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1.  Differential utilization of CARD9 by Dectin-1 in macrophages and dendritic cells12 
The pattern recognition receptors Toll-like receptor 2 (TLR2) and Dectin-1 play key roles in coordinating the responses of macrophages and dendritic cells (DC) to fungi. Induction of pro-inflammatory 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 pro-inflammatory cytokine induction in murine bone marrow-derived DC (bmDC) 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 (bmM). However, priming of bmM 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.
PMCID: PMC2718573  PMID: 19124758
Dendritic cells; Monocytes/Macrophages; Fungal infection; Phagocytosis; Signal transduction
2.  Dectin-1 Regulates IL-10 Production via a MSK1/2 and CREB Dependent Pathway and Promotes the Induction of Regulatory Macrophage Markers 
PLoS ONE  2013;8(3):e60086.
In response to infection by fungal pathogens, the innate immune system recognises specific fungal pathogen associated molecular patterns (PAMPs) via pattern recognition receptors including the C-type lectin dectin-1 and members of the Toll Like Receptor (TLR) family. Stimulation of these receptors leads to the induction of both pro- and anti-inflammatory cytokines. The protein kinases MSK1 and 2 are known to be important in limiting inflammatory cytokine production by macrophages in response to the TLR4 agonist LPS. In this study we show that MSKs are also activated in macrophages by the fungal derived ligand zymosan, as well as the dectin-1 specific agonists curdlan and depleted zymosan, via the ERK1/2 and p38α MAPK pathways. Furthermore, we show that MSKs regulate dectin-1 induced IL-10 production, and that this regulation is dependent on the ability of MSKs to phosphorylate the transcription factor CREB. IL-10 secreted in response to zymosan was able to promote STAT3 phosphorylation via an autocrine feedback loop. Consistent with the decreased IL-10 secretion in MSK1/2 knockout macrophages, these cells also had decreased STAT3 tyrosine phosphorylation relative to wild type controls after stimulation with zymosan. We further show that the reduction in IL-10 production in the MSK1/2 macrophages results in increased secretion of IL-12p40 in response to zymosan relative to wild type controls. The production of high levels of IL-10 but low levels of IL-12 has previously been associated with an M2b or ‘regulatory’ macrophage phenotype, which was initially described in macrophages stimulated with a combination of immune complexes and LPS. We found that zymosan, via dectin-1 activation, also leads to the expression of SphK1 and LIGHT, markers of a regulatory like phenotype in mouse macrophages. The expression of these makers was further reinforced by the high level of IL-10 secreted in response to zymosan stimulation.
doi:10.1371/journal.pone.0060086
PMCID: PMC3606242  PMID: 23533666
3.  Genetic Association Analysis of the Functional c.714T>G Polymorphism and Mucosal Expression of Dectin-1 in Inflammatory Bowel Disease 
PLoS ONE  2009;4(11):e7818.
Background
Dectin-1 is a pattern recognition receptor (PRR) expressed by myeloid cells that specifically recognizes β-1,3 glucan, a polysaccharide and major component of the fungal cell wall. Upon activation, dectin-1 signaling converges, similar to NOD2, on the adaptor molecule CARD9 which is associated with inflammatory bowel disease (IBD). An early stop codon polymorphism (c.714T>G) in DECTIN-1 results in a loss-of-function (p.Y238X) and impaired cytokine responses, including TNF-α, interleukin (IL)-1β and IL-17 upon in vitro stimulation with Candida albicans or β-glucan. The aim of the present study was to test the hypothesis that the DECTIN-1 c.714T>G (p.Y238X) polymorphism is associated with lower disease susceptibility or severity in IBD and to investigate the level of dectin-1 expression in inflamed and non-inflamed colon tissue of IBD patients.
Methodology
Paraffin embedded tissue samples from non-inflamed and inflamed colon of IBD patients and from diverticulitis patients were immunohistochemically stained for dectin-1 and related to CD68 macrophage staining. Genomic DNA of IBD patients (778 patients with Crohn's disease and 759 patients with ulcerative colitis) and healthy controls (n = 772) was genotyped for the c.714T>G polymorphism and genotype-phenotype interactions were investigated.
Principal Findings
Increased expression of dectin-1 was observed in actively inflamed colon tissue, as compared to non-inflamed tissue of the same patients. Also an increase in dectin-1 expression was apparent in diverticulitis tissue. No statistically significant difference in DECTIN-1 c.714T>G allele frequencies was observed between IBD patients and healthy controls. Furthermore, no differences in clinical characteristics could be observed related to DECTIN-1 genotype, neither alone, nor stratified for NOD2 genotype.
Conclusions
Our data demonstrate that dectin-1 expression is elevated on macrophages, neutrophils, and other immune cells involved in the inflammatory reaction in IBD. The DECTIN-1 c.714T>G polymorphism however, is not a major susceptibility factor for developing IBD.
doi:10.1371/journal.pone.0007818
PMCID: PMC2771910  PMID: 19915667
4.  Cytosolic Phospholipase A2 Activation by Candida albicans in Alveolar Macrophages 
Candida albicans is an increasingly important pulmonary fungal pathogen. Resident alveolar macrophages are important in host defense against opportunistic fungal infections. Activation of Group IVA cytosolic phospholipase A2α (cPLA2α) in macrophages initiates arachidonic acid (AA) release for production of eicosanoids, which regulate inflammation and immune responses. We investigated the ability of C. albicans to activate cPLA2α in unprimed alveolar macrophages and after priming with granulocyte macrophage colony-stimulating factor (GM-CSF), which regulates alveolar macrophage maturation. AA was released within minutes by GM-CSF–primed but not unprimed alveolar macrophages in response to C. albicans, and was blocked by soluble glucan phosphate (S-GP). The expression of the β-glucan receptor dectin-1 was increased in GM-CSF–primed macrophages, and AA release from GM-CSF–primed dectin-1−/− alveolar macrophages was reduced to basal levels. The enhanced activation of extracellular signal–regulated kinases and phosphorylation of cPLA2α on Ser-505 that occurred in GM-CSF–primed macrophages were reduced by MEK1 and Syk inhibitors, which also suppressed AA release. At later times after C. albicans infection (6 h), unprimed and GM-CSF–primed macrophages released similar levels of AA. The expression of cyclooxygenase 2 and prostanoid production at 6 hours was higher in GM-CSF–primed macrophages, but the responses were not dependent on dectin-1. However, dectin-1 contributed to the C. albicans–stimulated increase in TNF-α production that occurred in GM-CSF–primed macrophages. The results demonstrate that dectin-1 mediates the acute activation of cPLA2α in GM-CSF–primed alveolar macrophages, but not in the more delayed phase of AA release and GM-CSF–dependent prostanoid production.
doi:10.1165/rcmb.2009-0110OC
PMCID: PMC2848735  PMID: 19502385
cytosolic phospholipase A2; dectin-1; alveolar macrophages; granulocyte macrophage colony-stimulating factor; arachidonic acid
5.  The Beta-Glucan Receptor Dectin-1 Recognizes Specific Morphologies of Aspergillus fumigatus 
PLoS Pathogens  2005;1(4):e42.
