Exosomal circRNA BTG2 derived from RBP-J overexpressed-macrophages inhibits glioma progression via miR-25-3p/PTEN

Macrophage-derived exosomes (Mφ-Exos) are involved in tumor progression, but its role in glioma is not fully understood. RBP-J is related to macrophage activation. In this study, we assess the role of exosomes derived from RBP-J-overexpressed macrophages (RBP-J OE Mφ-Exos) in glioma. The circular RNA (circRNA) profiles in RBP-J OE Mφ-Exos and THP-1-like macrophages (WT Mφ)-Exos were evaluated using circRNA microarray. Then the functions of Mφ-Exo-circRNA in glioma cells were assessed via CCK-8, EdU, Transwell invasion, and nude mouse assays. Besides, luciferase reporter assay, RNA immunoprecipitation, and Pearson’s correlation analysis were adopted to confirm interactions. We found that circRNA BTG (circBTG2) is upregulated in RBP-J OE Mφ-Exos compared to WT Mφ-Exos. RBP-J OE Mφ-Exos co-culture and circBTG2 overexpression inhibited proliferation and invasion of glioma cells, whereas circBTG2 knockdown promotes tumor growth in vivo. The effects of RBP-J OE Mφ-Exos on glioma cells can be reversed by the circBTG2 knockdown. In conclusions, Exo-circBTG2 secreted from RBP-J OE Mφ inhibits tumor progression through the circBTG2/miR-25-3p/PTEN pathway, and circBTG2 is probably a diagnostic biomarker and potential target for glioma therapy.Subject terms: CNS cancer, CNS cancer     

Macrophage Subset Sensitivity to Endotoxin Tolerisation by Porphyromonas gingivalis

Macrophages (MΦs) determine oral mucosal responses; mediating tolerance to commensal microbes and food whilst maintaining the capacity to activate immune defences to pathogens. MΦ responses are determined by both differentiation and activation stimuli, giving rise to two distinct subsets; pro-inflammatory M1- and anti-inflammatory/regulatory M2- MΦs. M2-like subsets predominate tolerance induction whereas M1 MΦs predominate in inflammatory pathologies, mediating destructive inflammatory mechanisms, such as those in chronic P.gingivalis (PG) periodontal infection. MΦ responses can be suppressed to benefit either the host or the pathogen. Chronic stimulation by bacterial pathogen associated molecular patterns (PAMPs), such as LPS, is well established to induce tolerance. The aim of this study was to investigate the susceptibility of MΦ subsets to suppression by P. gingivalis. CD14^hi and CD14^lo M1- and M2-like MΦs were generated in vitro from the THP-1 monocyte cell line by differentiation with PMA and vitamin D₃, respectively. MΦ subsets were pre-treated with heat-killed PG (HKPG) and PG-LPS prior to stimulation by bacterial PAMPs. Modulation of inflammation was measured by TNFα, IL-1β, IL-6, IL-10 ELISA and NFκB activation by reporter gene assay. HKPG and PG-LPS differentially suppress PAMP-induced TNFα, IL-6 and IL-10 but fail to suppress IL-1β expression in M1 and M2 MΦs. In addition, P.gingivalis suppressed NFκB activation in CD14^lo and CD14^hi M2 regulatory MΦs and CD14^lo M1 MΦs whereas CD14^hi M1 pro-inflammatory MΦs were refractory to suppression. In conclusion, P.gingivalis selectively tolerises regulatory M2 MΦs with little effect on pro-inflammatory CD14^hi M1 MΦs; differential suppression facilitating immunopathology at the expense of immunity.     

