The sirtuin 1 activator SRT1720 alleviated endotoxin-induced fulminant hepatitis in mice

The metabolic sensor sirtuin 1 (SIRT1) also functions as a checkpoint in inflammation, and SRT1720 is a highly active and selective SIRT1 activator shown to alleviate inflammatory injury in several recent experimental studies. In the present study, the potential effects and underlying mechanisms of SRT1720 on lipopolysaccharide (LPS)-induced fulminant hepatitis in D-galactosamine (D-Gal)-sensitized mice were investigated. The results indicated that treatment with SRT1720 inhibited LPS/D-Gal-induced elevation of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), alleviated the histological abnormalities, suppressed the induction of tumor necrosis factor alpha (TNF-α) and IL-6, mitigated the phosphorylation of c-Jun N-terminal kinase (JNK), downregulated the activities of caspase 8, caspase 9 and caspase 3, decreased the level of cleaved caspase 3, reduced the TUNEL-positive cells, and improved the survival rate of the LPS/D-Gal-exposed mice. These data indicated that treatment with the SIRT1 activator SRT1720 alleviated LPS/D-Gal-induced fulminant hepatitis, which might be attributed to the suppressive effects of SRT1720 on TNF-α production and the subsequent activation of the apoptosis cascade.     

Glucose regulates expression of pro-inflammatory genes,IL-1β and IL-12, through a mechanism involving hexosamine biosynthesis pathway-dependent regulation of α-E catenin

High glucose levels are associated with changes in macrophage polarisation and evidence indicates that the sustained or even short-term high glucose levels modulate inflammatory responses in macrophages. However, the mechanism by which macrophages can sense the changes in glucose levels are not clearly understood. We find that high glucose levels rapidly increase the α-E catenin protein level in RAW264.7 macrophages. We also find an attenuation of glucose-induced increase in α-E catenin when hexosamine biosynthesis (HB) pathway is inhibited either with glutamine depletion or with the drugs azaserine and tunicamycin. This indicates the involvement of HB pathway in this process. Then, we investigated the potential role of α-E catenin in glucose-induced macrophage polarisation. We find that the reduction in α-E catenin level using siRNA attenuates the glucose-induced changes of both IL-1β and IL-12 mRNA levels under LPS-stimulated condition but does not affect TNF-α expression. Together this indicates that α-E catenin can sense the changes in glucose levels in macrophages via HB pathway and also can modulate the glucose-induced gene expression of inflammatory markers such as IL-1β and IL-12. This identifies a new part of the mechanism by which macrophages are able to respond to changes in glucose levels.     

The Therapeutic Potential and Mechanisms of Action of Quercetin in Relation to Lipopolysaccharide-Induced Sepsis In Vitro and In Vivo

Sepsis caused by Gram-negative bacterial infection is characterized by extensive inflammatory cytokine production, which leads to multiple organ failure and a high lethality rate. Therefore, compounds that are able to alleviate profound inflammatory responses may have therapeutic potential in relation to sepsis. Quercetin, one of the flavonoids found widely in the human diet, has been reported to have many health benefits, but the mechanisms underlying its biological effects remain obscure. In the present study, our aim was to investigate the molecular mechanisms by which quercetin inhibits lipopolysaccharide (LPS)-induced pro-inflammatory cytokine production and to evaluate the capacity of quercetin to attenuate the mortality rate in a mice model of lethal sepsis. Our results show that quercetin significantly attenuates LPS-induced production of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in RAW264.7 macrophages. The LPS-stimulated phosphorylations of the inhibitors of κB kinase (IKKs), Akt, and c-Jun N-terminal kinase (JNK) are also inhibited by quercetin. Quercetin causes a significant reduction in the phosphorylation and degradation of inhibitor of κBα (IκBα) and in the nuclear level of nuclear factor-κB (NF-κB), the latter being associated with decreased NF-κB binding activity. Most importantly, acute administration of quercetin reduces the lethality rate and circulating levels of TNF-α and IL-1β in C57BL/6J mice with endotoxemia induced by LPS, whereas chronic dietary supplementation with quercetin shows no inhibitory effect on serum TNF-α and IL-1β levels. These findings provide clues that quercetin may be a promising agent for the prevention of systemic inflammatory diseases such as sepsis.     

