cells infected with M. marinum ΔesxBA was significantly higher than that in cells infected with M. marinum WT and M. marinum ΔesxBA/pesxBA (P < 0.001 and P < 0.01, respectively) (Fig. 4A). This once again supports the results presented in Fig. 1, suggesting that the presence of esxBA has an inhibitory effect on the expression of miR-147, while in the presence of the inhibitor, the miR-147 levels in cells infected with M. marinum WT, M. marinum ΔesxBA, and M. marinum ΔesxBA/pesxBA generally showed a trend toward being lower than those in infected cells without inhibitors (Fig. 4A). The miR-147 mimic inhibited the production of the inflammatory factors IL-6 and IL-10, while the miR-147 inhibitor induced levels of production of IL-6 and IL-10 higher than those induced by the mimic (Fig. 4B and C). The result is consistent with the findings of previous studies that miR-147 is a negative regulator of IL-6 and TNF-α (30). Finally, transfection of the miR-147 mimic significantly reduced the intracellular survival of M. marinum, M. marinum ΔesxBA, and M. marinum ΔesxBA/pesxBA compared to that achieved by infection with those strains without the miR-147 mimic (P < 0.0001) (Fig. 4D). Transfection of the inhibitor modestly increased M. marinum WT intracellular survival but did not induce a significant increase in the intracellular survival of M. marinum ΔesxBA and M. marinum ΔesxBA/pesxBA (Fig. 4D), which could have been due to the low inhibition efficiency of the inhibitor for miR-147 expression (Fig. 3B).

FIG 4
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.

Effects of coregulation of miR-147 and miR-148 on mycobacterial survival in M. marinum-infected cells.Our previous study showed that the upregulation of miR-148 inhibits M. marinum intracellular survival in macrophages (29). It was thus of interest to test the effects of the coregulation of miR-148 and miR-147 on M. marinum intracellular survival. The coregulation of miR-148 and miR-147 was achieved by cotransfection of the cells with the mimics and inhibitors of miR-148 and miR-147 in the combinations indicated in Fig. 5A. Cotransfection of both inhibitors significantly affected the levels of the two miRNAs (P < 0.01 and P < 0.001), while cotransfection of both mimics dramatically upregulated the two miRNAs simultaneously, with a 4 × 105-fold increase for miR-148 and an ∼7,000-fold increase for miR-147 (P < 0.0001). When the miR-147 mimic and miR-148 inhibitor were cotransfected into the cells, the miR-147 level was increased and the miR-148 level was decreased (P < 0.001). Similarly, cotransfection of the miR-148 mimic and the miR-147 inhibitor resulted in the upregulation of miR-148 and the downregulation of miR-147 (P < 0.001 and P < 0.01). These results suggest that the mimics and inhibitors are highly specific to their own miRNA targets and do not have significant cross-interference. Subsequently, we tested the effects of the coregulation of miR-147 and miR-148 on M. marinum intracellular survival (Fig. 5B). As expected, transfection of the miR-147 mimic or the miR-148 mimic alone significantly inhibited M. marinum intracellular survival (P < 0.01). Interestingly, cotransfection of both mimics did not show an enhanced inhibitory effect compared to that achieved with the transfection of a single mimic (P > 0.05). Transfection of a single inhibitor or cotransfection of both inhibitors did not significantly affect M. marinum intracellular survival. Interestingly, the inhibitor of one miRNA reciprocally eliminated the inhibitory effect induced by the mimic of the other miRNA, suggesting that the two miRNAs share common downstream pathways in response to M. marinum infection.

