Search

Neutrophils are the first responders to infection and inflammation and are thus a critical component of innate immune defense. Understanding the behavior of neutrophils as they act within various inflammatory contexts has provided insights into their role in sterile and infectious diseases; however, the field of neutrophils in cancer is comparatively young. Here, we summarize key concepts and current knowledge gaps related to the diverse roles of neutrophils throughout cancer progression. We discuss sources of neutrophil heterogeneity in cancer and provide recommendations on nomenclature for neutrophil states that are distinct in maturation and activation. We address discrepancies in the literature that highlight a need for technical standards that ought to be considered between laboratories. Finally, we review emerging questions in neutrophil biology and innate immunity in cancer. Overall, we emphasize that neutrophils are a more diverse population than previously appreciated and that their role in cancer may present novel unexplored opportunities to treat cancer. Acknowledgments This consensus statement is dedicated to our esteemed colleagues, Zena Werb and Paul S. Frenette, who attended and participated in the Banbury Center meeting and encouraged a collective effort by attendees to write this report. Both made significant contributions to ideas and discussions that are represented here. The Banbury Center meeting was organized by M. Egeblad, P. Kubes, K.E. de Visser, R. Leshan, and Banbury Center staff. The meeting was supported financially by Cold Spring Harbor Laboratory Northwell Health Affiliation. The funder had no involvement with the writing of this consensus statement. D.F. Quail acknowledges funding from the Canadian Institutes of Health Research (PJT-159742, PJT-178306), Terry Fox Research Institute, and Tier II Canada Research Chair. B. Amulic acknowledges funding from the Medical Research Council (MR/R02149x/1). M. Aziz acknowledges funding from the National Institutes of Health (NIH; R01GM129633). B.J. Barnes acknowledges funding from the NIH National Institute of Arthritis and Musculoskeletal and Skin Diseases (AR065959-01) and Department of Defense (CDMRP LRP W81XWH-18-1-0674). E. Eruslanov acknowledges funding from the NIH National Cancer Institute (NCI; R01CA187392) and the Department of Defense (CDMRP W81XWH-15-1-0717). Z.G. Fridlender acknowledges funding from the the Israel Science Foundation (grant number 1708/20) and the Sasson and Luisa Naor Fund. H.S. Goodridge acknowledges funding from the NIH National Institute of Allergy and Infectious Diseases (R01AI134987). Z. Granot acknowledges funding from the Israel Science Foundation Grant 405/18, the Israel Cancer Research Fund, the Deutsche Forschungsgemeinschaft, and the Rosetrees Trust. A. Hidalgo acknowledges funding from FET-OPEN (861878) from the European Commission; the Centro Nacional de Investigaciones Cardiovasculares (CNIC) is supported by the Ministerio de Ciencia e Innovacion and the Pro-CNIC Foundation. M.J. Kaplan acknowledges funding from the Intramural Research Program of the NIH, National Institute of Arthritis and Musculoskeletal and Skin Diseases (ZIAAR041199). I. Malanchi acknowledges funding from the European Research Council grant (ERC CoG-H2020-725492) and from the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001112), the UK Medical Research Council (FC001112), and the Wellcome Trust (FC001112). T. Merghoub acknowledges funding from the NIH/NCI Cancer Center Support Grant (P30 CA008748/NCI R01 CA056821), Swim Across America, Ludwig Institute for Cancer Research, Ludwig Center for Cancer Immunotherapy at Memorial Sloan Kettering, Cancer Research Institute, and Parker Institute for Cancer Immunotherapy. E. Meylan acknowledges funding from the Swiss National Science Foundation (310030_179324) and Fonds de la Recherche Scientifique (MISU F.6003.22). M.J. Pittet acknowledges funding from the Institut Suisse de Recherche Experimentale sur le Cancer Foundation, Ludwig Cancer Research, and the NIH (P01CA240239, R01CA218579). I.A. Udalova acknowledges funding from the Wellcome Trust Investigator Award (209422/Z/17/Z). T.K. van den Berg acknowledges funding from Byondis BV, the Dutch Ministry of Health, and the Dutch Cancer Society (10300). D.D. Wagner acknowledges funding from the NIH National Health, Lung and Blood Institute (R35HL135765). P. Wang acknowledges funding from the NIH National Institute of General Medical Sciences (R35GM118337). A. Zychlinsky acknowledges funding from the Max Planck Society. K.E. de Visser acknowledges funding from the Dutch Cancer Society (KWF10623, KWF10083, and KWF13191), Oncode Institute, and the Netherlands Organization for Scientific Research (NWO-VICI 91819616). M. Egeblad acknowledges funding from the Department of Defense, Congressional Directed Medical Research Program (W81XWH2010753). P. Kubes acknowledges funding from the Canadian Institutes of Health Research. Sí

