Syk kinase is required for collaborative cytokine production induced through Dectin-1 and Toll-like receptors

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 beta-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-1alpha 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 kappaB (IkappaB), enhancing NFkappaB 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.     

Syk- and CARD9-dependent coupling of innate immunity to the induction of T helper cells that produce interleukin 17

The C-type lectin dectin-1 binds to yeast and signals through the kinase Syk and the adaptor CARD9 to induce production of interleukin 10 (IL-10) and IL-2 in dendritic cells (DCs). However, whether this pathway promotes full DC activation remains unclear. Here we show that dectin-1-Syk-CARD9 signaling induced DC maturation and the secretion of proinflammatory cytokines, including IL-6, tumor necrosis factor and IL-23, but little IL-12. Dectin-1-activated DCs 'instructed' the differentiation of CD4+ IL-17-producing effector T cells (T(H)-17 cells) in vitro, and a dectin-1 agonist acted as an adjuvant promoting the differentiation of T(H)-17 and T helper type 1 cells in vivo. Infection with Candida albicans induced CARD9-dependent T(H)-17 responses to the organism. Our data indicate that signaling through Syk and CARD9 can couple innate to adaptive immunity independently of Toll-like receptor signals and that CARD9 is required for the development of T(H)-17 responses to some pathogens.     

Blueprints of Signaling Interactions between Pattern Recognition Receptors: Implications for the Design of Vaccine Adjuvants

Innate immunity activation largely depends on recognition of microorganism structures by Pattern Recognition Receptors (PRRs). PRR downstream signaling results in production of pro- and anti-inflammatory cytokines and other mediators. Moreover, PRR engagement in antigen-presenting cells initiates the activation of adaptive immunity. Recent reports suggest that for the activation of innate immune responses and initiation of adaptive immunity, synergistic effects between two or more PRRs are necessary. No systematic analysis of the interaction between the major PRR pathways were performed to date. In this study, a systematical analysis of the interactions between PRR signaling pathways was performed. PBMCs derived from 10 healthy volunteers were stimulated with either a single PRR ligand or a combination of two PRR ligands. Known ligands for the major PRR families were used: Toll-like receptors (TLRs), C-type lectin receptors (CLRs), NOD-like receptors (NLRs), and RigI-helicases. After 24 h of incubation, production of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), IL-6, and IL-10 was measured in supernatants by enzyme-linked immunosorbent assay (ELISA). The consistency of the PRR interactions (both inhibitory and synergistic) between the various individuals was assessed. A number of PRR-dependent signaling interactions were found to be consistent, both between individuals and with regard to multiple cytokines. The combinations of TLR2 and NOD2, TLR5 and NOD2, TLR5 and TLR3, and TLR5 and TLR9 acted as synergistic combinations. Surprisingly, inhibitory interactions between TLR4 and TLR2, TLR4 and Dectin-1, and TLR2 and TLR9 as well as TLR3 and TLR2 were observed. These consistent signaling interactions between PRR combinations may represent promising targets for immunomodulation and vaccine adjuvant development.     

Syk-dependent ERK activation regulates IL-2 and IL-10 production by DC stimulated with zymosan

Zymosan is a particulate yeast preparation that elicits high levels of IL-2 and IL-10 from dendritic cells (DC) and engages multiple innate receptors, including the Syk-coupled receptor dectin-1 and the MyD88-coupled receptor TLR2. Here, we show that induction of IL-2 and IL-10 by zymosan requires activation of ERK MAP kinase in murine DC. Surprisingly, ERK activation in response to zymosan is completely blocked in Syk-deficient DC and unaffected by MyD88 deficiency. Conversely, ERK activation in response to the TLR2 agonist Pam3Cys is completely MyD88 dependent and unaffected by Syk deficiency. The inability of TLR2 ligands in zymosan to couple to ERK may explain the Syk dependence of the IL-2 and IL-10 response in DC and emphasises the importance of Syk-coupled pattern recognition receptors such as dectin-1 in the detection of yeasts. Furthermore, the lack of receptor compensation observed here suggests that responses induced by complex innate stimuli cannot always be predicted by the signalling pathways downstream of individual receptors.     

