Liver kinase B1 overexpression controls mycobacterial infection in macrophages via FOXO1/Wnt5a signaling

Mycobacterium tuberculosis (Mtb) is a primary cause of tuberculosis (TB), which has infected more than one-third of the world’s population. Mtb survival and subsequent inflammation in macrophages are important components of TB. Liver kinase B1 (LKB1) has demonstrated anti-inflammation effects, but its function and underlying mechanism in mycobacteria-infected macrophages remains unknown. In the current study, we discovered that LKB1 was markedly decreased in Mtb-infected THP-1 and U937 macrophages. Moreover, LKB1 overexpression inhibited Mtb survival in macrophages. Mtb infection increased expression of nitric oxide, inducible nitric oxide synthase, and inflammation-related cytokines interleukin (IL)-6, tumor necrosis factor-α, and IL-1β, whereas pcDNA3-LKB1 transfection inhibited the release of these cytokines in THP-1 and U937 cells. Furthermore, LKB1 overexpression significantly decreased protein expression of Wnt5a, which is dependent on the elevation of forkhead box protein O1 (FOXO1). Generally, we show that interruption of FOXO1 or overexpression of Wnt5a can reverse the effects of LKB1 on mycobacterial intracellular survival, nitric oxide, inducible nitric oxide synthase expression, and inflammatory cytokine release. These findings indicate important roles for LKB1, FOXO1, and Wnt5a in controlling mycobacteria and cell inflammation.     

Differential expression of NF-κB in mycobacteria infected THP-1 affects apoptosis

The present study was conducted to see the role of NF-κB in virulent (Mycobacterium tuberculosis H37Rv) and avirulent (M. tuberculosis H37Ra) mycobacterial infection in THP-1 cells. To inactivate NF-κB, pCMV-IκBαM dn containing THP-1 cell line was generated which showed marked increase in apoptosis with M. tuberculosis H37Rv and M. tuberculosis H37Ra. Infected THP-1-IκBαM dn cells showed decrease in mitochondrial membrane potential, cytochrome c release, activation of caspase-3 and enhanced TNF-α production. Increase in apoptosis of infected THP-1-IκBαM dn cells resulted in inhibition of intracellular mycobacterial growth. Differential NF-κB activation potential was observed with M. tuberculosis H37Rv and M. tuberculosis H37Ra. Both the strains activated NF-κB after 4 h in THP-1 cells however after 48 h only M. tuberculosis H37Rv activated NF-κB which lead to up-regulation of bcl-2 family anti-apoptotic member, bfl-1/A1. Our results indicated that NF-κB activation may be a determinant factor for the success of virulent mycobacteria within macrophages.     

Mycobacteria Exploit Host Hyaluronan for Efficient Extracellular Replication

In spite of the importance of hyaluronan in host protection against infectious organisms in the alveolar spaces, its role in mycobacterial infection is unknown. In a previous study, we found that mycobacteria interact with hyaluronan on lung epithelial cells. Here, we have analyzed the role of hyaluronan after mycobacterial infection was established and found that pathogenic mycobacteria can grow by utilizing hyaluronan as a carbon source. Both mouse and human possess 3 kinds of hyaluronan synthases (HAS), designated HAS1, HAS2, and HAS3. Utilizing individual HAS-transfected cells, we show that HAS1 and HAS3 but not HAS2 support growth of mycobacteria. We found that the major hyaluronan synthase expressed in the lung is HAS1, and that its expression was increased after infection with Mycobacterium tuberculosis. Histochemical analysis demonstrated that hyaluronan profoundly accumulated in the granulomatous legion of the lungs in M. tuberculosis-infected mice and rhesus monkeys that died from tuberculosis. We detected hyaluronidase activity in the lysate of mycobacteria and showed that it was critical for hyaluronan-dependent extracellular growth. Finally, we showed that L-Ascorbic acid 6-hexadecanoate, a hyaluronidase inhibitor, suppressed growth of mycobacteria in vivo. Taken together, our data show that pathogenic mycobacteria exploit an intrinsic host-protective molecule, hyaluronan, to grow in the respiratory tract and demonstrate the potential usefulness of hyaluronidase inhibitors against mycobacterial diseases.     