The intestinal immune system and microbiota are emerging as important contributors to the development of metabolic syndrome, but the role of intestinal dendritic cells (DCs) in this context is incompletely understood. BATF3 is a transcription factor essential in the development of mucosal conventional DCs type 1 (cDC1). We show that Batf3-/- mice developed metabolic syndrome and have altered localization of tight junction proteins in intestinal epithelial cells leading to increased intestinal permeability. Treatment with the glycolysis inhibitor 2-deoxy-D-glucose reduced intestinal inflammation and restored barrier function in obese Batf3-/- mice. High-fat diet further enhanced the metabolic phenotype and susceptibility to dextran sulfate sodium colitis in Batf3-/- mice. Antibiotic treatment of Batf3-/- mice prevented metabolic syndrome and impaired intestinal barrier function. Batf3-/- mice have altered IgA-coating of fecal bacteria and displayed microbial dysbiosis marked by decreased obesity protective Akkermansia muciniphila, and Bifidobacterium. Thus, BATF3 protects against metabolic syndrome and preserves intestinal epithelial barrier by maintaining beneficial microbiota.

Type I IFNs are key mediators of immune defense against viruses and bacteria. Type I IFNs were also previously implicated in protection against fungal infection, but their roles in antifungal immunity have not been thoroughly investigated. A recent study demonstrated that bacterial and fungal β-glucans stimulate IFN-β production by dendritic cells (DCs) following detection by the Dectin-1 receptor, but the effects of β-glucan–induced type I IFNs have not been defined. We investigated whether type I IFNs regulate CD8 T cell activation by fungal β-glucan particle–stimulated DCs. We demonstrate that β-glucan–stimulated DCs induce CD8 T cell proliferation, activation marker (CD44 and CD69) expression, and production of IFN-γ, IL-2, and granzyme B. Moreover, we show that type I IFNs support robust CD8 T cell activation (proliferation and IFN-γ and granzyme B production) by β-glucan–stimulated DCs in vitro and in vivo due to autocrine effects on the DCs. Specifically, type I IFNs promote Ag presentation on MHC I molecules, CD86 and CD40 expression, and the production of IL-12 p70, IL-2, IL-6, and TNF-α by β-glucan–stimulated DCs. We also demonstrate a role for autocrine type I IFN signaling in bacterial LPS-induced DC maturation, although, in the context of LPS stimulation, this mechanism is not so critical for CD8 T cell activation (promotes IFN-γ production but not proliferation or granzyme B production). This study provides insight into the mechanisms underlying CD8 T cell activation during infection, which may be useful in the rational design of vaccines directed against pathogens and tumors.

Mast cells have been thought to derive from bone marrow hematopoietic stem cells. In this issue of Immunity, Gentek et al. (2018) reveal that mast cells have dual developmental origins in primitive and definitive hematopoiesis and that adult mast cell maintenance is largely bone marrow independent.

