A20 Is Critical for the Induction of Pam3CSK4-Tolerance in Monocytic THP-1 Cells

A20 functions to terminate Toll-like receptor (TLR)-induced immune response, and play important roles in the induction of lipopolysacchride (LPS)-tolerance. However, the molecular mechanism for Pam3CSK4-tolerance is uncertain. Here we report that TLR1/2 ligand Pam3CSK4 induced tolerance in monocytic THP-1 cells. The pre-treatment of THP-1 cells with Pam3CSK4 down-regulated the induction of pro-inflammatory cytokines induced by Pam3CSK4 re-stimulation. Pam3CSK4 pre-treatment also down-regulated the signaling transduction of JNK, p38 and NF-κB induced by Pam3CSK4 re-stimulation. The activation of TLR1/2 induced a rapid and robust up-regulation of A20, suggesting that A20 may contribute to the induction of Pam3CSK4-tolerance. This hypothesis was proved by the observation that the over-expression of A20 by gene transfer down-regulated Pam3CSK4-induced inflammatory responses, and the down-regulation of A20 by RNA interference inhibited the induction of tolerance. Moreover, LPS induced a significant up-regulation of A20, which contributed to the induction of cross-tolerance between LPS and Pam3CSK4. A20 was also induced by the treatment of THP-1 cells with TNF-α and IL-1β. The pre-treatment with TNF-α and IL-1β partly down-regulated Pam3CSK4-induced activation of MAPKs. Furthermore, pharmacologic inhibition of GSK3 signaling down-regulated Pam3CSK4-induced A20 expression, up-regulated Pam3CSK4-induced inflammatory responses, and partly reversed Pam3CSK4 pre-treatment-induced tolerance, suggesting that GSK3 is involved in TLR1/2-induced tolerance by up-regulation of A20 expression. Taken together, these results indicated that A20 is a critical regulator for TLR1/2-induced pro-inflammatory responses.     

Failure of mycoplasma lipoprotein MALP-2 to induce NK cell activation through dendritic cell TLR2

Macrophage-activating lipopeptide 2 (MALP-2), a mycoplasmal diacylated lipopeptide with palmitic acid moiety (Pam2), activates Toll-like receptor (TLR) 2 to induce inflammatory cytokines. TLR2 is known to mature myeloid dendritic cells (mDC) to drive mDC contact-mediated natural killer (NK) cell activation. Here we tested if MALP-2 activates NK cells through stimulation of TLR2 on mDC. Although synthetic MALP-2 with 6 or 14 amino acids (a.a.) stretch (designated as s and f) matured mDC to induce IL-6, IL-12p40 and TNF-α to a similar extent, they far less activated NK cells than Pam2CSK4, a positive control of 6 a.a.-containing diacyl lipopeptide. MALP-2s and f were TLR2/6 agonists and activate the MyD88 pathway similar to Pam2CSK4, but MALP-2s having the CGNNDE sequence acted on mDC TLR2 to barely induce external NK activation. Even the s form, with slightly high induction of IL-6 compared to the f form, barely induced in vivo growth retardation of NK-sensitive implant tumor. Pam2CSK4 and MALP-2 have the common lipid moiety but different peptides, which are crucial for NK cell activation. The results infer that MALP-2 is applicable to a cytokine inducer but not to an adjuvant for antitumor NK immunotherapy.     

IFN regulatory factor 8 restricts the size of the marginal zone and follicular B cell pools