The Genetic Basis of Escherichia coli Pathoadaptation to Macrophages

Antagonistic interactions are likely important driving forces of the evolutionary process underlying bacterial genome complexity and diversity. We hypothesized that the ability of evolved bacteria to escape specific components of host innate immunity, such as phagocytosis and killing by macrophages (MΦ), is a critical trait relevant in the acquisition of bacterial virulence. Here, we used a combination of experimental evolution, phenotypic characterization, genome sequencing and mathematical modeling to address how fast, and through how many adaptive steps, a commensal Escherichia coli (E. coli) acquire this virulence trait. We show that when maintained in vitro under the selective pressure of host MΦ commensal E. coli can evolve, in less than 500 generations, virulent clones that escape phagocytosis and MΦ killing in vitro, while increasing their pathogenicity in vivo, as assessed in mice. This pathoadaptive process is driven by a mechanism involving the insertion of a single transposable element into the promoter region of the E. coli yrfF gene. Moreover, transposition of the IS186 element into the promoter of Lon gene, encoding an ATP-dependent serine protease, is likely to accelerate this pathoadaptive process. Competition between clones carrying distinct beneficial mutations dominates the dynamics of the pathoadaptive process, as suggested from a mathematical model, which reproduces the observed experimental dynamics of E. coli evolution towards virulence. In conclusion, we reveal a molecular mechanism explaining how a specific component of host innate immunity can modulate microbial evolution towards pathogenicity.     

The Type I NADH Dehydrogenase of Mycobacterium tuberculosis Counters Phagosomal NOX2 Activity to Inhibit TNF-α-Mediated Host Cell Apoptosis

The capacity of infected cells to undergo apoptosis upon insult with a pathogen is an ancient innate immune defense mechanism. Consequently, the ability of persisting, intracellular pathogens such as the human pathogen Mycobacterium tuberculosis (Mtb) to inhibit infection-induced apoptosis of macrophages is important for virulence. The nuoG gene of Mtb, which encodes the NuoG subunit of the type I NADH dehydrogenase, NDH-1, is important in Mtb-mediated inhibition of host macrophage apoptosis, but the molecular mechanism of this host pathogen interaction remains elusive. Here we show that the apoptogenic phenotype of MtbΔnuoG was significantly reduced in human macrophages treated with caspase-3 and -8 inhibitors, TNF-α-neutralizing antibodies, and also after infection of murine TNF^−/− macrophages. Interestingly, incubation of macrophages with inhibitors of reactive oxygen species (ROS) reduced not only the apoptosis induced by the nuoG mutant, but also its capacity to increase macrophage TNF-α secretion. The MtbΔnuoG phagosomes showed increased ROS levels compared to Mtb phagosomes in primary murine and human alveolar macrophages. The increase in MtbΔnuoG induced ROS and apoptosis was abolished in NOX-2 deficient (gp91^−/−) macrophages. These results suggest that Mtb, via a NuoG-dependent mechanism, can neutralize NOX2-derived ROS in order to inhibit TNF-α-mediated host cell apoptosis. Consistently, an Mtb mutant deficient in secreted catalase induced increases in phagosomal ROS and host cell apoptosis, both of which were dependent upon macrophage NOX-2 activity. In conclusion, these results serendipitously reveal a novel connection between NOX2 activity, phagosomal ROS, and TNF-α signaling during infection-induced apoptosis in macrophages. Furthermore, our study reveals a novel function of NOX2 activity in innate immunity beyond the initial respiratory burst, which is the sensing of persistent intracellular pathogens and subsequent induction of host cell apoptosis as a second line of defense.     

Protein Tyrosine Phosphatases Are Regulated by Mononuclear Iron Dicitrate

The involvement of macrophages (Mφs) as host, accessory, and effector cells in the development of infectious diseases, together with their central role in iron homeostasis, place these immune cells as key players in the interface between iron and infection. Having previously shown that the functional expression of NRAMP-1 results in increased protein phosphorylation mediated in part by an iron-dependent inhibition of Mφ protein-tyrosine phosphatase (PTP) activity, we sought to study the mechanism(s) underlying this specific event. Herein we have identified the mononuclear dicitrate iron complex [Fe(cit)₂H_4-x]^(1+x)− as the species responsible for the specific inhibition of Mφ PTP activity. By using biochemical and computational approaches, we show that [Fe(cit)₂]⁵⁻ targets the catalytic pocket of the PTP SHP-1, competitively inhibiting its interaction with an incoming phosphosubstrate. In vitro and in vivo inhibition of PTP activity by iron-citrate results in protein hyperphosphorylation and enhanced MAPK signaling in response to LPS stimulation. We propose that iron-citrate-mediated PTP inhibition represents a novel and biologically relevant regulatory mechanism of signal transduction.     