Classical and non-classical monocytes, and the macrophages and monocyte-derived dendritic cells they produce, play key roles in host defense against pathogens, immune regulation, tissue repair and many other processes throughout the body. Recent studies have revealed previously unappreciated heterogeneity among monocytes that may explain this functional diversity, but our understanding of mechanisms controlling the functional programming of distinct monocyte subsets remains incomplete. Resolving monocyte heterogeneity and understanding how their functional identity is determined holds great promise for therapeutic immune modulation. In this review, we examine how monocyte origins and developmental influences shape the phenotypic and functional characteristics of monocyte subsets during homeostasis and in the context of infection, inflammation, and cancer. We consider how extrinsic signals and transcriptional regulators impact monocyte production and functional programming, as well as the influence of epigenetic and metabolic mechanisms. We also examine the evidence that functionally distinct monocyte subsets are produced via different developmental pathways during homeostasis and that inflammatory stimuli differentially target progenitors during an emergency response. We highlight the need for a more comprehensive understanding of the relationship between monocyte ontogeny and heterogeneity, including multiparametric single-cell profiling and functional analyses. Studies defining mechanisms of monocyte subset production and maintenance of unique monocyte identities have the potential to facilitate the design of therapeutic interventions to target specific monocyte subsets in a variety of disease contexts, including infectious and inflammatory diseases, cancer, and aging.

An array of phenotypically diverse myeloid cells and macrophages (MC&M) resides in the tumor microenvironment, requiring multiplexed detection systems for visualization. Here we report an automated, multiplexed staining approach, named PLEXODY, that consists of five MC&M-related fluorescently-tagged antibodies (anti - CD68, - CD163, - CD206, - CD11b, and - CD11c), and three chromogenic antibodies, reactive with high- and low-molecular weight cytokeratins and CD3, highlighting tumor regions, benign glands and T cells. The staining prototype and image analysis methods which include a pixel/area-based quantification were developed using tissues from inflamed colon and tonsil and revealed a unique tissue-specific composition of 14 MC&M-associated pixel classes. As a proof-of-principle, PLEXODY was applied to three cases of pancreatic, prostate and renal cancers. Across digital images from these cancer types we observed 10 MC&M-associated pixel classes at frequencies greater than 3%. Cases revealed higher frequencies of single positive compared to multi-color pixels and a high abundance of CD68+/CD163+ and CD68+/CD163+/CD206+ pixels. Significantly more CD68+ and CD163+ vs. CD11b+ and CD11c+ pixels were in direct contact with tumor cells and T cells. While the greatest percentage (~70%) of CD68+ and CD163+ pixels was 0-20 microns away from tumor and T cell borders, CD11b+ and CD11c+ pixels were detected up to 240 microns away from tumor/T cell masks. Together, these data demonstrate significant differences in densities and spatial organization of MC&M-associated pixel classes, but surprising similarities between the three cancer types.

Granulocyte-monocyte progenitors (GMPs) and monocyte-dendritic cell progenitors (MDPs) produce monocytes during homeostasis and in response to increased demand during infection. Both progenitor populations are thought to derive from common myeloid progenitors (CMPs), and a hierarchical relationship (CMP-GMP-MDP-monocyte) is presumed to underlie monocyte differentiation. Here, however, we demonstrate that mouse MDPs arose from CMPs independently of GMPs, and that GMPs and MDPs produced monocytes via similar, but distinct, monocyte-committed progenitors. GMPs and MDPs yielded classical (Ly6Chi) monocytes with gene expression signatures that were defined by their origins and impacted their function. GMPs produced a subset of “neutrophil-like” monocytes, whereas MDPs gave rise to a subset of monocytes that yielded monocyte-derived dendritic cells. GMPs and MDPs were also independently mobilized to produce specific combinations of myeloid cell types following the injection of microbial components. Thus, the balance of GMP and MDP differentiation shapes the myeloid cell repertoire during homeostasis and following infection.