ApoE Production in Human Monocytes and Its Regulation by Inflammatory Cytokines

The apoE production by tissue macrophages is crucial for the prevention of atherosclerosis and the aim of this study was to further elucidate how this apolipoprotein is regulated by cytokines present during inflammation. Here we studied apoE production in peripheral blood mononuclear cells (PBMC) and analysis was made with a newly developed apoE ELISpot assay. In PBMC, apoE secretion was restricted to monocytes with classical (CD14⁺⁺CD16⁻) and intermediate (CD14⁺CD16⁺) monocytes being the main producers. As earlier described for macrophages, production was strongly upregulated by TGF-β and downregulated by bacterial lipopolysaccharide (LPS) and the inflammatory cytokines IFN-γ, TNF-α and IL-1β. We could here show that a similar down-regulatory effect was also observed with the type I interferon, IFN-α, while IL-6, often regarded as one of the more prominent inflammatory cytokines, did not affect TGF-β-induced apoE production. The TNF-α inhibitor Enbrel could partly block the down-regulatory effect of IFN-γ, IFN-α and IL-1β, indicating that inhibition of apoE by these cytokines may be dependent on or synergize with TNF-α. Other cytokines tested, IL-2, IL-4, IL-12, IL-13, IL-17A and IL-23, had no inhibitory effect on apoE production. In contrast to the effect on monocytes, apoE production by primary hepatocytes and the hepatoma cell line HepG2 was more or less unaffected by treatment with cytokines or LPS.     

Cross Regulation of Sirtuin 1, AMPK,and PPARγ in Conjugated Linoleic Acid Treated Adipocytes

Trans-10, cis-12 conjugated linoleic acid (t10c12 CLA) reduces triglyceride (TG) levels in adipocytes through multiple pathways, with AMP-activated protein kinase (AMPK) generally facilitating, and peroxisome proliferator-activated receptor γ (PPARγ) generally opposing these reductions. Sirtuin 1 (SIRT1), a histone/protein deacetylase that affects energy homeostasis, often functions coordinately with AMPK, and is capable of binding to PPARγ, thereby inhibiting its activity. This study investigated the role of SIRT1 in the response of 3T3-L1 adipocytes to t10c12 CLA by testing the following hypotheses: 1) SIRT1 is functionally required for robust TG reduction; and 2) SIRT1, AMPK, and PPARγ cross regulate each other. These experiments were performed by using activators, inhibitors, or siRNA knockdowns that affected these pathways in t10c12 CLA-treated 3T3-L1 adipocytes. Inhibition of SIRT1 amounts or activity using siRNA, sirtinol, nicotinamide, or etomoxir attenuated the amount of TG loss, while SIRT1 activator SRT1720 increased the TG loss. SRT1720 increased AMPK activity while sirtuin-specific inhibitors decreased AMPK activity. Reciprocally, an AMPK inhibitor reduced SIRT1 activity. Treatment with t10c12 CLA increased PPARγ phosphorylation in an AMPK-dependent manner and increased the amount of PPARγ bound to SIRT1. Reciprocally, a PPARγ agonist attenuated AMPK and SIRT1 activity levels. These results indicated SIRT1 increased TG loss and that cross regulation between SIRT1, AMPK, and PPARγ occurred in 3T3-L1 adipocytes treated with t10c12 CLA.     

Inhibition of Sirtuin 3 prevents titanium particle-induced bone resorption and osteoclastsogenesis via suppressing ERK and JNK signaling