This study examined the effect of TLR2 activation by its specific ligand, Pam3CSK4, on cerebral ischemia/reperfusion (I/R) injury. Mice (n = 8/group) were treated with Pam3CSK4 1 h before cerebral ischemia (60 min), followed by reperfusion (24 h). Pam3CSK4 was also given to the mice (n = 8) 30 min after ischemia. Infarct size was determined by triphenyltetrazolium chloride staining. The morphology of neurons in brain sections was examined by Nissl staining. Pam3CSK4 administration significantly reduced infarct size by 55.9% (p < 0.01) compared with untreated I/R mice. Therapeutic treatment with Pam3CSK4 also significantly reduced infarct size by 55.8%. Morphologic examination showed that there was less neuronal damage in the hippocampus of Pam3CSK4-treated mice compared with untreated cerebral I/R mice. Pam3CSK4 treatment increased the levels of Hsp27, Hsp70, and Bcl2, and decreased Bax levels and NF-κB-binding activity in the brain tissues. Administration of Pam3CSK4 significantly increased the levels of phospho-Akt/Akt and phospho-GSK-3β/GSK-3β compared with untreated I/R mice. More significantly, either TLR2 deficiency or PI3K inhibition with LY29004 abolished the protection by Pam3CSK4. These data demonstrate that activation of TLR2 by its ligand prevents focal cerebral ischemic damage through a TLR2/PI3K/Akt-dependent mechanism. Of greater significance, these data indicate that therapy with a TLR2-specific agonist during cerebral ischemia is effective in reducing injury.     

Differences in PGE2 Production between Primary Human Monocytes and Differentiated Macrophages: Role of IL-1β and TRIF/IRF3

Prostaglandin E2 (PGE₂) is induced in vivo by bacterial products including TLR agonists. To determine whether PGE₂ is induced directly or via IL-1β, human monocytes and macrophages were cultured with LPS or with Pam3CSK4 in presence of caspase-1 inhibitor, ZVAD, or IL-1R antagonist, Kineret. TLR agonists induced PGE₂ in macrophages exclusively via IL-1β-independent mechanisms. In contrast, ZVAD and Kineret reduced PGE₂ production in LPS-treated (but not in Pam3CSK4-treated) monocytes, by 30–60%. Recombinant human IL-1β augmented COX-2 and mPGES-1 mRNA and PGE₂ production in LPS-pretreated monocytes but not in un-primed or Pam3CSK4-primed monocytes. This difference was explained by the finding that LPS but not Pam3CSK4 induced phosphorylation of IRF3 in monocytes suggesting activation of the TRIF signaling pathway. Knocking down TRIF, TRAM, or IRF3 genes by siRNA inhibited IL-1β-induced COX-2 and mPGES-1 mRNA. Blocking of TLR4 endocytosis during LPS priming prevented the increase in PGE₂ production by exogenous IL-1β. Our data showed that TLR2 agonists induce PGE₂ in monocytes independently from IL-1β. In the case of TLR4, IL-1β augments PGE₂ production in LPS-primed monocytes (but not in macrophages) through a mechanism that requires TLR4 internalization and activation of the TRIF/IRF3 pathway. These findings suggest a key role for blood monocytes in the rapid onset of fever in animals and humans exposed to bacterial products and some novel adjuvants.     

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.     

Nod2 downregulates TLR2/1 mediated IL1β gene expression in mouse peritoneal macrophages

Nod2 is a cytosolic pattern recognition receptor. It has been implicated in many inflammatory conditions. Its signaling has been suggested to modulate TLR responses in a variety of ways, yet little is known about the mechanistic details of the process. We show in this study that Nod2 knockdown mouse peritoneal macrophages secrete more IL1β than normal macrophages when stimulated with peptidoglycan (PGN). Muramyl dipeptide (MDP, a Nod2 ligand) + PGN co-stimulated macrophages have lower expression of IL1β than PGN (TLR2/1 ligand) stimulated macrophages. MDP co-stimulation have similar effects on Pam3CSK4 (synthetic TLR2/1 ligand) mediated IL1β expression suggesting that MDP mediated down regulating effects are receptor dependent and ligand independent. MDP mediated down regulation was specific for TLR2/1 signaling as MDP does not affect LPS (TLR4 ligand) or zymosan A (TLR2/6 ligand) mediated IL1β expression. Mechanistically, MDP exerts its down regulating effects by lowering PGN/Pam3CSK4 mediated nuclear cRel levels. Lower nuclear cRel level were observed to be because of enhanced transporting back rather than reduced nuclear translocation of cRel in MDP + PGN stimulated macrophages. These results demonstrate that Nod2 and TLR2/1 signaling pathways are independent and do not interact at the level of MAPK or NF-κB activation.     

