Subhankar Mukhopadhyay

Scavenger receptors in innate immunity

Scavenger receptors (SR) are expressed by myeloid cells (macrophages and dendritic cells) and certain endothelial cells. They play an important role in uptake and clearance of effete components, such as modified host molecules and apoptotic cells. They bind and internalise micro-organisms and their products including Gram-positive bacteria (lipoteichoic acid), Gram-negative bacteria (lipopolysaccharide), intracellular bacteria and CpG DNA. SR can alter cell morphology and their expression is affected by various cytokines. SR are involved in lipid metabolism and bind modified low-density lipoproteins.

MARCO, TLR2, and CD14 Are Required for Macrophage Cytokine Responses to Mycobacterial Trehalose Dimycolate and Mycobacterium tuberculosis

Virtually all of the elements of Mycobacterium tuberculosis (Mtb) pathogenesis, including pro-inflammatory cytokine production, granuloma formation, cachexia, and mortality, can be induced by its predominant cell wall glycolipid, trehalose 6,6′-dimycolate (TDM/cord factor). TDM mediates these potent inflammatory responses via interactions with macrophages both in vitro and in vivo in a myeloid differentiation factor 88 (MyD88)-dependent manner via phosphorylation of the mitogen activated protein kinases (MAPKs), implying involvement of toll-like receptors (TLRs). However, specific TLRs or binding receptors for TDM have yet to be identified. Herein, we demonstrate that the macrophage receptor with collagenous structure (MARCO), a class A scavenger receptor, is utilized preferentially to “tether” TDM to the macrophage and to activate the TLR2 signaling pathway. TDM-induced signaling, as measured by a nuclear factor-kappa B (NF-κB)-luciferase reporter assay, required MARCO in addition to TLR2 and CD14. MARCO was used preferentially over the highly homologous scavenger receptor class A (SRA), which required TLR2 and TLR4, as well as their respective accessory molecules, in order for a slight increase in NF-κB signaling to occur. Consistent with these observations, macrophages from MARCO−/− or MARCO−/−SRA−/− mice are defective in activation of extracellular signal-related kinase 1/2 (ERK1/2) and subsequent pro-inflammatory cytokine production in response to TDM. These results show that MARCO-expressing macrophages secrete pro-inflammatory cytokines in response to TDM by cooperation between MARCO and TLR2/CD14, whereas other macrophage subtypes (e.g. bone marrow–derived) may rely somewhat less effectively on SRA, TLR2/CD14, and TLR4/MD2. Macrophages from MARCO−/− mice also produce markedly lower levels of pro-inflammatory cytokines in response to infection with virulent Mtb. These observations identify the scavenger receptors as essential binding receptors for TDM, explain the differential response to TDM of various macrophage populations, which differ in their expression of the scavenger receptors, and identify MARCO as a novel component required for TLR signaling.

The potential for Toll‐like receptors to collaborate with other innate immune receptors

Cells of the innate immune system express a large repertoire of germ‐line encoded cell‐surface glycoprotein receptors including Toll‐like receptors (TLRs). TLRs recognize conserved motifs on microbes and induce inflammatory signals. Evidence suggests that individual members of the TLR family or other non‐TLR surface antigens either physically or functionally interact with each other and cumulative effects of these interactions instruct the nature and outcome of the immune response to a particular pathogen.

Innate immunity to intracellular pathogens: macrophage receptors and responses to microbial entry

Summary:  Innate immunity to intracellular pathogens encompasses a range of interactions of cellular and humoral activities of the host with the invading microorganism, determining the outcome of infection. Here, we review the particular role of macrophage recognition receptors and effector responses in the uptake of microbes and their products. We place this in context and raise issues for discussion and further experimentation.

The interaction of macrophage receptors with bacterial ligands

Innate immune receptors play a key role in the early recognition of invading bacterial pathogens and initiate the crucial innate immune response. The diverse macrophage receptors recognise Gram-positive and Gram-negative bacteria via conserved structures on the bacterial surface and facilitate phagocytosis and/or signalling, providing the trigger for the adaptive immune response. These receptors include scavenger receptors, C-type lectins, integrins, Toll-like receptors and siglecs. The bacterial ligands generally recognised by these receptors range from lipopolysaccharides on Gram-negative bacteria to peptidoglycan and lipoteichoic acid on Gram-positive bacteria. However, emerging evidence indicates that bacterial proteins are also important ligands; for example, surface proteins from Neisseria meningitidis have been shown to be ligands for class A scavenger receptors. In addition, a group of cytosolic receptors, the NBS-LRR proteins, have been implicated in recognition of bacterial breakdown products. It is becoming increasingly apparent that macrophage receptors can act in conjunction with one another to deliver an appropriate response.

MARCO, an innate activation marker of macrophages, is a class A scavenger receptor for Neisseria meningitidis

The scavenger receptor‐A I/II (SR‐A) and macrophage receptor with collagenous domain (MARCO) share a common domain organisation and ligand repertoire, including selected polyanions and gram‐positive and ‐negative organisms, but differ in fine specificity of ligand binding, tissue distribution and regulation. Neisseria meningitidis (NM) is a selective ligand for SR‐A, but there is evidence for an additional SR‐A‐independent, polyanion‐sensitive component for NM recognition. We therefore studied the relative contribution of MARCO and SR‐A to binding of NM by resident and elicited peritoneal macrophages obtained from MARCO–/–, SR‐A–/– and SR‐A‐MARCO–/– mice. Results confirmed that both mouse and human MARCO are able to bind NM independently of NM LPS. MARCO and SR‐A contributed independently to NM binding, correlating with their expression levels in different cell populations, but neither of these two molecules was required for release of TNF‐α and nitric oxide. We propose that the TLR‐dependent induction of MARCO by innate immune stimulation enhances recognition and uptake of pathogenic organisms such as NM, thus contributing to host defence against infection.

