Reishi polysaccharides induce immunoglobulin production through the TLR4/TLR2-mediated induction of transcription factor Blimp-1

The polysaccharides of Ganoderma lucidum (Reishi) possess immunomodulation activities; however, their mode of molecular action in regulating each cellular subset in the immune system is still not clear. Here, we investigate the function of the main polysaccharide fraction of Reishi (Reishi-F3) in B lymphocyte activation/differentiation. We find that Reishi-F3 causes mouse splenic B cell activation and differentiation to IgM-secreting plasma cells, and the process depends on Reishi-F3-mediated induction of Blimp-1, a master regulator capable of triggering the changes of a cascade of gene expression during plasmacytic differentiation. In human peripheral B lymphocytes, although Reishi-F3 fails to induce their activation, it is able to enhance antibody secretion, which is associated with Blimp-1 mRNA induction. The function of Reishi-F3 depends on the Toll-like receptors TLR4/TLR2 as neutralizing antibodies against TLR4/TLR2 block Reishi-F3-mediated induction of Blimp-1 mRNA and Ig secretion. We have shown that interaction of Reishi-F3 with TLR4/TLR2 followed by signaling through p38 MAPK is involved in the induction of Blimp-1 mRNA, whereas signaling through ERK, p38 MAPK, JNK, and IKK complex is involved in Reishi-F3-mediated Ig secretion. Furthermore, the differential mechanism of Reishi-F3 in mouse and human B cell activation is probably due to the presence of Blimp-1 regulatory site in human CD86 promoter. These results establish the signaling and molecular mechanisms of Reishi-F3 on promoting antibody secretion.     

Kir3.1 channel is functionally involved in TLR4-mediated signaling

We aimed to study the involvement of Kir3.1 channel in TLR4-mediated signaling. LPS stimulation induced the recruitment of TLR4 and Kir3.1 into the lipid raft in THP-1 cells. Treatment with Tertiapin-Q, an inhibitor of Kir3.1, markedly abolished the recruitment of TLR4 into the lipid raft and inhibited the LPS-induced NF-κB activation, resulting in decreased production of TNF-α, IL-1β, and IL-6. To verify the specific role of the Kir3.1 channel, we generated Kir3.1-knockdown THP-1 cells. The Kir3.1^KD THP-1 cells exhibited inhibition of NF-κB activation and production of these pro-inflammatory cytokines in response to TLR4 stimulation. Taken together, our results demonstrate that the Kir3.1 channel is involved in the TLR4-mediated signal at an early event by facilitating the recruitment of TLR4 into lipid raft.     

Hyaluronic Acid Oligosaccharides Suppress TLR3-Dependent Cytokine Expression in a TLR4-Dependent Manner

The release of endogenous molecules from the skin after injury has been proposed to influence inflammation. Recent studies have found that pro-inflammatory signals can be generated by damaged endogenous self-RNA, and this event is detected by TLR3. Conversely, release of endogenous fragments of hyaluronic acid (HA) after injury has been proposed to inhibit LPS induced inflammation driven by TLR4. In this study we investigated if HA oligomers could also influence inflammation mediated by TLR3. A tetramer form of HA (oligo-HA) was added to MH-S cells (mouse alveolar macrophage cell line) that were then activated by poly(I:C). ELISA analysis of culture supernatants showed that the presence of oligo-HA suppressed the poly(I:C) induced release of IL-6 and TNFα. IL-6 mRNA expression was also suppressed as measured by quantitative RT-PCR. To determine the mechanism of action for oligo-HA to inhibit poly(I:C), macrophages derived from wild-type (WT), Tlr2−/− or Tlr4−/− mice were treated with oligo-HA and poly(I:C). Similar to WT cells, Tlr2−/− macrophages were inhibited by oligo-HA and retained suppression of cytokine release. In contrast, Tlr4−/− macrophages lost the capacity to be suppressed by oligo-HA. An increase in Traf1 (TLR negative regulator) mRNA was observed after oligo-HA treatment of WT but not in Tlr4−/− macrophages, and oligo-HA did not suppress cytokine responsiveness in Traf1−/− macrophages. These results show that oligo-HA acts through TLR4 and TRAF1 to inhibit TLR3-dependent inflammation. This observation illustrates the complex immunomodulatory action of endogenous products released after injury.     