Macrophage Receptors for Influenza A Virus: Role of the Macrophage Galactose-Type Lectin and Mannose Receptor in Viral Entry

Although sialic acid has long been recognized as the primary receptor determinant for attachment of influenza virus to host cells, the specific receptor molecules that mediate viral entry are not known for any cell type. For the infection of murine macrophages by influenza virus, our earlier study indicated involvement of a C-type lectin, the macrophage mannose receptor (MMR), in this process. Here, we have used direct binding techniques to confirm and characterize the interaction of influenza virus with the MMR and to seek additional macrophage surface molecules that may have potential as receptors for viral entry. We identified the macrophage galactose-type lectin (MGL) as a second macrophage membrane C-type lectin that binds influenza virus and is known to be endocytic. Binding of influenza virus to MMR and MGL occurred independently of sialic acid through Ca²⁺-dependent recognition of viral glycans by the carbohydrate recognition domains of the two lectins; influenza virus also bound to the sialic acid on the MMR. Multivalent ligands of the MMR and MGL inhibited influenza virus infection of macrophages in a manner that correlated with expression of these receptors on different macrophage populations. Influenza virus strain A/PR/8/34, which is poorly glycosylated and infects macrophages poorly, was not recognized by the C-type lectin activity of either the MMR or the MGL. We conclude that lectin-mediated interactions of influenza virus with the MMR or the MGL are required for the endocytic uptake of the virus into macrophages, and these lectins can thus be considered secondary or coreceptors with sialic acid for infection of this cell type.Infection of host cells by influenza virus is initiated by attachment of virus to sialic acid residues on the host cell surface through the receptor-binding site at the distal tip of the viral hemagglutinin (HA) (43). After attachment, the virus is internalized by endocytosis, and acidification of the endosome triggers a conformational change in viral HA that results in fusion of the viral envelope and host cell membrane (34). At the cell surface, sialic acid residues are commonly found at the termini of oligosaccharide chains that are attached in O or N linkage to cell surface proteins; they are also an essential component of acidic glycosphingolipids (gangliosides) that are present in all mammalian cell membranes. Although the abundance of sialic acid on mammalian cells provides influenza virus with multiple potential receptors, virus attachment does not always lead to virus entry (5, 8, 46). Furthermore, sialic acid-independent infection of Madin-Darby canine kidney (MDCK) cells by influenza virus has been reported (35). The specific host cell molecules that serve as functional receptors (or coreceptors) for the infectious entry of influenza virus have yet to be defined.We have studied the infectious entry of influenza virus into macrophages (Mφ), which represents an early event in recognition of the virus by the innate immune system (23, 44). After intranasal infection of mice, influenza virus replicates productively in cells of the respiratory epithelium. Mφ are also infected and viral proteins are produced, but replication is abortive and no live progeny are released (32); infection of Mφ is thus a dead-end for the virus leading to a reduction in viral load. In addition, influenza virus infection of Mφ stimulates production and release of proinflammatory cytokines and alpha/beta interferon (28), which may assist in further limiting viral replication and spread within the respiratory tract. Depletion of airway Mφ from mice prior to intranasal influenza virus infection leads to increased virus titers in the lung, attesting to the important role of Mφ in early host defense against the virus (38, 44).We observed in a previous study (30) that influenza A virus strains differed in their ability to infect murine Mφ, strains carrying a more highly glycosylated hemagglutinin (HA) molecule being more efficient at infecting Mφ than less glycosylated strains, although binding of viruses to the Mφ cell surface was equivalent. Our investigation of this phenomenon indicated involvement of the Mφ mannose receptor MMR (CD206), a C-type lectin, in infectious viral entry (29, 30). The involvement of other receptors was not excluded, and our subsequent observation that influenza virus can infect the RAW 264.7 Mφ cell line, which does not express the MMR, indeed points to the existence of other routes of infectious entry of the virus into Mφ.In the present study we used direct binding methods to confirm and characterize the interaction of influenza virus with the MMR and to seek additional Mφ surface molecules that may have potential as receptors for viral entry. We identify the Mφ galactose-type lectin (MGL) as a second Mφ membrane C-type lectin that binds influenza virus and investigate its involvement in the infectious process.     