The Toll-like receptor 2/1 (TLR2/1) complex initiates human platelet activation via the src/Syk/LAT/PLCγ2 signalling cascade

The specific TLR2/1 complex activator Pam3CSK4 has been shown to provoke prominent activation and aggregation of human non-nucleated platelets. As Pam3CSK4-evoked platelet activation does not employ the major signalling pathway established in nucleated immune cells, we investigated if the TLR2/1 complex on platelets may initiate signalling pathways known to be induced by physiological agonists such as collagen via GPVI or thrombin via PARs. We found that triggering TLR2/1 complex-signalling with Pam3CSK4, in common with that induced via GPVI, and in contrast to that provoked by PARs, involves tyrosine phosphorylation of the adaptor protein LAT as well as of PLCγ2 in a src- and Syk-dependent manner. In this respect, we provide evidence that Pam3CSK4 does not cross-activate GPVI.Further, by the use of platelets from a Glanzmann's thrombasthenia patient lacking β₃, in contrast to findings in nucleated immune cells, we show that the initiation of platelet activation by Pam3CSK4 does not involve integrin β₃ signalling; whereas the latter, subsequent to intermediate TXA2 synthesis and signalling, was found to be indispensable for proper dense granule secretion and full platelet aggregation. Together, our findings reveal that triggering the TLR2/1 complex with Pam3CSK4 initiates human platelet activation by engaging tyrosine kinases of the src family and Syk, the adaptor protein LAT, as well as the key mediator PLCγ2.     

Aquatic birnavirus-induced ER stress-mediated death signaling contribute to downregulation of Bcl-2 family proteins in salmon embryo cells

Aquatic birnavirus induces mitochondria-mediated cell death, but whether connects to endoplasmic reticulum (ER) stress is still unknown. In this present, we characterized that IPNV infection triggers ER stress-mediated cell death via PKR/eIF2α phosphorylation signaling for regulating the Bcl-2 family protein expression in fish cells. The IPNV infection can induce ER stress as follows: (1) ER stress sensor ATF6 cleavaged; (2) ER stress marker GRP78 upregulation, and (3) PERK/eIF2α phosphorylation. Then, the IPNV-induced ER stress signals can induce the CHOP expression at early (6 h p.i.) and middle replication (12 h p.i.) stages. Moreover, IPNV-induced CHOP upregulation dramatically correlates to apparently downregulate the Bcl-2 family proteins, Bcl-2, Mcl-1 and Bcl-xL at middle replication stage (12 h p.i.) and produces mitochondria membrane potential (MMP) loss and cell death. Furthermore, with GRP78 synthesis inhibitor momitoxin (VT) and PKR inhibitor 2-aminopurine (2-AP) treatment for blocking GRP78 expression and eIF2α phosphorylation, PKR/PERK may involve in eIF2α phosphorylation/CHOP upregulation pathway that enhances the downstream regulators Bcl-2 family proteins expression and increased cell survival. Taken together, our results suggest that IPNV infection activates PKR/PERK/eIF2α ER stress signals for regulating downstream molecules CHOP upregulation and Bcl-2 family downregulation that led to induce mitochondria-mediated cell death in fish cells, which may provide new insight into RNA virus pathogenesis and disease.     

Differential effects of cytokines and corticosteroids on Toll-like receptor 2 expression and activity in human airway epithelia

Background

The recognition of microbial molecular patterns via Toll-like receptors (TLRs) is critical for mucosal defenses.

Methods

Using well-differentiated primary cultures of human airway epithelia, we investigated the effects of exposure of the cells to cytokines (TNF-α and IFN-γ) and dexamethasone (dex) on responsiveness to the TLR2/TLR1 ligand Pam3CSK4. Production of IL-8, CCL20, and airway surface liquid antimicrobial activity were used as endpoints.