Role of TLR in B cell development: signaling through TLR4 promotes B cell maturation and is inhibited by TLR2

The role of TLR4 in mature B cell activation is well characterized. However, little is known about TLR4 role in B cell development. Here, we analyzed the effects of TLR4 and TLR2 agonists on B cell development using an in vitro model of B cell maturation. Highly purified B220(+)IgM(-) B cell precursors from normal C57BL/6 mouse were cultured for 72 h, and B cell maturation in the presence of the TLR agonists was evaluated by expression of IgM, IgD, CD23, and AA4. The addition of LPS or lipid A resulted in a marked increase in the percentage of CD23(+) B cells, while Pam3Cys had no effect alone, but inhibited the increase of CD23(+) B cell population induced by lipid A or LPS. The TLR4-induced expression of CD23 is not accompanied by full activation of the lymphocyte, as suggested by the absence of activation Ag CD69. Experiments with TLR2-knockout mice confirmed that the inhibitory effects of Pam3Cys depend on the expression of TLR2. We studied the effects of TLR-agonists on early steps of B cell differentiation by analyzing IL-7 responsiveness and phenotype of early B cell precursors: we found that both lipid A and Pam3Cys impaired IL-7-dependent proliferation; however, while lipid A up-regulates B220 surface marker, consistent with a more mature phenotype of the IgM(-) precursors, Pam3Cys keeps the precursors on a more immature stage. Taken together, our results suggest that TLR4 signaling favors B lymphocyte maturation, whereas TLR2 arrests/retards that process, ascribing new roles for TLRs in B cell physiology.     

The cardioprotection of the late phase of ischemic preconditioning is enhanced by postconditioning via a COX-2-mediated mechanism in conscious rats

The present study sought to determine whether the combination of late preconditioning (PC) with postconditioning enhances the reduction in infarct size. Chronically instrumented rats were assigned to a 45-min (subset 1) or 60-min (subset 2) coronary occlusion followed by 24 h of reperfusion. In each subset, rats received no further intervention (control) or were preconditioned 24 h before occlusion (PC), postconditioned at the onset of reperfusion following occlusion, or preconditioned and postconditioned without (PC + postconditioning) or with the COX-2 inhibitor celecoxib (3 mg/kg ip; PC + postconditioning + celecoxib) 10 min before postconditioning. Myocardial cyclooxygenase-2 (COX-2) protein expression and COX-2 activity (assessed as myocardial levels of PGE(2)) were measured 6 min after reperfusion in an additional five groups (control, PC, postconditioning, PC + postconditioning, and PC + postconditioning + celecoxib) subjected to a 45-min occlusion. PC alone reduced infarct size after a 45-min occlusion but not after a 60-min occlusion. Postconditioning alone did not reduce infarct size in either setting. However, the combination of late PC and postconditioning resulted in a robust infarct-sparing effect in both settings, suggesting additive cardioprotection. Celecoxib completely abrogated the infarct-sparing effect of the combined interventions in both settings. Late PC increased COX-2 protein expression and PGE(2) content. PGE(2) content (but not COX-2 protein) was further increased by the combination of both interventions, suggesting that postconditioning increases the activity of COX-2 induced by late PC. In conclusion, the combination of late PC and postconditioning produces additive protection, likely due to a postconditioning-induced enhancement of COX-2 activity.     

Role of TLR2- and TLR4-mediated signaling in Mycobacterium tuberculosis-induced macrophage death

Infection of macrophages with Mycobacterium tuberculosis (Mtb) induces cell death by apoptosis or necrosis. TLRs 2 and 4 recognition of mycobacterial ligands has been independently associated to apoptosis induction. To try to understand the particular contribution of these receptors to apoptotic or necrotic signaling upon infection with live Mtb H37Rv, we used macrophage lines derived from wild-type or TLR2-, TLR4-, and MyD88-deficient mouse strains. Mtb-infection triggered apoptosis depending on a TLR2/TLR4/MyD88/p38/ERK/PI-3K/NF-kB pathway; however, necrosis was favored in absence of TLR4 signaling independently of p38, ERK1/2, PI-3K or NF-κB activity. In conclusion, our results indicate that cooperation between TLR2- and TLR4-dependent mediated signals play a critical role in macrophage apoptosis induced by Mtb and the TLR4-mediated signaling has important role in the maintenance of the balance between apoptotic vs. necrotic cell death induced by macrophage infection with Mtb.     

