Szczepan Józefowski

Disparate Regulation and Function of the Class A Scavenger Receptors SR-AI/II and MARCO

The macrophage class A scavenger receptors, macrophage receptor with a collagenous structure (MARCO) and type I/II class A scavenger receptor (SR-AI/II), share structural features and roles in host defense, but little is known about their regulation and signaling properties. Ligation of MARCO on mouse thioglycollate-elicited peritoneal macrophages (PEMs) with immobilized mAb costimulated IL-12 production, in contrast to previously reported inhibition by SR-AI/II. PEMs from MARCO-deficient mice exhibited 2.7 times lower IL-12 production in responses to stimulation with LPS and IFN-γ and lack of significant IL-12 production on stimulation with LPS alone. Conversely, SR-AI/II-deficient PEMs produced 2.4 and 1.8 times more IL-12 than wild-type PEMs in response to LPS or LPS and IFN-γ, respectively. Corresponding differences in regulation of SR-A and MARCO expression were also observed. Th1 adjuvants (LPS, a CpG motif-containing oligodeoxynucleotide (CpG-ODN), IL-12, and GM-CSF) increased, whereas Th2-polarizing factors (IL-4, M-CSF, and non-CpG ODN) decreased expression of MARCO on J774 macrophage-like cells. Expression of SR-A was regulated in the opposite manner to MARCO or not affected. Whereas MARCO was involved in opsonin-independent phagocytosis in CpG-ODN-pretreated but not in IL-4-pretreated J774 cells, anti-SR-A Abs inhibited particle uptake in untreated and IL-4-pretreated but not in CpG-ODN-pretreated cells. SR-A and MARCO are regulated differently and mediate distinct negative and positive effects on IL-12 production in macrophages. These differences may contribute to sustained Th1 or Th2 polarization of ongoing immune responses.

Role of scavenger receptor MARCO in macrophage responses to CpG oligodeoxynucleotides

The macrophage Class A scavenger receptor MARCO (macrophage receptor with a collagenous structure) functions as a pattern‐recognition receptor for bacterial components, but its role in responses to CpG oligonucleotide sequences (CpG‐ODN) in microbial DNA has not been characterized. Phosphorothioate (PS)‐linked CpG‐ODN stimulated IL‐12 and NO production in wild‐type but not in MARCO‐deficient, thioglycollate‐elicited peritoneal macrophages. MARCO and the related class A receptor SR‐A belong to a redundant system of receptors for PS ODNs. The ability of MARCO to bind CpG‐ODNs and conversely, to costimulate IL‐12 and NO production upon specific ligation with immobilized mAb is consistent with MARCO being a signaling receptor for CpG‐ODNs, costimulating TLR9‐mediated NO and IL‐12 production in macrophages. In contrast to MARCO, SR‐A is likely to mediate negative regulation of macrophage responses to CpG‐ODNs. In particular, increased affinity toward SR‐A may contribute to decreased potency of oligo G‐modified CpG‐ODNs in stimulating IL‐12 production. The results suggest that differential involvement of activating and inhibitory membrane receptors, such as SR‐A and MARCO, may underlie profound differences observed in biological activities of different ODN sequences.

Scavenger receptor A mediates H2O2 production and suppression of IL-12 release in murine macrophages

Although class A type I/II scavenger receptor (SR‐A) is involved in numerous macrophage functions, its signaling ability remains uncertain. We used monoclonal antibodies (mAb) to specifically stimulate receptors on mouse alveolar (AMs) and peritoneal macrophages (PMs). Immobilized anti‐SR‐A (2F8) and anti‐FcγR II/III (2.4G2) mAb stimulated hydrogen peroxide (H2O2) production in normal C3H/HeJ AMs (by 55% and 98%, respectively) and resident PMs (66% and 128%). The 2F8 mAb‐stimulated H2O2 production resulted from specific stimulation of SR‐A, since this response was absent in AMs from SR‐A‐deficient or C57BL/6 mice—the latter strain expressing an allelic form of SR‐A, unrecognizable by 2F8 mAb. H2O2 production stimulated by anti‐SR‐A but not by anti‐FcγRII/III mAb was preserved in FcγRI/III‐deficient mice, ruling out involvement of FcγRs in the 2F8 mAb effect. In comparison with the FcγR‐stimulated respiratory burst, the response to anti‐SR‐A mAb was delayed and, unlike the former, inhibited by pertussis toxin. Ligation of SR‐A also inhibited lipopolysaccharide plus interferon‐γ‐stimulated interleukin‐12 (IL‐12) release, by 25% in AMs and by 68% in thioglycollate‐elicited PMs, consistent with different levels of SR‐A expression. Neither nitrite nor IL‐6 accumulation was affected by anti‐SR‐A mAb. SR‐A‐stimulated H2O2 does not seem to mediate the inhibition of IL‐12 release, since the inhibition was neither reversed by scavenging of H2O2 nor mimicked by exogenous H2O2. Our results indicate that SR‐A not only mediates endocytosis but can also generate signals such as H2O2, which may affect microbicidal or proinflammatory functions.

