Diverse effects of bacterial cell wall components on mast cell degranulation,cysteinyl leukotriene generation and migration

Nowadays there is more and more evidence that mast cells take part in antibacterial defence. Mast cells have the ability to kill bacteria via phagocytose‐dependent or phagocytose‐independent ways and express antimicrobial peptides that can directly kill pathogens at their site of entry. What is more, mast cells are capable of processing bacterial antigens for presentation through class I and II MHC molecules. Some data indicate that these cells can release various proinflammatory mediators in response to activation with bacteria and/or their products, however this information is still far from complete. Therefore, in this study we examined the ability of PGN from Staphylococcus aureus, LPS from Eschericha coli and LAM from Mycobacterium smegmatis to stimulate mature rat mast cell degranulation as well as cysteinyl LT generation. We also studied the influence of these bacterial components on mast cell migration. We found that PGN, LPS and LAM all failed to induce mast cell degranulation and histamine release. At the same time, activation of mast cells with these bacterial antigens resulted in generation and release of significant amounts of LT. Moreover, we documented that, even in the presence of laminin, none of the bacterial antigens used stimulated mast cell migration. However, PGN did induce migration of RANTES‐primed mast cells, and LPS did stimulate mast cell migratory response after priming with IL‐6. Our results show that PGN, LPS and LAM might be among the important bacterial antigens involved in mast cell activation during bacterial infection.     

The role of TLR2 in vivo following challenge with Staphylococcus aureus and prototypic ligands

Based on a wealth of in vitro macrophage studies, immunity to Staphylococcus aureus cell wall-derived peptidoglycan (PGN) and lipoteichoic acid has been attributed to TLR2. We investigated whether the in vitro paradigm of TLR2 dominance would hold true in vivo. Using an experimental peritonitis model, we challenged mice with PGN or lipoteichoic acid and found that only PGN resulted in significant leukocyte (primarily neutrophil) accumulation in the peritoneum at 4 h. PGN-mediated leukocyte recruitment was P-/E-selectin dependent but only partially TLR2 dependent, and also involved the C5aR. Concomitant inhibition of TLR2 and C5aR resulted in a further reduction in PGN-induced peritonitis. Peritoneal neutrophilia was partially mast cell dependent; however, the defect could not be reconstituted with TLR2(-/-) or C5aR(-/-) mast cells. Interestingly, macrophage-deficient mice did not have defective neutrophil recruitment. By 24 h, the response to PGN involved primarily monocytes and was TLR2 and C5aR independent. Finally, we challenged mice with live S. aureus and found a similar degree of TLR2 involvement in leukocyte recruitment to that observed with PGN. Most importantly, bacterial clearance from the spleen and peritoneum was not altered in TLR2(-/-) mice vs wild-type mice. Morbidity was only significantly increased in S. aureus-infected mice treated with a blocking Fab against C5aR. Taken together, these studies indicate that in vivo responses to prototypic TLR2 ligands do not necessarily recapitulate the absolute necessity for TLR2 observed in vitro, and additional receptors contribute, in a significant manner, to PGN and S. aureus-mediated immune responses.     

MD-2 is required for the full responsiveness of mast cells to LPS but not to PGN

To address the role played by MD-2 in mast cell recognition of LPS, we examined bone marrow-derived mast cells (BMMCs) from MD-2 gene-targeted mice. BMMCs from MD-2-/- mice showed impaired cytokine production (TNF-alpha, IL-6, IL-13, and IL-1beta) in response to LPS from Escherichia coli, but not to peptidoglycan (PGN) from Staphylococcus aureus. In a mast cell-dependent acute septic model, MD-2 deficiency of mast cell resulted in significantly higher mortality due to defective neutrophil recruitment and the production of cytokines in the peritoneal cavity, which was similar to mice with TLR4-deficient mast cells. The TLR2-dependent activation of skin mast cells by PGN was not altered by the absence of MD-2 in vivo. Collectively, MD-2 is essential for the recognition of LPS by TLR4 but not for that of PGN by TLR2 of mast cells.     

