Antinociceptive actions of honokiol and magnolol on glutamatergic and inflammatory pain

Background

Autophagy has been shown recently to play an important role in the intracellular survival of several pathogenic bacteria. In this study, we investigated the effect of a novel small-molecule autophagy-inducing agent, AR-12, on the survival of Francisella tularensis, the causative bacterium of tularemia in humans and a potential bioterrorism agent, in macrophages.

Methods and results

Our results show that AR-12 induces autophagy in THP-1 macrophages, as indicated by increased autophagosome formation, and potently inhibits the intracellular survival of F. tularensis (type A strain, Schu S4) and F. novicida in macrophages in association with increased bacterial co-localization with autophagosomes. The effect of AR-12 on intracellular F. novicida was fully reversed in the presence of the autophagy inhibitor, 3-methyl adenine or the lysosome inhibitor, chloroquine. Intracellular F. novicida were not susceptible to the inhibitory activity of AR-12 added at 12 h post-infection in THP-1 macrophages, and this lack of susceptibility was independent of the intracellular location of bacteria.

Conclusion

Together, AR-12 represents a proof-of-principle that intracellular F. tularensis can be eradicated by small-molecule agents that target innate immunity.     

Interactions of attenuated Mycobacterium tuberculosis phoP mutant with human macrophages

Background

Mycobacterium tuberculosis phoP mutant SO2 derived from a clinical isolate was shown to be attenuated in mouse bone marrow-derived macrophages and in vivo mouse infection model and has demonstrated a high potential as attenuated vaccine candidate against tuberculosis.

Methodology/Principal Findings

In this study, we analyze the adhesion and the intracellular growth and trafficking of SO2 in human macrophages. Our results indicate an enhanced adhesion to phagocitic cells and impaired intracellular replication of SO2 in both monocyte-derived macrophages and human cell line THP-1 in comparison with the wild type strain, consistent with murine model. Intracellular trafficking analysis in human THP-1 cells suggest that attenuation of SO2 within macrophages could be due to an impaired ability to block phagosome-lysosome fusion compared with the parental M. tuberculosis strain. No differences were found between SO2 and the wild-type strains in the release and mycobacterial susceptibility to nitric oxide (NO) produced by infected macrophages.

Conclusions/Significance

SO2 has enhanced ability to bind human macrophages and differs in intracellular trafficking as to wild-type M. tuberculosis. The altered lipid profile expression of the phoP mutant SO2 and its inability to secrete ESAT-6 is discussed.     

Nucleolin, a shuttle protein promoting infection of human monocytes by Francisella tularensis

Background

Francisella tularensis is a highly virulent facultative intracellular bacterium, disseminating in vivo mainly within host mononuclear phagocytes. After entry into macrophages, F. tularensis initially resides in a phagosomal compartment, whose maturation is then arrested. Bacteria escape rapidly into the cytoplasm, where they replicate freely. We recently demonstrated that nucleolin, an eukaryotic protein able to traffic from the nucleus to the cell surface, acted as a surface receptor for F. tularensis LVS on human monocyte-like THP-1 cells.

Methodology/Principal Findings

Here, we followed the fate of nucleolin once F. tularensis has been endocytosed. We first confirmed by siRNA silencing experiments that expression of nucleolin protein was essential for binding of LVS on human macrophage-type THP-1 cells. We then showed that nucleolin co-localized with intracellular bacteria in the phagosomal compartment. Strikingly, in that compartment, nucleolin also co-localized with LAMP-1, a late endosomal marker. Co-immunoprecipation assays further demonstrated an interaction of nucleolin with LAMP-1. Co-localization of nucleolin with LVS was no longer detectable at 24 h when bacteria were multiplying in the cytoplasm. In contrast, with an iglC mutant of LVS, which remains trapped into the phagosomal compartment, or with inert particles, nucleolin/bacteria co-localization remained almost constant.

Conclusions/Significance

We herein confirm the importance of nucleolin expression for LVS binding and its specificity as nucleolin is not involved in binding of another intracellular pathogen as L. monocytogenes or an inert particle. Association of nucleolin with F. tularensis during infection continues intracellularly after endocytosis of the bacteria. The present work therefore unravels for the first time the presence of nucleolin in the phagosomal compartment of macrophages.     

