Meiyu Li

MicroRNA-155 Promotes Autophagy to Eliminate Intracellular Mycobacteria by Targeting Rheb

Mycobacterium tuberculosis is a hard-to-eradicate intracellular pathogen that infects one-third of the global population. It can live within macrophages owning to its ability to arrest phagolysosome biogenesis. Autophagy has recently been identified as an effective way to control the intracellular mycobacteria by enhancing phagosome maturation. In the present study, we demonstrate a novel role of miR-155 in regulating the autophagy-mediated anti-mycobacterial response. Both in vivo and in vitro studies showed that miR-155 expression was significantly enhanced after mycobacterial infection. Forced expression of miR-155 accelerated the autophagic response in macrophages, thus promoting the maturation of mycobacterial phagosomes and decreasing the survival rate of intracellular mycobacteria, while transfection with miR-155 inhibitor increased mycobacterial survival. However, macrophage-mediated mycobacterial phagocytosis was not affected after miR-155 overexpression or inhibition. Furthermore, blocking autophagy with specific inhibitor 3-methyladenine or silencing of autophagy related gene 7 (Atg7) reduced the ability of miR-155 to promote autophagy and mycobacterial elimination. More importantly, our study demonstrated that miR-155 bound to the 3′-untranslated region of Ras homologue enriched in brain (Rheb), a negative regulator of autophagy, accelerated the process of autophagy and sequential killing of intracellular mycobacteria by suppressing Rheb expression. Our results reveal a novel role of miR-155 in regulating autophagy-mediated mycobacterial elimination by targeting Rheb, and provide potential targets for clinical treatment.

TREM-2 Promotes Host Resistance Against Pseudomonas aeruginosa Infection by Suppressing Corneal Inflammation via a PI3K/Akt Signaling Pathway

PURPOSE To explore the role of triggering receptor expressed on myeloid cells 2 (TREM-2) in Pseudomonas aeruginosa (PA) keratitis. METHODS BALB/c mice were routinely infected with PA and evaluated at various postinfection time points for corneal expression of TREM-2, by real-time PCR, Western blot, and flow cytometry. Next, BALB/c and C57BL/6 mice were respectively treated with TREM-2 siRNA or agonistic anti-TREM-2 antibody, to determine the role of TREM-2 in PA keratitis. Bacterial load and neutrophil infiltration were tested by plate count and myeloperoxidase assay, respectively. Th1-/Th2-type and proinflammatory cytokine expression were tested by real-time PCR and ELISA after in vivo and in vitro silencing of TREM-2. Moreover, phosphorylated Akt levels were tested by Western blot in murine macrophages after treatment with agonistic anti-TREM-2 antibody. mRNA levels of proinflammatory cytokines were examined in murine macrophages after TREM-2 activation and lipopolysaccharide stimulation, following pretreatment with inhibitors for PI3K or Akt, to determine whether PI3K/Akt is required in TREM-2-mediated immune modulation. In addition, BALB/c mice were treated with wortmannin and analyzed for bacterial load and proinflammatory cytokine expression. RESULTS TREM-2 expression was elevated in the infected BALB/c corneas at 3 or 5 days postinfection. Silencing of TREM-2 accelerated disease progression by enhancing bacterial load and corneal inflammation, whereas activation of TREM-2 promoted host resistance to PA keratitis. PI3K/Akt signaling is required in the TREM-2-mediated immune modulation, and inhibition of PI3K resulted in worsened disease after PA corneal infection. CONCLUSIONS TREM-2 promoted host resistance to PA infection by suppressing corneal inflammation via activation of the PI3K/Akt pathway.

MicroRNA-155 induction by Mycobacterium bovis BCG enhances ROS production through targeting SHIP1.

Macrophages play a critical role in the host immune response against mycobacterial infection. Our previous study has demonstrated that microRNA-155 (miR-155), one of the most important small non-coding RNAs in the immune system, promotes oxygen-independent mycobacterial killing in macrophages. However, little is known regarding the role of miR-155 in modulating oxygen-dependent mycobactericidal response in macrophages, including the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). In the present study, we demonstrated that miR-155 was increased in macrophages after Mycobacterium bovis bacille Calmette-Guérin (BCG) infection. Moreover, the BCG-induced upregulation of miR-155 in macrophages was dependent on TLR2, NF-κB and JNK signaling pathways. More importantly, our study explored that miR-155 significantly elevated ROS production in macrophages, although miR-155 had no influence on the inducible nitric oxide synthase (iNOS) expression or nitric oxide (NO) production. In addition, our study demonstrated that miR-155 repressed the expression of src homology 2 (SH2) containing inositol 5-phosphatase1 (SHIP1), and knockdown of SHIP1 greatly increased ROS production in BCG-infected macrophages. Collectively, these data indicate that miR-155 modulates ROS but not RNS production by targeting SHIP1, which may provide a better understanding of the host anti-mycobacterial response.

microRNA-146a promotes mycobacterial survival in macrophages through suppressing nitric oxide production.

