Endocytosis of Mycobacterium tuberculosis Heat Shock Protein 60 Is Required to Induce Interleukin-10 Production in Macrophages

Understanding the signaling pathways involved in the regulation of anti-inflammatory and pro-inflammatory responses in tuberculosis is extremely important in tailoring a macrophage innate response to promote anti-tuberculosis immunity in the host. Although the role of toll-like receptors (TLRs) in the regulation of anti-inflammatory and pro-inflammatory responses is known, the detailed molecular mechanisms by which the Mycobacterium tuberculosis bacteria modulate these innate responses are not clearly understood. In this study, we demonstrate that M. tuberculosis heat shock protein 60 (Mtbhsp60, Cpn60.1, and Rv3417c) interacts with both TLR2 and TLR4 receptors, but its interaction with TLR2 leads to clathrin-dependent endocytosis resulting in an increased production of interleukin (IL)-10 and activated p38 MAPK. Blockage of TLR2-mediated endocytosis inhibited IL-10 production but induced production of tumor necrosis factor (TNF)-α and activated ERK1/2. In contrast, upon interaction with TLR4, Mtbhsp60 remained predominantly localized on the cell surface due to poorer endocytosis of the protein that led to decreased IL-10 production and p38 MAPK activation. The Escherichia coli homologue of hsp60 was found to be retained mainly on the macrophage surface upon interaction with either TLR2 or TLR4 that triggered predominantly a pro-inflammatory-type immune response. Our data suggest that cellular localization of Mtbhsp60 upon interaction with TLRs dictates the type of polarization in the innate immune responses in macrophages. This information is likely to help us in tailoring the host protective immune responses against M. tuberculosis.     

Acylation determines the toll-like receptor (TLR)-dependent positive versus TLR2-, mannose receptor-, and SIGNR1-independent negative regulation of pro-inflammatory cytokines by mycobacterial lipomannan

Mycobacterium tuberculosis lipomannans (LMs) modulate the host innate immune response. The total fraction of Mycobacterium bovis BCG LM was shown both to induce macrophage activation and pro-inflammatory cytokines through Toll-like receptor 2 (TLR2) and to inhibit pro-inflammatory cytokine production by lipopolysaccharide (LPS)-activated macrophages through a TLR2-independent pathway. The pro-inflammatory activity was attributed to tri- and tetra-acylated forms of BCG LM but not the mono- and di-acylated ones. Here, we further characterize the negative activities of M. bovis BCG LM on primary murine macrophage activation. We show that di-acylated LMs exhibit a potent inhibitory effect on cytokine and NO secretion by LPS-activated macrophages. The inhibitory activity of mycobacterial mannose-capped lipoarabino-mannans on human phagocytes was previously attributed to their binding to the C-type lectins mannose receptor or specific intracellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN). However, we found that di-acylated LM inhibition of LPS-induced tumor necrosis factor secretion by murine macrophages was independent of TLR2, mannose receptor, or the murine ortholog SIGNR1. We further determined that tri-acyl-LM, an agonist of TLR2/TLR1, promoted interleukin-12 p40 and NO secretion through the adaptor proteins MyD88 and TIRAP, whereas the fraction containing tetra-acylated LM activated macrophages in a MyD88-dependent fashion, mostly through TLR4. TLR4-dependent pro-inflammatory activity was also seen with M. tuberculosis LM, composed mostly of tri-acylated LM, suggesting that acylation degree per se might not be sufficient to determine TLR2 versus TLR4 usage. Therefore, LM acylation pattern determines the anti-inflammatory versus pro-inflammatory effects of LM through different pattern recognition receptors or signaling pathways and may represent an additional mean of regulating the host innate immunity by mycobacteria.     