The Novel Deacetylase Inhibitor AR-42 Demonstrates Pre-Clinical Activity in B-Cell Malignancies In Vitro and In Vivo

Background

While deacetylase (DAC) inhibitors show promise for the treatment of B-cell malignancies, those introduced to date are weak inhibitors of class I and II DACs or potent inhibitors of class I DAC only, and have shown suboptimal activity or unacceptable toxicities. We therefore investigated the novel DAC inhibitor AR-42 to determine its efficacy in B-cell malignancies.

Principal Findings

In mantle cell lymphoma (JeKo-1), Burkitt''s lymphoma (Raji), and acute lymphoblastic leukemia (697) cell lines, the 48-hr IC₅₀ (50% growth inhibitory concentration) of AR-42 is 0.61 µM or less. In chronic lymphocytic leukemia (CLL) patient cells, the 48-hr LC₅₀ (concentration lethal to 50%) of AR-42 is 0.76 µM. AR-42 produces dose- and time-dependent acetylation both of histones and tubulin, and induces caspase-dependent apoptosis that is not reduced in the presence of stromal cells. AR-42 also sensitizes CLL cells to TNF-Related Apoptosis Inducing Ligand (TRAIL), potentially through reduction of c-FLIP. AR-42 significantly reduced leukocyte counts and/or prolonged survival in three separate mouse models of B-cell malignancy without evidence of toxicity.

Conclusions/Significance

Together, these data demonstrate that AR-42 has in vitro and in vivo efficacy at tolerable doses. These results strongly support upcoming phase I testing of AR-42 in B-cell malignancies.     

Listeriolysin O Is Necessary and Sufficient to Induce Autophagy during Listeria monocytogenes Infection

Background

Recent studies have suggested that autophagy is utilized by cells as a protective mechanism against Listeria monocytogenes infection.

Methodology/Principal Findings

However we find autophagy has no measurable role in vacuolar escape and intracellular growth in primary cultured bone marrow derived macrophages (BMDMs) deficient for autophagy (atg5^−/−). Nevertheless, we provide evidence that the pore forming activity of the cholesterol-dependent cytolysin listeriolysin O (LLO) can induce autophagy subsequent to infection by L. monocytogenes. Infection of BMDMs with L. monocytogenes induced microtubule-associated protein light chain 3 (LC3) lipidation, consistent with autophagy activation, whereas a mutant lacking LLO did not. Infection of BMDMs that express LC3-GFP demonstrated that wild-type L. monocytogenes was encapsulated by LC3-GFP, consistent with autophagy activation, whereas a mutant lacking LLO was not. Bacillus subtilis expressing either LLO or a related cytolysin, perfringolysin O (PFO), induced LC3 colocalization and LC3 lipidation. Further, LLO-containing liposomes also recruited LC3-GFP, indicating that LLO was sufficient to induce targeted autophagy in the absence of infection. The role of autophagy had variable effects depending on the cell type assayed. In atg5^−/− mouse embryonic fibroblasts, L. monocytogenes had a primary vacuole escape defect. However, the bacteria escaped and grew normally in atg5^−/− BMDMs.

Conclusions/Significance

We propose that membrane damage, such as that caused by LLO, triggers bacterial-targeted autophagy, although autophagy does not affect the fate of wild-type intracellular L. monocytogenes in primary BMDMs.     

Skeletal Muscle Insulin Resistance Associated with Cholesterol-Induced Activation of Macrophages Is Prevented by High Density Lipoprotein

Background

Emerging evidence suggests that high density lipoprotein (HDL) may modulate glucose metabolism through multiple mechanisms including pancreatic insulin secretion as well as insulin-independent glucose uptake into muscle. We hypothesized that HDL may also increase skeletal muscle insulin sensitivity via cholesterol removal and anti-inflammatory actions in macrophages associated with excess adiposity and ectopic lipid deposition.