Macrophages play a crucial role in host innate anti-mycobacterial defense, which is tightly regulated by multiple factors, including microRNAs. Our previous study showed that a panel of microRNAs was markedly up-regulated in macrophages upon mycobacterial infection. Here, we investigated the biological function of miR-146a during mycobacterial infection. miR-146a expression was induced both in vitro and in vivo after Mycobacterium bovis BCG infection. The inducible miR-146a could suppress the inducible nitric oxide (NO) synthase (iNOS) expression and NO generation, thus promoting mycobacterial survival in macrophages. Inhibition of endogenous miR-146a increased NO production and mycobacterial clearance. Moreover, miR-146a attenuated the activation of nuclear factor κB and mitogen-activated protein kinases signaling pathways during BCG infection, which in turn repressed iNOS expression. Mechanistically, miR-146a directly targeted tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) at post-transcriptional level. Silencing TRAF6 decreased iNOS expression and NO production in BCG-infected macrophages, while overexpression of TRAF6 reversed miR-146a-mediated inhibition of NO production and clearance of mycobacteria. Therefore, we demonstrated a novel role of miR-146a in the modulation of host defense against mycobacterial infection by repressing NO production via targeting TRAF6, which may provide a promising therapeutic target for tuberculosis.

Pseudomonas aeruginosa Triggers Macrophage Autophagy To Escape Intracellular Killing by Activation of the NLRP3 Inflammasome

ABSTRACT Assembly of the inflammasome has recently been identified to be a critical event in the initiation of inflammation. However, its role in bacterial killing remains unclear. Our study demonstrates that Pseudomonas aeruginosa infection induces the assembly of the NLRP3 inflammasome and the sequential secretion of caspase1 and interleukin-1β (IL-1β) in human macrophages. More importantly, activation of the NLRP3 inflammasome reduces the killing of P. aeruginosa in human macrophages, without affecting the generation of antimicrobial peptides, reactive oxygen species, and nitric oxide. In addition, our results demonstrate that P. aeruginosa infection increases the amount of the LC3-II protein and triggers the formation of autophagosomes in human macrophages. The P. aeruginosa-induced autophagy was enhanced by overexpression of NLRP3, ASC, or caspase1 but was reduced by knockdown of these core molecules of the NLRP3 inflammasome. Treatment with IL-1β enhanced autophagy in human macrophages. More importantly, IL-1β decreased the macrophage-mediated killing of P. aeruginosa, whereas knockdown of ATG7 or Beclin1 restored the IL-1β-mediated suppression of bacterial killing. Collectively, our study explores a novel mechanism employed by P. aeruginosa to escape from phagocyte killing and may provide a better understanding of the interaction between P. aeruginosa and host immune cells, including macrophages.

Mesenchymal stem cells detect and defend against gammaherpesvirus infection via the cGAS-STING pathway

Mesenchymal stem cells (MSCs) are widely used in clinical settings to treat tissue injuries and autoimmune disorders due to their multipotentiality and immunomodulation. Long-term observations reveal several complications after MSCs infusion, especially herpesviral infection. However, the mechanism of host defense against herpesviruses in MSCs remains largely unknown. Here we showed that murine gammaherpesvirus-68 (MHV-68), which is genetically and biologically related to human gammaherpesviruses, efficiently infected MSCs both in vitro and in vivo. Cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) was identified as the sensor of MHV-68 in MSCs for the first time. Moreover, the cytosolic DNA sensing pathway mediated a potent anti-herpesviral effect through the adaptor STING and downstream kinase TBK1. Furthermore, blockade of IFN signaling suggested that cytosolic DNA sensing triggered both IFN-dependent and -independent anti-herpesviral responses. Our findings demonstrate that cGAS-STING mediates innate immunity to gammaherpesvirus infection in MSCs, which may provide a clue to develop therapeutic strategy.

Macrophage-mediated inflammatory response decreases mycobacterial survival in mouse MSCs by augmenting NO production.

Mycobacterium tuberculosis (MTB) is a hard-to-eradicate intracellular microbe, which escapes host immune attack during latent infection. Recent studies reveal that mesenchymal stem cells (MSCs) provide a protective niche for MTB to maintain latency. However, the regulation of mycobacterial residency in MSCs in the infectious microenvironment remains largely unknown. Here, we found that macrophage-mediated inflammatory response during MTB infection facilitated the clearance of bacilli residing in mouse MSCs. Higher inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production were observed in mouse MSCs under macrophage-mediated inflammatory circumstance. Blocking NO production in MSCs increased the survival of intracellular mycobacteria, indicating NO-mediated antimycobacterial activity. Moreover, both nuclear factor κB (NF-κB) and Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways were involved in iNOS expression and NO production in inflammatory microenvironment. Furthermore, pro-inflammatory cytokine interleukin-1β could trigger NO production in MSCs and exert anti-mycobacterial activity via NF-κB signaling pathway. Neutralization of interleukin-1β in macrophage-mediated inflammatory microenvironment dampened the ability of mouse MSCs to produce NO. Together, our findings demonstrated that macrophage-mediated inflammatory response during mycobacterial infection promotes the clearance of bacilli in mouse MSCs by increasing NO production, which may provide a better understanding of latent MTB infection.

Corrigendum: microRNA-146a promotes mycobacterial survival in macrophages through suppressing nitric oxide production

Scientific Reports 6: Article number: 23351;10.1038/srep23351 published online: March302016; updated: April222016 This Article contains typographical errors in a grant number in the Acknowledgements section. “This work was supported by grants National Natural Science Foundation of China (331470877, 81172811),” should read: “This work was supported by grants National Natural Science Foundation of China (31470877, 81172811),”