Syk kinase-coupled C-type lectin receptors engage protein kinase C-σ to elicit Card9 adaptor-mediated innate immunity

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.     

树突状细胞相关凝集素受体1和2在真菌免疫领域的研究进展

树突状细胞相关凝集素受体1和2(Dectin1和2)是2C型凝集素受体家族(CLR)的重要成员,作为模式识别受体( PRRs),其有效地识别病原体相关分子模式(PAMPs);Dectin-1识别β-葡聚糖,通过自身免疫受体络氨酸激活基序( ITAM)向胞内转导信号;Dectin-2识别α-甘露聚糖,通过耦联FcRγ链上的ITAM结构转导信号.ITAM招募并激活非受体络氨酸蛋白激酶(Syk),后者激活MAPKs或介导CARD9-Malt1-Bcl10复合体组装,激活核因子-κB(NF-κB),诱导合成炎症因子等一系列细胞活动.其配体β-葡聚糖和甘露聚糖都是真菌细胞壁的主要成分.近年来研究表明,Dectin-1和Dectin-2受体在真菌免疫防御中具有重要作用.     

When signaling pathways collide: positive and negative regulation of toll-like receptor signal transduction

Toll-like receptor (TLR) signaling is subjected to crosstalk from other signals, with a resulting positive or negative effect. There is complex crosstalk between the NLR family of immune-regulatory molecules and TLRs, and C-type lectin receptors such as Dectin-1 synergize with TLR2 via the tyrosine kinase Syk. Bruton's tyrosine kinase plays an important positive role in TLR signaling, whereas the TAM family of receptor tyrosine kinases is inhibitory. The tyrosine phosphatase SHP1 has been shown to positively regulate induction of interferon-beta, whereas SHP2 inhibits the kinase TBK1, limiting this response. K63-linked polyubiquination has also been shown to be critical for the initiation of TLR signaling. Finally, glucocorticoids affect TLR signaling by inducing the phosphatase MKP1 and inhibiting TBK1 activation. These recent findings emphasize the importance of considering TLR signaling in the context of other signaling pathways, as is likely to occur in vivo during infection and inflammation.     

Dectin-1 on macrophages modulates the immune response to Fasciola hepatica products through the ERK signaling pathway

Fasciolosis is a zoonotic disease of increasing importance due to its worldwide distribution and elevated economic losses. Previously, we demonstrated that Fasciola hepatica excretory–secretory products (FhESP) induce immunomodulatory effects on peritoneal macrophages in a Dectin-1 dependent manner.In this study, we observed that peritoneal macrophages from naive BALB/c mice stimulated in vitro with FhESP presented increased expression levels of phosphorylated extracellular-signal-regulated kinase (ERK), and this effect was dependent on Syk, protein kinase C (PKC) and Dectin-1. In this sense, we observed increased levels of arginase activity, IL-10 and TGF-β in macrophages stimulated with FhESP, which were dependent on PKC and ERK. Furthermore, we observed that the increased arginase activity, as well as in TGF-β and IL-10 levels, was partially dependent on IL-10 receptor signaling in macrophages that were pre-incubated with anti-IL10R before being stimulated with FhESP.Taken together, these results suggest the participation of Dectin-1 and Syk in FhESP interaction with peritoneal macrophages and the possible role of ERK and IL-10 in downstream signaling pathways involved in the immunomodulatory effects induced by Fasciola hepatica products.     