Exosomal hsa_circ_0004658 derived from RBPJ overexpressed-macrophages inhibits hepatocellular carcinoma progression via miR-499b-5p/JAM3

Macrophage-derived exosomes (Mφ-Exo) have multidimensional involvement in tumor initiation, progression, and metastasis, but their regulation in hepatocellular carcinoma (HCC) is not fully understood. RBPJ has been implicated in macrophage activation and plasticity. In this study we assess the role of exosomes derived from RBPJ-overexpressed macrophages (RBPJ^+/+ Mφ-Exo) in HCC. The circular RNA (circRNA) profiles in RBPJ^+/+ Mφ-Exo and THP-1-like macrophages (WT Mφ)-Exo was evaluated using circRNA microarray. CCK-8, Transwell, and flow cytometry analyses were used to evaluate the function of Mφ-Exo-circRNA on HCC cells. Luciferase reporter assays, RNA immunoprecipitation, and Pearson’s correlation analysis were used to confirm interactions. A nude mouse xenograft model was used to further analyze the functional significance of Mφ-Exo-cirRNA in vivo. Our results shown that hsa_circ_0004658 is upregulated in RBPJ^+/+ Mφ-Exo compared to WT Mφ-Exo. RBPJ^+/+ Mφ-Exo and hsa_circ_0004658 inhibits proliferation and promotes apoptosis in HCC cells, whereas hsa_circ_0004658 knockdown stimulated cell proliferation and migration but restrained apoptosis in vitro and promotes tumor growth in vivo. The effects of RBPJ^+/+ Mφ-Exo on HCC cells can be reversed by the hsa_circ_0004658 knockdown. Mechanistic investigations revealed that hsa_circ_0004658 acts as a ceRNA of miR-499b-5p, resulting in the de-repression of JAM3. These results indicate that exosome circRNAs secreted from RBPJ^+/+ Mφ inhibits tumor progression through the hsa_circ_0004658/miR-499b-5p/JAM3 pathway and hsa_circ_0004658 may be a diagnostic biomarker and potential target for HCC therapy.Subject terms: Cancer stem cells, Liver cancer     

Mouse Macrophage Galactose-type Lectin (mMGL) is Critical for Host Resistance against Trypanosoma cruzi Infection

The C-type lectin receptor mMGL is expressed exclusively by myeloid antigen presenting cells (APC) such as dendritic cells (DC) and macrophages (Mφ), and it mediates binding to glycoproteins carrying terminal galactose and α- or β-N-acetylgalactosamine (Gal/GalNAc) residues. Trypanosoma cruzi (T. cruzi) expresses large amounts of mucin (TcMUC)-like glycoproteins. Here, we show by lectin-blot that galactose moieties are also expressed on the surface of T. cruzi. Male mMGL knockout (-/-) and wild-type (WT) C57BL/6 mice were infected intraperitoneally with 10⁴ T. cruzi trypomastigotes (Queretaro strain). Following T. cruzi infection, mMGL-/- mice developed higher parasitemia and higher mortality rates compared with WT mice. Although hearts from T. cruzi-infected WT mice presented few amastigote nests, mMGL-/- mice displayed higher numbers of amastigote nests. Compared with WT, Mφ from mMGL-/- mice had low production of nitric oxide (NO), interleukin (IL)-12 and tumor necrosis factor (TNF)-α in response to soluble T. cruzi antigens (TcAg). Interestingly, upon in vitro T. cruzi infection, mMGL-/- Mφ expressed lower levels of MHC-II and TLR-4 and harbored higher numbers of parasites, even when mMGL-/- Mφ were previously primed with IFN-γ or LPS/IFN-γ. These data suggest that mMGL plays an important role during T. cruzi infection, is required for optimal Mφ activation, and may synergize with TLR-4-induced pathways to produce TNF-α, IL-1β and NO during the early phase of infection.     