FIG 5
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.

miR-147 negatively regulated TLR4/NF-κB expression in M. marinum-infected cells.As one of the major receptors for pathogen-associated molecular patterns, TLR4 is involved in macrophage responses that influence both innate and adaptive immunity in many bacterial infections (43–45). A previous study has shown that the C terminus of EsxA mediates its inhibitory effects on TLR signaling, leading to the inhibition of NF-κB activation (46). Our previous study showed that miR-148 inhibits TLR4-mediated NF-κB activation (29). Thus, we hypothesize that the up- or downregulation of miR-147 affects the TLR4/NF-κB signaling pathway. The effects of miR-147 on the expression of TLR4 in M. marinum-infected RAW264.7 cells were detected by Western blotting (Fig. 6). 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. The expression of TLR4 was detected by Western blotting (Fig. 6A and B). M. marinum infection markedly increased the expression of TLR4, but transfection of the miR-147 mimic inhibited it, indicating that miR-147 downregulates TLR4 upon M. marinum infection. Consistent with the results in Fig. 6A, NF-κB p65, the downstream factor of TLR4 in the NF-κB pathway, was activated upon M. marinum infection, as evidenced by an increase in the level of phosphorylation, while the overexpression of miR-147 downregulated the phosphorylation of NF-κB p65 (Fig. 6C and D).

FIG 6
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.

DISCUSSION

As the key virulence factor essential for M. tuberculosis pathogenesis, EsxA is believed to antagonize the host defense system by inhibiting phagosome maturation and translocating M. tuberculosis to the cytosol for replication and cell-to-cell spread (6, 8, 9, 47–58). However, the role of EsxA in mediating phagosome rupture and mycobacterial cytosolic translocation has been questioned by two recent studies (59, 60). In a report from Conrad et al. (59), the authors successfully repeated our previous in vitro experiments and confirmed that the detergent-free EsxA disrupted liposomal membranes under acidic conditions (6, 61–63), but they found that M. marinum was still able to penetrate the phagosome and translocate to the cytosol in the presence of bafilomycin, a reagent that inhibits intracellular acidification. That study indicated that phagosome rupture does not occur through the acidic pH-dependent membrane permeabilizing activity of EsxA (59). Most recently, Lienard et al. employed a collection of M. marinum ESX-1 transposon mutants, including mutants that disrupt EsxA secretion, to infect macrophages and showed that the transposon mutants without EsxA secretion were still able to permeabilize phagosomes, suggesting that other factors independent of EsxAB play a role in cytosolic translocation (60). Since the EsxBA heterodimer is secreted through the ESX-1 system together with several codependently secreted factors (e.g., EspA, EspC, and, probably, EspB) (64–66), we cannot exclude the possibility that deletion of esxB-esxA affects the codependently secreted factors, which may be directly responsible for phagosome rupture and cytosolic translocation. The exact role of EsxBA and other factors warrants future studies.

EsxA also functions as a modulator by inhibiting macrophage activation and promoting inflammatory responses (5–11). Recently, miRNAs have been identified to be crucial biological regulators of gene expression at the posttranscriptional level, and their emerging roles in regulating immune responses by regulating cytokine production have attracted increasing attention (16). Numerous miRNAs have been found to be involved in mycobacterial infection, either enhancing or inhibiting infections through immune modulation (25, 27–29, 67–69). Moreover, several miRNAs, such as miR-155, miR-1et7f, and miR-148a, have been shown to respond to mycobacterial infection in an EsxA-dependent manner (27–29). In the present study, we characterized the role of miR-147, a negative regulator of proinflammatory responses, in M. marinum infection. We found that miR-147 is negatively regulated by the ESX-1 system and that the upregulation of miR-147 inhibits the production of IL-6 and IL-10, M. marinum intracellular survival, and the TLR4/NF-κB signaling pathway.