Alveolar macrophages represent a first-line innate host defense mechanism for clearing inhaled Aspergillus fumigatus from the lungs, yet contradictory data exist as to which alveolar macrophage recognition receptor is critical for innate immunity to A. fumigatus. Acknowledging that the A. fumigatus cell wall contains a high beta-1,3–glucan content, we questioned whether the beta-glucan receptor dectin-1 played a role in this recognition process. Monoclonal antibody, soluble receptor, and competitive carbohydrate blockage indicated that the alveolar macrophage inflammatory response, specifically the production of tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α), IL-1β, IL-6, CXCL2/macrophage inflammatory protein-2 (MIP-2), CCL3/macrophage inflammatory protein-1α (MIP-1α), granulocyte-colony stimulating factor (G-CSF), and granulocyte monocyte–CSF (GM-CSF), to live A. fumigatus was dependent on recognition via the beta-glucan receptor dectin-1. The inflammatory response was triggered at the highest level by A. fumigatus swollen conidia and early germlings and correlated to the levels of surface-exposed beta glucans, indicating that dectin-1 preferentially recognizes specific morphological forms of A. fumigatus. Intratracheal administration of A. fumigatus conidia to mice in the presence of a soluble dectin-Fc fusion protein reduced both lung proinflammatory cytokine/chemokine levels and cellular recruitment while modestly increasing the A. fumigatus fungal burden, illustrating the importance of beta-glucan–initiated dectin-1 signaling in defense against this pathogen. Collectively, these data show that dectin-1 is centrally required for the generation of alveolar macrophage proinflammatory responses to A. fumigatus and to our knowledge provides the first in vivo evidence for the role of dectin-1 in fungal innate defense.
Synopsis
Individuals with defective immune systems are highly susceptible to infection by parasites, bacteria, viruses, and fungi. Infection by the opportunistic fungal organism Aspergillus fumigatus can be particularly severe in this population. Because many pathogenic microorganisms, including A. fumigatus, enter the body through the lung, it is important to understand the function of its immune system. The alveolar macrophage is one of the first cell types to come in contact with inhaled pathogens. An intense area of research is how lung immune cells—i.e., alveolar macrophages—recognize inhaled pathogens and respond to them. Steele et al. recently discovered that alveolar macrophages express a receptor on their surface, dectin-1, that is essential in recognizing and responding to inhaled fungal pathogens. They now have investigated the interaction between dectin-1 and A. fumigatus to determine how the dectin-1 receptor orchestrates the alveolar macrophage response. They found that alveolar macrophages respond poorly to A. fumigatus when the dectin-1 receptor is blocked. Also, in animal experiments, blocking dectin-1 renders the animals more susceptible to infection with A. fumigatus. This study may lay the foundation for developing new and novel strategies to combat infections caused by A. fumigatus.
doi:10.1371/journal.ppat.0010042
PMCID: PMC1311140  PMID: 16344862
6.  Candida albicans β-Glucan Exposure Is Controlled by the Fungal CEK1-Mediated Mitogen-Activated Protein Kinase Pathway That Modulates Immune Responses Triggered through Dectin-1 ▿ †  
Infection and Immunity  2010;78(4):1426-1436.
Innate immunity to Candida albicans depends upon the recognition of molecular patterns on the fungal cell wall. However, the masking of major components such as β-glucan seems to be a mechanism that fungi have evolved to avoid immune cell recognition through the dectin-1 receptor. Although the role of C. albicans mitogen-activated protein kinase (MAPK) pathways as virulence determinants has been established previously with animal models, the mechanism involved in this behavior is largely unknown. In this study we demonstrate that a disruption of the C. albicans extracellular signal-regulated kinase (ERK)-like 1 (CEK1)-mediated MAPK pathway causes enhanced cell wall β-glucan exposure, triggering immune responses more efficiently than the wild type, as measured by dectin-1-mediated specific binding and human dendritic cell (hDC)- and macrophage-mediated phagocytosis, killing, and activation of intracellular signaling pathways. At the molecular level, the disruption of CEK1 resulted in altered spleen tyrosine kinase (Syk), Raf-1, and ERK1/2 activations together with IκB degradation on hDCs and increased dectin-1-dependent activator protein 1 (AP-1) activation on transfected cells. In addition, concurring with these altered pathways, we detected increased reactive oxygen species production and cytokine secretion. In conclusion, the CEK1-mediated MAPK pathway is involved in β-glucan exposure in a fungal pathogen, hence influencing dectin-1-dependent immune cell recognition, thus establishing this fungal intracellular signaling route as a promising novel therapeutic target.
doi:10.1128/IAI.00989-09
PMCID: PMC2849429  PMID: 20100861
7.  Dectin-1 Stimulation Selectively Reinforces LPS-driven IgG1 Production by Mouse B Cells 
Immune Network  2013;13(5):205-212.
Dectin-1, which specifically recognizes β-glucan of fungal cell walls, is a non-Toll-like receptor (TLR) pattern recognition receptor and a representative of C-type lectin receptors (CLRs). The importance of Dectin-1 in innate immune cells, such as dendritic cells and macrophages, has previously been well studied. However, the function of Dectin-1 in B cells is very poorly understood. To determine the role of Dectin-1 in B cell activation, we first investigated whether mouse B cells express Dectin-1 and then assessed the effect of Dectin-1 stimulation on B cell proliferation and antibody production. Mouse B cells express mRNAs encoding CLRs, including Dectin-1, and surface Dectin-1 was expressed in B cells of C57BL/6 rather than BALB/c strain. Dectin-1 agonists, heat-killed Candida albicans (HKCA) and heat-killed Saccharomyces cerevisiae (HKSC), alone induced B cell proliferation but not antibody production. Interestingly, HKSC, HKCA, and depleted zymosan (a selective Dectin-1 agonist) selectively enhanced LPS-driven IgG1 production. Taken together, these results suggest that, during fungal infection, β-glucan-stimulated Dectin-1 may cooperate with TLR4 to specifically enhance IgG1 production by mouse B cells.
doi:10.4110/in.2013.13.5.205
PMCID: PMC3817302  PMID: 24198746
Dectin-1; B cell; IgG1; C-type lectin receptor; Fungal immunity
8.  MARCO, TLR2, and CD14 Are Required for Macrophage Cytokine Responses to Mycobacterial Trehalose Dimycolate and Mycobacterium tuberculosis 
PLoS Pathogens  2009;5(6):e1000474.