Mycobacterium tuberculosis surface protein Rv0227c contains high activity binding peptides which inhibit cell invasion

Mycobacterium tuberculosis surface proteins involved in target cell invasion may be identified as a strategy for developing subunit-based, chemically-synthesized vaccines. The Rv0227c protein was thus selected to assess its role in the invasion and infection of Mycobacterium tuberculosis target cells. Results revealed Rv0227c localization on mycobacterial surface by immunoelectron microscopy and Western blot. Receptor–ligand assays using 20-mer, non-overlapping peptides covering the complete Rv0227c protein sequence revealed three high activity binding peptides for U937 phagocytic cells and seven for A549 cells. Peptide 16944 significantly inhibited mycobacterial entry to both cell lines while 16943 and 16949 only managed to inhibit entrance to U937 cells and 16951 to A549 cells. The Jnet bioinformatics tool predicted secondary structure elements for the complete protein, agreeing with elements determined for such chemically-synthesized peptides. It was thus concluded that high activity binding peptides which were able to inhibit mycobacterial entry to target cells are of great importance when selecting peptide candidates for inclusion in an anti-tuberculosis vaccine.     

Interferon gamma activated macrophages kill mycobacteria by nitric oxide induced apoptosis

Mycobacterium tuberculosis is an intracellular pathogen of macrophages and escapes the macrophages' bactericidal effectors by interfering with phagosome-lysosome fusion. IFN-γ activation renders the macrophages capable of killing intracellular mycobacteria by overcoming the phagosome maturation block, nutrient deprivation and exposure to microbicidal effectors including nitric oxide (NO). While the importance about NO for the control of mycobacterial infection in murine macrophages is well documented, the underlying mechanism has not been revealed yet. In this study we show that IFN-γ induced apoptosis in mycobacteria-infected macrophages, which was strictly dependent on NO. Subsequently, NO-mediated apoptosis resulted in the killing of intracellular mycobacteria independent of autophagy. In fact, killing of mycobacteria was susceptible to the autophagy inhibitor 3-methyladenine (3-MA). However, 3-MA also suppressed NO production, which is an important off-target effect to be considered in autophagy studies using 3-MA. Inhibition of caspase 3/7 activation, as well as NO production, abolished apoptosis and elimination of mycobacteria by IFN-γ activated macrophages. In line with the finding that drug-induced apoptosis kills intracellular mycobacteria in the absence of NO, we identified NO-mediated apoptosis as a new defense mechanism of activated macrophages against M. tuberculosis.     

Exosomal Hsp70 Induces a Pro-Inflammatory Response to Foreign Particles Including Mycobacteria

Background

Exosomes are endosome-derived vesicles that are released when multi-vesicular bodies (MVBs) fuse with the plasma membrane. Exosomes released from mycobacteria-infected cells have recently been shown to be pro-inflammatory. A prominent host molecule that is found within these exosomes is Hsp70, a member of the heat-shock family of proteins.

Methodology/Principal Findings

We first characterized the exosomes purified from control and mycobacteria-infected cells. We found that relative to uninfected cells, macrophages infected with M. smegmatis and M. avium release more exosomes and the exosomes they released had more Hsp70 on their surface. Both exosomes and exogenous Hsp70 treatment of macrophages led to NF-κB activation and TNFα release in uninfected macrophages; Hsp70 levels were elevated in mycobacteria-infected cells. Macrophage treatment with Hsp70 also led to increase in the phagocytosis and maturation of latex-bead phagosomes. Finally, Hsp70 pre-incubation of M. smegmatis- and M. avium-infected cells led to increased phago-lysosome fusion, as well as more killing of mycobacteria within macrophages.