Implant-derived wear particles can be phagocytosed by local macrophages, triggering an inflammatory cascade that can drive the activation and recruitment of osteoclasts, thereby inducing peri-prosthetic osteolysis. Efforts to suppress pro-inflammatory cytokine release and osteoclastsogenesis thus represent primary approaches to treating and preventing such osteolysis. Sirtuin 3 (SIRT3) is a NAD⁺-dependent deacetylases that control diverse metabolic processes. However, whether SIRT3 could mitigate wear debris-induced osteolysis has not been reported. Herein we explored the impact of the SIRT3 on titanium particle-induced osteolysis. Tartrate resistant acid phosphatase (TRAP) staining revealed that the inhibition of SIRT3 suppressed nuclear factor-κB ligand (RANKL)-mediated osteoclasts activation in a dose-dependent fashion. Notably, inhibition of SIRT3 also suppressed matrix metallopeptidase 9 (MMP9) and nuclear factor of activated T‐cell cytoplasmic 1 (NFATc1) expression at the mRNA and protein levels, while also inhibiting the mRNA expression of dendritic cell-specific transmembrane protein (DC-STAMP), ATPase H⁺ Transporting V0 Subunit D2 (Atp6v0d2), TRAP and Cathepsin K (CTSK) . In addition, inhibition of SIRT3 suppressed titanium particle-induced tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) expression and prevented titanium particle-induced osteolysis and bone loss in vivo. This inhibition of osteoclasts differentiation was found to be linked to the downregulation and reduced phosphorylation of JNK and ERK. Taken together, inhibition of SIRT3 may be a potential target for titanium particle-induced bone loss.     

Alveolar Macrophages Play a Key Role in Cockroach-Induced Allergic Inflammation via TNF-α Pathway

The activity of the serine protease in the German cockroach allergen is important to the development of allergic disease. The protease-activated receptor (PAR)-2, which is expressed in numerous cell types in lung tissue, is known to mediate the cellular events caused by inhaled serine protease. Alveolar macrophages express PAR-2 and produce considerable amounts of tumor necrosis factor (TNF)-α. We determined whether the serine protease in German cockroach extract (GCE) enhances TNF-α production by alveolar macrophages through the PAR-2 pathway and whether the TNF-α production affects GCE-induced pulmonary inflammation. Effects of GCE on alveolar macrophages and TNF-α production were evaluated using in vitro MH-S and RAW264.6 cells and in vivo GCE-induced asthma models of BALB/c mice. GCE contained a large amount of serine protease. In the MH-S and RAW264.7 cells, GCE activated PAR-2 and thereby produced TNF-α. In the GCE-induced asthma model, intranasal administration of GCE increased airway hyperresponsiveness (AHR), inflammatory cell infiltration, productions of serum immunoglobulin E, interleukin (IL)-5, IL-13 and TNF-α production in alveolar macrophages. Blockade of serine proteases prevented the development of GCE induced allergic pathologies. TNF-α blockade also prevented the development of such asthma-like lesions. Depletion of alveolar macrophages reduced AHR and intracellular TNF-α level in pulmonary cell populations in the GCE-induced asthma model. These results suggest that serine protease from GCE affects asthma through an alveolar macrophage and TNF-α dependent manner, reflecting the close relation of innate and adaptive immune response in allergic asthma model.     

Effect of short-time treatment with TNF-α on stem cell activity and barrier function in enteroids

Although tumor necrosis factor-α (TNF-α) is a known major inflammatory mediator in inflammatory bowel disease (IBD) and has various effects on intestinal epithelial cell (IEC) homeostasis, the changes in IECs in the early inflammatory state induced during short-time treatment (24 h) with TNF-α remain unclear. In this study, we investigated TNF-α-induced alterations in IECs in the early inflammatory state using mouse jejunal organoids (enteroids). Of the inflammatory cytokines, i.e., TNF-α, IL-1β, IL-6, and IL-17, only TNF-α markedly increased the mRNA level of macrophage inflammatory protein 2 (MIP-2; the mouse homologue of interleukin-8), which is induced in the early stages of inflammation. TNF-α stimulation (3 h and 6 h) decreased the mRNA level of the stem cell markers leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) and polycomb group ring finger 4 and the progenitor cell marker prominin-1, which is also known as CD133. In addition, TNF-α treatment (24 h) decreased the number of Lgr5-positive cells and enteroid proliferation. TNF-α stimulation at 3 h and 6 h also decreased the mRNA level of chromogranin A and mucin 2, which are respective markers of enteroendocrine and goblet cells. Moreover, enteroids treated with TNF-α (24 h) not only decreased the integrity of tight junctions and cytoskeletal components but also increased intercellular permeability in an influx test with fluorescent dextran, indicating disrupted intestinal barrier function. Taken together, our findings indicate that short-time treatment with TNF-α promotes the inflammatory response and decreases intestinal stem cell activity and barrier function.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10616-021-00487-y.     