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.
Author Summary
The causative agent of tuberculosis, Mycobacterium tuberculosis, has a lipid-rich cell wall that contains a high percentage of mycolic acids. These mycolic acids contribute to both the impermeable nature of the cell wall and to the immunostimulatory properties of the bacterium. Indeed, it has been known for over 50 years that trehalose 6,6′-dimycolate (TDM/cord factor) is the major immunogenic lipid of M. tuberculosis, which induces potent pro-inflammatory responses from macrophages, although the receptor has not been identified. We have demonstrated that the toll-like receptor (TLR) pathway is required for pro-inflammatory cytokine production in response to TDM; however, the TLRs alone, or in conjunction with known co-receptors, are not sufficient to induce a response. We demonstrate that the macrophage receptor MARCO, a scavenger receptor, is utilized preferentially to “tether” TDM to the macrophage and activate the TLR2 signaling pathway, and is used preferentially over the related SRA. Macrophages from MARCO−/− mice are defective in activation of TDM-induced signaling and subsequent pro-inflammatory cytokine production in response to both TDM-coated beads and virulent M. tuberculosis. By identifying the macrophage receptors involved in initial recognition we can now explain variable responses to TDM between different macrophage populations (which differ in scavenger receptor expression), and have identified a novel co-receptor that may be involved in lipid presentation to TLRs.
doi:10.1371/journal.ppat.1000474
PMCID: PMC2688075  PMID: 19521507
9.  The Response of Human Macrophages to β-Glucans Depends on the Inflammatory Milieu 
PLoS ONE  2013;8(4):e62016.
Background
β-glucans are fungal cell wall components that bind to the C-type lectin-like receptor dectin-1. Polymorphisms of dectin-1 gene are associated with susceptibility to invasive fungal infection and medically refractory ulcerative colitis. The purpose of this study has been addressing the response of human macrophages to β-glucans under different conditions mimicking the composition of the inflammatory milieu in view of the wide plasticity and large range of phenotypical changes showed by these cells, and the relevant role of dectin-1 in several pathophysiological conditions.
Principal Findings
Serum-differentiated macrophages stimulated with β-glucans showed a low production of TNFα and IL-1β, a high production of IL-6 and IL-23, and a delayed induction of cyclooxygenase-2 and PGE2 biosynthesis that resembled the responses elicited by crystals and those produced when phagosomal degradation of the phagocytic cargo increases ligand access to intracellular pattern recognition receptors. Priming with a low concentration of LPS produced a rapid induction of cyclooxygenase-2 and a synergistic release of PGE2. When the differentiation of the macrophages was carried out in the presence of M-CSF, an increased expression of dectin-1 B isoform was observed. In addition, this treatment made the cells capable to release arachidonic acid in response to β-glucan.
Conclusions
These results indicate that the macrophage response to fungal β-glucans is strongly influenced by cytokines and microbial-derived factors that are usual components of the inflammatory milieu. These responses can be sorted into three main patterns i) an elementary response dependent on phagosomal processing of pathogen-associated molecular patterns and/or receptor-independent, direct membrane binding linked to the immunoreceptor tyrosine-based activation motif-bearing transmembrane adaptor DNAX-activating protein 12, ii) a response primed by TLR4-dependent signals, and iii) a response dependent on M-CSF and dectin-1 B isoform expression that mainly signals through the dectin-1 B/spleen tyrosine kinase/cytosolic phospholipase A2 route.
doi:10.1371/journal.pone.0062016
PMCID: PMC3634770  PMID: 23637950
10.  Dectin-2 Recognition of House Dust Mite Triggers Cysteinyl Leukotriene Generation by Dendritic Cells1 
House dust mites are a significant source of airborne allergen worldwide, but there is little understanding of how they so potently generate allergic inflammation. We found that extracts from the house dust mites Dermatophagoides farinae (Df) and Dermatophagoides pteronyssinus and from the mold Aspergillus fumigatus stimulated a rapid and robust production of cysteinyl leukotrienes (cys-LTs), proinflammatory lipid mediators, from mouse bone marrow-derived dendritic cells (BMDCs). Con A affinity chromatography of the Df extract revealed that the relevant ligand is a glycan(s), suggesting stimulation via a dendritic cell (DC) lectin receptor. Cys-LT production in BMDCs from wild-type mice was inhibited by spleen tyrosine kinase (Syk) inhibitors and was abolished in BMDCs from FcRγ−/− mice, implicating either Dectin-2 or DC immunoactivating receptor. Transfection of each receptor in bone marrow-derived mast cells revealed that only Dectin-2 mediates cys-LT production by Df, Dermatophagoides pteronyssinus, and Aspergillus fumigatus. Lentiviral knockdown of Dectin-2 in BMDCs attenuated Df extract-elicited cys-LT generation, thereby identifying Dectin-2 as the receptor. Lung CD11c+ cells, but not peritoneal or alveolar macrophages, also generated cys-LTs in response to Df. These findings place Dectin-2 among the C-type lectin receptors that activate arachidonic acid metabolism and identify the Dectin-2/FcRγ/Syk/cys-LT axis as a novel mechanism by which three potent indoor allergens may activate innate immune cells to promote allergic inflammation.
PMCID: PMC3682801  PMID: 19124755
11.  Dectin-1 Is Required for Resistance to Coccidioidomycosis in Mice 
mBio  2013;4(1):e00597-12.