The cytosolic pattern recognition receptor NOD1 induces inflammatory interleukin-8 during Chlamydia trachomatis infection

Inflammation is a hallmark of chlamydial infections, but how inflammatory cytokines are induced is not well understood. Pattern recognition receptors (PRR) of the host innate immune system recognize pathogen molecules and activate intracellular signaling pathways that modulate immune responses. The role of PRR such as Toll-like receptors (TLR) and nucleotide-binding oligomerization domain (NOD) proteins in the endogenous interleukin-8 (IL-8) response induced during Chlamydia trachomatis infection is not known. We hypothesized that a PRR is essential for the IL-8 response induced by C. trachomatis infection. RNA interference was used to knock down the TLR signaling partner MyD88 as well as NOD1 and its signaling molecule receptor-interacting protein 2 (RIP2). IL-8 induced at 30 h postinfection by C. trachomatis was dependent on NOD1 signaling through RIP2; however, the IL-8 response was independent of MyD88-dependent TLR signaling. Activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase cellular signaling pathway, which is essential for up-regulation of IL-8 in response to C. trachomatis infection, was independent of NOD1 or RIP2. We conclude that the endogenous IL-8 response induced by C. trachomatis infection is dependent upon NOD1 PRR signaling through RIP2 as part of a signal system requiring multiple inputs for optimal IL-8 induction. Since ERK is not activated through this pathway, a concomitant interaction between the host and bacteria is additionally required for full activation of the endogenous IL-8 response.     

The role of Syk/CARD9 coupled C-type lectins in antifungal immunity

Fungal infections are affecting an increasing number of people, and the failure of current therapies in treating systemic infection has resulted in an unacceptably high mortality rate. It is therefore of importance that we understand immune mechanisms operating during fungal infections, in order to facilitate development of adjunctive immunotherapies for the treatment of these diseases. C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that are critical for immune responses to fungi. Many of these receptors are coupled to Syk kinase, which allows these receptors to signal via CARD9 leading to NF-κB activation, which in turn contributes to the induction of both innate and adaptive immunity. Dectin-1, Dectin-2 and Mincle are all CLRs that share this common signalling mechanism and have been shown to play key roles in antifungal immunity. This review aims to update existing paradigms and summarise the most recent findings on these CLRs, their signal transduction mechanisms and the collaborations between these CLRs and other PRRs.     

Soluble β-glucan from Grifola frondosa induces proliferation and Dectin-1/Syk signaling in resident macrophages via the GM-CSF autocrine pathway

MD-Fraction, a highly purified, soluble β-(1,3) (1,6)-glucan obtained from Grifola frondosa (an oriental edible mushroom), has been reported to inhibit tumor growth by modulating host immunity. β-Glucan, a major component of the fungal cell wall, is generally recognized by PRRs expressed on macrophages and DCs, such as Dectin-1, and the ability of β-glucans to modulate host immunity is influenced by their structure and purity. Most cellular studies have used particulate β-glucans, such as yeast zymosan (crude β-glucan) and curdlan (purified β-glucan). However, little is known about the cellular mechanism of soluble β-glucans, including MD-Fraction, despite significant therapeutic implications. In this study, we investigated the cellular mechanism of MD-Fraction in murine resident macrophages and compared it with two well-known β-glucan particles. MD-Fraction induced GM-CSF production rapidly through Dectin-1-independent ERK and p38 MAPK activation. Subsequently, MD-Fraction-induced GM-CSF enhanced proliferation and Dectin-1 expression, which permitted Dectin-1-mediated TNF-α induction through the Syk pathway. Curdlan induced not only the proliferation and activation of Dectin-1/Syk signaling in a manner similar to MD-Fraction but also the uncontrolled, proinflammatory cytokine response. Contrastingly, zymosan reduced proliferation and Dectin-1 expression significantly, indicating that the mechanism of macrophage activation by MD-Fraction differs from that of zymosan. This is the first study to demonstrate that purified β-glucans, such as MD-Fraction and curdlan, induce GM-CSF production directly, resulting in Dectin-1/Syk activation in resident macrophages. In conclusion, we demonstrated that MD-Fraction induces cell proliferation and cytokine production without excessive inflammation in resident macrophages, supporting its immunotherapeutic potential.     