Methods

Human primary and THP-1 macrophages were treated with vehicle (PBS) or acetylated low density lipoprotein (acLDL) with or without HDL for 18 hours. Treatments were then removed, and macrophages were incubated with fresh media for 4 hours. This conditioned media was then applied to primary human skeletal myotubes derived from vastus lateralis biopsies taken from patients with type 2 diabetes to examine insulin-stimulated glucose uptake.

Results

Conditioned media from acLDL-treated primary and THP-1 macrophages reduced insulin-stimulated glucose uptake in primary human skeletal myotubes compared with vehicle (primary macrophages, 168±21% of basal uptake to 104±19%; THP-1 macrophages, 142±8% of basal uptake to 108±6%; P<0.05). This was restored by co-treatment of macrophages with HDL. While acLDL increased total intracellular cholesterol content, phosphorylation of c-jun N-terminal kinase and secretion of pro- and anti-inflammatory cytokines from macrophages, none were altered by co-incubation with HDL. Insulin-stimulated Akt phosphorylation in human skeletal myotubes exposed to conditioned media was unaltered by either treatment condition.

Conclusion

Inhibition of insulin-stimulated glucose uptake in primary human skeletal myotubes by conditioned media from macrophages pre-incubated with acLDL was restored by co-treatment with HDL. However, these actions were not linked to modulation of common pro- or anti-inflammatory mediators or insulin signaling via Akt.     

Endoplasmic reticulum stress pathway-mediated apoptosis in macrophages contributes to the survival of Mycobacterium tuberculosis

Background

Apoptosis is thought to play a role in host defenses against intracellular pathogens, including Mycobacterium tuberculosis (Mtb), by preventing the release of intracellular components and the spread of mycobacterial infection. This study aims to investigate the role of endoplasmic reticulum (ER) stress mediated apoptosis in mycobacteria infected macrophages.

Methodology/Principal Findings

Here, we demonstrate that ER stress-induced apoptosis is associated with Mtb H37Rv-induced cell death of Raw264.7 murine macrophages. We have shown that Mtb H37Rv induced apoptosis are involved in activation of caspase-12, which resides on the cytoplasmic district of the ER. Mtb infection increase levels of other ER stress indicators in a time-dependent manner. Phosphorylation of eIF2α was decreased gradually after Mtb H37Rv infection signifying that Mtb H37Rv infection may affect eIF2α phosphorylation in an attempt to survive within macrophages. Interestingly, the survival of mycobacteria in macrophages was enhanced by silencing CHOP expression. In contrast, survival rate of mycobacteria was reduced by phosphorylation of the eIF2α. Futhermore, the levels of ROS, NO or CHOP expression were significantly increased by live Mtb H37Rv compared to heat-killed Mtb H37Rv indicating that live Mtb H37Rv could induce ER stress response.

Conclusion/Significance

These findings indicate that eIF2α/CHOP pathway may influence intracellular survival of Mtb H37Rv in macrophages and only live Mtb H37Rv can induce ER stress response. The data support the ER stress pathway plays an important role in the pathogenesis and persistence of mycobacteria.     

Prohibitin 1 modulates mitochondrial stress-related autophagy in human colonic epithelial cells

Introduction

Autophagy is an adaptive response to extracellular and intracellular stress by which cytoplasmic components and organelles, including damaged mitochondria, are degraded to promote cell survival and restore cell homeostasis. Certain genes involved in autophagy confer susceptibility to Crohn''s disease. Reactive oxygen species and pro-inflammatory cytokines such as tumor necrosis factor α (TNFα), both of which are increased during active inflammatory bowel disease, promote cellular injury and autophagy via mitochondrial damage. Prohibitin (PHB), which plays a role in maintaining normal mitochondrial respiratory function, is decreased during active inflammatory bowel disease. Restoration of colonic epithelial PHB expression protects mice from experimental colitis and combats oxidative stress. In this study, we investigated the potential role of PHB in modulating mitochondrial stress-related autophagy in intestinal epithelial cells.