Exogenous but not endogenous prostanoids regulate cytokine secretion from murine bone marrow dendritic cells: EP2, DP, and IP but not EP1, EP3, and FP prostanoid receptors are involved

Murine bone marrow-derived dendritic cells (DC), stimulated with lipopolysaccharide (LPS) and/or LPS+interferon-gamma (IFN-gamma), secrete a variety of inflammatory mediators which may modulate their functions. We have examined the potential for exogenous prostanoids, acting in a paracrine fashion, and endogenous prostanoids, acting in an autocrine fashion, to regulate secretion of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), IL-10, and IL-12 in DC. In order to identify receptors mediating these effects, DC were treated in vitro with receptor-selective prostanoids. Agonists of cyclic AMP-elevating receptors, namely, prostaglandin E(2) (PGE(2)), butaprost (EP(2) receptor), iloprost (IP receptor), and BW245C (DP receptor), dose-dependently inhibited the release of IL-6, TNF-alpha, and IL-12 and enhanced the release of IL-10 from LPS-stimulated DC, with TNF-alpha secretion being the most strongly affected. In contrast, 15-deoxy-Delta(12,14)-PGJ(2)-an activator of nuclear peroxisome proliferator-activated receptor-gamma (PPAR-gamma) receptors-inhibited release of all tested cytokines. Exogenous prostanoids, cyclic AMP-elevating analogs, lost their ability to modulate cytokine release in cells pre-incubated for 4 h with LPS, indicating that prostanoids may affect DC functions during initial phases of LPS stimulation only. Sulprostone and (+)-fluprostenol failed to modulate any of responses tested, suggesting lack of involvement/expression of EP(1), EP(3), and FP receptors in DC activation. In order to examine the role of endogenous prostanoids, DC were treated with inhibitors of cyclooxygenases (COX). At concentrations that completely blocked PGE(2) release, neither indomethacin (nonselective inhibitor) nor rofecoxib (COX-2-selective inhibitor) influenced cytokine release from LPS-stimulated DC. Thus, cytokine release from LPS-stimulated DC does not seem to be autoregulated by endogenous prostanoids, whereas in vivo regulatory function may be fulfilled in a paracrine manner by PGD(2), PGE(2), and PGI(2) released from neighboring cells.

Leukotrienes modulate cytokine release from dendritic cells

Leukotriene B4 (LTB4) and cysteinyl leukotrienes (CysLTs) are known as potent mediators of inflammation, whereas their role in the regulation of adaptive immunity remains poorly characterized. Dendritic cells (DCs) are specialized antigen‐presenting cells, uniquely capable to initiate primary immune responses. We have found that zymosan, but not lipopolysaccharide (LPS) stimulates murine bone marrow‐derived dendritic cells (BM‐DCs) to produce large amounts of CysLTs and LTB4 from endogenous substrates. A selective inhibitor of leukotriene synthesis MK886 as well as an antagonist of the high affinity LTB4 receptor (BLT1) U‐75302 slightly inhibited zymosan‐, but not LPS‐stimulated interleukin (IL)‐10 release from BM‐DCs. In contrast, U‐75302 increased zymosan‐stimulated release of IL‐12 p40 by ∼23%. Pre‐treatment with transforming growth factor‐β1 enhanced both stimulated leukotriene synthesis and the inhibitory effect of U‐75302 and MK886 on IL‐10 release from DCs. Consistent with the effects of leukotriene antagonists, exogenous LTB4 enhanced LPS‐stimulated IL‐10 release by ∼39% and inhibited IL‐12 p40 release by ∼22%. Both effects were mediated by the BLT1 receptor. Ligands of the high affinity CysLTs receptor (CysLT1), MK‐571 and LTD4 had little or no effect on cytokine release. Agonists of the nuclear LTB4 receptor peroxisome proliferator‐activated receptor‐α, 8(S)‐hydroxyeicosatetraenoic acid and 5,8,11,14‐eicosatetraynoic acid, inhibited release of both IL‐12 p40 and IL‐10. Our results indicate that both autocrine and paracrine leukotrienes may modulate cytokine release from DCs, in a manner that is consistent with previously reported T helper 2‐polarizing effects of leukotrienes.