ABSTRACT
We assessed the role of Dectin-1 in the immune response to the pathogenic fungus Coccidioides, both in vitro and in vivo, using mice with a targeted mutation in Clec7a. Elicited peritoneal macrophages responded to formalin-killed spherules (FKS) and alkali-treated FKS by secreting proinflammatory cytokines in a Dectin-1- and β-glucan-dependent manner. The responses of bone marrow-derived dendritic cells (BMDC) to the same stimulants were more complex; interleukin 1β (IL-1β) and tumor necrosis factor alpha (TNF-α) secretion was independent of Dectin-1, while IL-6, IL-10, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were largely but not entirely dependent on Dectin-1. After intranasal infection, Dectin-1−/− mice had lower concentrations of IL-12p70, gamma interferon (IFN-γ), IL-1β, and the Th17 cytokines IL-22, IL-23, and 17A in the lung lavage fluid, which may explain why they were significantly more susceptible to pulmonary coccidioidomycosis two weeks after infection. The Dectin-1 mutation was even more deleterious in (B6 × DBA/2)F2 mice, which are more resistant to coccidioidomycosis than B6 mice by virtue of protective genes from DBA/2, a genetically resistant strain. We also found that two susceptible strains of mice (B6 and BALB/c) expressed much less Dectin-1 in their lungs than did resistant DBA/2 mice. We conclude that Dectin-1 is necessary for resistance to Coccidioides immitis, that Dectin-1 promotes both Th1 and Th17 protective immune responses to this infection, and that there is a correlation between expression of Dectin-1 by the inflammatory infiltrate and resistance to coccidioidomycosis.
IMPORTANCE
Coccidioidomycosis is a fungal infection endemic in the southwestern United States and neighboring Mexico, causing ~150,000 lung infections in people and resulting in ~17,000 hospitalizations annually in California alone. Very little is known about innate immunity to this fungus. This paper shows that Dectin-1, the primary β-glucan receptor on myeloid cells, is required for resistance to this pathogen. Dectin-1 is part of the innate immune system, and it is needed to direct the acquired immune response toward into a pathway that will lead to macrophage activation. Lungs from infected mice lacking Dectin-1 had lower concentrations of Th1 and Th17 cytokines, two cytokine pathways that are very important for acquired T cell immunity to Coccidioides spp. This is the first demonstration that Dectin-1 is required for host resistance to a dimorphic, primary pathogenic fungus.
doi:10.1128/mBio.00597-12
PMCID: PMC3562125  PMID: 23386437
12.  Mycobacterium massiliense Induces Inflammatory Responses in Macrophages Through Toll-Like Receptor 2 and c-Jun N-Terminal Kinase 
Journal of Clinical Immunology  2014;34(2):212-223.
Mycobacterium massiliense (Mmass) is an emerging, rapidly growing mycobacterium (RGM) that belongs to the M. abscessus (Mabc) group, albeit clearly differentiated from Mabc. Compared with M. tuberculosis, a well-characterized human pathogen, the host innate immune response against Mmass infection is largely unknown. In this study, we show that Mmass robustly activates mRNA and protein expression of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in murine bone marrow-derived macrophages (BMDMs). Toll-like receptor (TLR)-2 and myeloid differentiation primary response gene 88 (MyD88), but neither TLR4 nor Dectin-1, are involved in Mmass-induced TNF-α or IL-6 production in BMDMs. Mmass infection also activates the mitogen-activated protein kinase (MAPKs; c-Jun N-terminal kinase (JNK), ERK1/2 and p38 MAPK) pathway. Mmass-induced TNF-α and IL-6 production was dependent on JNK activation, while they were unaffected by either the ERK1/2 or p38 pathway in BMDMs. Additionally, intracellular reactive oxygen species (ROS), NADPH oxidase-2, and nuclear factor-κB are required for Mmass-induced proinflammatory cytokine generation in macrophages. Furthermore, the S morphotype of Mmass showed lower overall induction of pro-inflammatory (TNF-α, IL-6, and IL-1β) and anti-inflammatory (IL-10) cytokines than the R morphotype, suggesting fewer immunogenic characteristics for this clinical strain. Together, these results suggest that Mmass-induced activation of host proinflammatory cytokines is mediated through TLR2-dependent JNK and ROS signaling pathways.
doi:10.1007/s10875-013-9978-y
PMCID: PMC3937545  PMID: 24402617
Mycobacterium massiliense; Dectin-1; TLR; MyD88; ROS; NF-kB; TNF-α; IL-6
13.  Calcium-Activated Pathways and Oxidative Burst Mediate Zymosan-Induced Signaling and IL-10 Production in Human Macrophages 
Outside of the TLR paradigm, there is little understanding of how pathogen recognition at the cell surface is linked to functional responses in cells of the innate immune system. Recent work in this area demonstrates that the yeast particle zymosan, by binding to the β-glucan receptor Dectin-1, activates an ITAM-Syk–dependent pathway in dendritic cells, which is required for optimal cytokine production and generation of an oxidative burst. It remains unclear how activation of Syk is coupled to effector mechanisms. In human macrophages, zymosan rapidly activated a calcium-dependent pathway downstream of Dectin-1 and Syk that led to activation of calmodulin-dependent kinase II and Pyk2. Calmodulin-dependent kinase and Pyk2 transduced calcium signals into activation of the ERK–MAPK pathway, CREB, and generation of an oxidative burst, leading to downstream production of IL-10. These observations identify a new calcium-mediated signaling pathway activated by zymosan and link this pathway to both inflammatory and anti-inflammatory responses in macrophages.
doi:10.4049/jimmunol.0901293
PMCID: PMC3016855  PMID: 20400701
14.  Immune Recognition of Candida albicans β-glucan by Dectin-1 
The Journal of infectious diseases  2007;196(10):1565-1571.
β(1,3)-glucans represent 40% of the cell wall of the yeast Candida albicans. The dectin-1 lectin-like receptor has shown to recognize fungal β(1,3)-glucans and induce innate immune responses. The importance of β-glucan-dectin-1 pathways for the recognition of C. albicans by human primary blood cells has not been firmly established. In this study we demonstrate that cytokine production by both human peripheral blood mononuclear cells and murine macrophages is dependent on the recognition of β-glucans by dectin-1. Heat killing of C. albicans resulted in exposure of β-glucans on the surface of the cell wall and subsequent recognition by dectin-1, whereas live yeasts stimulated monocytes mainly via recognition of cell-surface mannans. Dectin-1 induced cytokine production through the following 2 pathways: Syk-dependent production of the T-helper (Th) 2-type anti-inflammatory cytokine interleukin-10 and Toll-like receptor-Myd88-dependent stimulation of monocyte-derived proinflammatory cytokines, such as tumor necrosis factor-α. In contrast, stimulation of Th1-type cytokines, such as interferon-γ, by C. albicans was independent of the recognition of β-glucans by dectin-1. In conclusion, C. albicans induces production of monocyte-derived and T cell-derived cytokines through distinct pathways dependent on or independent of dectin-1.
doi:10.1086/523110
PMCID: PMC2655640  PMID: 18008237
15.  Syk kinase is required for collaborative cytokine production induced through Dectin-1 and Toll-like receptors 
European journal of immunology  2008;38(2):500-506.