Protein kinase Cδ is a critical component of Dectin-1 signaling in primary human monocytes

Zymosan, a mimic of fungal pathogens, and its opsonized form (ZOP) are potent stimulators of monocyte NADPH oxidase, resulting in the production of O(2)(.-), which is critical for host defense against fungal and bacterial pathogens and efficient immune responses; however, uncontrolled O(2)(.-) production may contribute to chronic inflammation and tissue injury. Our laboratory has focused on characterizing the signal transduction pathways that regulate NADPH oxidase activity in primary human monocytes. In this study, we examined the involvement of various pattern recognition receptors and found that Dectin-1 is the primary receptor for zymosan stimulation of O(2)(.-) via NADPH oxidase in human monocytes, whereas Dectin-1 and CR3 mediate the activation by ZOP. Further studies identified Syk and Src as important signaling components downstream of Dectin-1 and additionally identified PKCδ as a novel downstream signaling component for zymosan-induced O(2)(.-) as well as phagocytosis. Our results show that Syk and Src association with Dectin-1 is dependent on PKCδ activity and expression and demonstrate direct binding between Dectin-1 and PKCδ. Finally, our data show that PKCδ and Syk but not Src are required for Dectin-1-mediated phagocytosis. Taken together, our data identify Dectin-1 as the major PRR for zymosan in primary human monocytes and identify PKCδ as a novel downstream signaling kinase for Dectin-1-mediated regulation of monocyte NADPH oxidase and zymosan phagocytosis.     

Intracellular signaling and cytokine induction upon interactions of Porphyromonas gingivalis fimbriae with pattern-recognition receptors

Toll-like receptors (TLRs) and other pattern-recognition receptors (PRRs) of the innate immune system form functional receptor complexes that recognize and respond to pathogen-associated molecular patterns (PAMPs). Porphyromonas gingivalis is an important pathogen in human periodontitis and has also been implicated in atherosclerosis. A major virulence factor of this pathogen is the fimbriae, which function as a surface adhesin. Here we present evidence that fimbriae also constitute a predominant P. gingivalis proinflammatory molecule which activates the TLR signaling pathway resulting in induction of proinflammatory cytokines (IL-1beta, IL-6, and TNF-alpha) and chemokines (IL-8) in monocytic cells. Although TLR2 and TLR4 mediate cellular activation in response to fimbriae, other PRRs, namely CD14 and CD11b/CD18, are involved in the recognition of fimbriae. We thus propose that fimbriae function as a PAMP which interacts with a PRR multi-receptor complex, where CD14 and CD11b/CD18 function as recruiting receptors and TLRs function as signaling receptors. In addition to cytokine induction, TLR activation by fimbriae also results in upregulation of the CD40, CD80, and CD86 costimulatory molecules in antigen-presenting cells, suggesting that fimbriae are sensed as a potential "danger" to the host immune system. Moreover, proinflammatory cytokine induction is attenuated upon repeated cellular stimulation with P. gingivalis fimbriae. This mechanism of tolerance induction which serves to mitigate excessive and potentially harmful inflammatory reactions appears to be due partly to fimbria-induced downregulation of the expression of interleukin-1 receptor-associated kinase-1 (IRAK-1), an important signaling intermediate of the TLR pathway. Understanding the molecular basis of how the host recognizes and responds to P. gingivalis fimbriae is essential for developing molecular approaches to control P. gingivalis-induced inflammatory responses in periodontal disease and perhaps atherosclerosis.     

Toll-like receptors: critical proteins linking innate and acquired immunity

Recognition of pathogens is mediated by a set of germline-encoded receptors that are referred to as pattern-recognition receptors (PRRs). These receptors recognize conserved molecular patterns (pathogen-associated molecular patterns), which are shared by large groups of microorganisms. Toll-like receptors (TLRs) function as the PRRs in mammals and play an essential role in the recognition of microbial components. The TLRs may also recognize endogenous ligands induced during the inflammatory response. Similar cytoplasmic domains allow TLRs to use the same signaling molecules used by the interleukin 1 receptors (IL-1Rs): these include MyD88, IL-1R--associated protein kinase and tumor necrosis factor receptor--activated factor 6. However, evidence is accumulating that the signaling pathways associated with each TLR are not identical and may, therefore, result in different biological responses.     