As the major target of M. tuberculosis, macrophages produce a complex pattern of inflammatory cytokines in M. tuberculosis infection. The pro- and anti-inflammatory cytokines stimulated by M. tuberculosis infection are believed to be double-edged swords (70). An earlier study showed that miR-147 has potent anti-inflammatory properties and functions as a negative regulator of proinflammatory cytokines (TNF-α and IL-6) (30, 34–36). IL-10, as an important regulatory factor, was highly secreted in immune regulation during M. tuberculosis infection (37–39). In our study, we found that the difference in miR-147 expression between the three groups was not obvious at the early stage of infection (Fig. 1A), while the expression of cytokines showed a difference at 6 hpi (Fig. 1B and C). At 12 hpi, miR-147 expression increased significantly, while IL-6 and IL-10 levels decreased sharply in the M. marinum ΔesxBA group compared with those in the M. marinum WT or M. marinum ΔesxBA/pesxBA group. There was an inverse correlation between miR-147 expression and IL-6 and IL-10 levels at 12 hpi, but this was not true at 6 hpi (Fig. 1). Consistent with the previous results, our present study found that the upregulation of miR-147 downregulates inflammatory cytokines (IL-6 and IL-10) and inhibits mycobacterial intracellular survival (Fig. 4). The recombinant M. tuberculosis EsxA protein purified from M. smegmatis has been reported to interfere with the immune response in macrophages (71, 72). In our study, the genes encoding M. tuberculosis EsxA and EsxB were amplified by PCR from a genomic DNA extract of M. tuberculosis (H37Rv) (6). The pMyNT plasmid, containing the native operon of M. tuberculosis esxB::esxA was transformed into Mycobacterium smegmatis. The detergent-free recombinant proteins EsxA and EsxB and the EsxBA heterodimer were all purified from M. smegmatis, as previously described (29), and were used to challenge the macrophages. The results showed that EsxA, EsxB, and the EsxBA heterodimer could directly regulate miR-147 expression over time (Fig. 2).

M. marinum infection downregulated both miR-147 and miR-148 in an ESX-1-dependent manner, and overexpression of either miR-147 or miR-148 inhibited M. marinum intracellular survival. We hypothesize that miR-147 and miR-148 may share common downstream pathways in response to M. marinum infection. When the mimics and inhibitors of both miRNAs were cotransfected in various combinations, we found that the mimics and inhibitors specifically targeted their corresponding miRNAs with little cross-reactivity (Fig. 5A). Compared to the transfection of either of the two miRNA mimics, cotransfection of the mimics for both miRNAs did not enhance the inhibitory effect on M. marinum intracellular survival, suggesting that the inhibition effect is saturated (Fig. 5B). Relative to the survival seen by transfection of a single inhibitor, cotransfection of both inhibitors did not significantly increase M. marinum intracellular survival, suggesting a similar saturation effect. Interestingly, cotransfection of any combination of a mimic and an inhibitor eliminated the mimic-induced inhibitory effect. Thus, the inhibitor of one miRNA functions as a dominant negative inhibitor of the mimic of another miRNA, indicating that miR-147 and miR-148 share common downstream pathways. In line with this notion, the overexpression of miR-147 downregulated the NF-κB signaling pathway, evidenced by reducing the TLR4 expression and phosphorylation of NF-κB p65 (Fig. 6), which is consistent with the results obtained in a previous study showing that the upregulation of miR-148 inhibits the TLR4/NF-κB pathway (29).

Only a few studies have reported on the combinatorial effects of multiple miRNAs within cellular systems. For instance, Forrest et al. (73) have reported a model whereby multiple miRNAs are induced in conjunction with various transcription factors to cooperatively promote differentiation and inhibit cellular proliferation. Zhang et al. (74) have reported that miR-143, miR-218, and miR-338-3p have coregulatory effects on the metastasis and invasion of gastric cancer. Another study explored the combination of miR-143 and miR-145 as a potential biomarker for the earlier diagnosis and prognosis of esophageal cancer (75). Based on our best knowledge, the present study is the first to characterize the combinatory effects of two miRNAs in mycobacterial infection. The mechanism of combinatory effects of the mimics and inhibitors for both miRNAs warrants further studies.

MATERIALS AND METHODS

M. marinum infection of macrophages.M. marinum WT, M. marinum ΔesxBA, and M. marinum ΔesxBA/pesxBA (the complemented M. marinum ΔesxBA strain) were grown in Middlebrook 7H9 liquid medium supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC) at 30°C and 150 rpm with shaking. RAW264.7 macrophages (ATCC) were cultured in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum in 5% CO2 at 37°C. The RAW264.7 cells were infected with M. marinum WT, M. marinum ΔesxBA, or M. marinum ΔesxBA/pesxBA at an MOI of 10. At 6 and 12 h postinfection (hpi), the cells were collected and subjected to qRT-PCR and Western blot analysis.