Methods

We measured autophagy activation in response to knockdown of PHB expression by RNA interference in Caco2-BBE and HCT116 WT and p53 null cells. The effect of exogenous PHB expression on TNFα- and IFNγ-induced autophagy was assessed. Autophagy was inhibited using Bafilomycin A₁ or siATG16L1 during PHB knockdown and the affect on intracellular oxidative stress, mitochondrial membrane potential, and cell viability were determined. The requirement of intracellular ROS in siPHB-induced autophagy was assessed using the ROS scavenger N-acetyl-L-cysteine.

Results

TNFα and IFNγ-induced autophagy inversely correlated with PHB protein expression. Exogenous PHB expression reduced basal autophagy and TNFα-induced autophagy. Gene silencing of PHB in epithelial cells induces mitochondrial autophagy via increased intracellular ROS. Inhibition of autophagy during PHB knockdown exacerbates mitochondrial depolarization and reduces cell viability.

Conclusions

Decreased PHB levels coupled with dysfunctional autophagy renders intestinal epithelial cells susceptible to mitochondrial damage and cytotoxicity. Repletion of PHB may represent a therapeutic approach to combat oxidant and cytokine-induced mitochondrial damage in diseases such as inflammatory bowel disease.     

Role of PPE18 Protein in Intracellular Survival and Pathogenicity of Mycobacterium tuberculosis in Mice

Background

Ever since its discovery the mycobacterial proline-proline-glutamic acid (PPE) family of proteins has generated a huge amount of interest. Understanding the role of these proteins in the pathogenesis of Mycobacterium tuberculosis (Mtb) is important. We have demonstrated earlier that the PPE18 protein of Mtb induces IL-10 production in macrophages with subsequent downregulation of pro-inflammatory cytokines like IL-12 and TNF-α and favors a T-helper (Th) 2-type of immune response.

Methodology/Principal Findings

Using a ppe18 genetic knock-out Mtb strain, we have now carried out infection studies in mice to understand the role of PPE18 in Mtb virulence. The studies reveal that lack of PPE18 leads to attenuation of Mtb in vivo. Mice infected with the ppe18 deleted strain have reduced infection burden in lung, liver and spleen and have better survival rates compared to mice infected with the wild-type Mtb strain.

Conclusions/Significance

Taken together our data suggest that PPE18 could be a crucial virulence factor for intracellular survival of Mtb.     

Effects of Benzalkonium Chloride on THP-1 Differentiated Macrophages In Vitro

Purpose

To characterize the effects of benzalkonium chloride (BAK) in THP-1 differentiated cells in vitro.

Methods

Macrophages were obtained after differentiation of THP-1 cells, a human monocytic leukemia cell line. Macrophages were exposed for 24 h to 33 nM (10⁻⁵%) benzalkonium chloride (BAK), 10 nM dinitrochlorobenzene (DNCB), 100 ng/mL lipopolysaccharide (LPS), 5 ng/mL tumor necrosis factor alpha (TNF-α) or phosphate buffered saline (PBS) as controls. The expression of CD11b, CD11c, CD33 and CD54 was evaluated using immunohistochemistry and flow cytometry (FCM). Phagocytosis function was analyzed using carboxylate-modified fluorescent microspheres and quantified by FCM. Migration was evaluated in cocultures with conjunctival epithelial cells. Cytokine production was detected and quantified in culture supernatants using a human cytokine array.

Results

Stimulation of THP-1-derived macrophages with a low concentration of BAK increased CD11b and CD11c expression and decreased CD33. Macrophages exposed to BAK, LPS and TNF-α had increased phagocytosis. In contrast to LPS, BAK and TNF-α increased macrophage migration. Cytokines in supernatants of macrophages exposed to BAK revealed an increased release of CCL1, CCL4/MIP-1β, TNF-α, soluble CD54/ICAM-1 and IL-1β.

Conclusion

In vitro, BAK has a direct stimulating effect on macrophages, increasing phagocytosis, cytokine release, migration and expression of CD11b and CD11c. Long-term exposure to low concentrations of BAK should be considered as a stimulating factor responsible for inflammation through macrophage activation.     