Toll-like receptor 2 (TLR2)-dependent-positive and TLR2-independent-negative regulation of proinflammatory cytokines by mycobacterial lipomannans

Lipoarabinomannans (LAM) and lipomannans (LM) are integral parts of the mycobacterial cell wall recognized by cells involved in the innate immune response and have been found to modulate the cytokine response. Typically, mannosylated LAM from pathogenic mycobacteria have been reported to be anti-inflammatory, whereas phosphoinositol-substituted LAM from nonpathogenic species are proinflammatory molecules. In this study, we show that LM from several mycobacterial species, including Mycobacterium chelonae, Mycobacterium kansasii, and Mycobacterium bovis bacillus Calmette-Guérin, display a dual function by stimulating or inhibiting proinflammatory cytokine synthesis through different pathways in murine primary macrophages. LM, but none of the corresponding LAM, induce macrophage activation characterized by cell surface expression of CD40 and CD86 and by TNF and NO secretion. This activation is dependent on the presence of Toll-like receptor (TLR) 2 and mediated through the adaptor protein myeloid differentiation factor 88 (MyD88), but independent of either TLR4 or TLR6 recognition. Surprisingly, LM exerted also a potent inhibitory effect on TNF, IL-12p40, and NO production by LPS-activated macrophages. This TLR2-, TLR6-, and MyD88-independent inhibitory effect is also mediated by LAM from M. bovis bacillus Calmette-Guérin but not by LAM derived from M. chelonae and M. kansasii. This study provides evidence that mycobacterial LM bear structural motifs susceptible to interact with different pattern recognition receptors with pro- or anti-inflammatory effects. Thus, the ultimate response of the host may therefore depend on the prevailing LM or LAM in the mycobacterial envelope and the local host cell receptor availability.     

Differential Effects of the Toll-Like Receptor 2 Agonists,PGN and Pam3CSK4 on Anti-IgE Induced Human Mast Cell Activation

Mast cells are pivotal in the pathogenesis of allergy and inflammation. In addition to the classical IgE-dependent mechanism involving crosslinking of the high-affinity receptor for IgE (FcεRI), mast cells are also activated by Toll-like receptors (TLRs) which are at the center of innate immunity. In this study, we demonstrated that the response of LAD2 cells (a human mast cell line) to anti-IgE was altered in the presence of the TLR2 agonists peptidoglycan (PGN) and tripalmitoyl-S-glycero-Cys-(Lys)4 (Pam3CSK4). Pretreatment of PGN and Pam3CSK4 inhibited anti-IgE induced calcium mobilization and degranulation without down-regulation of FcεRI expression. Pam3CSK4 but not PGN acted in synergy with anti-IgE for IL-8 release when the TLR2 agonist was added simultaneously with anti-IgE. Studies with inhibitors of key enzymes implicated in mast cell signaling revealed that the synergistic release of IL-8 induced by Pam3CSK4 and anti-IgE involved ERK and calcineurin signaling cascades. The differential modulations of anti-IgE induced mast cell activation by PGN and Pam3CSK4 suggest that dimerization of TLR2 with TLR1 or TLR6 produced different modulating actions on FcεRI mediated human mast cell activation.     

Staphylococcal peptidoglycan co-localizes with Nod2 and TLR2 and activates innate immune response via both receptors in primary murine keratinocytes