Recombinant MPT83 derived from Mycobacterium tuberculosis induces cytokine production and upregulates the function of mouse macrophages through TLR2

TLR2 recognizes components of Mycobacterium tuberculosis and initiates APC activities that influence both innate and adaptive immunity. M. tuberculosis lipoproteins are an important class of TLR2 ligands. In this study, we focused on recombinant MPT83 (rMPT83) to determine its effects on mouse macrophages. We demonstrated that rMPT83 induced the production of TNF-α, IL-6, and IL-12 p40 and that cytokine induction depended on activated MAPKs, because we observed the rapid phosphorylation of ERK1/2, p38, and JNK in macrophages. Additionally, neutralizing Abs against TLR2 significantly inhibited cytokine secretion and reduced or attenuated the rMPT83-induced activation of p38 and JNK in RAW264.7 cells, a mouse macrophage cell line. Furthermore, rMPT83-induced cytokine production was significantly lower in macrophages from TLR2(-/-) mice than in macrophages from wild-type mice. We further found that prolonged exposure (>24 h) of RAW264.7 cells or macrophages from wild-type and TLR2(-/-) mice to rMPT83 resulted in a significant enhancement of IFN-γ-induced MHC class II expression and an enhanced ability of macrophages to present the rMPT83 peptide to CD4(+) T cells. These results indicated that rMPT83 is a TLR2 agonist that induces the production of cytokines by macrophages and upregulates macrophage function.     

Fine Tuning Inflammation at the Front Door: Macrophage Complement Receptor 3-mediates Phagocytosis and Immune Suppression for Francisella tularensis

Complement receptor 3 (CR3, CD11b/CD18) is a major macrophage phagocytic receptor. The biochemical pathways through which CR3 regulates immunologic responses have not been fully characterized. Francisella tularensis is a remarkably infectious, facultative intracellular pathogen of macrophages that causes tularemia. Early evasion of the host immune response contributes to the virulence of F. tularensis and CR3 is an important receptor for its phagocytosis. Here we confirm that efficient attachment and uptake of the highly virulent Type A F. tularensis spp. tularensis strain Schu S4 by human monocyte-derived macrophages (hMDMs) requires complement C3 opsonization and CR3. However, despite a>40-fold increase in uptake following C3 opsonization, Schu S4 induces limited pro-inflammatory cytokine production compared with non-opsonized Schu S4 and the low virulent F. novicida. This suggests that engagement of CR3 by opsonized Schu S4 contributes specifically to the immune suppression during and shortly following phagocytosis which we demonstrate by CD11b siRNA knockdown in hMDMs. This immune suppression is concomitant with early inhibition of ERK1/2, p38 MAPK and NF-κB activation. Furthermore, TLR2 siRNA knockdown shows that pro-inflammatory cytokine production and MAPK activation in response to non-opsonized Schu S4 depends on TLR2 signaling providing evidence that CR3-TLR2 crosstalk mediates immune suppression for opsonized Schu S4. Deletion of the CD11b cytoplasmic tail reverses the CR3-mediated decrease in ERK and p38 activation during opsonized Schu-S4 infection. The CR3-mediated signaling pathway involved in this immune suppression includes Lyn kinase and Akt activation, and increased MKP-1, which limits TLR2-mediated pro-inflammatory responses. These data indicate that while the highly virulent F. tularensis uses CR3 for efficient uptake, optimal engagement of this receptor down-regulates TLR2-dependent pro-inflammatory responses by inhibiting MAPK activation through outside-in signaling. CR3-linked immune suppression is an important mechanism involved in the pathogenesis of F. tularensis infection.     