Immunological Interactions between 2 Common Pathogens,Th1-Inducing Protozoan Toxoplasma gondii and the Th2-Inducing Helminth Fasciola hepatica

Background

The nature of the immune response to infection is dependent on the type of infecting organism. Intracellular organisms such as Toxoplasma gondii stimulate a Th1-driven response associated with production of IL-12, IFN-γ, nitric oxide and IgG2a antibodies and classical activation of macrophages. In contrast, extracellular helminths such as Fasciola hepatica induce Th2 responses characterised by the production of IL-4, IL-5, IL-10 and IgG1 antibodies and alternative activation of macrophages. As co-infections with these types of parasites commonly exist in the field it is relevant to examine how the various facets of the immune responses induced by each may influence or counter-regulate that of the other.

Principal Findings

Regardless, of whether F. hepatica infection preceded or succeeded T. gondii infection, there was little impact on the production of the Th1 cytokines IL-12, IFN-γ or on the development of classically-activated macrophages induced by T. gondii. By contrast, the production of helminth-specific Th2 cytokines, such as IL-4 and IL-5, was suppressed by infection with T. gondii. Additionally, the recruitment and alternative activation of macrophages by F. hepatica was blocked or reversed by subsequent infection with T. gondii. The clinical symptoms of toxoplasmosis and the survival rate of infected mice were not significantly altered by the helminth.

Conclusions

Despite previous studies showing that F. hepatica suppressed the classical activation of macrophages and the Th1-driven responses of mice to bystander microbial infection, as well as reduced their ability to reject these, here we found that the potent immune responses to T. gondii were capable of suppressing the responses to helminth infection. Clearly, the outcome of particular infections in polyparasitoses depends on the means and potency by which each pathogen controls the immune response.     

Legionella pneumophila induces cathepsin B-dependent necrotic cell death with releasing high mobility group box1 in macrophages

Background

Legionella pneumophila (LPN) can cause a lethal infectious disease with a marked inflammatory response in humans. However, the mechanism of this severe inflammation remains poorly understood. Since necrosis is known to induce inflammation, we investigated whether LPN induces necrosis in macrophages. We also analyzed the involvement of lysosomal cathepsin B in LPN-induced cell death.

Methods

The human monocytic cell line THP-1 was infected with LPN, NUL1 strain. MG132-treated cells were used as apoptotic control cells. After infection, the type of cell death was analyzed by using microscopy, LDH release and flow cytometry. As a proinflammatory mediator, high-mobility group box 1 (HMGB-1), was measured. Cathepsin B activity was also measured and the inhibitory effects of cathepsin B on LPN-induced cell death were analyzed.

Results

THP-1 cells after treatment with high dose of LPN showed necrotic features with releasing HMGB-1. This necrosis and the HMGB-1 release were inhibited by a specific lysosomal cathepsin B inhibitor and were characterized by a rapid and high activation of cathepsin B that was not observed in apoptotic control cells. The necrosis was also accompanied by cathepsin B-dependent poly(ADP-ribose) polymerase (PARP) cleavage.

Conclusions

We demonstrate here that L. pneumophila rapidly induces cathepsin B-dependent necrosis in a dose-dependent manner and releases a proinflammatory mediator, HMGB-1, from macrophages. This report describes a novel aspect of the pathogenesis of Legionnaires'' disease and provides a possible therapeutic target for the regulation of inflammation.     

Modulation of biogenesis of the Francisella tularensis subsp. novicida-containing phagosome in quiescent human macrophages and its maturation into a phagolysosome upon activation by IFN-gamma