Macrophage-specific responses to human- and animal-adapted tubercle bacilli reveal pathogen and host factors driving multinucleated cell formation

The Mycobacterium tuberculosis complex (MTBC) is a group of related pathogens that cause tuberculosis (TB) in mammals. MTBC species are distinguished by their ability to sustain in distinct host populations. While Mycobacterium bovis (Mbv) sustains transmission cycles in cattle and wild animals and causes zoonotic TB, M. tuberculosis (Mtb) affects human populations and seldom causes disease in cattle. The host and pathogen determinants underlying host tropism between MTBC species are still unknown. Macrophages are the main host cell that encounters mycobacteria upon initial infection, and we hypothesised that early interactions between the macrophage and mycobacteria influence species-specific disease outcome. To identify factors that contribute to host tropism, we analysed blood-derived primary human and bovine macrophages (hMϕ or bMϕ, respectively) infected with Mbv and Mtb. We show that Mbv and Mtb reside in different cellular compartments and differentially replicate in hMϕ whereas both Mbv and Mtb efficiently replicate in bMϕ. Specifically, we show that out of the four infection combinations, only the infection of bMϕ with Mbv promoted the formation of multinucleated giant cells (MNGCs), a hallmark of tuberculous granulomas. Mechanistically, we demonstrate that both MPB70 from Mbv and extracellular vesicles released by Mbv-infected bMϕ promote macrophage multinucleation. Importantly, we extended our in vitro studies to show that granulomas from Mbv-infected but not Mtb-infected cattle contained higher numbers of MNGCs. Our findings implicate MNGC formation in the contrasting pathology between Mtb and Mbv for the bovine host and identify MPB70 from Mbv and extracellular vesicles from bMϕ as mediators of this process.     

Stimulation of allergen-loaded macrophages by TLR9-ligand potentiates IL-10-mediated suppression of allergic airway inflammation in mice

BackgroundPreviously, we demonstrated that OVA-loaded macrophages (OVA-Mφ) partially suppress OVA-induced airway manifestations of asthma in BALB/c mice. In vitro studies showed that OVA-Mφ start to produce IL-10 upon interaction with allergen-specific T cells, which might mediate their immunosuppressive effects. Herein, we examined whether IL-10 is essential for the immunosuppressive effects of OVA-Mφ in vivo, and whether ex vivo stimulation of the IL-10 production by OVA-Mφ could enhance these effects.MethodsPeritoneal Mφ were loaded with OVA and stimulated with LPS or immunostimulatory sequence oligodeoxynucleotide (ISS-ODN) in vitro. The increase of IL-10 production was examined and, subsequently, ex vivo stimulated OVA-Mφ were used to treat (i.v.) OVA-sensitized mice. To further explore whether Mφ-derived IL-10 mediates the immunosuppressive effects, Mφ isolated from IL-10^-/- mice were used for treatment.ResultsWe found that stimulation with LPS or ISS-ODN highly increased the IL-10 production by OVA-Mφ (2.5-fold and 4.5-fold increase, respectively). ISS-ODN stimulation of OVA-Mφ significantly potentiated the suppressive effects on allergic airway inflammation. Compared to sham-treatment, ISS-ODN-stimulated OVA-Mφ suppressed the airway eosinophilia by 85% (vs. 30% by unstimulated OVA-Mφ), IL-5 levels in bronchoalveolar lavage fluid by 80% (vs. 50%) and serum OVA-specific IgE levels by 60% (vs. 30%). Importantly, IL-10^-/-Mφ that were loaded with OVA and stimulated with ISS-ODN ex vivo, failed to suppress OVA-induced airway inflammation.ConclusionsThese results demonstrate that Mφ-derived IL-10 mediates anti-inflammatory responses in a mouse model of allergic asthma, which both can be potentiated by stimulation with ISS-ODN.     