In mammalian host cells staphylococcal peptidoglycan (PGN) is recognized by Nod2. Whether PGN is also recognized by TLR2 is disputed. Here we carried out PGN co-localization and stimulation studies with TLR2 and Nod2 in wild type and mutant host cells. To exclude contamination with lipoproteins, polymeric staphylococcal PGN (PGN(pol)) was isolated from Staphylococcus aureus Δlgt (lacking lipidated prelipoproteins). PGN(pol) was biotinylated (PGN-Bio) for fluorescence monitoring with specific antibodies. Keratinocytes from murine oral epithelium (MK) readily internalized PGN-Bio in an endocytosis-like process. In wt MK, PGN(pol) induced intracellular accumulation of Nod2 and TLR2 and co-localized with Nod2 and TLR2, but not with TLR4. In TLR2-deficient MK Nod2 and in Nod2-deficient MK TLR2 was induced, indicating that PGN(pol) recognition by Nod2 is independent of TLR2 and vice versa. In both mutants IL-6 and IL-1B release was decreased by approximately 50% compared to wt MK, suggesting that the immune responses induced by Nod2 and TLR2 are comparable and that the two receptors act additively in MK. In TLR2-transfected HEK293 cells PGN(pol) induced NFkB-promoter fused luciferase expression. To support the data, co-localization and signaling studies were carried out with SHL-PGN, a lipase protein covalently tethered to PGN-fragments of varying sizes at its C-terminus. SHL-PGN also co-localized with Nod2 or TLR2 and induced their accumulation, while SHL without PGN did not. The results show that staphylococcal PGN not only co-localizes with Nod2 but also with TLR2. PGN is able to stimulate the immune system via both receptors.     

Mast cells have a pivotal role in TNF-independent lymph node hypertrophy and the mobilization of Langerhans cells in response to bacterial peptidoglycan

Peptidoglycan (PGN) from Gram-positive bacteria, activates multiple immune effector cells. PGN-induced lymph node (LN) hypertrophy and dendritic cell mobilization in vivo were investigated following PGN injection into the skin. Both LN activation and the migration of Langerhans cells (LCs) to draining LNs were dependent on the presence of mast cells as demonstrated using mast cell deficient W/W(v) mice. However, these responses did not require TLR2, TLR4, or MYD88. TNF-deficient mice exhibited normal increases in LN cellularity but significantly reduced LC migration. In contrast, responses to IgE-mediated mast cell activation were highly TNF dependent. Complement component C3-deficient mice showed decreased LN hypertrophy and abrogated LC migration in response to PGN. These data demonstrate a critical role for mast cells and complement in LN responses to PGN and illustrate a novel TNF-independent mechanism whereby mast cells participate in the initiation of immunity.     

Deficiency in TLR2 but not in TLR4 impairs dendritic cells derived IL-10 responses to schistosome antigens

The purpose of this study was to observe the diverse functions of Toll-like receptors (TLRs) in responses to specific schistosome antigens. Bone marrow-derived dendritic cells (BMDCs) from TLR2-deficient (TLR2(-/-)) or TLR4-deficient (TLR4(-/-)) mice were activated with soluble schistosomule antigen (SSA) or soluble egg antigen (SEA). TLR2 mRNA expression was significantly increased in B6 BMDCs following SEA stimulation. TLR2-deficient BMDCs showed enhanced MHCII expression following SSA and SEA stimulation. TLR2-deficient but not TLR4-deficient BMDC failed to produce IL-12p70 and IL-10 in response to schistosome antigens. TLR2-deficient BMDCs induced a stronger CD4(+) T cell proliferative response. IL-4 and IL-10 expression was inhibited in CD4(+) T cells primed with TLR2-deficient BMDCs, while enhanced in TLR4-deficient BMDCs-primed CD4(+) T cells. These results suggest that TLR2 is essential for the establishment of the DC production of IL-12p70 and IL-10.     

TLR3-, TLR7-, and TLR9-mediated production of proinflammatory cytokines and chemokines from murine connective tissue type skin-derived mast cells but not from bone marrow-derived mast cells