Francisella tularensis is a highly virulent facultative intracellular pathogen that has been categorized as a class A bioterrorism agent, and is classified into four subsp, tularensis, holarctica, mediasiatica and novicida. Although the ability of F. tularensis subsp. novicida to cause tularemia in mice is similar to the virulent subsp. tularensis and holarctica, it is attenuated in humans. It is not known whether attenuation of F. tularensis subsp. novicida in humans is resulting from a different route of trafficking within human macrophages, compared with the tularensis or holarctica subsp. Here we show that in quiescent human monocytes-derived macrophages (hMDMs), the F. tularensis subsp. novicida containing phagosome (FCP) matures into a late endosome-like stage that acquires the late endosomal marker LAMP-2 but does not fuse to lysosomes. This modulation of phagosome biogenesis by F. tularensis is followed by disruption of the phagosome at 4-12 h and subsequent bacterial escape into cytoplasm where the organism replicates. In IFN-gamma-activated hMDMs, intracellular replication of F. tularensis is completely inhibited, and is associated with failure of the organism to escape from the phagosome into the cytoplasm for up to 24 h after infection. In IFN-gamma-activated hMDMs, the FCPs acquire the lysosomal enzymes Cathepsin D, which is excluded in quiescent hMDMs. When the lysosomes of IFN-gamma-activated hMDMs are preload with Texas Red Ovalbumin or BSA-gold, the FCPs acquire both lysosomal tracers. In contrast, both lysosomal tracers are excluded from the FCPs within quiescent hMDMs. We conclude that although F. tularensis subsp. novicida is attenuated in humans, it modulates biogenesis of its phagosome into a late endosome-like compartment followed by bacterial escape into the cytoplasm within quiescent hMDMs, similar to the virulent subsp. tularensis. In IFN-gamma-activated hMDMs, the organism fails to escape into the cytoplasm and its phagosome fuses to lysosomes, similar to inert particles.     

Host-adaptation of Francisella tularensis alters the bacterium's surface-carbohydrates to hinder effectors of innate and adaptive immunity

Background

The gram-negative bacterium Francisella tularensis survives in arthropods, fresh water amoeba, and mammals with both intracellular and extracellular phases and could reasonably be expected to express distinct phenotypes in these environments. The presence of a capsule on this bacterium has been controversial with some groups finding such a structure while other groups report that no capsule could be identified. Previously we reported in vitro culture conditions for this bacterium which, in contrast to typical methods, yielded a bacterial phenotype that mimics that of the bacterium''s mammalian, extracellular phase.

Methods/Findings

SDS-PAGE and carbohydrate analysis of differentially-cultivated F. tularensis LVS revealed that bacteria displaying the host-adapted phenotype produce both longer polymers of LPS O-antigen (OAg) and additional HMW carbohydrates/glycoproteins that are reduced/absent in non-host-adapted bacteria. Analysis of wildtype and OAg-mutant bacteria indicated that the induced changes in surface carbohydrates involved both OAg and non-OAg species. To assess the impact of these HMW carbohydrates on the access of outer membrane constituents to antibody we used differentially-cultivated bacteria in vitro to immunoprecipitate antibodies directed against outer membrane moieties. We observed that the surface-carbohydrates induced during host–adaptation shield many outer membrane antigens from binding by antibody. Similar assays with normal mouse serum indicate that the induced HMW carbohydrates also impede complement deposition. Using an in vitro macrophage infection assay, we find that the bacterial HMW carbohydrate impedes TLR2-dependent, pro-inflammatory cytokine production by macrophages. Lastly we show that upon host-adaptation, the human-virulent strain, F. tularensis SchuS4 also induces capsule production with the effect of reducing macrophage-activation and accelerating tularemia pathogenesis in mice.

Conclusion

F. tularensis undergoes host-adaptation which includes production of multiple capsular materials. These capsules impede recognition of bacterial outer membrane constituents by antibody, complement, and Toll-Like Receptor 2. These changes in the host-pathogen interface have profound implications for pathogenesis and vaccine development.     

Francisella novicida LPS has greater immunobiological activity in mice than F. tularensis LPS,and contributes to F. novicida murine pathogenesis

To further understand the role of LPS in the pathogenesis of Francisella infection, we characterized murine infection with F. novicida, and compared immunobiological activities of F. novicida LPS and the LPS from F. tularensis live vaccine strain (LVS). F. novicida had a lower intradermal LD(50) in BALB/cByJ mice than F. tularensis LVS, and mice given a lethal F. novicida dose intraperitoneally died faster than those given the same lethal F. tularensis LVS dose. However, the pattern of in vivo dissemination was similar, and in vitro growth of both bacteria in bone marrow-derived macrophages was comparable. F. novicida LPS stimulated very modest in vitro proliferation of mouse splenocytes at high doses, but F. tularensis LVS LPS did not. Murine bone marrow macrophages treated in vitro with F. novicida LPS produced IL12 and TNF-alpha, but did not produce detectable interferon-gamma, IL10, or nitric oxide; in contrast, murine macrophages treated with F. tularensis LVS LPS produced none of these mediators. In contrast to clear differences in stimulation of proliferation and especially cytokines, both types of purified LPS stimulated early protection against lethal challenge of mice with F. tularensis LVS, but not against lethal challenge with F. novicida. Thus, although LPS recognition may not be a major factor in engendering protection, the ability of F. novicida LPS to stimulate the production of proinflammatory cytokines including TNF-alpha likely contributes to the increased virulence for mice of F. novicida compared to F. tularensis LVS.     