Disruption of CD36 impairs cytokine response to Plasmodium falciparum glycosylphosphatidylinositol and confers susceptibility to severe and fatal malaria in vivo

CD36 is a scavenger receptor that has been implicated in malaria pathogenesis as well as innate defense against blood-stage infection. Inflammatory responses to Plasmodium falciparum GPI (pfGPI) anchors are believed to play an important role in innate immune response to malaria. We investigated the role of CD36 in pfGPI-induced MAPK activation and proinflammatory cytokine secretion. Furthermore, we explored the role of this receptor in an experimental model of acute malaria in vivo. We demonstrate that ERK1/2, JNK, p38, and c-Jun became phosphorylated in pfGPI-stimulated macrophages. In contrast, pfGPI-induced phosphorylation of JNK, ERK1/2, and c-Jun was reduced in Cd36(-/-) macrophages and Cd36(-/-) macrophages secreted significantly less TNF-alpha in response to pfGPI than their wild-type counterparts. In addition, we demonstrate a role for CD36 in innate immune response to malaria in vivo. Compared with wild-type mice, Cd36(-/-) mice experienced more severe and fatal malaria when challenged with Plasmodium chabaudi chabaudi AS. Cd36(-/-) mice displayed a combined defect in cytokine induction and parasite clearance with a dysregulated cytokine response to infection, earlier peak parasitemias, higher parasite densities, and higher mortality rates than wild-type mice. These results provide direct evidence that pfGPI induces TNF-alpha secretion in a CD36-dependent manner and support a role for CD36 in modulating host cytokine response and innate control of acute blood-stage malaria infection in vivo.     

The TLR2-MyD88-NOD2-RIPK2 signalling axis regulates a balanced pro-inflammatory and IL-10-mediated anti-inflammatory cytokine response to Gram-positive cell walls

Systemic infection with Streptococcus pneumoniae is associated with a vigorous pro-inflammatory response to structurally complex cell wall fragments (PnCW) that are shed during cell growth and antibiotic-induced autolysis. Consistent with previous studies, inflammatory cytokine production induced by PnCW was dependent on TLR2 but independent of NOD2, a cytoplasmic NLR protein. However, in parallel with the pro-inflammatory response, we found that PnCW also induced prodigious secretion of anti-inflammatory IL-10 from macrophages. This response was dependent on TLR2, but also involved NOD2 as absence of NOD2-reduced IL-10 secretion in response to cell wall and translated into diminished downstream effects on IL-10-regulated target gene expression. PnCW-mediated production of IL-10 via TLR2 required RIPK2 a kinase required for NOD2 function, and MyD88 but differed from that known for zymosan in that ERK pathway activation was not detected. As mutations in NOD2 are linked to aberrant immune responses, the temporal and quantitative effects of activation of the TLR2-NOD2-RIPK2 pathway on IL-10 secretion may affect the balance between pro- and anti-inflammatory responses to Gram-positive bacteria.     

Innate immune responses to bacterial ligands in the peripheral human lung--role of alveolar epithelial TLR expression and signalling

It is widely believed that the alveolar epithelium is unresponsive to LPS, in the absence of serum, due to low expression of TLR4 and CD14. Furthermore, the responsiveness of the epithelium to TLR-2 ligands is also poorly understood. We hypothesised that human alveolar type I (ATI) and type II (ATII) epithelial cells were responsive to TLR2 and TLR4 ligands (MALP-2 and LPS respectively), expressed the necessary TLRs and co-receptors (CD14 and MD2) and released distinct profiles of cytokines via differential activation of MAP kinases. Primary ATII cells and alveolar macrophages and an immortalised ATI cell line (TT1) elicited CD14 and MD2-dependent responses to LPS which did not require the addition of exogenous soluble CD14. TT1 and primary ATII cells expressed CD14 whereas A549 cells did not, as confirmed by flow cytometry. Following LPS and MALP-2 exposure, macrophages and ATII cells released significant amounts of TNFα, IL-8 and MCP-1 whereas TT1 cells only released IL-8 and MCP-1. P38, ERK and JNK were involved in MALP-2 and LPS-induced cytokine release from all three cell types. However, ERK and JNK were significantly more important than p38 in cytokine release from macrophages whereas all three were similarly involved in LPS-induced mediator release from TT1 cells. In ATII cells, JNK was significantly more important than p38 and ERK in LPS-induced MCP-1 release. MALP-2 and LPS exposure stimulated TLR4 protein expression in all three cell types; significantly more so in ATII cells than macrophages and TT1 cells. In conclusion, this is the first study describing the expression of CD14 on, and TLR2 and 4 signalling in, primary human ATII cells and ATI cells; suggesting that differential activation of MAP kinases, cytokine secretion and TLR4 expression by the alveolar epithelium and macrophages is important in orchestrating a co-ordinated response to inhaled pathogens.     