Recent studies have revealed that murine bone marrow-derived cultured mast cells (BMMC), which are phenotypically immature mast cells, express functional TLR2 and TLR4 that recognize distinct pathogen-associated molecules. However, it remains relatively uncertain whether mast cells express other TLR. We recently established a method to obtain large numbers of murine fetal skin-derived cultured mast cells (FSMC); these cells exhibit important features of connective tissue type mast cells. Working with FSMC and BMMC, the TLR mRNA expression profiles were compared between both cell types. Although TLR2 and TLR4 mRNA were detected in both cells at comparable levels, TLR3, TLR7, and TLR9 mRNA were expressed by FSMC at higher levels than by BMMC, suggesting distinct TLR expression profiles among different mast cell populations. With respect to their functional aspects, FSMC, but not BMMC, dose dependently produced proinflammatory cytokines (TNF-alpha and IL-6) and chemokines (RANTES, MIP-1alpha, and MIP-2) in response to poly(I:C), R-848, and CpG oligodeoxynucleotide, which are TLR3, TLR7, and TLR9 activators, respectively. Interestingly, these TLR activators failed to induce degranulation and IL-13 production by both mast cells, although peptidoglycan and LPS (TLR2 and TLR4 activators, respectively) induced IL-13 production by both cells. Mast cells, thus, may have potential to recruit other immune cells to the infected sites by responding to various bacterial and viral components through TLR signaling pathways, presumably being involved in initiating innate immunity and subsequently linking innate and acquired immune responses.     

Mast cell TLR2 signaling is crucial for effective killing of Francisella tularensis

TLR signaling is critical for early host defense against pathogens, but the contributions of mast cell TLR-mediated mechanisms and subsequent effector functions during pulmonary infection are largely unknown. We have previously demonstrated that mast cells, through the production of IL-4, effectively control Francisella tularensis replication. In this study, the highly human virulent strain of F. tularensis SCHU S4 and the live vaccine strain were used to investigate the contribution of mast cell/TLR regulation of Francisella. Mast cells required TLR2 for effective bacterial killing, regulation of the hydrolytic enzyme cathepsin L, and for coordination and trafficking of MHC class II and lysosomal-associated membrane protein 2. Infected TLR2(-/-) mast cells, in contrast to wild-type and TLR4(-/-) cells, lacked detectable IL-4 and displayed increased cell death with a 2-3 log increase of F. tularensis replication, but could be rescued with rIL-4 treatment. Importantly, MHC class II and lysosomal-associated membrane protein 2 localization with labeled F. tularensis in the lungs was greater in wild-type than in TLR2(-/-) mice. These results provide evidence for the important effector contribution of mast cells and TLR2-mediated signaling on early innate processes in the lung following pulmonary F. tularensis infection and provide additional insight into possible mechanisms by which intracellular pathogens modulate respiratory immune defenses.     

Cutting edge: TLR2-mediated proinflammatory cytokine and chemokine production by microglial cells in response to herpes simplex virus

Recent studies indicate that TLRs are critical in generating innate immune responses during infection with HSV-1. In this study, we investigated the role of TLR2 signaling in regulating the production of neuroimmune mediators by examining cytokine and chemokine expression using primary microglial cells obtained from TLR2-/- as well as wild-type mice. Data presented here demonstrate that TLR2 signaling is required for the production of proinflammatory cytokines and chemokines: TNF-alpha, IL-1beta, IL-6, IL-12, CCL7, CCL8, CCL9, CXCL1, CXCL2, CXCL4, and CXCL5. CXCL9 and CXCL10 were also induced by HSV, but their production was not dependent upon TLR2 signaling. Because TLR2-/- mice display significantly reduced mortality and diminished neuroinflammation in response to brain infection with HSV, the TLR2-dependent cytokines identified here might function as key players influencing viral neuropathogenesis.     

Cutting edge: distinct Toll-like receptor 2 activators selectively induce different classes of mediator production from human mast cells

Mast cells play a critical role in host defense against bacterial infection. Murine mast cells produce cytokines in response to bacterial peptidoglycan and LPS via Toll-like receptor (TLR) TLR2- and TLR4-dependent mechanisms. The expression of TLRs by human mast cells and responses to known TLR activators was examined. Human mast cells expressed mRNA for TLR1, TLR2, and TLR6 but not TLR4. Bacterial peptidoglycan and yeast zymosan were potent inducers of GM-CSF and IL-1beta and also induced substantial short-term cysteinyl leukotriene generation. In contrast, a synthetic triacylated lipopeptide induced short-term degranulation but failed to induce cysteinyl leukotriene production. The TLR4 activator Escherichia coli LPS did not induce a GM-CSF, IL-1beta leukotriene, or degranulation response. These data demonstrate highly selective production of different classes of mast cell mediators in response to distinct TLR activators of potential importance to the host response to bacterial or fungal pathogens.     