CD1d deficiency limits tolerogenic properties of peritoneal macrophages

Invariant natural killer T (iNKT) cells are involved in various autoimmune diseases. Although iNKT cells are arthritogenic, transforming growth factor beta (TGFβ)-treated tolerogenic peritoneal macrophages (Tol-pMφ) from wild-type (WT) mice are more tolerogenic than those from CD1d knock-out iNKT cell-deficient mice in a collagen-induced arthritis (CIA) model. The underlying mechanism by which pMφ can act as tolerogenic antigen presenting cells (APCs) is currently unclear. To determine cellular mechanisms underlying CD1d-dependent tolerogenicity of pMφ, in vitro and in vivo characteristics of pMφ were investigated. Unlike dendritic cells or splenic Mφ, pMφ from CD1d^+/− mice showed lower expression levels of costimulatory molecule CD86 and produced lower amounts of inflammatory cytokines upon lipopolysaccharide (LPS) stimulation compared to pMφ from CD1d-deficient mice. In a CIA model of CD1d-deficient mice, adoptively transferred pMφ from WT mice reduced the severity of arthritis. However, pMφ from CD1d-deficient mice were unable to reduce the severity of arthritis. Hence, the tolerogenicity of pMφ is a cell-intrinsic property that is probably confer-red by iNKT cells during pMφ development rather than by interactions of pMφ with iNKT cells during antigen presentation to cognate T cells.     

CD14-Dependent Monocyte Isolation Enhances Phagocytosis of Listeria monocytogenes by Proinflammatory,GM-CSF-Derived Macrophages

Macrophages are an important line of defence against invading pathogens. Human macrophages derived by different methods were tested for their suitability as models to investigate Listeria monocytogenes (Lm) infection and compared to macrophage-like THP-1 cells. Human primary monocytes were isolated by either positive or negative immunomagnetic selection and differentiated in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) into pro- or anti-inflammatory macrophages, respectively. Regardless of the isolation method, GM-CSF-derived macrophages (GM-Mφ) stained positive for CD206 and M-CSF-derived macrophages (M-Mφ) for CD163. THP-1 cells did not express CD206 or CD163 following incubation with PMA, M- or GM-CSF alone or in combination. Upon infection with Lm, all primary macrophages showed good survival at high multiplicities of infection whereas viability of THP-1 was severely reduced even at lower bacterial numbers. M-Mφ generally showed high phagocytosis of Lm. Strikingly, phagocytosis of Lm by GM-Mφ was markedly influenced by the method used for isolation of monocytes. GM-Mφ derived from negatively isolated monocytes showed low phagocytosis of Lm whereas GM-Mφ generated from positively selected monocytes displayed high phagocytosis of Lm. Moreover, incubation with CD14 antibody was sufficient to enhance phagocytosis of Lm by GM-Mφ generated from negatively isolated monocytes. By contrast, non-specific phagocytosis of latex beads by GM-Mφ was not influenced by treatment with CD14 antibody. Furthermore, phagocytosis of Lactococcus lactis, Escherichia coli, human cytomegalovirus and the protozoan parasite Leishmania major by GM-Mφ was not enhanced upon treatment with CD14 antibody indicating that this effect is specific for Lm. Based on these observations, we propose macrophages derived by ex vivo differentiation of negatively selected human primary monocytes as the most suitable model to study Lm infection of macrophages.     