Scavenger receptors and β-glucan receptors participate in the recognition of yeasts by murine macrophages

ObjectivesNumerous receptors have been implicated in recognition of pathogenic fungi by macrophages, including the β-glucan receptor dectin-1. The role of scavenger receptors (SRs) in anti-fungal immunity is not well characterized.MethodsWe studied uptake of unopsonized Saccharomycetes cerevisiae (zymosan) and live Candida albicans yeasts as well as zymosan-stimulated H2O2 production in J774 macrophage-like cells and peritoneal exudate macrophages (PEMs). The role of different receptors was assessed with the use of competitive ligands, transfected cells and receptor-deficient macrophages.ResultsThe uptake of zymosan by untreated J774 cells was mediated approximately half by SRs and half by a β-glucan receptor which was distinct from dectin-1 and not linked to stimulation of H2O2 production. Ligands of β-glucan receptors and of SRs also inhibited uptake of C. albicans by macrophages (J774 cells and PEMs). In macrophages pretreated with a CpG motif-containing oligodeoxynucleotide (CpG-ODN) the relative contribution of SRs to yeast uptake increased and that of β-glucan receptors decreased. Whereas the class A SR MARCO participated in the uptake of both zymosan and C. albicans by CpG-ODN-pretreated, but not untreated macrophages, the related receptor SR-A/CD204 was involved in the uptake of zymosan, but not of C. albicans. The reduction of zymosan-stimulated H2O2 production observed in DS-pretreated J774 cells and in class A SRs-deficient PEMs suggest that class A SRs mediate part of this process.ConclusionsOur results revealed that SRs belong to a redundant system of receptors for yeasts. Binding of yeasts to different receptors in resting versus CpG-ODN-pre-exposed macrophages may differentially affect polarization of adaptive immune responses.

Female resistance to pneumonia identifies lung macrophage nitric oxide Synthase-3 as a therapeutic target

To identify new approaches to enhance innate immunity to bacterial pneumonia, we investigated the natural experiment of gender differences in resistance to infections. Female and estrogen-treated male mice show greater resistance to pneumococcal pneumonia, seen as greater bacterial clearance, diminished lung inflammation, and better survival. In vitro, lung macrophages from female mice and humans show better killing of ingested bacteria. Inhibitors and genetically altered mice identify a critical role for estrogen-mediated activation of lung macrophage nitric oxide synthase-3 (NOS3). Epidemiologic data show decreased hospitalization for pneumonia in women receiving estrogen or statins (known to activate NOS3). Pharmacologic targeting of NOS3 with statins or another small-molecule compound (AVE3085) enhanced macrophage bacterial killing, improved bacterial clearance, and increased host survival in both primary and secondary (post-influenza) pneumonia. The data identify a novel mechanism for host defense via NOS3 and suggest a potential therapeutic strategy to reduce secondary bacterial pneumonia after influenza. DOI: http://dx.doi.org/10.7554/eLife.03711.001

Oxidation by Neutrophils-Derived HOCl Increases Immunogenicity of Proteins by Converting Them into Ligands of Several Endocytic Receptors Involved in Antigen Uptake by Dendritic Cells and Macrophages