TRAF6-dependent mitogen-activated protein kinase activation differentially regulates the production of interleukin-12 by macrophages in response to Toxoplasma gondii

The production of interleukin-12 (IL-12) is critical to the development of innate and adaptive immune responses required for the control of intracellular pathogens. Many microbial products signal through Toll-like receptors (TLR) and activate NF-kappaB family members that are required for the production of IL-12. Recent studies suggest that components of the TLR pathway are required for the production of IL-12 in response to the parasite Toxoplasma gondii; however, the production of IL-12 in response to this parasite is independent of NF-kappaB activation. The adaptor molecule TRAF6 is involved in TLR signaling pathways and associates with serine/threonine kinases involved in the activation of both NF-kappaB and mitogen-activated protein kinase (MAPK). To elucidate the intracellular signaling pathways involved in the production of IL-12 in response to soluble toxoplasma antigen (STAg), wild-type and TRAF6(-/-) mice were inoculated with STAg, and the production of IL-12(p40) was determined. TRAF6(-/-) mice failed to produce IL-12(p40) in response to STAg, and TRAF6(-/-) macrophages stimulated with STAg also failed to produce IL-12(p40). Studies using Western blot analysis of wild-type and TRAF6(-/-) macrophages revealed that stimulation with STAg resulted in the rapid TRAF6-dependent phosphorylation of p38 and extracellular signal-related kinase, which differentially regulated the production of IL-12(p40). The studies presented here demonstrate for the first time that the production of IL-12(p40) in response to toxoplasma is dependent upon TRAF6 and p38 MAPK.     

CLEC-2 signaling via Syk in myeloid cells can regulate inflammatory responses

Myeloid cells express a plethora of C-type lectin receptors (CLRs) that can regulate immune responses. CLEC-2 belongs to the Dectin-1 sub-family of CLRs that possess an extracellular C-type lectin-like domain and a single intracellular hemITAM motif. CLEC-2 is highly expressed on mouse and human platelets where it signals via Syk to promote aggregation. We generated a monoclonal antibody (mAb) against mouse CLEC-2 and found that CLEC-2 is additionally widely expressed on leukocytes and that its expression is upregulated during inflammation. MAb-mediated crosslinking of CLEC-2 leads to hemITAM-dependent signaling via Syk, Ca(2+) and NFAT and, in myeloid cells, modulates the effect of toll-like receptor (TLR) agonists to selectively potentiate production of IL-10. A macrophage/dendritic cell-dependent increase in IL-10 is also observed in mice given anti-CLEC-2 mAb together with LPS. Collectively, these data indicate that CLEC-2 is expressed in myeloid cells and acts as a Syk-coupled CLR able to modulate TLR signaling and inflammatory responses.     

Toll-Like Receptors’ Pathway Disturbances are Associated with Increased Susceptibility to Infections in Humans

Toll-like receptors (TLRs) sense microbial products and play an important role in innate immunity. Currently, 11 members of TLRs have been identified in humans, with important function in host defense in early steps of the inflammatory response. TLRs are present in the plasma membrane (TLR1, TLR2, TLR4, TLR5, TLR6) and endosome (TLR3, TLR7, TLR8, TLR9) of leukocytes. TLRs and IL-1R are a family of receptors related to the innate immune response that contain an intracellular domain known as the Toll-IL-1R (TIR) domain that recruits the TIR-containing cytosolic adapters MyD88, TRIF, TIRAP and TRAM. The classical pathway results in the activation of both nuclear factor κB and MAPKs via the IRAK complex, with two active kinases (IRAK-1 and IRAK-4) and two non-catalytic subunits (IRAK-2 and IRAK-3/M). The classical pro-inflammatory TLR signaling pathway leads to the synthesis of inflammatory cytokines and chemokines, such as IL-1β, IL-6, IL-8, IL-12 and TNF-α. In humans, genetic defects have been identified that impair signaling of the TLR pathway and this may result in recurrent pyogenic infections, as well as virus and fungi infections. In this review, we discuss the main mechanisms of microbial recognition and the defects involving TLRs.     

Mycobacterium massiliense Induces Inflammatory Responses in Macrophages Through Toll-Like Receptor 2 and c-Jun N-Terminal Kinase

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.