The sst1 Resistance Locus Regulates Evasion of Type I Interferon Signaling by Chlamydia pneumoniae as a Disease Tolerance Mechanism

The sst1, “supersusceptibility to tuberculosis,” locus has previously been shown to be a genetic determinant of host resistance to infection with the intracellular pathogen, Mycobacterium tuberculosis. Chlamydia pneumoniae is an obligate intracellular bacterium associated with community acquired pneumonia, and chronic infection with C. pneumoniae has been linked to asthma and atherosclerosis. C. pneumoniae is a highly adapted pathogen that can productively infect macrophages and inhibit host cell apoptosis. Here we examined the role of sst1 in regulating the host response to infection with C. pneumoniae. Although mice carrying the sst1 susceptible (sst1^S) locus were not impaired in their ability to clear the acute infection, they were dramatically less tolerant of the induced immune response, displaying higher clinical scores, more severe lung inflammation, exaggerated macrophage and neutrophil influx, and the development of fibrosis compared to wild type mice. This correlated with increased activated caspase-3 in the lungs of infected sst1^S mice. Infection of sst1^S macrophages with C. pneumoniae resulted in a shift in the secreted cytokine profile towards enhanced production of interferon-β and interleukin-10, and induced apoptotic cell death, which was dependent on secretion of interferon-β. Intriguingly macrophages from the sst1^S mice failed to support normal chlamydial growth, resulting in arrested development and failure of the organism to complete its infectious cycle. We conclude that the sst1 locus regulates a shared macrophage-mediated innate defense mechanism against diverse intracellular bacterial pathogens. Its susceptibility allele leads to upregulation of type I interferon pathway, which, in the context of C. pneumoniae, results in decreased tolerance, but not resistance, to the infection. Further dissection of the relationship between type I interferons and host tolerance during infection with intracellular pathogens may provide identification of biomarkers and novel therapeutic targets.     

Salmonella Induced IL-23 and IL-1β Allow for IL-12 Production by Monocytes and M?1 through Induction of IFN-γ in CD56+ NK/NK-Like T Cells

Background

The type-1 cytokine pathway plays a pivotal role in immunity against intracellular bacterial pathogens such as Salmonellae and Mycobacteria. Bacterial stimulation of pattern recognition receptors on monocytes, macrophages and dendritic cells initiates this pathway, and results in the production of cytokines that activate lymphocytes to produce interferon (IFN)-γ. Interleukin (IL)-12 and IL-23 are thought to be the key cytokines required for initiating a type-1 cytokine immune response to Mycobacteria and Salmonellae. The relative contribution of IL-23 and IL-12 to this process is uncertain.

Methodology/Principal Findings

We show that various TLR agonists induce the production of IL-23 but not IL-12 in freshly isolated human monocytes and cultured human macrophages. In addition, type 1 pro-inflammatory macrophages (Mϕ1) differentiated in the presence of GM-CSF and infected with live Salmonella produce IL-23, IL-1β and IL-18, but not IL-12. Supernatants of Salmonella-infected Mϕ1 contained more IL-18 and IL-1β as compared with supernatants of Mϕ1 stimulated with isolated TLR agonists, and induced IFN-γ production in human CD56⁺ cells in an IL-23 and IL-1β-dependent but IL-12-independent manner. In addition, IL-23 together with IL-18 or IL-1β led to the production of GM-CSF in CD56⁺ cells. Both IFN-γ and GM-CSF enhanced IL-23 production by monocytes in response to TLR agonists, as well as induced IL-12 production.

Conclusions/Significance

The findings implicate a positive feedback loop in which IL-23 can enhance its release via induction of IFN-γ and GM-CSF. The IL-23 induced cytokines allow for the subsequent production of IL-12 and amplify the IFN-γ production in the type-1 cytokine pathway.     

