Lipopolysaccharide- and lipoteichoic acid-induced tolerance and cross-tolerance: distinct alterations in IL-1 receptor-associated kinase

Human Toll-like receptor (TLR) 4 and TLR2 receptors recognize LPS or lipoteichoic acid (LTA), respectively. Prolonged exposure of human macrophages/monocytes to bacterial LPS induces a state of adaptation/tolerance to subsequent LPS challenge. Inflammatory gene expressions such as IL-1beta and TNF-alpha are selectively repressed, while certain anti-inflammatory genes such as secretory IL-1R antagonist are still induced in LPS-adapted/tolerant cells. In this report, we demonstrate that LPS-tolerized human promonocytic THP-1 cells develop cross-tolerance and no longer respond to LTA-induced IL-1beta/TNF-alpha production, indicating that disruption of common intracellular signaling is responsible for the decreased IL-1beta/TNF-alpha production. We observe that down-regulation of IL-1R-associated kinase (IRAK) protein level and kinase activity closely correlates with the development of cross-tolerance. IRAK protein levels and kinase activities in LPS-tolerized cells remain low and hyporesponsive to subsequent LPS or LTA challenges. We also demonstrate that THP-1 cells with prolonged LTA treatment develop LTA tolerance and do not express IL-1beta/TNF-alpha upon further LTA challenge. Strikingly, cells tolerized with LTA are only refractory to subsequent LTA challenge and can still respond to LPS stimulation. Correspondingly, stimulation of TLR2 by LTA, although activating IRAK, does not cause IRAK degradation. IRAK from LTA-tolerized cells can be subsequently activated and degraded by further LPS challenge, but not LTA treatment. Our studies reveal that LTA-induced tolerance is distinct compared with that of LPS tolerance, and is likely due to disruption of unique TLR2 signaling components upstream of MyD88/IRAK.     

IL-6 and maturation govern TLR2 and TLR4 induced TLR agonist tolerance and cross-tolerance in dendritic cells

Stimulation of naive mouse dendritic cells (DC) with LPS or Pam(3)CSK(4) (P3C) induces production of TNF-alpha via TLR4- or TLR2-signaling. Although tolerance in macrophages has been studied in detail, we investigated the role of TLR agonist concentration and IL-6 for tolerance in DC. P3C- or LPS-primed DC were nonresponsive to P3C or LPS restimulation in terms of TNF-alpha but not IL-6 production. The mechanisms involved in tolerance were dependent on the concentration of the TLR ligand used for DC priming. DC primed with LPS or P3C at high concentrations developed a maturation dependent, IL-6 independent tolerance associated with inhibition of TLR signaling upstream of IkappaB as indicated by decreased IkappaB degradation. In contrast, priming of DC with LPS or P3C at low concentrations resulted in IL-6-dependent tolerance, which was abolished in IL-6 deficient DC, and was not accompanied by maturation of DC or by down-regulation of TLR2 or TLR4. In homotolerogenic DC primed with LPS or P3C at high concentrations, degradation of IkappaB upon restimulation with LPS or P3C was inhibited suggesting tolerance mechanism(s) upstream of IkappaB; in contrast, cross-tolerance in DC primed with LPS or P3C at low concentrations was not associated with reduced IkappaB degradation suggesting tolerance mechanisms downstream of IkappaB. Our data indicate that in naive DC TLR4- and TLR2-stimulation results in homo- and cross-tolerance; the mechanisms involved in tolerance depend on the concentration of the TLR agonist used for DC priming and are governed by IL-6 and maturation.     

Induction of in vitro reprogramming by Toll-like receptor (TLR)2 and TLR4 agonists in murine macrophages: effects of TLR "homotolerance" versus "heterotolerance" on NF-kappa B signaling pathway components