The ability of macrophages to respond to chemoattractants and inflammatory signals is important for their migration to sites of inflammation and immune activity and for host responses to infection. Macrophages differentiated from the bone marrow (BM) of UV-irradiated mice, even after activation with LPS, migrated inefficiently toward CSF-1 and CCL2. When BM cells were harvested from UV-irradiated mice and transplanted into naive mice, the recipient mice (UV-chimeric) had reduced accumulation of elicited monocytes/macrophages in the peritoneal cavity in response to inflammatory thioglycollate or alum. Macrophages differentiating from the BM of UV-chimeric mice also had an inherent reduced ability to migrate toward chemoattractants in vitro, even after LPS activation. Microarray analysis identified reduced reticulon-1 mRNA expressed in macrophages differentiated from the BM of UV-chimeric mice. By using an anti-reticulon-1 Ab, a role for reticulon-1 in macrophage migration toward both CSF-1 and CCL2 was confirmed. Reticulon-1 subcellular localization to the periphery after exposure to CSF-1 for 2.5 min was shown by immunofluorescence microscopy. The proposal that reduced reticulon-1 is responsible for the poor inherent ability of macrophages to respond to chemokine gradients was supported by Western blotting. In summary, skin exposure to erythemal UV radiation can modulate macrophage progenitors in the BM such that their differentiated progeny respond inefficiently to signals to accumulate at sites of inflammation and immunity.

Recent research has shown that (i) Toll-like receptor (TLR) agonists drive hematopoietic stem and progenitor cells (HSPCs) to proliferate and differentiate along the myeloid lineage in vitro, and (ii) direct TLR-mediated stimulation of HSPCs also promotes macrophage differentiation in vivo following infection. These new insights demonstrate that TLR signaling in HSPCs, in addition to other TLR-dependent mechanisms, can contribute to HSPC expansion and myeloid differentiation after infection. Evidence is, therefore, mounting that direct TLR-induced programming of hematopoiesis plays a key role in host defense by rapidly replenishing the innate immune system with the cells needed to deal with pathogens.

Several groups have shown that detection of microbial components by TLRs on hematopoietic stem and progenitor cells (HSPCs) instructs myeloid cell generation, raising interest in the possibility of targeting TLRs on HSPCs to boost myelopoiesis. However, although "TLR-derived" cells exhibit myeloid cell characteristics (phagocytosis, cytokine production, antigen presentation), it is not clear whether they are functionally equivalent to macrophages derived in the absence of TLR activation. Our in vitro and in vivo studies show that macrophages derived from mouse and human HSPC subsets (including stem cells) exposed to a TLR2 agonist prior to or during macrophage differentiation produce lower levels of inflammatory cytokines (TNF-α, IL-6, and IL-1β) and reactive oxygen species. This is in contrast to prior exposure of differentiated macrophages to the TLR2 agonist ("tolerance"), which suppresses inflammatory cytokine production, but elevates reactive oxygen species. Soluble factors produced following exposure of HSPCs to a TLR2 agonist can also act in a paracrine manner to influence the function of macrophages derived from unexposed HSPCs. Our data demonstrate that macrophage function can be influenced by TLR signaling in the HSPCs from which they are derived, and that this may impact the clinical utility of targeting TLRs on HSPCs to boost myelopoiesis.

Lineage bias among early hematopoietic progenitor cells is specified by transcription-factor programming, and lineage switching reduces the quantity of cells produced.

Lnk physically interacts with c-Fms and blunts its activity in-cluding proliferation of macrophage progenitor cells, M-CSF stimulated migration, and generaton of ROS. The M-CSFR (c-Fms) participates in proliferation, differentiation, and survival of macrophages and is involved in the regulation of distinct macrophage functions. Interaction with the ligand M-CSF results in phosphorylation of tyrosine residues on c-Fms, thereby creating binding sites for molecules containing SH2 domains. Lnk is a SH2 domain adaptor protein that negatively regulates hematopoietic cytokine receptors. Here, we show that Lnk binds to c-Fms. Biological and functional effects of this interaction were examined in macrophages from Lnk-deficient (KO) and WT mice. Clonogenic assays demonstrated an elevated number of M-CFUs in the bone marrow of Lnk KO mice. Furthermore, the M-CSF-induced phosphorylation of Akt in Lnk KO macrophages was increased and prolonged, whereas phosphorylation of Erk was diminished. Zymosan-stimulated production of ROS was increased dramatically in a M-CSF-dependent manner in Lnk KO macrophages. Lastly, Lnk inhibited M-CSF-induced migration of macrophages. In summary, we show that Lnk binds to c-Fms and can blunt M-CSF stimulation. Modulation of levels of Lnk in macrophages may provide a unique therapeutic approach to increase innate host defenses.