The effect of myeloperoxidase-oxidized LDL on THP-1 macrophage polarization and repolarization

Macrophages (Mφs) play a crucial role in the development of atherosclerosis by engulfing modified LDL particles and forming foam cells, the hallmark of atherosclerosis. Many studies suggest that myeloperoxidase-oxidized LDL (Mox-LDL) is an important pathophysiological model for LDL modification in vivo. Classically (M1) and alternatively activated (M2) Mφs are both implicated in the process of atherogenesis. Mφs are highly plastic cells whereby they undergo repolarization from M1 to M2 and vice versa. Since little is known about the effects of Mox-LDL on Mφ polarization and repolarization, our study aimed at evaluating the in vitro effects of Mox-LDL at this level through making use of the well-established model of human THP-1-derived Mφs. Resting M0-Mφs were polarized toward M1- and M2-Mφs, then M0-, M1- and M2-Mφs were all treated with physiological concentrations of Mox-LDL to assess the effect of Mox-LDL treatment on Mφ polarization and repolarization. Treatment of M0-Mφs with a physiological concentration of Mox-LDL had no significant effects at the level of their polarization. However, treatment of M1-Mφs with Mox-LDL resulted in a significant reduction in their IL-10 cytokine secretion. Our results point to a potential role of Mox-LDL in increasing the pro-inflammatory state in Mφs through reducing the release of the anti-inflammatory cytokine, IL-10.     

Chlamydia pneumoniae Hides inside Apoptotic Neutrophils to Silently Infect and Propagate in Macrophages

Background

Intracellular pathogens have developed elaborate strategies for silent infection of preferred host cells. Chlamydia pneumoniae is a common pathogen in acute infections of the respiratory tract (e.g. pneumonia) and associated with chronic lung sequelae in adults and children. Within the lung, alveolar macrophages and polymorph nuclear neutrophils (PMN) are the first line of defense against bacteria, but also preferred host phagocytes of chlamydiae.

Methodology/Principal Findings

We could show that C. pneumoniae easily infect and hide inside neutrophil granulocytes until these cells become apoptotic and are subsequently taken up by macrophages. C. pneumoniae infection of macrophages via apoptotic PMN results in enhanced replicative activity of chlamydiae when compared to direct infection of macrophages, which results in persistence of the pathogen. Inhibition of the apoptotic recognition of C. pneumoniae infected PMN using PS- masking Annexin A5 significantly lowered the transmission of chlamydial infection to macrophages. Transfer of apoptotic C. pneumoniae infected PMN to macrophages resulted in an increased TGF-ß production, whereas direct infection of macrophages with chlamydiae was characterized by an enhanced TNF-α response.

Conclusions/Significance

Taken together, our data suggest that C. pneumoniae uses neutrophil granulocytes to be silently taken up by long-lived macrophages, which allows for efficient propagation and immune protection within the human host.     

Arabidopsis Heterotrimeric G-Proteins Play a Critical Role in Host and Nonhost Resistance against Pseudomonas syringae Pathogens

Heterotrimeric G-proteins have been proposed to be involved in many aspects of plant disease resistance but their precise role in mediating nonhost disease resistance is not well understood. We evaluated the roles of specific subunits of heterotrimeric G-proteins using knock-out mutants of Arabidopsis Gα, Gβ and Gγ subunits in response to host and nonhost Pseudomonas pathogens. Plants lacking functional Gα, Gβ and Gγ1Gγ2 proteins displayed enhanced bacterial growth and disease susceptibility in response to host and nonhost pathogens. Mutations of single Gγ subunits Gγ1, Gγ2 and Gγ3 did not alter bacterial disease resistance. Some specificity of subunit usage was observed when comparing host pathogen versus nonhost pathogen. Overexpression of both Gα and Gβ led to reduced bacterial multiplication of nonhost pathogen P. syringae pv. tabaci whereas overexpression of Gβ, but not of Gα, resulted in reduced bacterial growth of host pathogen P. syringae pv. maculicola, compared to wild-type Col-0. Moreover, the regulation of stomatal aperture by bacterial pathogens was altered in Gα and Gβ mutants but not in any of the single or double Gγ mutants. Taken together, these data substantiate the critical role of heterotrimeric G-proteins in plant innate immunity and stomatal modulation in response to P. syringae.