Phosphatidylinositol 3-kinase activation attenuates the TLR2-mediated macrophage proinflammatory cytokine response to Francisella tularensis live vaccine strain

An inadequate innate immune response appears to contribute to the virulence of Francisella tularensis following pulmonary infection. Studies in mice suggest that this poor response results from suppression of proinflammatory cytokine production early during infection, but the mechanisms involved are not understood. PI3K is known to regulate proinflammatory cytokine expression, but its exact role (positive versus negative) is controversial. We sought to clarify the role of PI3K in regulating proinflammatory signaling and cytokine production during infection with F. tularensis live vaccine strain (LVS). In this study, we demonstrate that the induction of TNF and IL-6 expression by LVS in mouse bone marrow-derived macrophages was markedly enhanced when PI3K activity was inhibited by either of the well-known chemical inhibitors, wortmannin or LY294002. The enhanced cytokine expression was accompanied by enhanced activation of p38 MAPK and ERK1/2, both of which were critical for LVS-induced expression of TNF and IL-6. LVS-induced MAPK activation and cytokine production were TLR2- and MyD88- dependent. PI3K/Akt activation was MyD88-dependent, but was surprisingly TLR2-independent. LVS infection also rapidly induced MAPK phosphatase-1 (MKP-1) expression; PI3K and TLR2 signaling were required. Peak levels of MKP-1 correlated closely with the decline in p38 MAPK and ERK1/2 phosphorylation. These data suggest that infection by LVS restrains the TLR2-triggered proinflammatory response via parallel activation of PI3K, leading to enhanced MKP-1 expression, accelerated deactivation of MAPKs, and suppression of proinflammatory cytokine production. This TLR2-independent inhibitory pathway may be an important mechanism by which Francisella suppresses the host's innate immune response.     

Lactoferrin activates macrophages via TLR4-dependent and -independent signaling pathways

Lactoferrin (LF) is a component of innate immunity and is known to interact with accessory molecules involved in the TLR4 pathway, including CD14 and LPS binding protein, suggesting that LF may activate components of the TLR4 pathway. In the present study, we have asked whether bovine LF (bLF)-induced macrophage activation is TLR4-dependent. Both bLF and LPS stimulated IL-6 production and CD40 expression in RAW 264.7 macrophages and in BALB/cJ peritoneal exudate macrophages. However, in macrophages from congenic TLR4(-/-) C.C3-Tlr4(lps-d) mice, CD40 was not expressed while IL-6 secretion was increased relative to wild-type cells. The signaling components NF-kappaB, p38, ERK and JNK were activated in RAW 264.7 cells and BALB/cJ macrophages after bLF or LPS stimulation, demonstrating that the TLR4-dependent bLF activation pathway utilizes signaling components common to LPS activation. In TLR4 deficient macrophages, bLF-induced activation of NF-kappaB, p38, ERK and JNK whereas LPS-induced cell signaling was absent. We conclude from these studies that bLF induces limited and defined macrophage activation and cell signaling events via TLR4-dependent and -independent mechanisms. bLF-induced CD40 expression was TLR4-dependent whereas bLF-induced IL-6 secretion was TLR4-independent, indicating potentially separate pathways for bLF mediated macrophage activation events in innate immunity.     