Results

Microarray expression profiling in human airway epithelia revealed that first response cytokines markedly induced TLR2 expression. Real-time PCR confirmed that cytokines (TNF-α and IFN-γ), dexamethasone (dex), or cytokines + dex increased TLR2 mRNA abundance. A synergistic increase was seen with cytokines + dex. To assess TLR2 function, epithelia pre-treated with cytokines ± dex were exposed to the TLR2/TLR1 ligand Pam3CSK4 for 24 hours. While cells pre-treated with cytokines alone exhibited significantly enhanced IL-8 and CCL20 secretion following Pam3CSK4, mean IL-8 and CCL20 release decreased in Pam3CSK4 stimulated cells following cytokines + dex pre-treatment. This marked increase in inflammatory gene expression seen after treatment with cytokines followed by the TLR2 ligand did not correlate well with NF-κB, Stat1, or p38 MAP kinase pathway activation. Cytokines also enhanced TLR2 agonist-induced beta-defensin 2 mRNA expression and increased the antimicrobial activity of airway surface liquid. Dex blocked these effects.

Conclusion

While dex treatment enhanced TLR2 expression, co-administration of dex with cytokines inhibited airway epithelial cell responsiveness to TLR2/TLR1 ligand over cytokines alone. Enhanced functional TLR2 expression following exposure to TNF-α and IFN-γ may serve as a dynamic means to amplify epithelial innate immune responses during infectious or inflammatory pulmonary diseases.     

Scavenger receptor A (SR-A) is required for LPS-induced TLR4 mediated NF-κB activation in macrophages

Recent evidence suggests that the macrophage scavenger receptor class A (SR-A, aka, CD204) plays a role in the induction of innate immune and inflammatory responses. We investigated whether SR-A will cooperate with Toll-like receptors (TLRs) in response to TLR ligand stimulation. Macrophages (J774/a) were treated with Pam2CSK4, (TLR2 ligand), Polyinosinic:polycytidylic acid (Poly I:C) (TLR3 ligand), and Lipopolysaccharides (LPS) (TLR4 ligand) for 15 min in the presence or absence of fucoidan (the SR-A ligand). The levels of phosphorylated IκBα (p-IκBα) were examined by Western blot. We observed that Poly I:C and LPS alone, but not Pam2CSK4 or fucoidan increased the levels of p-IκBα. However, LPS-induced increases in p-IκBα levels were further enhanced when presence of the fucoidan. Immunoprecipitation and double fluorescent staining showed that LPS stimulation promotes SR-A association with TLR4 in the presence of fucoidan. To further confirm our observation, we isolated peritoneal macrophages from SR-A deficient (SR-A(-/-)), TLR4(-/-) and wild type (WT) mice, respectively. The peritoneal macrophages were treated with LPS for 15min in the presence and absence of fucoidan. We observed that LPS-stimulated TNFα and IL-1β production was further enhanced in the WT macrophages, but did not in either TLR4(-/-) or SR-A(-/-) macrophages, when fucoidan was present. Similarly, in the presence of fucoidan, LPS-induced IκBα phosphorylation, NF-κB binding activity, and association between TLR4 and SR-A were significantly enhanced in WT macrophages compared with LPS stimulation alone. The data suggests that SR-A is needed for LPS-induced inflammatory responses in macrophages.     

Polo-Like Kinase 1 (PLK1) Is Involved in Toll-like Receptor (TLR)-Mediated TNF-α Production in Monocytic THP-1 Cells

Polo-like kinases (PLKs) have been reported to be essential components of anti-viral pathways. However, the role of PLKs in the production of pro-inflammatory cytokines induced by TLR activation is uncertain. We report here that monocytic THP-1 cells expressed PLK1, PLK2, PLK3 and PLK4. When THP-1 cells were treated with GW843682X, an inhibitor of PLK1 and PLK3, the results showed that GW843682X down-regulated Pam3CSK4- and LPS-induced TNF-α at both the gene and protein levels. GW843682X did not impact Pam3CSK4-induced IL-1β and IL-8 or LPS-induced IL-1β, but it down-regulated LPS-induced IL-8 significantly. Moreover, western blot results showed that TLRs activated PLK1, and PLK1 inhibition by RNA interference down-regulated Pam3CSK4-induced TNF-α production, suggesting the involvement of PLK1 in TNF-α up-regulation. In addition, GW843682X treatment for 12 to 24 h induced cell death and down-regulated MyD88, but neither of these roles contributed to the down-regulation of TNF-α, as TNF-α gene expression was up-regulated at 1 h. Furthermore, GW843682X inhibited Pam3CSK4-induced activation of ERK and NF-κB, which contributed to Pam3CSK4-induced up-regulation of TNF-α. GW843682X also inhibited LPS-induced activation of ERK, p38 and NF-κB, which contributed to LPS-induced up-regulation of TNF-α. Taken together, these results suggested that PLK1 is involved in TLR2- and TLR4-induced inflammation, and GW843682X may be valuable for the regulation of the inflammatory response.     