In this study, tolerance induction by preexposure of murine macrophages to Toll-like receptor (TLR)2 and TLR4 agonists was revisited, focusing on the major signaling components associated with NF-kappaB activation. Pretreatment of macrophages with a pure TLR4 agonist (protein-free Escherichia coli (Ec) LPS) or with TLR2 agonists (Porphyromonas gingivalis LPS or synthetic lipoprotein Pam3Cys) led to suppression of TNF-alpha secretion, IL-1R-associated kinase-1, and IkappaB kinase (IKK) kinase activities, c-jun N-terminal kinase, and extracellular signal-regulated kinase phosphorylation, and to suppression of NF-kappaB DNA binding and transactivation upon challenge with the same agonist (TLR4 or TLR2 "homotolerance," respectively). Despite inhibited NF-kappaB DNA binding, increased levels of nuclear NF-kappaB were detected in agonist-pretreated macrophages. For all the intermediate signaling elements, heterotolerance was weaker than TLR4 or TLR2 homotolerance with the exception of IKK kinase activity. IKK kinase activity was unperturbed in heterotolerance. TNF-alpha secretion was also suppressed in P. gingivalis LPS-pretreated, Ec LPS-challenged cells, but not vice versa, while Pam3Cys and Ec LPS did not induce a state of cross-tolerance at the level of TNF-alpha. Experiments designed to elucidate novel mechanisms of NF-kappaB inhibition in tolerized cells revealed the potential contribution of IkappaBepsilon and IkappaBxi inhibitory proteins and the necessity of TLR4 engagement for induction of tolerance to Toll receptor-IL-1R domain-containing adapter protein/MyD88-adapter-like-dependent gene expression. Collectively, these data demonstrate that induction of homotolerance affects a broader spectrum of signaling components than in heterotolerance, with selective modulation of specific elements within the NF-kappaB signaling pathway.     

Induction of tolerance to lipopolysaccharide and mycobacterial components in Chinese hamster ovary/CD14 cells is not affected by overexpression of Toll-like receptors 2 or 4

Down-regulation of cell surface expression of Toll-like receptor (TLR) 4 following LPS stimulation has been suggested to underlie endotoxin tolerance. In this study, we examined whether overexpression of TLR2 or TLR4 would affect the ability of cells to become tolerant to LPS or the mycobacterial components, arabinose-capped lipoarabinomannan (LAM) and soluble tuberculosis factor (STF). To this end, Chinese hamster ovary/CD14 cells stably transfected with a NF-kappaB-dependent reporter construct, endothelial leukocyte adhesion molecule CD25 (the 3E10 clone), were engineered to overexpress either human TLR2 or TLR4. Transfected TLRs exhibited proper signaling functions, as evidenced by increased LPS responsiveness of 3E10/TLR4 cells and acquisition of sensitivity to TLR2-specific ligands upon transfection of TLR2 into TLR2-negative 3E10 cells. Pretreatment of cells with LPS, LAM, or STF did not modulate TLR2 or TLR4 cell surface expression. Following LPS exposure, 3E10, 3E10/TLR2, and 3E10/TLR4 cells exhibited comparable decreases in LPS-mediated NF-kappaB activation and mitogen-activated protein (MAP) kinase phosphorylation. Likewise, LPS pretreatment profoundly inhibited LPS-induced NF-kappaB translocation in Chinese hamster ovary cells that concomitantly overexpressed human TLR4 and myeloid differentiation protein-2 (MD-2), but failed to modulate TLR4 or MD-2 cell surface expression. Pretreatment of 3E10/TLR2 cells with LAM or STF decreased their NF-kappaB responses induced by subsequent stimulation with these substances or LPS. Conversely, prior exposure of 3E10/TLR2 cells to LPS led to hyporesponsiveness to LPS, LAM, and STF, indicating that LPS and mycobacterial products induce cross-tolerance. Thus, tolerance to LPS and mycobacterial components cannot be attributed solely to a decrease in TLR/MD-2 expression levels, suggesting inhibition of expression or function of other signaling intermediates.     

Gram-negative flagellin-induced self-tolerance is associated with a block in interleukin-1 receptor-associated kinase release from toll-like receptor 5

Flagellin from a number of Gram-negative bacteria induces cytokine and nitric oxide production by inflammatory cell types. In view of the evidence that flagellin responsiveness is subject to modulation, we explored the possibilities that a prior exposure to flagellin might result in a state of reduced flagellin responsiveness or tolerance and that lipopolysaccharide (LPS) and flagellin may induce a state of cross-tolerance to each other. Our results demonstrate that a prior exposure to flagellin results in a subsequent state of flagellin tolerance in human monocytes, THP1 cells, Jurkat cells, and COS-1 cells. Tolerance occurs within 2 h after addition of flagellin and does not require protein synthesis. Flagellin did not induce tolerance to LPS in monocytes and THP1 cells; however, LPS treatment of monocytes and THP1 cells resulted in a state of flagellin cross-tolerance. Flagellin-induced self-tolerance is not the result of a decrease in the steady-state level of toll-like receptor 5 (TLR5) or interleukin-1 receptor associated kinase (IRAK), but it is associated with a block in the release of IRAK from the TLR5 complex in flagellin-tolerant cells. Release is essential for IRAK activity because the TLR5-associated IRAK lacks kinase activity. LPS-induced cross-tolerance to flagellin is also associated with a block in IRAK release from TLR5. These results provide evidence for a novel mechanism of TLR signaling control.     