Synergy and cross-tolerance between toll-like receptor (TLR) 2- and TLR4-mediated signaling pathways

A family of Toll-like receptor (TLR) mediates the cellular response to bacterial cell wall components; murine TLR2 and TLR4 recognize mycoplasmal lipopeptides (macrophage-activating lipopeptides, 2 kDa (MALP-2)) and LPS, respectively. Costimulation of mouse peritoneal macrophages with MALP-2 and LPS results in a marked increase in TNF-alpha production, showing the synergy between TLR2- and TLR4-mediated signaling pathways. Macrophages pretreated with LPS show hyporesponsiveness to the second LPS stimulation, termed LPS tolerance. The LPS tolerance has recently been shown to be primarily due to the down-regulation of surface expression of the TLR4-MD2 complex. When macrophages were treated with MALP-2, the cells showed hyporesponsiveness to the second MALP-2 stimulation, like LPS tolerance. Furthermore, macrophages pretreated with MALP-2 showed reduced production of TNF-alpha in response to LPS. LPS-induced activation of both NF-kappaB and c-Jun NH(2)-terminal kinase was severely impaired in MALP-2-pretreated cells. However, MALP-2-pretreated macrophages did not show any reduction in surface expression of the TLR4-MD2 complex. These findings indicate that LPS-induced LPS tolerance mainly occurs through the down-regulation of surface expression of the TLR4-MD2 complex; in contrast, MALP-2-induced LPS tolerance is due to modulation of the downstream cytoplasmic signaling pathways.     

Protection against myocardial ischemia/reperfusion injury in TLR4-deficient mice is mediated through a phosphoinositide 3-kinase-dependent mechanism

TLRs play a critical role in the induction of innate and adaptive immunity. However, TLRs have also been reported to mediate the pathophysiology of organ damage following ischemia/reperfusion (I/R) injury. We have reported that TLR4(-/-) mice show decreased myocardial injury following I/R; however, the protective mechanisms have not been elucidated. We examined the role of the PI3K/Akt signaling pathway in TLR4(-/-) cardioprotection following I/R injury. TLR4(-/-) and age-matched wild-type (WT) mice were subjected to myocardial ischemia for 45 min, followed by reperfusion for 4 h. Pharmacologic inhibitors of PI3K (wortmannin or LY294002) were administered 1 h before myocardial I/R. Myocardial infarct size/area at risk was reduced by 51.2% in TLR4(-/-) vs WT mice. Cardiac myocyte apoptosis was also increased in WT vs TLR4(-/-) mice following I/R. Pharmacologic blockade of PI3K abrogated myocardial protection in TLR4(-/-) mice following I/R. Specifically, heart infarct size/area at risk was increased by 98% in wortmannin and 101% in LY294002-treated TLR4(-/-) mice, when compared with control TLR4(-/-) mice. These data indicate that protection against myocardial I/R injury in TLR4(-/-) mice is mediated through a PI3K/Akt-dependent mechanism. The mechanisms by which PI3K/Akt are increased in the TLR4(-/-) myocardium may involve increased phosphorylation/inactivation of myocardial phosphatase and tensin homolog deleted on chromosome 10 as well as increased phosphorylation/inactivation of myocardial glycogen synthase kinase-3beta. These data implicate innate immune signaling pathways in the pathology of acute myocardial I/R injury. These data also suggest that modulation of TLR4/PI3K/Akt-dependent signaling pathways may be a viable strategy for reducing myocardial I/R injury.     

TLR2- and TLR4-mediated signals determine attenuation or augmentation of inflammation by acute alcohol in monocytes

Most pathogens express ligands for multiple TLRs that share common downstream signaling. In this study, we investigated the effects of acute alcohol on inflammatory pathways induced by TLR2 or TLR4 ligands and their combination. In human monocytes, alcohol attenuated TLR4- but not TLR2-induced TNF-alpha protein and mRNA levels and NF-kappaB activation. In contrast, acute alcohol augmented TNF-alpha production when both TLR2 and TLR4 ligands were present. IL-1R-associated kinase (IRAK)-1 activity was reduced by alcohol in TLR4, but it was augmented in TLR2- plus TLR4-stimulated cells. IRAK-monocyte, an inhibitor of IRAK-1, was induced in TLR4, but it was reduced in TLR2- plus TLR4-stimulated monocytes by alcohol. This was supported by decreased IRAK-1:TRAF6 association in TLR4 induced but sustained presence of IRAK-1:TRAF6 complexes in TLR2- plus TLR4-stimulated monocytes after alcohol treatment. Phosphorylation of MAPKs such as ERK1/2 was selectively inhibited by acute alcohol in TLR4-stimulated cells. In contrast, JNK phosphorylation as well as AP-1 nuclear binding were augmented by acute alcohol in the presence of combined TLR4 and TLR2 stimulation. Consistent with this result, the JNK inhibitor prevented alcohol-induced augmentation of TNF-alpha production. These results suggest that acute alcohol attenuates TLR4-induced inflammation via inhibition of IRAK-1 and ERK1/2 kinases and increases in IRAK-monocyte levels in monocytes. Conversely, in the presence of TLR2 and TLR4 ligands, acute alcohol augments inflammatory responses via IRAK-1 activation and JNK phosphorylation. Thus, the complexity of TLR-mediated signals may determine attenuation or augmentation of inflammatory responses by acute alcohol.     