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.     

Endoplasmic reticulum stress pathway-mediated apoptosis in macrophages contributes to the survival of Mycobacterium tuberculosis

Background

Apoptosis is thought to play a role in host defenses against intracellular pathogens, including Mycobacterium tuberculosis (Mtb), by preventing the release of intracellular components and the spread of mycobacterial infection. This study aims to investigate the role of endoplasmic reticulum (ER) stress mediated apoptosis in mycobacteria infected macrophages.

Methodology/Principal Findings

Here, we demonstrate that ER stress-induced apoptosis is associated with Mtb H37Rv-induced cell death of Raw264.7 murine macrophages. We have shown that Mtb H37Rv induced apoptosis are involved in activation of caspase-12, which resides on the cytoplasmic district of the ER. Mtb infection increase levels of other ER stress indicators in a time-dependent manner. Phosphorylation of eIF2α was decreased gradually after Mtb H37Rv infection signifying that Mtb H37Rv infection may affect eIF2α phosphorylation in an attempt to survive within macrophages. Interestingly, the survival of mycobacteria in macrophages was enhanced by silencing CHOP expression. In contrast, survival rate of mycobacteria was reduced by phosphorylation of the eIF2α. Futhermore, the levels of ROS, NO or CHOP expression were significantly increased by live Mtb H37Rv compared to heat-killed Mtb H37Rv indicating that live Mtb H37Rv could induce ER stress response.

Conclusion/Significance

These findings indicate that eIF2α/CHOP pathway may influence intracellular survival of Mtb H37Rv in macrophages and only live Mtb H37Rv can induce ER stress response. The data support the ER stress pathway plays an important role in the pathogenesis and persistence of mycobacteria.     

Implication of NOD1 and NOD2 for the differentiation of multipotent mesenchymal stem cells derived from human umbilical cord blood

Toll-like receptors (TLRs) and Nod-like receptors (NLRs) are known to trigger an innate immune response against microbial infection. Although studies suggest that activation of TLRs modulate the function of mesenchymal stem cells (MSCs), little is known about the role of NLRs on the MSC function. In this study, we investigated whether NOD1 and NOD2 regulate the functions of human umbilical cord blood-derived MSCs (hUCB-MSCs). The genes of TLR2, TLR4, NOD1, and NOD2 were expressed in hUCB-MSCs. Stimulation with each agonist (Pam(3)CSK(4) for TLR2, LPS for TLR4, Tri-DAP for NOD1, and MDP for NOD2) led to IL-8 production in hUCB-MSC, suggesting the expressed receptors are functional in hUCB-MSC. CCK-8 assay revealed that none of agonist influenced proliferation of hUCB-MSCs. We next examined whether TLR and NLR agonists affect osteogenic-, adipogenic-, and chondrogenic differentiation of hUCB-MSCs. Pam(3)CSK(4) and Tri-DAP strongly enhanced osteogenic differentiation and ERK phosphorylation in hUCB-MSCs, and LPS and MDP also slightly did. Treatment of U0126 (MEK1/2 inhibitor) restored osteogenic differentiation enhanced by Pam(3)CSK(4). Tri-DAP and MDP inhibited adipogenic differentiation of hUCB-MSCs, but Pam(3)CSK(4) and LPS did not. On chondrogenic differentiation, all TLR and NLR agonists could promote chondrogenesis of hUCB-MSCs with difference in the ability. Our findings suggest that NOD1 and NOD2 as well as TLRs are involved in regulating the differentiation of MSCs.