The initiation of adaptive immune responses to protein antigens has to be preceded by their uptake by antigen presenting cells and intracellular proteolytic processing. Paradoxically, endocytic receptors involved in antigen uptake do not bind the majority of proteins, which may be the main reason why purified proteins stimulate at most weak immune responses. A shared feature of different types of adjuvants, capable of boosting immunogenicity of protein vaccines, is their ability to induce acute inflammation, characterized by early influx of activated neutrophils. Neutrophils are also rapidly recruited to sites of tissue injury or infection. These cells are the source of potent oxidants, including hypochlorous acid (HOCl), causing oxidation of proteins present in inflammatory foci. We demonstrate that oxidation of proteins by endogenous, neutrophils-derived HOCl increases their immunogenicity. Upon oxidation, different, randomly chosen simple proteins (yeast alcohol dehydrogenase, human and bovine serum albumin) and glycoproteins (human apo-transferrin, ovalbumin) gain the ability to bind with high affinity to several endocytic receptors on antigen presenting cells, which seems to be the major mechanism of their increased immunogenicity. The mannose receptor (CD206), scavenger receptors A (CD204) and CD36 were responsible for the uptake and presentation of HOCl-modified proteins by murine dendritic cells and macrophages. Other scavenger receptors, SREC-I and LOX-1, as well as RAGE were also able to bind HOCl-modified proteins, but they did not contribute significantly to these ligands uptake by dendritic cells because they were either not expressed or exhibited preference for more heavily oxidised proteins. Our results indicate that oxidation by neutrophils-derived HOCl may be a physiological mechanism of conferring immunogenicity on proteins which in their native forms do not bind to endocytic receptors. This mechanism might enable the immune system to detect infections caused by pathogens not recognized by pattern recognition receptors.

The class A scavenger receptor SR-A/CD204 and the class B scavenger receptor CD36 regulate immune functions of macrophages differently

SR-A/CD204 and CD36 are major receptors responsible for oxidized lipoproteins uptake by macrophages in atherosclerotic plaques. Both receptors also share the role as receptors for different pathogens, but studies on their signaling have been hampered by the lack of selective ligands. We report that, upon specific ligation by Ab, SR-A does not induce cytokine production, but mediates inhibition of LPS-stimulated production of IL-6 and IL-12/23p40, enhancement of IL-10 release, and has no effect on TNF-α and RANTES production in murine macrophages. In contrast, anti-CD36 Ab alone stimulated production of all these cytokines, with IL-10 production being exceptionally high. Effects of anti-CD36 Ab, except of IL-10 production, were mediated by CD14 and TLR2, whereas those of SR-A ligation by heterotrimeric Gi/o proteins and by phosphatidylinositol 3-kinases. Surprisingly, we found that LPS uptake by macrophages was mediated in part by CD36 cooperating with CD14, whereas SR-A was not involved in this process. Finely, during in vitro Ag presentation to naïve CD4+ lymphocytes, pre-incubation of macrophages with anti-CD36 Ab enhanced IFN-γ production in the co-culture, but exerted the opposite effect under conditions enabling IL-10 accumulation. In contrast, anti-SR-A Ab was ineffective alone, but reversed the Th1-polarizing effect of LPS.

CD36 Differently Regulates Macrophage Responses to Smooth and Rough Lipopolysaccharide

Lipopolysaccharide (LPS) is the major pathogen-associated molecular pattern of Gram-negative bacterial infections, and includes smooth (S-LPS) and rough (R-LPS) chemotypes. Upon activation by LPS through CD14, TLR4/MD-2 heterodimers sequentially induce two waves of intracellular signaling for macrophage activation: the MyD88-dependent pathway from the plasma membrane and, following internalization, the TRIF-dependent pathway from endosomes. We sought to better define the role of scavenger receptors CD36 and CD204/SR-A as accessory LPS receptors that can contribute to pro-inflammatory and microbicidal activation of macrophages. We have found that CD36 differently regulates activation of mouse macrophages by S-LPS versus R-LPS. The ability of CD36 to substitute for CD14 in loading R-LPS, but not S-LPS onto TLR4/MD-2 allows CD14-independent macrophage responses to R-LPS. Conversely, S-LPS, but not R-LPS effectively stimulates CD14 binding to CD36, which favors S-LPS transfer from CD14 onto TLR4/MD-2 under conditions of low CD14 occupancy with S-LPS in serum-free medium. In contrast, in the presence of serum, CD36 reduces S-LPS binding to TLR4/MD-2 and the subsequent MyD88-dependent signaling, by mediating internalization of S-LPS/CD14 complexes. Additionally, CD36 positively regulates activation of TRIF-dependent signaling by both S-LPS and R-LPS, by promoting TLR4/MD-2 endocytosis. In contrast, we have found that SR-A does not function as a S-LPS receptor. Thus, by co-operating with CD14 in both R- and S-LPS loading onto TLR4/MD-2, CD36 can enhance the sensitivity of tissue-resident macrophages in detecting infections by Gram-negative bacteria. However, in later phases, following influx of serum to the infection site, the CD36-mediated negative regulation of MyD88-dependent branch of S-LPS-induced TLR4 signaling might constitute a mechanism to prevent an excessive inflammatory response, while preserving the adjuvant effect of S-LPS for adaptive immunity.