Conclusions/Significance

Our results fit into an emerging concept whereby exosomes-containing Hsp70 are effective inducers of inflammation, also in response to mycobacterial infection.     

The Mycobacterium tuberculosis Proteasome Active Site Threonine Is Essential for Persistence Yet Dispensable for Replication and Resistance to Nitric Oxide

Previous work revealed that conditional depletion of the core proteasome subunits PrcB and PrcA impaired growth of Mycobacterium tuberculosis in vitro and in mouse lungs, caused hypersusceptibility to nitric oxide (NO) and impaired persistence of the bacilli during chronic mouse infections. Here, we show that genetic deletion of prcBA led to similar phenotypes. Surprisingly, however, an active site mutant proteasome complemented the in vitro and in vivo growth defects of the prcBA knockout (ΔprcBA) as well as its NO hypersensitivity. In contrast, long-term survival of M. tuberculosis in stationary phase and during starvation in vitro and in the chronic phase of mouse infection required a proteolytically active proteasome. Inhibition of inducible nitric oxide synthase did not rescue survival of ΔprcBA, revealing a function beyond NO defense, by which the proteasome contributes to M. tuberculosis fitness during chronic mouse infections. These findings suggest that proteasomal proteolysis facilitates mycobacterial persistence, that M. tuberculosis faces starvation during chronic mouse infections and that the proteasome serves a proteolysis-independent function.     

Mycobacterium bovis bacilli Calmette-Guerin regulates leukocyte recruitment by modulating alveolar inflammatory responses

Leukocyte migration into the epithelial compartment is an important feature in the active phase of mycobacterial infections. In this study, we used the Transwell model to investigate the mechanisms behind mycobacteria-induced leukocyte recruitment and investigated the role of TLR2 and TLR4 in this process. Infection of epithelial cells resulted in significantly increased secretion of the neutrophil chemotactic CXCL8 and IL-6, but no secretion of monocyte chemotactic CCL2 or TNF-α was observed. In contrast to epithelial response, mycobacteria-infected neutrophils and monocytes secreted all these cytokines. Corresponding with epithelial cytokine response, mycobacterial infection of the epithelial cells increased neutrophil diapedesis, but decreased monocyte recruitment. However, monocyte recruitment towards mycobacteria infected epithelial cells significantly increased following addition of neutrophil pre-conditioned medium. Mycobacterial infection also increases alveolar epithelial expression of TLR2, but not TLR4, as analyzed by flow cytometry, Western blotting and visualized by confocal microscopy. Blocking of TLR2 inhibited neutrophil recruitment and cytokine secretion, while blocking of TLR4 had a lesser effect. To summarize, we found that primary alveolar epithelial cells produced a selective TLR2-dependent cytokine secretion upon mycobacterial infection. Furthermore, we found that cooperation between cells of the innate immunity is required in mounting proper antimicrobial defence.     

Syndecans promote mycobacterial internalization by lung epithelial cells

Pulmonary tuberculosis (TB) is an airborne disease caused by the intracellular bacterial pathogen Mycobacterium tuberculosis (Mtb). Alveolar epithelial cells and macrophages are the first point of contact for Mtb in the respiratory tract. However, the mechanisms of mycobacterial attachment to, and internalization by, nonprofessional phagocytes, such as epithelial cells, remain incompletely understood. We identified syndecan 4 (Sdc4) as mycobacterial attachment receptor on alveolar epithelial cells. Sdc4 mRNA expression was increased in human and mouse alveolar epithelial cells after mycobacterial infection. Sdc4 knockdown in alveolar epithelial cells or blocking with anti‐Sdc4 antibody reduced mycobacterial attachment and internalization. At the molecular level, interactions between epithelial cells and mycobacteria involved host Sdc and the mycobacterial heparin‐binding hemagglutinin adhesin. In vivo, Sdc1/Sdc4 double‐knockout mice were more resistant to Mtb colonization of the lung. Our work reveals a role for distinct Sdcs in promoting mycobacterial entry into alveolar epithelial cells with impact on outcome of TB disease.     