Beta-glucans are recognized by the innate immune system. This recognition plays important roles in host defense and presents specific opportunities for clinical modulation of the host immune response. Neutrophils, macrophages, and dendritic cells among others express several receptors capable of recognizing beta-glucan in its various forms. This review explores what is currently known about beta-glucan recognition and how this recognition stimulates immune responses. Special emphasis is placed on Dectin-1, as we know the most about how this key beta-glucan receptor translates recognition into intracellular signaling, stimulates cellular responses, and participates in orchestrating the adaptive immune response.

The pattern recognition receptors TLR2 and Dectin-1 play key roles in coordinating the responses of macrophages and dendritic cells (DC) to fungi. Induction of proinflammatory cytokines is instructed by signals from both TLR2 and Dectin-1. A recent report identified a role for CARD9 in innate anti-fungal responses, demonstrating CARD9-Bcl10-mediated activation of NF-κB and proinflammatory cytokine induction in murine bone marrow-derived DC stimulated via Dectin-1. We now report that Dectin-1-CARD9 signals fail to activate NF-κB and drive TNF-α induction in murine bone marrow-derived macrophages. However, priming of bone marrow-derived macrophages with GM-CSF or IFN-γ permits Dectin-1-CARD9-mediated TNF-α induction. Analysis of other macrophage/DC populations revealed further variation in the ability of Dectin-1-CARD9 signaling to drive TNF-α production. Resident peritoneal cells and alveolar macrophages produce TNF-α upon Dectin-1 ligation, while thioglycollate-elicited peritoneal macrophages and Flt3L-derived DC do not. We present data demonstrating that CARD9 is recruited to phagosomes via its CARD domain where it enhances TLR-induced cytokine production even in cells in which Dectin-1 is insufficient to drive cytokine production. In such cells, Dectin-1, CARD9, and Bcl10 levels are not limiting, and data indicate that these cells express additional factors that restrict Dectin-1-CARD9 signaling for TNF-α induction.

Innate and adaptive immune responses are regulated by receptors that signal through immunoreceptor tyrosine-based activation motifs (ITAMs). The molecular basis of ITAM signaling has been extensively characterized and serves as a model for receptor-mediated signal transduction. Src family kinases typically phosphorylate ITAMs on dual tyrosines, which enable recruitment and activation of Syk family kinases through binding to dual SH2 domains on these kinases. Examples of ITAM-based signaling that do not conform precisely to the standard model are becoming increasingly common. ITAMs that suppress signaling under specific conditions and activate under others have been described, as have ITAM-like signaling mechanisms using nonstandard sequence motifs. Elucidating the diversity of ITAM-based signaling mechanisms will clarify how activating signals generated by ITAMs are tightly regulated and will open opportunities for specific therapeutic manipulation of ITAM-based signaling pathways.

Bacterial CpG-containing (CpG) DNA promotes survival of murine macrophages and triggers production of proinflammatory mediators. The CpG DNA-induced inflammatory response is mediated via TLR9, whereas a recent study reported that activation of the Akt prosurvival pathway occurs via DNA-dependent protein kinase (DNA-PK) and independently of TLR9. We show, in this study, that Akt activation and survival of murine bone marrow-derived macrophages (BMM) triggered by CpG-containing phosphodiester oligodeoxynucleotides or CpG-containing phosphorothioate oligodeoxynucleotides was completely dependent on TLR9. In addition, survival triggered by CpG-containing phosphodiester oligodeoxynucleotides was not compromised in BMM from SCID mice that express a catalytically inactive form of DNA-PK. CpG DNA-induced survival of BMM was inhibited by the PI3K inhibitor, LY294002, but not by the MEK1/2 inhibitor, PD98059. The effect of LY294002 was specific to survival, because treatment of BMM with LY294002 affected CpG DNA-induced TNF-α production only modestly. Therefore, CpG DNA activates macrophage survival via TLR9 and the PI3K-Akt pathway and independently of DNA-PK and MEK-ERK.