Protein expression and purification.The pMyNT plasmid containing the native operon of M. tuberculosis esxB::esxA with a His6 tag at the N terminus of EsxB was a generous gift from Matthias Wilmanns (76–78). The plasmid was transformed into Mycobacterium smegmatis by electroporation at 2.5 kV. Protein expression and purification were done as previously described (29). The bacterial colonies were grown in 7H9 medium plates supplemented with 10% OADC, 0.5% glycerol, and 100 μg/ml of hygromycin. A single colony was picked up and precultured in 50 ml of 7H9 medium supplemented with 10% OADC, 0.5% glycerol, 0.05% Tween 80, and 100 μg/ml of hygromycin with shaking at 37°C. The preculture was transferred to 1 liter of 7H9 medium supplemented with 0.2% glycerol, 0.05% Tween 80, 0.2% glucose, and 100 μg/ml of hygromycin to a final optical density at 600 nm (OD600) of 0.05 with shaking at 37°C. Protein expression was induced at an OD600 of 1.5 by adding acetamide (final concentration, 33 mM) for 48 h, and the cells were harvested by centrifugation. The pellet was lysed by sonication in the soluble buffer (20 mM Tris-HCl, 150 mM NaCl, 10 mM imidazole, pH 7.4), and the supernatant was collected by centrifugation at 15,200 × g for 1 h at 4°C. The supernatant was loaded onto an Ni2+-charged Sepharose column that had been preequilibrated with the soluble buffer. The bound proteins were eluted from the column by a linear gradient (10 to 100%) of the elution buffer (20 mM Tris-HCl, 150 mM NaCl, 500 mM imidazole, pH 7.4). The eluted heterodimer was further clarified by gel filtration chromatography, using a Sephadex 75 column, in gel filtration buffer (20 mM Tris-HCl, 100 mM NaCl, pH 7.3). The EsxBA heterodimer was treated with 6 M guanidine. The solution was then concentrated to 1 ml and injected into a 5-ml HisTrap column, which was preequilibrated with 25 mM Tris-HCl, 150 mM NaCl, 10 mM imidazole, 6 M guanidine. The His-tagged EsxB protein was bound to the column, while EsxA flowed through the column. The EsxA protein was collected, extensively dialyzed in 20 mM Tris-HCl, 100 mM NaCl, pH 7.3, and stored at −80°C. The EsxB protein was eluted from the column by a linear gradient (10 to 100%) of the elution buffer (20 mM Tris-HCl, 150 mM NaCl, 500 mM imidazole, 6 M guanidine, pH 7.4). The eluted EsxB protein was dialyzed in 20 mM Tris-HCl, 100 mM NaCl, pH 7.3, and stored at −80°C.

qRT-PCR.Total RNA, including miRNA, was extracted with the TRIzol reagent (Applied Biosystems). The transcription level of the miRNA was quantified by using a TaqMan microRNA reverse transcription (RT) kit (Applied Biosystems). Two hundred nanograms of total RNA was used for the reverse transcription reaction in the miRNA-specific stem-loop in a 15-μl RT reaction mixture system following the instructions supplied with the kit (100 M. marinum bacteria, 0.15 μl deoxynucleoside triphosphates [dNTPs], 1.00 μl reverse transcriptase, 1.50 μl 10× RT buffer, 0.19 μl RNase inhibitor, 3 μl RT primer pool, 5 μl total RNA, 4.16 μl RNA-free water). The relative expression of miRNA was normalized to that of U6 by the 2−ΔΔCT cycle threshold (CT) method. For the transcription of mRNA, a commercial high-capacity cDNA reverse transcription kit (Applied Biosystems) was used. Total RNA (1,000 ng) was used for the synthesis of first-strand cDNA in a 20-μl RT reaction mixture using oligonucleotide primers according to the instructions supplied with the kit (0.8 μl 100 mM 25× dNTPs, 1.00 μl reverse transcriptase, 2 μl 10× RT buffer, 1 μl RNase inhibitor, 2 μl 10× RT random primers, 5 μl total RNA, RNase-free water to 20 μl). For the expression of IL-6 and IL-10 mRNA, 13.3 ng of cDNA for each sample was used for qRT-PCR analysis using a SYBR Select Master Mix kit (Applied Biosystems). The primers used in this study are described in Tables 1 and 2.