Recognition of microbial components by germ-line encoded pattern recognition receptors (PRR) initiates immune responses to infectious agents. We and others have proposed that pairs or sets of PRR mediate host immunity. One such pair comprises the fungal β-glucan receptor, Dectin-1, which collaborates through an undefined mechanism with Toll-like receptor 2 (TLR2) to induce optimal cytokine responses in macrophages. We show here that Dectin-1 signaling through the spleen tyrosine kinase (Syk) pathway is required for this collaboration, which can also occur with TLR4, 5, 7 and 9. Deficiency of either Syk or the TLR adaptor MyD88 abolished collaborative responses, which include TNF, MIP-1α and MIP-2 production, and which are comparable to the previously described synergy between TLR2 and TLR4. Collaboration of the Syk and TLR/MyD88 pathways results in sustained degradation of the inhibitor of kB (IkB), enhancing NFkB nuclear translocation. These findings establish the first example of Syk- and MyD88-coupled PRR collaboration, further supporting the concept that paired receptors collaborate to control infectious agents.
doi:10.1002/eji.200737741
PMCID: PMC2430329  PMID: 18200499
C-type lectin; Innate immunity; Macrophage; Syk; TLR
16.  Syk kinase is required for collaborative cytokine production induced through Dectin-1 and Toll-like receptors 
European Journal of Immunology  2008;38(2):500-506.
Recognition of microbial components by germ-line encoded pattern recognition receptors (PRR) initiates immune responses to infectious agents. We and others have proposed that pairs or sets of PRR mediate host immunity. One such pair comprises the fungal β-glucan receptor, Dectin-1, which collaborates through an undefined mechanism with Toll-like receptor 2 (TLR2) to induce optimal cytokine responses in macrophages. We show here that Dectin-1 signaling through the spleen tyrosine kinase (Syk) pathway is required for this collaboration, which can also occur with TLR4, 5, 7 and 9. Deficiency of either Syk or the TLR adaptor MyD88 abolished collaborative responses, which include TNF, MIP-1α and MIP-2 production, and which are comparable to the previously described synergy between TLR2 and TLR4. Collaboration of the Syk and TLR/MyD88 pathways results in sustained degradation of the inhibitor of kB (IkB), enhancing NFkB nuclear translocation. These findings establish the first example of Syk- and MyD88-coupled PRR collaboration, further supporting the concept that paired receptors collaborate to control infectious agents.
doi:10.1002/eji.200737741
PMCID: PMC2430329  PMID: 18200499
C-type lectin; Innate immunity; Macrophage; Syk; TLR
17.  Dectin-2 is a Syk-coupled pattern recognition receptor crucial for Th17 responses to fungal infection 
The Journal of Experimental Medicine  2009;206(9):2037-2051.
Innate immune cells detect pathogens via pattern recognition receptors (PRRs), which signal for initiation of immune responses to infection. Studies with Dectin-1, a PRR for fungi, have defined a novel innate signaling pathway involving Syk kinase and the adaptor CARD9, which is critical for inducing Th17 responses to fungal infection. We show that another C-type lectin, Dectin-2, also signals via Syk and CARD9, and contributes to dendritic cell (DC) activation by fungal particles. Unlike Dectin-1, Dectin-2 couples to Syk indirectly, through association with the FcRγ chain. In a model of Candida albicans infection, blockade of Dectin-2 did not affect innate immune resistance but abrogated Candida-specific T cell production of IL-17 and, in combination with the absence of Dectin-1, decreased Th1 responses to the organism. Thus, Dectin-2 constitutes a major fungal PRR that can couple to the Syk–CARD9 innate signaling pathway to activate DCs and regulate adaptive immune responses to fungal infection.
doi:10.1084/jem.20082818
PMCID: PMC2737172  PMID: 19703985
18.  The phosphoproteome of toll-like receptor-activated macrophages 
First global and quantitative analysis of phosphorylation cascades induced by toll-like receptor (TLR) stimulation in macrophages identifies nearly 7000 phosphorylation sites and shows extensive and dynamic up-regulation and down-regulation after lipopolysaccharide (LPS).In addition to the canonical TLR-associated pathways, mining of the phosphorylation data suggests an involvement of ATM/ATR kinases in signalling and shows that the cytoskeleton is a hotspot of TLR-induced phosphorylation.Intersecting transcription factor phosphorylation with bioinformatic promoter analysis of genes induced by LPS identified several candidate transcriptional regulators that were previously not implicated in TLR-induced transcriptional control.
Toll-like receptors (TLR) are a family of pattern recognition receptors that enable innate immune cells to sense infectious danger. Recognition of microbial structures, like lipopolysaccharide (LPS) of Gram-negative bacteria by TLR4, causes within hours substantial re-programming of macrophage gene expression, including up-regulation of chemokines driving inflammation, anti-microbial effector molecules and cytokines directing adaptive immune responses. TLR signalling is initiated by the adapter protein Myd88 and leads to the activation of kinase cascades that result in activation of the MAPK and NFkB pathways. Phosphorylation has an essential role in these early steps of TLR signalling, and in addition regulates critical transcription factors (TFs). Although TLR signalling has been extensively studied, a comprehensive analysis of phosphorylation events in TLR-activated macrophages is lacking. It is therefore unknown whether the canonical MAPK and NFkB pathways comprise the main phosphorylation events and which other molecular functions and processes are regulated by phosphorylation after stimulation with LPS.
Recent progress in mass spectrometry-based proteomics has opened the possibility to quantitatively investigate global changes in protein abundance and post-translational modifications. Stable isotope labelling with amino acids in cell culture (SILAC) allows highly accurate quantification, and has proved especially useful for direct comparison of phosphopeptide abundance in time-course or treatment analyses.
