Minhao Wu

Activation of Toll-like receptor 3 impairs the dengue virus serotype 2 replication through induction of IFN-β in cultured hepatoma cells.

Toll-like receptors (TLRs) play an important role in innate immunity against invading pathogens. Although TLR signaling has been indicated to protect cells from infection of several viruses, the role of TLRs in Dengue virus (DENV) replication is still unclear. In the present study, we examined the replication of DENV serotype 2 (DENV2) by challenging hepatoma cells HepG2 with different TLR ligands. Activation of TLR3 showed an antiviral effect, while pretreatment of other TLR ligands (including TLR1/2, TLR2/6, TLR4, TLR5 or TLR7/8) did not show a significant effect. TLR3 ligand poly(I∶C) treatment prior to viral infection or simultaneously, but not post-treatment, significantly down-regulated virus replication. Pretreatment with poly(I∶C) reduced viral mRNA expression and viral staining positive cells, accompanying an induction of the type I interferon (IFN-β) and type III IFN (IL-28A/B). Intriguingly, neutralization of IFN-β alone successfully restored the poly(I∶C)-inhibited replication of DENV2. The poly(I∶C)-mediated effects, including IFN induction and DENV2 suppression, were significantly reversed by IKK inhibitor, further suggesting that IFN-β is the dominant factor involved in the poly(I∶C) mediated antiviral effect. Our study presented the first evidence to show that activation of TLR3 is effective in blocking DENV2 replication via IFN-β, providing an experimental clue that poly(I∶C) may be a promising immunomodulatory agent against DENV infection and might be applicable for clinical prevention.

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.

TREM‐2 Promotes Macrophage‐Mediated Eradication of Pseudomonas aeruginosa via a PI3K/Akt Pathway

Triggering receptor expressed on myeloid cells 2 (TREM‐2) is a cell surface receptor abundantly expressed on myeloid lineage cells such as macrophages and dendritic cells. It is reported that TREM‐2 functions as an inflammatory inhibitor in macrophages and dendritic cells. However, the role of TREM‐2 in bacterial killing remains unclear. This study explored the role of TREM‐2 in bacterial eradication of Pseudomonas aeruginosa (PA), a Gram‐negative bacterium which causes various opportunistic infections. Phagocytosis assay assessed by flow cytometry suggested that TREM‐2 was not involved in the uptake of PA by macrophages, while bacterial plate count data showed that TREM‐2 was required for macrophage‐mediated intracellular killing of PA. Moreover, our results demonstrated that TREM‐2 promoted macrophage killing by enhancing reactive oxygen species (ROS), but not nitric oxygen (NO) production. Treatment with N‐acetylcysteine, a ROS scavenger, diminished the TREM‐2‐mediated intracellular killing of PA. To further investigate the underlined mechanisms of TREM‐2‐promoted bacterial killing, we examined the activation of downstream mitogen‐activated protein kinases and PI3K/Akt pathway. Western blot data showed that silencing of TREM‐2 inhibited phosphorylation of Akt, but not ERK, JNK or P38. In addition, pretreatment with PI3K active product PIP3 DiC16 reversed the elevation of intracellular bacterial load in TREM‐2‐silenced macrophages, while PI3K inhibitor wortmannin restored the decline of bacterial load in TREM‐2‐overexpressed macrophages. These data together suggested that the TREM‐2‐mediated bacterial killing is dependent on the activation of PI3K/Akt signalling, which may provide a better understanding of the host antibacterial immune defence.

MRP8/14 Enhances Corneal Susceptibility to Pseudomonas aeruginosa Infection by Amplifying Inflammatory Responses