Interaction of Salmonella enterica Serovar Typhimurium with Intestinal Organoids Derived from Human Induced Pluripotent Stem Cells

ABSTRACT The intestinal mucosa forms the first line of defense against infections mediated by enteric pathogens such as salmonellae. Here we exploited intestinal “organoids” (iHOs) generated from human induced pluripotent stem cells (hIPSCs) to explore the interaction of Salmonella enterica serovar Typhimurium with iHOs. Imaging and RNA sequencing were used to analyze these interactions, and clear changes in transcriptional signatures were detected, including altered patterns of cytokine expression after the exposure of iHOs to bacteria. S. Typhimurium microinjected into the lumen of iHOs was able to invade the epithelial barrier, with many bacteria residing within Salmonella-containing vacuoles. An S. Typhimurium invA mutant defective in the Salmonella pathogenicity island 1 invasion apparatus was less capable of invading the iHO epithelium. Hence, we provide evidence that hIPSC-derived organoids are a promising model of the intestinal epithelium for assessing interactions with enteric pathogens.

Immune Inhibitory Ligand CD200 Induction by TLRs and NLRs Limits Macrophage Activation to Protect the Host from Meningococcal Septicemia

Macrophage activation is essential for protection against bacterial pathogens but needs to be regulated to prevent damage to the host. We show a key role for the immune inhibitory receptor CD200R and its ligand CD200 in the context of infection with the Gram-negative human pathogen Neisseria meningitidis. N. meningitidis induced CD200 but downregulated CD200R on macrophages in a manner dependent on Neisserial lipopolysaccharide, Toll-like receptor-4 (TLR-4), and the MyD88 pathway but independent of a known Neisserial receptor, scavenger receptor A (SR-A). Agonists of the pattern-recognition receptors nucleotide oligomerization domain 2 (NOD2) and NACHT-LRR protein 3 (NALP3) also induced CD200. The NF-κB member c-Rel was essential for TLR-, NOD2-, and NALP3-mediated induction of CD200. CD200(-/-) animals showed higher lethality in response to experimental meningococcal septicemia, induced higher levels of proinflammatory cytokines, and recruited increased numbers of activated leukocytes, despite comparable bacterial clearance. Thus CD200 is induced by TLR-, NOD2-, and NALP3-mediated pathways, limiting their function and protecting the host from excessive inflammation.

Macrophage Scavenger Receptor A Promotes Tumor Progression in Murine Models of Ovarian and Pancreatic Cancer

Alternatively activated macrophages express the pattern recognition receptor scavenger receptor A (SR-A). We demonstrated previously that coculture of macrophages with tumor cells upregulates macrophage SR-A expression. We show in this study that macrophage SR-A deficiency inhibits tumor cell migration in a coculture assay. We further demonstrate that coculture of tumor-associated macrophages and tumor cells induces secretion of factors that are recognized by SR-A on tumor-associated macrophages. We tentatively identified several potential ligands for the SR-A receptor in tumor cell–macrophage cocultures by mass spectrometry. Competing with the coculture-induced ligand in our invasion assay recapitulates SR-A deficiency and leads to similar inhibition of tumor cell invasion. In line with our in vitro findings, tumor progression and metastasis are inhibited in SR-A−/− mice in two in vivo models of ovarian and pancreatic cancer. Finally, treatment of tumor-bearing mice with 4F, a small peptide SR-A ligand able to compete with physiological SR-A ligands in vitro, recapitulates the inhibition of tumor progression and metastasis observed in SR-A−/− mice. Our observations suggest that SR-A may be a potential drug target in the prevention of metastatic cancer progression.

Activation of murine macrophages by Neisseria meningitidis and IFN-γ in vitro: distinct roles of class A scavenger and toll-like pattern recognition receptors in selective modulation of surface phenotype

Innate and adaptive immune activation of macrophages (Mφ) by microorganisms and antigen‐activated lymphoid cells, respectively, plays an important role in host defense and immunopathology. Antigen‐presenting cells express a range of pattern recognition receptors including the class A types I and II scavenger receptors (SR‐A) and Toll‐like receptors (TLR). Recognition of microbial products by SR‐A and TLR controls uptake, killing, altered gene expression, and the adaptive immune response; however, the contribution of each receptor and interplay with cytokine stimuli such as interferon‐γ (IFN‐γ) are not defined. We used Neisseria meningitidis (NM), a potent activator of innate immunity, and IFN‐γ, a protoypic T helper cell type 1 proinflammatory cytokine, to compare surface antigens, secretion of mediators, and receptor functions in elicited peritoneal Mφ from wild‐type and genetically modified mouse strains. We show that these stimuli regulate major histocompatibility complex type II (MHC‐II) and costimulatory molecules differentially, as well as expression of the mannose receptor and of Mφ receptor with collagenous structure (MARCO), a distinct SR‐A, which provides a selective marker for innate activation. In combination, NM inhibited up‐regulation of MHC‐II by IFN‐γ while priming enhanced release of tumor necrosis factor α and nitric oxide. The SR‐A contributes to phagocytosis of the organisms but not to their ability to induce CD80, CD86, and MARCO or to inhibit MHC‐II. Conversely, studies with lipopolysaccharide (LPS)‐deficient organisms and/or TLR‐4 mutant mice showed that LPS and TLR‐4 are at least partially required to induce CD80, CD86, and MARCO, but LPS is not required to inhibit MHC‐II. These studies provide an experimental model and identify surface markers for analysis of innate and acquired immune activation of Mφ.