Here, we adapted SILAC to primary mouse macrophages, and performed a global, quantitative and kinetic analysis of the macrophage phosphoproteome after LPS stimulation. Bioinformatic analyses were used to identify kinases, pathways and biological processes enriched in the LPS-regulated phosphoproteome. To connect TF phosphorylation with transcription, we generated a parallel dataset of nascent RNA and used in silico promoter analysis to identify transcriptional regulators with binding site enrichment among the LPS-regulated gene set.
After establishing SILAC conditions for efficient labelling of primary bone marrow-derived macrophages in two independent experiments 1850 phosphoproteins with a total of 6956 phosphorylation sites were reproducibly identified. Phosphoproteins were detected from all cellular compartments, with a clear enrichment for nuclear and cytoskeleton-associated proteins. LPS caused major regulation of a large fraction of phosphopeptides, with 24% of all sites up-regulated and 9% down-regulated after stimulation (Figure 3A and B). These changes were highly dynamic, as the majority of the regulated phosphopeptides were up-regulated or down-regulated transiently or in a delayed manner (Figure 3C). Overall, the extent of changes in the phosphoproteome was comparable to the transcriptional re-programming, underscoring the importance of phosphorylation cascades in TLR signalling. Our parallel transcriptome data also showed that widespread phosphorylation precedes massive transcriptional changes.
To obtain footprints of kinase activation in response to TLR ligation, we searched phosphopeptide sequences for known linear sequence motifs of 33 kinases and identified kinase motifs enriched among LPS-regulated phosphorylation sites (compared to non-regulated phosphorylation sites) (Table I). Motif ERK/MAPK was highly enriched, in accordance with the essential role of the MAPK module in TLR signalling. Other kinases with motif enrichment have also recently been linked to TLR signalling (e.g. PKD; AKT and its targets GSK3 and mTOR). However, the DNA damage-actviated kinases ATM/ATR and the cell cycle-associated kinases AURORA and CHK1/2 have not been associated with the macrophage response to TLR activation yet. These finding shed new light on older data on the effect of TLR on macrophage proliferation in response to macrophage colony stimulating factor. Of interest, in follow-up experiments using pharmacological inhibitors of the kinases with motif enrichment, we observed that inhibition of ATM kinase activity caused increased LPS-induced expression of several cytokines and chemokines, suggesting that this pathway regulates inflammatory responses.
In further bioinformatic analyses, the Gene Ontology and signalling pathway annotations of phosphoproteins were used to identify signalling pathways and cellular processes targeted by TLR4-controlled phosphorylation (Table II). Among the expected hits, based on the known TLR pathways, were TLR signalling, MAPK and AKT as well as mTOR signalling. Of interest, the annotation terms ‘Rho GTPase cycle' and ‘cytoskeleton' were significantly enriched among LPS-regulated phosphoproteins, indicating a more prominent role for cytoskeletal proteins in the transduction of TLR signals or in the biological response to it.
We were especially interested in the phosphorylation of TFs and its regulation by LPS (Figure 6A). We hypothesised that functionally important TFs should have an increased frequency of binding sites in the promoters of LPS-regulated genes (Figure 6B). To identify transcriptionally regulated genes with high sensitivity, we isolated nascent RNA after metabolic labelling (Figure 6C–E). In silico promoter scanning using Genomatix software for binding sites for all 50 TF families with phosphorylated members was used to test for enrichment in transciptionally induced genes (Figure 6F). At the early time point, binding site enrichment for the canonical TLR-associated TF NFkB was detected, and in addition we found that several other TF families with an established role in the transcription of individual LPS-target genes showed binding site enrichment (CEBP, MEF2, NFAT and HEAT). In addition, enrichment for OCT and HOXC binding sites at the early time point and SORY matrices later after stimulation indicated an involvement of the phosphorylated members of the respective TF families in the execution of TLR-induced transcriptional responses. An initial test of the function for a few of these candidate transcriptional regulators was performed using siRNA knockdown in primary macrophages. These experiments suggested that knock down of the SORY binding phosphoprotein Capicua homolog (Cic) and to a lesser extent of the CREB family member Atf7 selectively attenuates LPS-induced expression of Il1a and Il1b.
In summary, this study provides a novel and global perspective on innate immune activation by TLR signalling (Figure 5). We quantitatively detected a large number of previously unknown site-specific phosphorylation events, which are now publicly available through the Phosida database. By combining different data mining approaches, we consistently identified canonical and newly implicated TLR-activated signalling modules. In particular, the PI3K/AKT and the related mTOR pathway were highlighted; furthermore, DNA damage–response associated ATM/ATR kinases and the cytoskeleton emerged as unexpected hotspots for phosphorylation. Finally, weaving together corresponding phophoproteome and nascent transcriptome datasets through the loom of in silico promoter analysis we identified TFs with a likely role in mediating TLR-induced gene expression programmes.
Recognition of microbial danger signals by toll-like receptors (TLR) causes re-programming of macrophages. To investigate kinase cascades triggered by the TLR4 ligand lipopolysaccharide (LPS) on systems level, we performed a global, quantitative and kinetic analysis of the phosphoproteome of primary macrophages using stable isotope labelling with amino acids in cell culture, phosphopeptide enrichment and high-resolution mass spectrometry. In parallel, nascent RNA was profiled to link transcription factor (TF) phosphorylation to TLR4-induced transcriptional activation. We reproducibly identified 1850 phosphoproteins with 6956 phosphorylation sites, two thirds of which were not reported earlier. LPS caused major dynamic changes in the phosphoproteome (24% up-regulation and 9% down-regulation). Functional bioinformatic analyses confirmed canonical players of the TLR pathway and highlighted other signalling modules (e.g. mTOR, ATM/ATR kinases) and the cytoskeleton as hotspots of LPS-regulated phosphorylation. Finally, weaving together phosphoproteome and nascent transcriptome data by in silico promoter analysis, we implicated several phosphorylated TFs in primary LPS-controlled gene expression.
doi:10.1038/msb.2010.29
PMCID: PMC2913394  PMID: 20531401
macrophage; nascent RNA; phosphoproteome; SILAC; toll-like receptors
19.  Syk Kinase-Coupled C-type Lectin Receptors Engage Protein Kinase C-δ to Elicit Card9 Adaptor-Mediated Innate Immunity 
Immunity  2012;36(1-2):32-42.