Sphingosine kinase, phosphatidylinositol 3-kinase, Akt, NF-kappaB, and p300 are required for CCL5 production in Mycobacterium bovis Bacillus Calmette-Guérin (BCG)-infected epithelial cells

CCL5 is a key in limiting mycobacterial infection. Although NF-κB has been implicated, signaling cascades involved in CCL5 production by epithelial cells following infection with Mycobacterium bovis BCG are still not defined. Here we show that using pharmacological inhibition of sphingosine kinase (SPK), striking inhibition of M. bovis BCG-induced CCL5 protein was observed. Phosphatidylinositol 3-kinase (PI3K) and Akt were also important for CCL5 production by epithelial cells infected with M. bovis BCG. Moreover, there was increased activation of PI3K, IKK/αβ and NF-κB in A549 cells infected with M. bovis BCG. Importantly, the PI3K activation was dependent on SPK. Finally, M. bovis BCG increases the recruitment of p300 with NF-κB in A549 cells. Together, these studies are the first to show that M. bovis BCG-induced CCL5 secretion is dependent on the SPK/PI3K/Akt/NF-κB and p300 signaling pathway. The regulatory pathways of M. bovis BCG-induced CCL5 production can potentially be exploited therapeutically.     

Functional characterization of Mycobacterium tuberculosis Rv2969c membrane protein

Identifying Mycobacterium tuberculosis membrane proteins involved in binding to and invasion of host cells is important in designing subunit-based anti-tuberculosis vaccines. The Rv2969c gene sequence was identified by PCR in M. tuberculosis complex strains, being transcribed in M. tuberculosis H37Rv, M. tuberculosis H37Ra, and M. bovis BCG. Rabbits immunized with synthetic peptides from highly specific conserved regions of this protein produced antibodies recognizing 27 and 29 kDa bands in M. tuberculosis lysate, which is consistent with the molecular weight of the Rv2969c gene product in M. tuberculosis H37Rv. Immunoelectron microscopy revealed the protein was localized on the bacillus surface. Four and three specific high activity binding peptides (HABPs) to the A549 alveolar epithelial and U937 monocyte cell lines were found, respectively. Two of the HABPs found inhibited M. tuberculosis invasion of A549 cells, suggesting that these peptides might be good candidates to be included in a multiepitopic, subunit-based anti-tuberculosis vaccine.     

水杨酸诱发的NO介导了丹参悬浮培养细胞中丹酚酸B的生物合成

水杨酸(SA)可诱导丹参悬浮培养细胞中一氧化氮(NO)产生、苯丙氨酸解氨酶(PAL)活化及丹酚酸B(Sal B)的生物合成。为了阐明NO对丹参悬浮培养细胞中Sal B生物合成的影响及作用机理,本实验利用NO供体硝普钠(SNP)、NO合成酶抑制剂L-NNA(Nω-nitro-L-arginine)、NO淬灭剂c PITO(carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide)以及PAL抑制剂L-AOPP(L-2-aminooxygen-3-phenyl acrylic acid)分别处理丹参悬浮培养细胞,并对其胞内NO水平、PAL活性和Sal B积累量进行了检测。结果表明,硝普钠(SNP)处理显著促进了NO产生、PAL活性和Sal B的积累,而L-NNA和c PITO抑制上述过程,说明NO诱发PAL活性提高并参与了SA诱导的Sal B生物合成;L-AOPP显著抑制了PAL活性及Sal B积累,却对NO产生没有显著影响,揭示NO位于PAL的上游。这说明SA诱发的NO产生、PAL活化及Sal B合成之间存在因果关系,即NO通过激活PAL触发Sal B生物合成。     

Nitric oxide confers cisplatin resistance in human lung cancer cells through upregulation of aldo-keto reductase 1B10 and proteasome