Bruton's tyrosine kinase is required for TLR2 and TLR4-induced TNF, but not IL-6, production

Bruton's tyrosine kinase (Btk), the gene mutated in the human immunodeficiency X-linked agammaglobulinemia, is activated by LPS and is required for LPS-induced TNF production. In this study, we have investigated the role of Btk both in signaling via another TLR (TLR2) and in the production of other proinflammatory cytokines such as IL-1beta, IL-6, and IL-8. Our data show that in X-linked agammaglobulinemia PBMCs, stimulation with TLR4 (LPS) or TLR2 (N-palmitoyl-S-[2, 3-bis(palmitoyloxy)-(2R)-propyl]-(R)-cysteine) ligands produces significantly less TNF and IL-1beta than in normal controls. In contrast, a lack of Btk has no impact on the production of IL-6, IL-8, or the anti-inflammatory cytokine, IL-10. Our previous data suggested that Btk lies within a p38-dependent pathway that stabilizes TNF mRNA. Accordingly, TaqMan quantitative PCR analysis of actinomycin D time courses presented in this work shows that overexpression of Btk is able to stabilize TNF, but not IL-6 mRNA. Furthermore, using the p38 inhibitor SB203580, we show that the TLR4-induced production of TNF, but not IL-6, requires the activity of p38 MAPK. These data provide evidence for a common requirement for Btk in TLR2- and TLR4-mediated induction of two important proinflammatory cytokines, TNF and IL-1beta, and reveal important differences in the TLR-mediated signals required for the production of IL-6, IL-8, and IL-10.     

Synergistic augmentation of inflammatory cytokine productions from murine mast cells by monomeric IgE and toll-like receptor ligands

Simultaneous activation of murine mast cells by monomeric IgE and toll-like receptor (TLR) ligands was examined. Inflammatory cytokine production elicited by the binding of IgE in the absence of antigen, was further enhanced by the addition of lipopolysaccharide (LPS) or peptidoglycan (PGN). Enhancement by LPS or PGN on cytokine production was mediated by TLR4 and TLR2, respectively, since TLR4- and TLR2-deficient mast cells did not show synergistic activation by monomeric IgE and LPS/PGN. Synergistic activation of mast cells was obtained via phosphorylation of several mitogen-activated protein kinases (MAPK). Furthermore, MAPK inhibitors, significantly attenuated the augmentation of inflammatory cytokine production by monomeric IgE and LPS or PGN. Altogether, these results suggest that simultaneous TLR activation of mast cells with IgE molecules, particularly highly cytokinergic (HC) IgE, might contribute to the exacerbation of allergic diseases associated with infection even in the absence of a specific antigen.     

SCF and TLR4 ligand cooperate to augment the tumor-promoting potential of mast cells

Mast cells may have either antitumor or tumor-promoting potential. Nevertheless, mast cells in tumor microenvironment have been found to promote tumor growth. So far the mechanisms underlying the modulation of mast cell function in tumor microenvironment remains to be fully elucidated. Here, we report that tumor-promoting potential of mast cells could be augmented by molecules released from damaged tumor cells through cooperative stimulation of stem cell factor (SCF) and ligand for Toll-like receptor 4 (TLR4). Co-simulation with SCF and TLR4 ligand inhibited mast cell degranulation, but efficiently induced the production and secretion of VEGF, PDGF, and IL-10. Although TLR4 ligand alone may induce IL-12 expression in mast cells, co-stimulation with SCF and TLR4 ligand induced the expression of IL-10, but not IL-12, in mast cells. The phosphorylation of GSK3β was crucial for the effect of SCF and TLR4 ligand. In addition to inducing phosphorylation of GSK3β at Ser9 through PI3K pathway, SCF and TLR4 ligand cooperated to induce phosphorylation of GSK3β at Tyr216 by simultaneous activation of ERK and p38MAPK pathways. Both phospho-Ser9 and phospho-Tyr216 of GSK3β were required for IL-10 expression induced by SCF/TLR4 ligand, whereas suppressive effect of SCF/TLR4 ligand on mast cell degranulation was related to phospho-Tyr216. Importantly, the effect of SCF and TLR4 ligand on mast cells could be abrogated by inhibiting phosphorylation of GSK3β at Tyr216. These findings disclose the mechanisms underlying the modulation of mast cell function in tumor microenvironment, and suggest that inhibiting GSK3β in mast cells will be beneficial to the treatment of cancer.     