The role of toll-like receptors (TLRs) in bacteria-induced maturation of murine dendritic cells (DCS). Peptidoglycan and lipoteichoic acid are inducers of DC maturation and require TLR2

Toll-like receptors (TLRs) have been found to be key elements in pathogen recognition by the host immune system. Dendritic cells (DCs) are crucial for both innate immune responses and initiation of acquired immunity. Here we focus on the potential involvement of TLR ligand interaction in DC maturation. TLR2 knockout mice and mice carrying a TLR4 mutation (C3H/HeJ) were investigated for DC maturation induced by peptidoglycan (PGN), lipopolysaccharide (LPS), or lipoteichoic acids (LTAs). All stimuli induced maturation of murine bone marrow-derived DCs in control mice. TLR2(-)/- mice lacked maturation upon stimulation with PGN, as assessed by expression of major histocompatibility complex class II, CD86, cytokine, and chemokine production, fluorescein isothiocyanate-dextran uptake, and mixed lymphocyte reactions, while being completely responsive to LPS. A similar lack of maturation was observed in C3H/HeJ mice upon stimulation with LPS. DC maturation induced by LTAs from two different types of bacteria was severely impaired in TLR2(-)/-, whereas C3H/HeJ mice responded to LTAs in a manner similar to wild-type mice. We demonstrate that DC maturation is induced by stimuli from Gram-positive microorganisms, such as PGN and LTA, with similar efficiency as by LPS. Finally, we provide evidence that TLR2 and TLR4 interaction with the appropriate ligand is essential for bacteria-induced maturation of DCs.     

Extracellular Hsp70 induces inflammation and modulates LPS/LTA-stimulated inflammatory response in THP-1 cells

Extracellular Hsp70 (eHsp70) can act as damage-associated molecular pattern (DAMP) via Toll-like receptors TLR2 and TLR4, and stimulate immune and inflammatory responses leading to sterile inflammation and propagation of already existing inflammation. It was found elevated in the blood of patients with chronic obstructive pulmonary disease (COPD), who might suffer occasional bacterial colonizations and infections. We used a monocytic THP-1 cell line as a cellular model of systemic compartment of COPD to assess inflammatory effects of eHsp70 when present alone or together with bacterial products lypopolysaccharide (LPS) and lypoteichoic acid (LTA). THP-1 cells were differentiated into macrophage-like cells and treated with various concentrations of recombinant human Hsp70 protein (rhHsp70), LPS (TLR4 agonist), LTA (TLR2 agonist), and their combinations for 4, 12, 24, and 48 h. Concentrations of IL-1α, IL-6, IL-8, and TNF-α were determined by ELISA. Cell viability was assessed by MTS assay, and mode of cell death by luminometric measurements of caspases-3/7, -8, and -9 activities. rhHsp70 showed cell protecting effect by suppressing caspases-3/7 activation, while LPS provoked cytotoxicity through caspases-8 and -3/7 pathway. Regarding inflammatory processes, rhHsp70 alone induced secretion of IL-1α and IL-8, but had modulatory effects on release of all four cytokines when applied together with LPS or LTA. Combined effect with LPS was mainly synergistic, and with LTA mainly antagonistic, although it was cytokine- and time-dependent. Our results confirmed pro-inflammatory function of extracellular Hsp70, and suggest its possible implication in COPD exacerbations caused by bacterial infection through desensitization or inappropriate activation of TLR2 and TLR4 receptors.     

Induction of bacterial lipoprotein tolerance is associated with suppression of toll-like receptor 2 expression