View this table:
TABLE 1

Cytokine-specific primers used for RT-qPCR

View this table:
TABLE 2

MicroRNA-specific primers used for RT-qPCR

Western blot analysis.RAW264.7 cells were cultured in 12-well plates overnight. The cells were transiently transfected with miR-147 mimic or inhibitor for 24 h, which was followed by M. marinum infection at an MOI of 10. At 12 hpi, the culture medium was discarded. The cells were then washed with phosphate-buffered saline (PBS) and lysed by 200 μl radioimmunoprecipitation assay buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS in PBS) with 4 μl Pierce protease inhibitor (1/2 tablet/ml). The cell lysates were incubated on ice for 30 min with periodic mixing (every 5 min). Then, 50 μl of SDS was added to each well (final SDS concentration, 70 mM). The samples were boiled at 100°C for 10 min and then stored at −20°C for use. The cellular total protein was harvested in a lysis solution containing the phosphatase repressor. The protein concentration was determined by use of a bicinchoninic acid assay kit (Pierce). The samples with the same amount of protein were applied to SDS-PAGE gels, followed by transfer to a polyvinylidene difluoride (PVDF) membrane with a transfer voltage of 100 mV for 45 to 65 min. After blocking with 5% skim milk overnight, the membrane was incubated with the corresponding primary antibodies and kept at 4°C overnight. The PVDF membrane was washed with Tris-buffered saline–Tween 20 (TBST) three times (for 5 min each time). The corresponding secondary antibodies were supplemented and incubated at room temperature for 45 min. The PVDF membranes were washed again three times (for 5 min each time) and subjected to analysis with a chemiluminescence system (SuperSignal). β-Actin was used as a loading control. The density of the target protein bands was measured with ImageJ software.

Assay for counting of the number of CFU.RAW264.7 cells were transiently transfected with the mimic (catalog number 4464066) or inhibitor (catalog number 4464084) of miR-147 or miR-148a (Invitrogen) for 24 h as described above. The culture medium was discarded and the cells were washed with 1× PBS, followed by infection with M. marinum WT, M. marinum ΔesxBA, or M. marinum ΔesxBA/pesxBA at an MOI of 10. The plates were centrifuged at 800 rpm for 10 min to allow the bacteria to settle down to the cells, and incubation was continued for 30 min at 30°C with 5% CO2. The cells were washed twice with 1× PBS and treated with amikacin (100 mg/ml) for 2 h. The culture medium was discarded, and the cells were washed with 1× PBS and incubated in the presence of a low concentration of amikacin (50 mg/ml) maintenance solution for 24 h. At 24 hpi, the culture medium was discarded and the cells were washed with 1× PBS 3 times. The infected cells were harvested and lysed with 0.1% Triton X-100. The cell lysates containing the bacteria were plated in a series of 10-fold dilutions onto Middlebrook 7H10 plates supplemented with 10% OADC and incubated at 37°C for 5 to 7 days. The colonies were counted, and the numbers of CFU were determined using standard procedures. The data were collected in triplicate for each sample. Three independent biological replicates were performed.

Statistical analysis.Statistical data are represented as the mean ± standard error of the mean from three independent experiments. The data were analyzed using Student's t test or one-way analysis of variance (ANOVA). P values of <0.05 were considered statistically significant difference.

ACKNOWLEDGMENTS

The study is supported by the grants from the National Center for Research Resources (grant 5G12RR008124) and the National Institute on Minority Health and Health Disparities (grant G12MD007592). Xiaoshu Zuo was supported by scholarships from the China Scholarship Council (grant 201806275121).

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

We declare that we have no conflicts of interest.

FOOTNOTES

    • Received 13 February 2020.
    • Returned for modification 5 March 2020.
    • Accepted 30 March 2020.
    • Accepted manuscript posted online 6 April 2020.

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KEYWORDS

Mycobacterium tuberculosis
Mycobacterium marinum
EsxA
ESAT-6
EsxB
CFP-10
miR-147
miR-147-3p

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