Secretion of immunomodulatory molecules is a key strategy employed by pathogens to enable their survival in host organisms. For example, arthropod-transmitted filarial nematodes, which achieve longevity within the infected host by suppressing and modulating the host immune response, produce excretory–secretory (ES) products that have been demonstrated to possess immunomodulatory properties. In this review we discuss the immunomodulatory effects of the phosphorylcholine-containing filarial nematode-secreted glycoprotein ES-62 and describe the intracellular signal transduction pathways it targets to achieve these effects.

Discussion of how imprinting of bone marrow progenitors impacts the function of the dendritic cells that they produce.

Macrophage activation by cytokines or microbial products such as LPS results in the induction and release of several key immune effector molecules including NO and IL-12. These have been shown to play crucial roles in the development of immunity to intracellular pathogens such as Leishmania. The molecular mechanisms underlying the induction of these effector molecules are not fully understood. We now show that the extracellular signal-related kinase (ERK) and p38 mitogen-activated protein (MAP) kinases play differential roles in the regulation of LPS-stimulated inducible NO synthase and IL-12 gene expression. In macrophages, LPS stimulates the simultaneous activation of all three classes of MAP kinases, ERK, c-jun N-terminal kinase, and p38, albeit with differential activation kinetics. However, studies using inhibitors selective for ERK (PD98059) and p38 (SB203580) show that while p38 plays an essential role in the induction of inducible NO synthase, ERK MAP kinases play only a minor role in promoting NO generation. In contrast, while p38 promotes induction of IL-12 (p40) mRNA, ERK activation suppresses LPS-mediated IL-12 transcription. The biological relevance of these regulatory signals is demonstrated by our finding that Leishmania lipophosphoglycans, which promote parasite survival, act by stimulating ERK MAP kinase to inhibit macrophage IL-12 production. Thus, as ERK and p38 MAP kinases differentially regulate the induction of the macrophage effector molecules, inducible NO synthase and IL-12, these kinases are potential targets not only for the development of novel strategies to combat intracellular pathogens but also for therapeutic immunomodulation.

Innate immune pattern recognition receptors play critical roles in pathogen detection and initiation of antimicrobial responses. We and others have previously demonstrated the importance of the β-glucan receptor Dectin-1 in the recognition of pathogenic fungi by macrophages and dendritic cells and have elucidated some of the mechanisms by which Dectin-1 signals to coordinate the antifungal response. While Dectin-1 signals alone are sufficient to trigger phagocytosis and Src-Syk-mediated induction of antimicrobial reactive oxygen species, collaboration with TLR2 signaling enhances NF-κB activation and regulates cytokine production. In this study we demonstrate that Dectin-1 signaling can also directly modulate gene expression via activation of NFAT. Dectin-1 ligation by zymosan particles or live Candida albicans yeast triggers NFAT activation in macrophages and dendritic cells. Dectin-1-triggered NFAT activation plays a role in the induction of early growth response 2 and early growth response 3 transcription factors, and cyclooxygenase-2. Furthermore, we show that NFAT activation regulates IL-2, IL-10 and IL-12 p70 production by zymosan-stimulated dendritic cells. These data establish NFAT activation in myeloid cells as a novel mechanism of regulation of the innate antimicrobial response.

The plant 2S seed albumins Ber e 1 and SFA8, although structurally very similar, vary with respect to their allergenic properties. Whereas the former represents a major allergen, the latter appears to promote only weak allergenic responses. The aim of this investigation was to determine whether the allergenic properties of Ber e 1 and SFA8 reflected differential polarization of dendritic cell (DC) and Th cell responses. We thus investigated the effect of recombinant forms of both allergens on DC and Th cell responses as indicated by cell surface phenotype and cytokine production. Exposure of murine DCs to SFA8, but not Ber e 1, resulted in production of the cytokines IL-12 p40 and TNF-α by a mechanism independent of recognition by TLRs. Furthermore, depending on the mouse strain used, increased expression of MHC class II and costimulatory molecules such as CD40, CD80, and CD86 was associated with exposure to SFA8, but not Ber e 1. In coculture experiments using the DO11.10 transgenic T cell that recognizes OVA peptide, DCs exposed to both allergens induced T cells to produce IFN-γ, but only Ber e 1 could induce significant production of IL-4 and IL-5. Likewise, analysis of transcription factors shows increased T-bet with respect to both allergens, but also GATA-3 with respect to Ber e 1. Overall, our data are consistent with the idea that the ability of Ber e 1, but not SFA8, to act as a potent allergen may reflect differences in their ability to induce IL-12 production.