Egr-1 regulates autophagy in cigarette smoke-induced chronic obstructive pulmonary disease

Background

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease characterized by abnormal cellular responses to cigarette smoke, resulting in tissue destruction and airflow limitation. Autophagy is a degradative process involving lysosomal turnover of cellular components, though its role in human diseases remains unclear.

Methodology and Principal Findings

Increased autophagy was observed in lung tissue from COPD patients, as indicated by electron microscopic analysis, as well as by increased activation of autophagic proteins (microtubule-associated protein-1 light chain-3B, LC3B, Atg4, Atg5/12, Atg7). Cigarette smoke extract (CSE) is an established model for studying the effects of cigarette smoke exposure in vitro. In human pulmonary epithelial cells, exposure to CSE or histone deacetylase (HDAC) inhibitor rapidly induced autophagy. CSE decreased HDAC activity, resulting in increased binding of early growth response-1 (Egr-1) and E2F factors to the autophagy gene LC3B promoter, and increased LC3B expression. Knockdown of E2F-4 or Egr-1 inhibited CSE-induced LC3B expression. Knockdown of Egr-1 also inhibited the expression of Atg4B, a critical factor for LC3B conversion. Inhibition of autophagy by LC3B-knockdown protected epithelial cells from CSE-induced apoptosis. Egr-1 ^−/− mice, which displayed basal airspace enlargement, resisted cigarette-smoke induced autophagy, apoptosis, and emphysema.

Conclusions

We demonstrate a critical role for Egr-1 in promoting autophagy and apoptosis in response to cigarette smoke exposure in vitro and in vivo. The induction of autophagy at early stages of COPD progression suggests novel therapeutic targets for the treatment of cigarette smoke induced lung injury.     

Berberine chloride mediates its anti-leishmanial activity via differential regulation of the mitogen activated protein kinase pathway in macrophages

Background

A complex interplay between Leishmania and macrophages influences parasite survival and necessitates disruption of signaling molecules, eventually resulting in impairment of macrophage function. In this study, we demonstrate the immunomodulatory activity of Berberine chloride in Leishmania infected macrophages.

Principal Findings

The IC₅₀ of Berberine chloride, a quaternary isoquinoline alkaloid was tested in an amastigote macrophage model and its safety index measured by a cell viability assay. It eliminated intracellular amastigotes, the IC₅₀ being 2.8 fold lower than its IC₅₀ in promastigotes (7.10 µM vs. 2.54 µM) and showed a safety index >16. Levels of intracellular and extracellular nitric oxide (NO) as measured by flow cytometry and Griess assay respectively showed that Berberine chloride in Leishmania infected macrophages increased production of NO. Measurement of the mRNA expression of iNOS, IL-12 and IL-10 by RT-PCR along with levels of IL-12p40 and IL-10 by ELISA showed that in infected macrophages, Berberine chloride enhanced expression of iNOS and IL-12p40, concomitant with a downregulation of IL-10. The phosphorylation status of extracellular signal related kinase (ERK1/2) and p38 mitogen activated protein kinase (p38 MAPK) was studied by western blotting. In infected macrophages, Berberine chloride caused a time dependent activation of p38 MAPK along with deactivation of ERK1/2; addition of a p38 MAPK inhibitor SB203580 inhibited the increased generation of NO and IL-12p40 by Berberine chloride as also prevented its decrease of IL-10.

Conclusions

Berberine chloride modulated macrophage effector responses via the mitogen activated protein kinase (MAPK) pathway, highlighting the importance of MAPKs as an antiparasite target.