Tolerance to bacterial cell wall components including lipopolysaccharide (LPS) may represent an essential regulatory mechanism during bacterial infection. Two members of the Toll-like receptor (TLR) family, TLR2 and TLR4, recognize the specific pattern of bacterial cell wall components. TLR4 has been found to be responsible for LPS tolerance. However, the role of TLR2 in bacterial lipoprotein (BLP) tolerance and LPS tolerance is unclear. Pretreatment of human THP-1 monocytic cells with a synthetic bacterial lipopeptide induced tolerance to a second BLP challenge with diminished tumor necrosis factor-alpha and interleukin-6 production, termed BLP tolerance. Furthermore, BLP-tolerized THP-1 cells no longer responded to LPS stimulation, indicating a cross-tolerance to LPS. Induction of BLP tolerance was CD14-independent, as THP-1 cells that lack membrane-bound CD14 developed tolerance both in serum-free conditions and in the presence of a specific CD14 blocking monoclonal antibody (MEM-18). Pre-exposure of THP-1 cells to BLP suppressed mitogen-activated protein kinase phosphorylation and nuclear factor-kappaB activation in response to subsequent BLP and LPS stimulation, which is comparable with that found in LPS-tolerized cells, indicating that BLP tolerance and LPS tolerance may share similar intracellular pathways. However, BLP strongly enhanced TLR2 expression in non-tolerized THP-1 cells, whereas LPS stimulation had no effect. Furthermore, a specific TLR2 blocking monoclonal antibody (2392) attenuated BLP-induced, but not LPS-induced, tumor necrosis factor-alpha and interleukin-6 production, indicating BLP rather than LPS as a ligand for TLR2 engagement and activation. More importantly, pretreatment of THP-1 cells with BLP strongly inhibited TLR2 activation in response to subsequent BLP stimulation. In contrast, LPS tolerance did not prevent BLP-induced TLR2 overexpression. These results demonstrate that BLP tolerance develops through down-regulation of TLR2 expression.     

Toll-like receptor 4 is needed to restrict the invasion of Escherichia coli P4 into mammary gland epithelial cells in a murine model of acute mastitis

Mastitis, an inflammatory response of the mammary tissue to invading pathogenic bacteria, is a common disease in breast-feeding women and dairy animals. Escherichia coli is a leading cause of mastitis in dairy animals. During the course of the disease the host mounts a strong inflammatory response, but specific bacterial virulence factors have not yet been identified. Here we report the use of a murine mastitis model to investigate the innate inflammatory reaction of the mammary gland. We show that lipopolysaccharide (LPS) infusion induces mastitis in wild-type mice (C3H/HeN), but not in mice expressing mutated Toll-like receptor 4 (TLR4) (C3H/HeJ). The wild-type phenotype was restored by adoptive transfer of TLR4-expressing macrophages into the alveolar milk space of C3H/HeJ mice. In contrast to the LPS treatment, infection with E. coli P4 (ECP4) resulted in inflammation even in the absence of LPS/TLR4 signalling, indicating that additional factors play a role in the pathogenesis of the intact bacteria. Furthermore, in the absence of functional TLR4 the infecting ECP4 invade the epithelial cells with high efficiency, forming intracellular microcolonies. However, adoptive transfer with TLR4-expressing macrophages drastically reduced the epithelial invasion. Taken together, these results indicate that ECP4 has an invasive potential, which is restricted by alveolar macrophages in response to the LPS/TLR4 signalling.     

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.     

Lymphotoxin β receptor activation on macrophages induces cross-tolerance to TLR4 and TLR9 ligands

Our previous studies indicated that lymphotoxin β receptor (LTβR) activation controls and downregulates inflammatory reactions. In this study, we report that LTβR activation on primary mouse macrophages results in induction of tripartite motif containing (TRIM) 30α, which negatively regulates NF-κB activation induced by TLR signaling. LTβR activation results in a downregulation of proinflammatory cytokine and mediator expression upon TLR restimulation, demonstrating that LTβR signaling is involved in the induction of TLR cross-tolerance. Specific knockdown experiments using TRIM30α-specific small interfering RNA abolished the LTβR-dependent induction of TRIM30α and LTβR-mediated TLR cross-tolerance. Concordantly, LTβR activation on bone marrow-derived macrophages induced cross-tolerance to TLR4 and TLR9 ligands in vitro. Furthermore, we have generated cell type-specific LTβR-deficient mice with ablation of LTβR expression on macrophages/neutrophils (LTβR(flox/flox) × LysM-Cre). In bone marrow-derived macrophages derived from these mice LTβR-induced cross-tolerance to TLR4 and TLR9 ligands was impaired. Additionally, mice with a conditional ablation of LTβR expression on macrophages (LTβR(flox/flox) × LysM-Cre) are resistant to LTβR-induced TLR4 tolerance in vivo. Collectively, our data indicate that LTβR activation on macrophages by T cell-derived lymphotoxin α(1)β(2) controls proinflammatory responses by activation of a TRIM30α-controlled, counterregulatory signaling pathway to protect against exacerbating inflammatory reactions.     