PURPOSE We explored the role of myeloid-related protein 8 and 14 (MRP8/14) in Pseudomonas aeruginosa (PA) keratitis. METHODS MRP8/14 mRNA levels in human corneal scrapes and mouse corneas infected by PA were tested using real-time PCR. MRP8/14 protein expression in C57BL/6 (B6) corneas was confirmed using Western blot assay and immunohistochemistry. B6 mice were injected subconjunctivally with siRNA for MRP8/14, and then infected with PA. Bacterial plate counts and myeloperoxidase assays were used to determine the bacterial load and polymorphonuclear neutrophil (PMN) infiltration in infected B6 corneas. Pro-inflammatory cytokine levels in vivo and in vitro were examined with PCR and ELISA. In murine macrophage-like RAW264.7 cells, phagocytosis and bacterial killing were assessed using plate count assays, and reactive oxygen species (ROS) and nitric oxide (NO) levels were tested with flow cytometry and Griess assay, respectively. RESULTS MRP8/14 expression levels were increased significantly in human corneal scrapes and B6 corneas after PA infection. Silencing of MRP8/14 in B6 corneas significantly reduced the severity of corneal disease, bacterial clearance, PMN infiltration, and pro-inflammatory cytokine expression after PA infection. In vitro studies demonstrated further that silencing of MRP8/14 suppressed pro-inflammatory cytokine production, bacterial killing, and ROS production, but not phagocytosis or NO production. CONCLUSIONS Our study demonstrated a dual role for MRP8/14 in bacterial keratitis. Although MRP8/14 promotes bacterial clearance by enhancing ROS production, it functions more importantly as an inflammatory amplifier at the ocular surface by enhancing pro-inflammatory cytokine expression, thus contributing to the corneal susceptibility.

Toll-like receptors in innate immunity and infectious diseases

The protective ability of host defense system is largely dependent on germ-line encoded pattern-recognition receptors (PRRs). These PRRs respond to a variety of exogenous pathogens or endogenous danger signals, by recognizing some highly conserved structures such as pathogen-associated molecular patterns (PAMPs) and danger/damage associated molecular patterns (DAMPs). The most studied PRRs are Toll-like receptors (TLRs). Activation of TLRs triggers production of inflammatory cytokines and type I interferons (IFNs) via myeloid differentiation primary response gene 88 (MyD88)-dependent or -independent signaling respectively, thereby modulating innate and adaptive immunity, as well as inflammatory responses. This review introduces the classification, structure, and specific ligands of TLRs, and focuses on their signal pathways and biological activities, as well as clinical relevance. These studies of TLRs in the innate immune system have implications for the prevention and treatment of a variety of infectious diseases, including tuberculosis (TB), microbial keratitis, and hepatitis B and C.

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.

Inhibition of TREM-1 and Dectin-1 Alleviates the Severity of Fungal Keratitis by Modulating Innate Immune Responses

Purpose To explore the possibility that inhibiting triggering receptor expressed on myeloid cells-1 (TREM-1) and Dendritic cell-associated C-type lectin-1(Dectin-1) could modulate the innate immune response and alleviate the severity of corneal fungal keratitis. Method TREM-1 and Dectin-1 expression was detected in fungus-infected human corneal specimens by real-time PCR. C57BL/6 (B6) mice were injected with Aspergillus fumigatus and divided into 4 groups that received subconjunctival injections of PBS and IgG as a control (group I), mTREM-1/IgG fusion protein (group II), the soluble β-glucan antagonist laminarin (group III), or mTREM-1/Fc and laminarin (group IV). Corneal virulence was evaluated based on clinical scores. TREM-1 and Dectin-1 mRNA levels were assayed using real-time PCR. The distribution patterns of TREM-1, Dectin-1 and cellular infiltrates in fungus-infected corneas were examined by immunohistochemistry. Moreover, changes in T Helper Type1 (Th1)-/ T Helper Type1 (Th2)- type cytokines and proinflammatory cytokines were measured. Results The expression of TREM-1 and Dectin-1 increased significantly and correlated positively with the progression of fungal keratitis. Most infiltrated cells were neutrophils and secondarily macrophages in infected cornea. The clinical scores decreased after interfering with TREM-1 and Dectin-1 expression in infected mouse corneas. Levels of Th1-type cytokines including interleukin-12 (IL-12), IL-18 and interferon-γ (IFN-γ) were decreased in the cornea, while the levels of Th2-type cytokines, including IL-4, IL-5 and IL-10, showed obvious increases. Conclusion TREM-1 and Dectin-1 function concurrently in the corneal innate immune response by regulating inflammatory cytokine expression in fungal keratitis. Inhibition of TREM-1 and Dectin-1 can alleviate the severity of corneal damage by downregulating the excessive inflammatory response.