Role of apoptosis-regulating signal kinase 1 in innate immune responses by Mycobacterium bovis bacillus Calmette-Guérin

Mycobacterium bovis bacillus Calmette-Guérin (BCG) induces innate immune responses through Toll-like receptor (TLR) 2 and TLR4. We investigated the role of apoptosis-regulating signal kinase (ASK) 1 in reactive oxygen species (ROS)-mediated innate immune responses induced by BCG mycobacterial infection. In macrophages, M. bovis BCG stimulation resulted in rapid activation of mitogen-activated protein kinases (MAPKs), secretion of inflammatory cytokines, such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-6, and ROS generation in a TLR2- and TLR4-dependent manner. M. bovis BCG-induced ROS production led to robust activation of ASK1 upstream of the c-jun-N-terminal kinase and p38 MAPK, but not extracellular-regulated kinase 1/2. Blocking ASK1 activity markedly attenuated M. bovis BCG-induced TNF-alpha and IL-6 production by macrophages. Both TLR2 and TLR4 were required for optimal activation of ASK1 in response to M. bovis BCG. Furthermore, we present evidence that TNF receptor-associated factor (TRAF) 6 activities were essential for ROS-mediated ASK1 activation by M. bovis BCG. Finally, ASK1 activities were required for effective control of intracellular mycobacterial survival. Thus, the results of this study suggest a novel role of the TLR-ROS-TRAF6-ASK1 axis in the innate immune response to mycobacteria as a signaling intermediate.     

In vivo ethanol exposure down-regulates TLR2-, TLR4-, and TLR9-mediated macrophage inflammatory response by limiting p38 and ERK1/2 activation

Ethanol is known to increase susceptibility to infections, in part, by suppressing macrophage function. Through TLRs, macrophages recognize pathogens and initiate inflammatory responses. In this study, we investigated the effect of acute ethanol exposure on murine macrophage activation mediated via TLR2, TLR4, and TLR9. Specifically, the study focused on the proinflammatory cytokines IL-6 and TNF-alpha and activation of p38 and ERK1/2 MAPKs after a single in vivo exposure to physiologically relevant level of ethanol followed by ex vivo stimulation with specific TLR ligands. Acute ethanol treatment inhibited IL-6 and TNF-alpha synthesis and impaired p38 and ERK1/2 activation induced by TLR2, TLR4, and TLR9 ligands. We also addressed the question of whether ethanol treatment modified activities of serine/threonine-specific, tyrosine-specific phosphatases, and MAPK phosphatase type 1. Inhibitors of three families of protein phosphatases did not restore ethanol-impaired proinflammatory cytokine production nor p38 and ERK1/2 activation. However, inhibitors of serine/threonine protein phosphatase type 1 and type 2A significantly increased IL-6 and TNF-alpha levels, and prolonged activation of p38 and ERK1/2 when triggered by TLR4 and TLR9 ligands. In contrast, with TLR2 ligand stimulation, TNF-alpha production was reduced, whereas IL-6 levels, and p38 and ERK1/2 activation were not affected. In conclusion, acute ethanol exposure impaired macrophage responsiveness to multiple TLR agonists by inhibiting IL-6 and TNF-alpha production. Mechanism responsible for ethanol-induced suppression involved inhibition of p38 and ERK1/2 activation. Furthermore, different TLR ligands stimulated IL-6 and TNF-alpha production via signaling pathways, which showed unique characteristics.     

Mycobacterium tuberculosis lipoprotein-induced association of TLR2 with protein kinase C zeta in lipid rafts contributes to reactive oxygen species-dependent inflammatory signalling in macrophages