Gingipains inactivate a cell surface ligand on Porphyromonas gingivalis that induces TLR2-and TLR4-independent signaling

Arginine-specific gingipain and lysine-specific gingipain are two major cysteine proteinases produced by Porphyromonas gingivalis. To clarify the role of gingipains in the interaction between P. gingivalis and the innate immune system, CHO reporter cells expressing TLR2 or TLR4 were stimulated with wildtype or gingipain-deficient P. gingivalis cells and activation of nuclear factor-kappaB in these cells was examined. While CHO/CD14 cells and 7.19 cells, an MD-2-defective mutant derived from CHO/CD14 cells, failed to respond to wild-type P. gingivalis, they responded to gingipain-deficient P. gingivalis. On the other hand, CHO/CD14/TLR2 cells responded to both wild-type and gingipain-deficient P. gingivalis. These results suggested that gingipains have no effects on TLR2-dependent signaling from P. gingivalis but have inhibitory effects on TLR2-and TLR4-independent signaling in CHO cells. Indeed, the activity of gingipain-deficient P. gingivalis to induce the activation of 7.19 cells was diminished after treatment of the bacterial cells with gingipains. We next partially purified bacterial cell components activating 7.19 cells from gingipain-deficient P. gingivalis. The activity of the partially purified components was diminished by treatment with heat or gingipains. It is also noteworthy that anti-CD14 mAb inhibited the activation of 7.19 cells induced by the partially purified components. These results indicated that the components of P. gingivalis that were able to induce TLR2-and TLR4-independent signaling were inactivated by gingipains before being recognized by CD14. The inactivation of the components would be helpful for P. gingivalis to escape from the innate immune system.     

TLR4 signaling via MyD88 and TRIF differentially shape the CD4+ T cell response to Porphyromonas gingivalis hemagglutinin B

Recombinant hemagglutinin B (rHagB), a virulence factor of the periodontal pathogen Porphyromonas gingivalis, has been shown to induce protective immunity against bacterial infection. Furthermore, we have demonstrated that rHagB is a TLR4 agonist for dendritic cells. However, it is not known how rHagB dendritic cell stimulation affects the activation and differentiation of T cells. Therefore, we undertook the present study to examine the role of TLR4 signaling in shaping the CD4(+) T cell response following immunization of mice with rHagB. Immunization with this Ag resulted in the induction of specific CD4(+) T cells and Ab responses. In TLR4(-/-) and MyD88(-/-) but not Toll/IL-1R domain-containing adapter inducing IFN-β-deficient (TRIF(Lps2)) mice, there was an increase in the Th2 CD4(+) T cell subset, a decrease in the Th1 subset, and higher serum IgG(1)/IgG(2) levels of HagB-specific Abs compared with those in wild-type mice. These finding were accompanied by increased GATA-3 and Foxp3 expression and a decrease in the activation of CD4(+) T cells isolated from TLR4(-/-) and MyD88(-/-) mice. Interestingly, TLR4(-/-) CD4(+) T cells showed an increase in IL-2/STAT5 signaling. Whereas TRIF deficiency had minimal effects on the CD4(+) T cell response, it resulted in increased IFN-γ and IL-17 production by memory CD4(+) T cells. To our knowledge, these results demonstrate for the first time that TLR4 signaling, via the downstream MyD88 and TRIF molecules, exerts a differential regulation on the CD4(+) T cell response to HagB Ag. The gained insight from the present work will aid in designing better therapeutic strategies against P. gingivalis infection.