TLR4-mediated survival of macrophages is MyD88 dependent and requires TNF-alpha autocrine signalling

Modulation of macrophage survival is a critical factor in the resolution of inflammatory responses. Exposure to LPS protects innate immune cells against apoptosis, although the precise pathways responsible for prolongation of macrophage survival remain to be fully established. The goal of this study was to characterize the mechanism of TLR4-mediated survival of murine bone marrow-derived macrophages upon M-CSF withdrawal in more detail. Using a combination of knockout mice and pharmacological inhibitors allowed us to show that TLR4 and TLR2 stimulation promotes long-term survival of macrophages in a MyD88-, PI3K-, ERK-, and NF-kappaB-dependent manner. LPS-induced long-term, but not short-term, survival requires autocrine signaling via TNF-alpha and is facilitated by a general cytoprotective program, similar to that mediated by M-CSF. TLR4-mediated macrophage survival is accompanied by a remarkable up-regulation of specific cell surface markers, suggesting that LPS stimulation leads to the differentiation of macrophages toward a mixed macrophage/dendritic cell-like phenotype.     

The Effects of Oral and Enteric Campylobacter concisus Strains on Expression of TLR4, MD-2, TLR2, TLR5 and COX-2 in HT-29 Cells

Campylobacter concisus, a Gram-negative bacterium that colonizes the human oral cavity, has been shown to be associated with inflammatory bowel diseases (IBD). The effects of different C. concisus strains on intestinal epithelial expression of Toll like receptors (TLR) have not been investigated. This study examined the effects of C. concisus strains isolated from patients with IBD and controls on expression of TLR4, its co-receptor myeloid differentiation factor (MD)-2; TLR2, TLR5, cyclooxygenase-2 (COX-2) and interleukin (IL)-8 in HT-29 cells.Fourteen oral and enteric C. concisus strains isolated from patients with IBD and healthy controls were co-incubated with HT-29 cells. Expression of TLR4, MD-2, TLR2, TLR5 and COX-2 in HT-29 cells in response to C. concisus infection was examined by Western blot, flow cytometry analysis and immunofluorescent staining visualized by confocal microscope. Production of IL-8 was evaluated by enzyme-linked immunosorbent assay.Both oral and enteric C. concisus strains upregulated expression of TLR4 in HT-29 cells. The levels of glycosylated TLR4 (Gly-TLR4) and surface TLR4 induced by C. concisus strains isolated from patients with IBD were significantly higher than those induced by C. concisus strains isolated from the healthy controls. Four C. concisus strains isolated from patients with IBD induced more than two-fold increase of surface expression of MD-2. C. concisus did not affect expression of TLR2 and TLR5. All C. concisus strains induced production of IL-8 and COX-2 in HT-29 cells.This study shows that some C. concisus strains, most from patients with IBD, upregulate surface expression of TLR4 and MD-2 in HT-29 cells. These data suggest that a potential role of specific C. concisus strains in modulating the intestinal epithelial responses to bacterial LPS needs to be investigated.     

T-cell glucocorticoid receptor is required to suppress COX-2-mediated lethal immune activation

Glucocorticoids, acting through the glucocorticoid receptor, potently modulate immune function and are a mainstay of therapy for treatment of inflammatory conditions, autoimmune diseases, leukemias and lymphomas. Moreover, removal of systemic glucocorticoids, by adrenalectomy in animal models or adrenal insufficiency in humans, has shown that endogenous glucocorticoid production is required for regulation of physiologic immune responses. These effects have been attributed to suppression of cytokines, although the crucial cellular and molecular targets remain unknown. In addition, considerable controversy remains as to whether glucocorticoids are required for thymocyte development. To assess the role of the glucocorticoid receptor in immune system development and function, we generated T-cell-specific glucocorticoid receptor knockout mice. Here we show that the T-cell is a critical cellular target of glucocorticoid receptor signaling, as immune activation in these mice resulted in significant mortality. This lethal activation is rescued by cyclooxygenase-2 (COX-2) inhibition but not steroid administration or cytokine neutralization. These studies indicate that glucocorticoid receptor suppression of COX-2 is crucial for curtailing lethal immune activation, and suggest new therapeutic approaches for regulation of T-cell-mediated inflammatory diseases.     