Abstract

In this study, we show that exposure of human lung cancer A549 cells to cisplatin (cis-diamminedichloroplatinum, CDDP) promotes production of nitric oxide (NO) through generation of reactive oxygen species (ROS) and resulting upregulation of inducible NO synthase (iNOS). The incubation of the cells with a NO donor, diethylenetriamine NONOate, not only reduced the CDDP-induced cell death and apoptotic alterations (induction of CCAAT-enhancer-binding protein homologous protein and caspase-3 activation), but also elevated proteolytic activity of 26S proteasome, suggesting that the activation of proteasome function contributes to the reduction of CDDP sensitivity by NO. Monitoring expression levels of six aldo-keto reductases (AKRs) (1A1, 1B1, 1B10, 1C1, 1C2, and 1C3) during the treatment with the NO donor and subsequent CDDP sensitivity test using the specific inhibitors also proposed that upregulation of AKR1B10 by NO is a key process for acquiring the CDDP resistance in A549 cells. Treatment with CDDP and NO increased amounts of nitrotyrosine protein adducts, indicative of peroxynitrite formation, and promoted the induction of AKR1B10, inferring a relationship between peroxynitrite formation and the enzyme upregulation in the cells. The treatment with CDDP or a ROS-related lipid aldehyde, 4-hydroxy-2-nonenal, facilitated the iNOS upregulation, which was restored by increasing the AKR1B10 expression. In contrast, the facilitation of NO production by CDDP treatment was hardly observed in AKR1B10-overexpressing A549 cells and established CDDP-resistant cancer cells (A549, LoVo, and PC3). Collectively, these results suggest the NO functions as a key regulator controlling AKR1B10 expression and 26S proteasome function leading to gain of the CDDP resistance.     

Endoplasmic reticulum stress response is involved in Mycobacterium tuberculosis protein ESAT-6-mediated apoptosis

Mycobacterium tuberculosis (Mtb) infection leads to the induction of the apoptotic response, which is associated with bacilli killing. The early secreted mycobacterial antigen ESAT-6 of Mtb has been shown to induce apoptosis in human macrophages and epithelial cells. In the present study, we demonstrate that the stimulation of human epithelial A549 cells by ESAT-6 induces the endoplasmic reticulum (ER) stress response. We observed that ESAT-6 treatment increases intracellular Ca²⁺ concentration, which results in ROS accumulation, and therefore induces the onset of ER stress-induced apoptosis. Our results uncover a novel apoptotic mechanism of ESAT-6 through ER stress responses in pathologic conditions such as tuberculosis.     

CD14 Mediates Toll-like Receptor 4 (TLR4) Endocytosis and Spleen Tyrosine Kinase (Syk) and Interferon Regulatory Transcription Factor 3 (IRF3) Activation in Epithelial Cells and Impairs Neutrophil Infiltration and Pseudomonas aeruginosa Killing in Vivo

In the current study, we examined the role of CD14 in regulating LPS activation of corneal epithelial cells and Pseudomonas aeruginosa corneal infection. Our findings demonstrate that LPS induces Toll-like receptor 4 (TLR4) internalization in corneal epithelial cells and that blocking with anti-CD14 selectively inhibits TLR4 endocytosis, spleen tyrosine kinase (Syk) and IRF3 phosphorylation, and production of CCL5/RANTES and IFN-β, but not IL-8. Using a murine model of P. aeruginosa corneal infection, we show that although infected CD14^−/− corneas produce less CCL5, they exhibit significantly increased CXC chemokine production, neutrophil recruitment to the corneal stroma, and bacterial clearance than C57BL/6 mice. We conclude that CD14 has a critical role in mediating TLR4 signaling through IRF3 in resident corneal epithelial cells and macrophages and thereby modulates TLR4 cell surface activation of the MyD88/NF-κB/AP-1 pathway and production of CXC chemokines and neutrophil infiltration to infected tissues.     

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.