Membrane lipid rafts are enriched in cholesterol and play an important role as signalling platforms. However, the roles of lipid rafts and associated signalling molecules in the innate immune responses to mycobacteria remain unknown. Here we show that stimulation with Mycobacterium tuberculosis 19 kDa lipoprotein, a TLR2/1 agonist, results in translocation of TLR2 to lipid rafts, coalescence of lipid rafts and production of reactive oxygen species (ROS) that drive pro-inflammatory responses. Disruption of lipid raft organization markedly reduced lipoprotein-induced ROS and inflammatory responses. Remarkably, the atypical protein kinase C (PKC) ζ was specifically recruited into detergent-resistant membrane fractions and associated with TLR2. PKCζ activity was critical for lipoprotein-dependent ROS generation, raft coalescence and the pro-inflammatory responses by macrophages. Moreover, lipid raft organization was required for 19 kDa mediated PKCζ activation. These results demonstrate that TLR2 trafficking and raft coalescence play an essential role for the initiation of lipoprotein-induced innate immune responses via TLR2 and ROS signalling. In addition, PKCζ targets to lipid rafts and may act as a critical adaptor molecule to regulate lipid raft dynamics during TLR2 signalling.     

Essential role of MAPK phosphatase-1 in the negative control of innate immune responses

TLR-induced innate immunity and inflammation are mediated by signaling cascades leading to activation of the MAPK family of Ser/Thr protein kinases, including p38 MAPK, which controls cytokine release during innate and adoptive immune responses. Failure to terminate such inflammatory reactions may lead to detrimental systemic effects, including septic shock and autoimmunity. In this study, we provide genetic evidence of a critical and nonredundant role of MAPK phosphatase (MKP)-1 in the negative control of MAPK-regulated inflammatory reactions in vivo. MKP-1-/- mice are hyperresponsive to low-dose LPS-induced toxicity and exhibit significantly increased serum TNF-alpha, IL-6, IL-12, MCP-1, IFN-gamma, and IL-10 levels after systemic administration of LPS. Furthermore, absence of MKP-1 increases systemic levels of proinflammatory cytokines and exacerbates disease development in a mouse model of rheumatoid arthritis. When activated through TLR2, TLR3, TLR4, TLR5, and TLR9, bone marrow-derived MKP-1-/- macrophages exhibit increased cytokine production and elevated expression of the differentiation markers B7.2 (CD86) and CD40. MKP-1-deficient macrophages also show enhanced constitutive and TLR-induced activation of p38 MAPK. Based on these findings, we propose that MKP-1 is an essential component of the intracellular homeostasis that controls the threshold and magnitude of p38 MAPK activation in macrophages, and inflammatory conditions accentuate the significance of this regulatory function.     

Cutting edge: critical role of intracellular osteopontin in antifungal innate immune responses

We found that absence of osteopontin (OPN) in immunocompromised Rag2(-/-) mice, which lack T and B cells, made the mice extremely susceptible to an opportunistic fungus Pneumocystis, although immunocompetent OPN-deficient mice could clear Pneumocystis as well as wild-type mice. OPN has been studied as an extracellular protein, and the role of an intracellular isoform of OPN (iOPN) is still largely unknown. In this study, we elucidated the mechanism by which iOPN was involved in antifungal innate immunity. First, iOPN was essential for cluster formation of fungal receptors that detect Pneumocystis, including dectin-1, TLR2, and mannose receptor. Second, iOPN played a role as an adaptor molecule in TLR2 and dectin-1 signaling pathways and mediated ERK activation and cytokine production by zymosan, which simultaneously activates TLR2 and dectin-1 pathways. Third, iOPN enhanced phagocytosis and clearance of Pneumocystis. Our study suggests the critical involvement of iOPN in antifungal innate immunity.     

Aspergillus fumigatus induces innate immune responses in alveolar macrophages through the MAPK pathway independently of TLR2 and TLR4