Summary
C-type lectin receptors (CLRs) that couple with the kinase Syk are major pattern recognition receptors for the activation of innate immunity and host defense. CLRs recognize fungi and other forms of microbial or sterile danger, and they induce inflammatory responses through the adaptor protein Card9. The mechanisms relaying CLR proximal signals to the core Card9 module are unknown. Here we demonstrated that protein kinase C-δ (PKCδ) was activated upon Dectin-1-Syk signaling, mediated phosphorylation of Card9 at Thr231, and was responsible for Card9-Bcl10 complex assembly and canonical NF-κB control. Prkcd−/− dendritic cells, but not those lacking PKCα, PKCβ, or PKCθ, were defective in innate responses to Dectin-1, Dectin-2, or Mincle stimulation. Moreover, Candida albicans-induced cytokine production was blocked in Prkcd−/− cells, and Prkcd−/− mice were highly susceptible to fungal infection. Thus, PKCδ is an essential link between Syk activation and Card9 signaling for CLR-mediated innate immunity and host protection.
Highlights
► Dectin-1-Syk stimulation activates PKCδ in dendritic cells ► PKCδ controls Card9-Bcl10 complex assembly for canonical NF-κB activation ► Prkcd−/− cells are defective in Dectin-1-, Dectin-2-, or Mincle-triggered responses ► PKCδ is essential for innate antifungal immunity and host protection in vivo
doi:10.1016/j.immuni.2011.11.015
PMCID: PMC3477316  PMID: 22265677
20.  Susceptibility to Coccidioides species in C57BL/6 mice is associated with expression of a truncated splice variant of Dectin-1 (Clec7a) 
Genes and immunity  2008;9(4):338-348.
Coccidioides posadasii spherules stimulate macrophages to make cytokines via TLR-2 and Dectin-1. We used formalin-killed spherules and 1,3-β-glucan purified from spherules to stimulate elicited peritoneal macrophages and myeloid dendritic cells (mDCs) from susceptible (C57BL/6) and resistant (DBA/2) mouse strains. DBA/2 macrophages produced more TNF-α and IL-6 than macrophages from C57BL/6 mice, and the amount of TNF-α made was dependent on both TLR2 and Dectin-1. DCs from C57BL/6 mice made more IL-10 and less IL-23p19 and IL-12p70 than did DBA/2 DC. These responses were inhibited by a monoclonal antibody to Dectin-1. DBA/2 mice expressed full-length Dectin-1, whereas C57BL/6 mice spliced out exon 3, which encodes most of the stalk. RAW cells transduced to express the full-length Dectin-1 responded better to FKS than cells expressing truncated Dectin-1. We compared the isoform of Dectin-1 expressed by 34 C57BL/6 X DBA/2 recombinant inbred (BXD RI) lines with their susceptibility to Coccidioides immitis. In 25 of 34 RI lines susceptibility or resistance corresponded to short or full-length isoforms, respectively. These results suggest that alternative splicing of the Dectin-1 gene contributes to susceptibility of C57BL/6 mice to coccidioidomycosis, and affects the cytokine responses of macrophages and mDCs to spherules.
doi:10.1038/gene.2008.23
PMCID: PMC3681288  PMID: 18418396
cytokines; fungal; inflammation; monocytes/macrophages; dendritic cells
21.  Functional consequences of DECTIN-1 early stop codon polymorphism Y238X in rheumatoid arthritis 
Introduction
Dectin-1, a pattern recognition receptor expressed by the innate immune system, is known to be a major receptor inducing Th17-type adaptive immune responses that have been demonstrated to mediate autoimmunity. In this study, dectin-1 mRNA and protein expression, as well as the recently characterized DECTIN-1 Y238X early stop codon polymorphism, were studied in relation to rheumatoid arthritis (RA) susceptibility and severity.
Methods
Dectin-1 mRNA expression was measured in synovial tissue specimens of RA, osteoarthritis (OA), and nonrheumatic patients. Dectin-1 protein expression and localization were assessed in RA synovial tissue specimens. Macrophages from individuals with different DECTIN-1 genotypes were examined for differences in cytokine responses on dectin-1 stimulation. Furthermore, clinical parameters of inflammation and bone destruction of 262 RA patients were correlated with the presence of the DECTIN-1 Y238X polymorphism.
Results
Evaluation of dectin-1 mRNA expression in synovial tissue biopsies revealed an increased expression in RA specimens, compared with biopsies from OA and nonrheumatic patients. Accordingly, dectin-1 protein expression in RA synovial tissue biopsies was moderate to high, especially on macrophage-like cells. Cytokine production capacity of macrophages bearing the DECTIN-1 Y238X polymorphism was demonstrated to be impaired on dectin-1 stimulation. However, the presence of the DECTIN-1 Y238X polymorphism was not associated with RA susceptibility or disease severity.
Conclusions
Although expression of dectin-1 was high in synovial tissue of RA patients, and reduced cytokine production was observed in macrophages of individuals bearing the DECTIN-1 Y238X polymorphism, loss of one functional allele of DECTIN-1 is not associated with either susceptibility to or severity of RA.
doi:10.1186/ar2933
PMCID: PMC2875660  PMID: 20158887
22.  Diverse Toll-Like Receptors Mediate Cytokine Production by Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans in Macrophages 
Infection and Immunity  2014;82(5):1914-1920.
Toll-like receptors (TLRs) orchestrate a repertoire of immune responses in macrophages against various pathogens. Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans are two important periodontal pathogens. In the present study, we investigated TLR signaling regulating cytokine production of macrophages in response to F. nucleatum and A. actinomycetemcomitans. TLR2 and TLR4 are redundant in the production of cytokines (interleukin-6 [IL-6] and tumor necrosis factor alpha [TNF-α]) in F. nucleatum- and A. actinomycetemcomitans-infected macrophages. The production of cytokines by macrophages in response to F. nucleatum and A. actinomycetemcomitans infection was impaired in MyD88-deficient macrophages. Moreover, cytokine concentrations were lower in MyD88-deficient macrophages than in TLR2/TLR4 (TLR2/4) double-deficient cells. An endosomal TLR inhibitor, chloroquine, reduced cytokine production in TLR2/4-deficient macrophages in response to F. nucleatum and A. actinomycetemcomitans, and DNA from F. nucleatum or A. actinomycetemcomitans induced IL-6 production in bone marrow-derived macrophages (BMDMs), which was abolished by chloroquine. Western blot analysis revealed that TLR2/4 and MyD88 were required for optimal activation of NF-κB and mitogen-activated protein kinases (MAPKs) in macrophages in response to F. nucleatum and A. actinomycetemcomitans, with different kinetics. An inhibitor assay showed that NF-κB and all MAPKs (p38, extracellular signal-regulated kinase [ERK], and Jun N-terminal protein kinase [JNK]) mediate F. nucleatum-induced production of cytokines in macrophages, whereas NF-κB and p38, but not ERK and JNK, are involved in A. actinomycetemcomitans-mediated cytokine production. These findings suggest that multiple TLRs may participate in the cytokine production of macrophages against periodontal bacteria.