Filarial nematodes, parasites of vertebrates, including humans, secrete immunomodulatory molecules into the host environment. We have previously demonstrated that one such molecule, the phosphorylcholine-containing glycoprotein ES-62, acts to bias the immune response toward an anti-inflammatory/Th2 phenotype that is conducive to both worm survival and host health. For example, although ES-62 initially induces macrophages to produce low levels of IL-12 and TNF-α, exposure to the parasite product ultimately renders the cells unable to produce these cytokines in response to classic stimulators such as LPS/IFN-γ. We have investigated the possibility that a TLR is involved in the recognition of ES-62 by target cells, because phosphorylcholine, a common pathogen-associated molecular pattern, appears to be responsible for many of the immunomodulatory properties of ES-62. We now demonstrate that ES-62-mediated, low level IL-12 and TNF-α production by macrophages and dendritic cells is abrogated in MyD88 and TLR4, but not TLR2, knockout, mice implicating TLR4 in the recognition of ES-62 by these cells and MyD88 in the transduction of the resulting intracellular signals. We also show that ES-62 inhibits IL-12 induction by TLR ligands other than LPS, bacterial lipopeptide (TLR2) and CpG (TLR9), via this TLR4-dependent pathway. Surprisingly, macrophages and dendritic cells from LPS-unresponsive, TLR4-mutant C3H/HeJ mice respond normally to ES-62. This is the first report to demonstrate that modulation of cytokine responses by a pathogen product can be abrogated in cells derived from TLR4 knockout, but not C3H/HeJ mice, suggesting the existence of a novel mechanism of TLR4-mediated immunomodulation.

We have previously shown in an in vitro study that the filarial nematode phosphorylcholine (PC)-containing glycoprotein ES-62 promotes a murine dendritic cell (DC) phenotype that induces T helper type 2 (Th2) responses. We now show that, in addition to directly priming Th2 responses, ES-62 can act to dampen down the pro-inflammatory DC responses elicited by lipopolysaccharide. Furthermore, we also demonstrate that murine DCs and macrophages derived ex vivo from bone marrow cells exposed in vivo to ES-62 by release from osmotic pumps are hyporesponsive to subsequent stimulation with lipopolysaccharide. These effects can be largely mimicked by exposure to the PC moiety of ES-62 conjugated to an irrelevant protein. The data we provide are, as far as we aware, the first to show that a defined pathogen product can modulate the developmental pathway of bone marrow cells of the immune system in vivo. Such a finding could have important implications for the use of pathogen products or their derivatives for immunotherapy.

Bioactive interleukin (IL)-12 is a 70 000-molecular weight (MW) heterodimeric cytokine comprising p40 and p35 chains. However, p40 can also form homodimers that antagonize bioactive IL-12 or heterodimerize with p19 to form IL-23, which exhibits overlapping yet distinct functions to that of IL-12. We now define distinct signalling mechanisms that regulate lipopolysaccharide (LPS)-mediated induction of IL-12 p40 and p35 in macrophages and which may therefore provide therapeutic targets for precise and specific fine-tuning of cytokine responses. Thus, whilst LPS-induced p38 mitogen-activated protein kinase (MAPkinase) activation is required for the induction of both p40 and p35 subunits, Erk MAPkinase signalling mediates negative feedback regulation of p40, but not p35, production. Such Erk MAPkinase activation is downstream of calcium influx and targets LPS-induced IL-12 p40 transcription by suppressing the synthesis of the transcription factor, interferon regulatory factor-1 (IRF-1). In contrast, negative regulation of the p35 subunit of IL-12 occurs via a calcium-dependent, but Erk-independent, mechanism, which is likely to involve nuclear factor (NF)-kappa B signalling. Finally, the importance of both Erk and p38 MAPkinases in differentially regulating IL-12 p40 and p35 production is underscored by each being targeted by ES-62, a product secreted by parasitic filarial nematodes to polarize the immune system towards an anti-inflammatory phenotype conducive to their survival.