Aspergillus fumigatus causes invasive aspergillosis in immunosuppressed patients. In the immunocompetent host, inhaled conidia are cleared by alveolar macrophages. The signaling pathways of the alveolar macrophage involved in the clearance of A. fumigatus are poorly understood. Therefore, we investigated the role of TLRs in the immune response against A. fumigatus and their contribution to the signaling events triggered in murine alveolar macrophages upon infection with A. fumigatus conidia. Specifically, we examined the MAPKs and NF-kappaB activation and cytokine signaling. Our investigations revealed that immunocompetent TLR2, TLR4, and MyD88 knockout mice were not more susceptible to invasive aspergillosis as compared with wild-type mice and that the in vitro phosphorylation of the MAPKs ERK and p38 was not affected in TLR2, TLR4, or MyD88 knockout mice following stimulation with conidia. In vivo experiments suggest that ERK was an essential MAPK in the defense against A. fumigatus, whereas the activation of NF-kappaB appeared to play only a secondary role. In conclusion, our findings demonstrate that TLR2/4 recognition and MyD88 signaling are dispensable for the clearance of A. fumigatus under immunocompetent situations. Furthermore, our data stress the important role of ERK activation in innate immunity to A. fumigatus.     

Adenovirus vector-induced innate inflammatory mediators, MAPK signaling, as well as adaptive immune responses are dependent upon both TLR2 and TLR9 in vivo

Adenovirus (Ad) vectors are promising candidates for both gene transfer and vaccine applications. In this study, we investigated the role of TLR2 in innate and adaptive immune responses to Ad and/or the transgene it expresses following systemic injection. We found that Ad directly activates ERK1/2 in vivo, but that initiation of ERK1/2 activation is primarily a MyD88/TLR2-independent, but Kupffer cell-dependent, event. The complexity of Ad-induced innate immune responses was confirmed when we also found that both TLR2 and MyD88 functions are required for the sustained activation of ERK1/2. Although we found that the initial activation of NF-kappaB by Ads is dependent upon MyD88, but independent of TLR2 in (non-Kupffer cells) the liver, TLR2 significantly influenced the Ad-induced late phase NF-kappaB activation. These very rapid responses were positively correlated with subsequent innate immune responses to the Ad vector, as our results confirmed that the induction of several cytokines and chemokines, and the expression of innate immune response genes following Ad injection were TLR2 dependent in vivo. The requirement of TLR2 in Ad-induced innate responses also correlated with significantly altered adaptive immune responses. For example, our results demonstrate that the generation of Ad-neutralizing Abs, and anti-transgene-specific Abs elicited subsequent to Ad vector treatments, are both dependent upon TLR2 functionality. Finally, we found that several Ad-induced innate immune responses are dependent on both TLR2 and TLR9. Therefore, this study confirms that several (but not all) Ad-induced innate and adaptive immune responses are TLR dependent.     

Dectin-1 synergizes with TLR2 and TLR4 for cytokine production in human primary monocytes and macrophages

The beta-glucan receptor dectin-1 and Toll-like receptors TLR2 and TLR4 are the main receptors for recognition of Candida albicans by the innate immune system. It has been reported that dectin-1 amplifies TLR2-dependent induction of cytokines in mouse models. In the present study we hypothesized that dectin-1 has potent synergistic effects with both TLR2 and TLR4 in human PBMCs and macrophages. Human PBMCs and monocyte-derived macrophages were stimulated with curdlan, a linear beta-1,3-glucan-polymer derived from Alcaligenes faecalis with specific ligand affinity for dectin-1, in combination with the synthetic TLR2 ligand Pam3Cys and the ultrapure TLR4 ligand LPS. TNF-alpha and IL-10 production was measured in the supernatants with ELISA. Curdlan is a specific dectin-1 ligand without TLR2- or TLR4-stimulating properties. Human primary monocytes and macrophages express dectin-1 on the cell membrane. Stimulation of human PBMCs with curdlan in combination with Pam3Cys or LPS leads to synergistic increase in TNF-alpha production that was inhibited by GE2, a neutralizing dectin-1 antibody. Dectin-1-dependent synergy between curdlan and TLR agonists was also apparent in human monocyte-derived macrophages. Conclusively, dectin-1 synergizes with both TLR2 and TLR4 pathways for the production of TNF-alpha in human primary PBMCs and in monocyte-derived macrophages.