doi:10.1128/IAI.01226-13
PMCID: PMC3993440  PMID: 24566622
23.  Ficus carica Polysaccharides Promote the Maturation and Function of Dendritic Cells 
Various polysaccharides purified from plants are considered to be biological response modifiers and have been shown to enhance immune responses. Ficus carica L. is a Chinese traditional plant and has been widely used in Asian countries for its anti-tumor properties. Ficus carica polysaccharides (FCPS), one of the most essential and effective components in Ficus carica L., have been considered to be a beneficial immunomodulator and may be used in immunotherapy. However, the immunologic mechanism of FCPS is still unclear. Dectin-1 is a non-toll-like pattern recognition receptor, predominately expressed on dendritic cells (DCs). Activation of DCs through dectin-1 signaling can lead to the maturation of DC, thus inducing both innate and adaptive immune responses against tumor development and microbial infection. In our study, we found that FCPS could effectively stimulate DCs, partially through the dectin-1/Syk pathway, and promote their maturation, as shown by the up-regulation of CD40, CD80, CD86, and major histocompatibility complex II (MHCII). FCPS also enhanced the production of cytokines by DCs, including IL-12, IFN-γ, IL-6, and IL-23. Moreover, FCPS-treated DCs showed an enhanced capability to stimulate T cells and promote T cell proliferation. Altogether, these results demonstrate that FCPS are able to activate and maturate DCs, thereby up-regulating the immunostimulatory capacity of DCs, which leads to enhanced T cell responses.
doi:10.3390/ijms150712469
PMCID: PMC4139854  PMID: 25026176
polysaccharide; dendritic cells; dectin-1; Syk; immunomodulators
24.  Reactive oxygen species and p47phox activation are essential for the Mycobacterium tuberculosis-induced pro-inflammatory response in murine microglia 
Background
Activated microglia elicits a robust amount of pro-inflammatory cytokines, which are implicated in the pathogenesis of tuberculosis in the central nervous system (CNS). However, little is known about the intracellular signaling mechanisms governing these inflammatory responses in microglia in response to Mycobacterium tuberculosis (Mtb).
Methods
Murine microglial BV-2 cells and primary mixed glial cells were stimulated with sonicated Mtb (s-Mtb). Intracellular ROS levels were measured by staining with oxidative fluorescent dyes [2',7'-Dichlorodihydrofluorescein diacetate (H2DCFDA) and dihydroethidium (DHE)]. NADPH oxidase activities were measured by lucigenin chemiluminescence assay. S-Mtb-induced MAPK activation and pro-inflammatory cytokine release in microglial cells were measured using by Western blot analysis and enzyme-linked immunosorbent assay, respectively.
Results
We demonstrate that s-Mtb promotes the up-regulation of reactive oxygen species (ROS) and the rapid activation of mitogen-activated protein kinases (MAPKs), including p38 and extracellular signal-regulated kinase (ERK) 1/2, as well as the secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-12p40 in murine microglial BV-2 cells and primary mixed glial cells. Both NADPH oxidase and mitochondrial electron transfer chain subunit I play an indispensable role in s-Mtb-induced MAPK activation and pro-inflammatory cytokine production in BV-2 cells and mixed glial cells. Furthermore, the activation of cytosolic NADPH oxidase p47phox and MAPKs (p38 and ERK1/2) is mutually dependent on s-Mtb-induced inflammatory signaling in murine microglia. Neither TLR2 nor dectin-1 was involved in s-Mtb-induced inflammatory responses in murine microglia.
Conclusion
These data collectively demonstrate that s-Mtb actively induces the pro-inflammatory response in microglia through NADPH oxidase-dependent ROS generation, although the specific pattern-recognition receptors involved in these responses remain to be identified.
doi:10.1186/1742-2094-4-27
PMCID: PMC2235845  PMID: 18036262
25.  Ionizing radiation affects the expression of Toll-like receptors 2 and 4 in human monocytic cells through c-Jun N-terminal kinase activation 
Journal of Radiation Research  2014;55(5):876-884.
Pattern recognition receptors recognize pathogen-associated molecular patterns. Among these, Toll-like receptors (TLRs) have well-characterized roles in antibacterial and antiviral immunity. In the present study, the effects of ionizing radiation on the expression of TLRs and cellular responses to ligands were investigated in THP1 monocytes (human monocytic leukemia cells) and THP1-derived macrophage cells (macrophage-like cells), which are induced by culturing in the presence of phorbol 12-myristate 13-acetate. TLR2 and TLR4 expression was detected in THP1 and macrophage-like cells. X-irradiation caused increased expression of these TLRs in THP1 and decreased expression in macrophage-like cells. Responses to FSL-1 (TLR2 ligand) and lipopolysaccharide (LPS, TLR4 ligand) were estimated by determining the induction of tumor necrosis factor-α (TNF-α). After FSL-1 or LPS stimulation, TNF-α induction was greater in X-irradiated THP1 monocytes than in non-irradiated cells. However, although TNF-α expression was not affected by X-irradiation in macrophage-like cells, the expression of LPS-inducible interferon-β was lower following X-irradiation of macrophage-like cells. To clarify the mechanisms of TLR2 and TLR4 regulation by X-irradiation, expression of mitogen-activated protein kinase was investigated. These experiments showed that c-Jun N-terminal kinase (JNK) mediated increases in TLR expression in X-irradiated THP1 monocytes and decreases in TLR expression in X-irradiated macrophage-like cells. This study demonstrates that ionizing radiation modulates ligand-responsive TLR expression through the JNK pathway, depending on differentiation state.
doi:10.1093/jrr/rru040
PMCID: PMC4202298  PMID: 24927726
Toll-like receptor 2; Toll-like receptor 4; ionizing radiation; c-Jun N-terminal kinase

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