Procyanidin dimer B2-mediated IRAK-M induction negatively regulates TLR4 signaling in macrophages

Polyphenolic compounds have been found to possess a wide range of physiological activities that may contribute to their beneficial effects against inflammation-related diseases; however, the molecular mechanisms underlying this anti-inflammatory activity are not completely characterized, and many features remain to be elucidated. In this study, we investigated the molecular basis for the down-regulation of toll-like receptor 4 (TLR4) signal transduction by procyanidin dimer B2 (Pro B2) in macrophages. Pro B2 markedly elevated the expression of the interleukin (IL)-1 receptor-associated kinase (IRAK)-M protein, a negative regulator of TLR signaling. Lipopolysaccharide (LPS)-induced expression of cell surface molecules (CD80, CD86, and MHC class I/II) and production of pro-inflammatory cytokines (tumor necrosis factor-α, IL-1β, IL-6, and IL-12p70) were inhibited by Pro B2, and this action was prevented by IRAK-M silencing. In addition, Pro B2-treated macrophages inhibited LPS-induced activation of mitogen-activated protein kinases such as extracellular signal-regulated kinase 1/2, p38, and c-Jun N-terminal kinase and the translocation of nuclear factor κB and p65 through IRAK-M. We also found that Pro B2-treated macrophages inactivated naïve T cells by inhibiting LPS-induced interferon-γ and IL-2 secretion through IRAK-M. These novel findings provide new insights into the understanding of negative regulatory mechanisms of the TLR4 signaling pathway and the immune-pharmacological role of Pro B2 in the immune response against the development and progression of many chronic diseases.     

Interaction of soluble form of recombinant extracellular TLR4 domain with MD-2 enables lipopolysaccharide binding and attenuates TLR4-mediated signaling

TLRs have been implicated in recognition of pathogen-associated molecular patterns. TLR4 is a signaling receptor for LPS, but requires MD-2 to respond efficiently to LPS. The purposes of this study were to examine the interactions of the extracellular TLR4 domain with MD-2 and LPS. We generated soluble forms of rTLR4 (sTLR4) and TLR2 (sTLR2) lacking the putative intracellular and transmembrane domains. sTLR4 consisted of Glu(24)-Lys(631). MD-2 bound to sTLR4, but not to sTLR2 or soluble CD14. BIAcore analysis demonstrated the direct binding of sTLR4 to MD-2 with a dissociation constant of K(D) = 6.29 x 10(-8) M. LPS-conjugated beads precipitated MD-2, but not sTLR4. However, LPS beads coprecipitated sTLR4 and MD-2 when both proteins were coincubated. The addition of sTLR4 to the medium containing the MD-2 protein significantly attenuated LPS-induced NF-kappaB activation and IL-8 secretion in wild-type TLR4-expressing cells. These results indicate that the extracellular TLR4 domain-MD-2 complex is capable of binding LPS, and that the extracellular TLR4 domain consisting of Glu(24)-Lys(631) enables MD-2 binding and LPS recognition to TLR4. In addition, the use of sTLR4 may lead to a new therapeutic strategy for dampening endotoxin-induced inflammation.     

Extracellular histones are mediators of death through TLR2 and TLR4 in mouse fatal liver injury

We previously reported that extracellular histones are major mediators of death in sepsis. Infusion of extracellular histones leads to increased cytokine levels. Histones activate TLR2 and TLR4 in a process that is enhanced by binding to DNA. Activation of TLR4 is responsible for the histone-dependent increase in cytokine levels. To study the impact of histone release on pathology we used two models: a Con A-triggered activation of T cells to mimic sterile inflammation, and acetaminophen to model drug-induced tissue toxicity. Histones were released in both models and anti-histone Abs were protective. TLR2- or TLR4-null mice were also protected. These studies imply that histone release contributes to death in inflammatory injury and in chemical-induced cellular injury, both of which are mediated in part through the TLRs.     