Gene expressions of Toll-like receptor 2, but not Toll-like receptor 4, is induced by LPS and inflammatory cytokines in mouse macrophages

Toll-like receptors (TLRs) are a family of mammalian homologues of Drosophila Toll and play important roles in host defense. Two of the TLRs, TLR2 and TLR4, mediate the responsiveness to LPS. Here the gene expression of TLR2 and TLR4 was analyzed in mouse macrophages. Mouse splenic macrophages responded to an intraperitoneal injection or in vitro treatment of LPS by increased gene expression of TLR2, but not TLR4. Treatment of a mouse macrophage cell line with LPS, synthetic lipid A, IL-2, IL-15, IL-1beta, IFN-gamma, or TNF-alpha significantly increased TLR2 mRNA expression, whereas TLR4 mRNA expression remained constant. TLR2 mRNA increase in response to synthetic lipid A was severely impaired in splenic macrophages isolated from TLR4-mutated C3H/HeJ mice, suggesting that TLR4 plays an essential role in the process. Specific inhibitors of mitogen-activated protein/extracellular signal-regulated kinase kinase and p38 kinase did not significantly inhibit TLR2 mRNA up-regulation by LPS. In contrast, LPS-mediated TLR2 mRNA induction was abrogated by pretreatment with a high concentration of curcumin, suggesting that NF-kappaB activation may be essential for the process. Taken together, our results indicate that TLR2, in contrast to TLR4, can be induced in macrophages in response to bacterial infections and may accelerate the innate immunity against pathogens.     

Wolbachia endosymbiotic bacteria of Brugia malayi mediate macrophage tolerance to TLR- and CD40-specific stimuli in a MyD88/TLR2-dependent manner

Lymphatic filarial nematodes are able to down-regulate parasite-specific and nonspecific responses of lymphocytes and APC. Lymphatic filariae are reliant on Wolbachia endosymbiotic bacteria for development and survival. We tested the hypothesis that repeated exposure to Wolbachia endosymbionts would drive macrophage tolerance in vitro and in vivo. We pre-exposed murine peritoneal-elicited macrophages to soluble extracts of Brugia malayi female worms (BMFE) before restimulating with BMFE or TLR agonists. BMFE tolerized macrophages (in terms of IFN-beta, IL-1beta, IL-6, IL-12p40, and TNF-alpha inflammatory cytokine production) in a dose-dependent manner toward self, LPS, MyD88-dependent TLR2 or TLR9 ligands (peptidoglycan, triacyl lipopeptide, CpG DNA) and the MyD88-independent/TRIF-dependent TLR3 ligand, polyinosinic-polycytidylic acid. This was accompanied with down-regulation in surface expression of TLR4 and up-regulation of CD14, CD40, and TLR2. BMFE tolerance extended to CD40 activation in vitro and systemic inflammation following lethal challenge in an in vivo model of endotoxin shock. The mechanism of BMFE-mediated macrophage tolerance was dependent on MyD88 and TLR2 but not TLR4. Evidence that desensitization was driven by Wolbachia-specific ligands was determined by use of extracts from Wolbachia-depleted B. malayi, aposymbiotic filarial species, and a cell line stably infected with Wolbachia pipientis. Our data promote a role for Wolbachia in contributing toward the dysregulated and tolerized immunological phenotype that accompanies the majority of human filarial infections.     

Toll-Like Receptor 4 Stimulation before or after Streptococcus pneumoniae Induced Sepsis Improves Survival and Is Dependent on T-Cells

Introduction

Endotoxin tolerance improves outcomes from gram negative sepsis but the underlying mechanism is not known. We determined if endotoxin tolerance before or after pneumococcal sepsis improved survival and the role of lymphocytes in this protection.

Methods

Mice received lipopolysaccharide (LPS) or vehicle before or after a lethal dose of Streptococcus pneumoniae. Survival, quantitative bacteriology, liver function, and cytokine concentrations were measured. We confirmed the necessity of Toll-like receptor 4 (TLR4) for endotoxin tolerance using C3H/HeN (TLR4 replete) and C3H/HeJ (TLR4 deficient) mice. The role of complement was investigated through A/J mice deficient in C5 complement. CBA/CaHN-Btk^xid//J mice with dysfunctional B cells and Rag-1 knockout (KO) mice deficient in T and B cells delineated the role of lymphocytes.

Results

Endotoxin tolerance improved survival from pneumococcal sepsis in mice with TLR4 that received LPS pretreatment or posttreatment. Survival was associated with reduced bacterial burden and serum cytokine concentrations. Death was associated with abnormal liver function and blood glucose concentrations. Endotoxin tolerance improved survival in A/J and CBA/CaHN-Btk^xid//J mice but not Rag-1 KO mice.

Conclusions

TLR4 stimulation before or after S. pneumoniae infection improved survival and was dependent on T-cells but did not require an intact complement cascade or functional B cells.     

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.