Parasite survival and host health may depend on the ability of the parasite to modulate the host immune response by the release of immunomodulatory molecules. Excretory-secretory (ES)-62, one such well-defined molecule, is a major secreted protein of the rodent filarial nematode Acanthocheilonema viteae, and has homologues in human filarial nematodes. Previously we have shown that ES-62 is exclusively associated with a Th2 Ab response in mice. Here we provide a rationale for this polarized immune response by showing that the parasite molecule suppresses the IFN-γ/LPS-induced production, by macrophages, of bioactive IL-12 (p70), a key cytokine in the development of Th1 responses. This suppression of the induction of a component of the host immune response extends to the production of the proinflammatory cytokines IL-6 and TNF-α, but not NO. The molecular mechanism underlying these findings awaits elucidation but, intriguingly, the initial response of macrophages to ES-62 is to demonstrate a low and transient release of these cytokines before becoming refractory to further release induced by IFN-γ/LPS. The relevance of our observations is underscored by the finding that macrophages recovered from mice exposed to “physiological” levels of ES-62 by the novel approach of continuous release from implanted osmotic pumps in vivo were similarly refractory to release of IL-12, TNF-α, IL-6, but not NO, ex vivo. Therefore, our results suggest that exposure to ES-62 renders macrophages subsequently unable to produce Th1/proinflammatory cytokines. This likely contributes to the generation of immune responses with an anti-inflammatory Th2 phenotype, a well-documented feature of filarial nematode infection.

The β-glucan receptor Dectin-1 is a central player in the anti-fungal responses of myeloid phagocytes. Detection of β-glucan-containing particles by Dectin-1, which signals via an ITAM-like motif in its cytoplasmic tail, triggers phagocytosis, the induction of an oxidative burst, and production of a variety of inflammatory mediators by macrophages and dendritic cells. In contrast, soluble β-glucans, which are too small to trigger phagocytosis, fail to activate Dectin-1 signaling. However, soluble β-glucans immobilized on latex beads are capable of inducing Dectin-1-mediated responses. We used immunofluorescence microscopy to study the rearrangement of macrophage surface proteins upon binding of β-glucan particles to Dectin-1, and observed the formation of a synapse analogous to the immunological synapse that forms between a T cell and an antigen presenting cell. Our data indicate that the formation of this "phagocytic synapse" is required to initiate Dectin-1 signaling, and that the inability of soluble β-glucans to trigger such reorganization accounts for their failure to activate Dectin-1 signaling. This work is supported by grants to DMU from the NIH (AI071116, GM085796) and AHA (0640100N); HSG is a CCFA Research Fellow

IL-1β produced by phagocytes is important for protection against Staphylococcus aureus, a bacterium that causes serious skin and invasive diseases and is contributing to a current public health crisis due to the emergence of methicillin-resistant strains. Secretion of IL-1β requires both activation of a transcriptional signal to stimulate production of pro- IL-1β, (usually provided by TLR signaling) and a second signal to stimulate processing by inflammasome complexes and release of the mature cytokine. We show here that phagocytosis and lysozyme-based degradation of bacterial cell walls are functionally coupled to activation of Nalp3 inflammasomes and secretion of IL-1β in response to live S. aureus and to S. aureus peptidoglycan. Further, a S. aureus peptidoglycan-modifying enzyme that makes its peptidoglycan resistant to lysozyme strongly suppresses inflammasome activation. This is the first demonstration of a case whereby a bacterium avoids IL-1β secretion through chemical modification of its cell wall peptidoglycan, and we hypothesize that this reduced IL-1β production is key to successful mucosal colonization and infection. This research is supported by grants from NIGMS and the American Heart Association.