Mastoparan, a G protein agonist peptide, differentially modulates TLR4- and TLR2-mediated signaling in human endothelial cells and murine macrophages

Previous studies have implicated a role for heterotrimeric G protein-coupled signaling in B cells, monocytes, and macrophages stimulated with LPS and have shown that G proteins coimmunoprecipitate with membrane-bound CD14. In this study, we have extended these observations in human dermal microvessel endothelial cells (HMEC) that lack membrane-bound CD14 and in murine macrophages to define further the role of heterotrimeric G proteins in TLR signaling. Using the wasp venom-derived peptide, mastoparan, to disrupt G protein-coupled signaling, we identified a G protein-dependent signaling pathway in HMEC stimulated with TLR4 agonists that is necessary for the activation of p38 phosphorylation and kinase activity, NF-kappaB and IL-6 transactivation, and IL-6 secretion. In contrast, HMEC activation by TLR2 agonists, TNF-alpha, or IL-1beta was insensitive to mastoparan. In the murine macrophage cell line, RAW 264.7, and in primary murine macrophages, G protein dysregulation by mastoparan resulted in significant inhibition of LPS-induced signaling leading to both MyD88-dependent and MyD88-independent gene expression, while TLR2-mediated gene expression was not significantly inhibited. In addition to inhibition of TLR4-mediated MAPK phosphorylation in macrophages, mastoparan blunted IL-1R-associated kinase-1 kinase activity induced by LPS, but not by TLR2 agonists, yet failed to affect phosphorylation of Akt by phosphoinositol-3-kinase induced by either TLR2- or TLR4-mediated signaling. These data confirm the importance of heterotrimeric G proteins in TLR4-mediated responses in cells that use either soluble or membrane-associated CD14 and reveal a level of TLR and signaling pathway specificity not previously appreciated.     

Hsp72 induces inflammation and regulates cytokine production in airway epithelium through a TLR4- and NF-kappaB-dependent mechanism

Heat shock proteins are generally regarded as intracellular proteins acting as molecular chaperones; however, Hsp72 is also detected in the extracellular compartment. Hsp72 has been identified in the bronchoalveolar lavage fluid (BALF) of patients with acute lung injury. To address whether Hsp72 directly activated airway epithelium, human bronchial epithelial cells (16HBE14o-) were treated with recombinant Hsp72. Hsp72 induced a dose-dependent increase in IL-8 expression, which was inhibited by the NF-kappaB inhibitor parthenolide. Hsp72 induced activation of NF-kappaB, as evidenced by NF-kappaB trans-activation and by p65 RelA and p50 NF-kappaB1 binding to DNA. Endotoxin contamination of the Hsp72 preparation was not responsible for these effects. Next, BALB/c mice were challenged with a single intratracheal inhalation of Hsp72 and killed 4 h later. Hsp72 induced significant up-regulation of KC, TNF-alpha, neutrophil recruitment, and myeloperoxidase in the BALF. A similar challenge with Hsp72 in TLR4 mutant mice did not stimulate the inflammatory response, stressing the importance of TLR4 in Hsp72-mediated lung inflammation. Last, cultured mouse tracheal epithelial cells (MTEC) from BALB/c and TLR4 mutant and wild-type mice were treated ex vivo with Hsp72. Hsp72 induced a significant increase in KC expression from BALB/c and wild-type MTEC in an NF-kappaB-dependent manner; however, TLR4 mutant MTEC had minimal cytokine release. Taken together, these data suggest that Hsp72 is released and biologically active in the BALF and can regulate airway epithelial cell cytokine expression in a TLR4 and NF-kappaB-dependent mechanism.     

Type II arabinogalactan from Anoectochilus formosanus induced dendritic cell maturation through TLR2 and TLR4

BackgroundPolysaccharides, considered as immunomodulators with the capacity to activate immunity against microbial pathogens and tumors, have been employed for their dietary and medical benefits.PurposeThis study investigated the immunomodulatory effect of polysaccharide such as type II arabinogalactan from Anoectochilus formosanus (AGAF) on dendritic cell (DC) maturation and the underlying molecular mechanisms.Methods and resultsExposing DCs to AGAF induces cell maturation, which is characterized by the upregulation of CD86, CD83, CD80, CD40, and MHC class I and class II expression through flow cytometry analysis and morphological change without cytotoxicity. In addition, AGAF-triggered DC2.4 cells were involved in priming T-cell activation in vitro and in vivo. Transfection of toll-like receptor (TLR) 2 proteins and TLR4 siRNA suppressed DC maturation, suggesting that AGAF induced DC maturation through TLR2 and TLR4.ConclusionThese findings indicate that AGAF may be a potentially effective immunomodulator in stimulating DC maturation.     

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.