SIRT6 stabilization and cytoplasmic localization in macrophages regulates acute and chronic inflammation in mice

Acute and chronic inflammations are key homeostatic events in health and disease. Sirtuins (SIRTs), a family of NAD-dependent protein deacylases, play a pivotal role in the regulation of these inflammatory responses. Indeed, SIRTs have anti-inflammatory effects through a myriad of signaling cascades, including histone deacetylation and gene silencing, p65/RelA deacetylation and inactivation, and nucleotide‑binding oligomerization domain, leucine rich repeat, and pyrin domain‑containing protein 3 inflammasome inhibition. Nevertheless, recent findings show that SIRTs, specifically SIRT6, are also necessary for mounting an active inflammatory response in macrophages. SIRT6 has been shown to positively regulate tumor necrosis factor alpha (TNFα) secretion by demyristoylating pro-TNFα in the cytoplasm. However, how SIRT6, a nuclear chromatin-binding protein, fulfills this function in the cytoplasm is currently unknown. Herein, we show by Western blot and immunofluorescence that in macrophages and fibroblasts there is a subpopulation of SIRT6 that is highly unstable and quickly degraded via the proteasome. Upon lipopolysaccharide stimulation in Raw 264.7, bone marrow, and peritoneal macrophages, this population of SIRT6 is rapidly stabilized and localizes in the cytoplasm, specifically in the vicinity of the endoplasmic reticulum, promoting TNFα secretion. Furthermore, we also found that acute SIRT6 inhibition dampens TNFα secretion both in vitro and in vivo, decreasing lipopolysaccharide-induced septic shock. Finally, we tested SIRT6 relevance in systemic inflammation using an obesity-induced chronic inflammatory in vivo model, where TNFα plays a key role, and we show that short-term genetic deletion of SIRT6 in macrophages of obese mice ameliorated systemic inflammation and hyperglycemia, suggesting that SIRT6 plays an active role in inflammation-mediated glucose intolerance during obesity.     

Berberine Inhibits HIV Protease Inhibitor-Induced Inflammatory Response by Modulating ER Stress Signaling Pathways in Murine Macrophages

Background

HIV protease inhibitor (PI)-induced inflammatory response plays an important role in HIV PI-associated dyslipidemia and cardiovascular complications. This study examined the effect of berberine, a traditional herb medicine, on HIV PI-induced inflammatory response and further investigated the underlying cellular/molecular mechanisms in macrophages.

Methodology and Principal Findings

Cultured mouse J774A.1 macrophages and primary mouse macrophages were used in this study. The expression of TNF-α and IL-6 were detected by real-time RT-PCR and ELISA. Activations of ER stress and ERK signaling pathways were determined by Western blot analysis. Immunofluorescent staining was used to determine the intracellular localization of RNA binding protein HuR. RNA-pull down assay was used to determine the association of HuR with endogenous TNF-α and IL-6. Berberine significantly inhibited HIV PI-induced TNF-α and IL-6 expression by modulating ER stress signaling pathways and subsequent ERK activation, in turn preventing the accumulation of the RNA binding protein HuR in cytosol and inhibiting the binding of HuR to the 3′-UTRs of TNF-α and IL-6 in macrophages.

Conclusions and Significance

Inhibition of ER stress represents a key mechanism by which berberine prevents HIV PI-induced inflammatory response. Our findings provide a new insight into the molecular mechanisms of berberine and show the potential application of berberine as a complimentary therapeutic agent for HIV infection.     

African Swine Fever Virus Infection Induces Tumor Necrosis Factor Alpha Production: Implications in Pathogenesis

We have analyzed the production of tumor necrosis factor alpha (TNF-α) induced by in vitro infection with African swine fever (ASF) virus (ASFV) and the systemic and local release of this inflammatory cytokine upon in vivo infection. An early increase in TNF-α mRNA expression was detected in ASFV-infected alveolar macrophages, and high levels of TNF-α protein were detected by ELISA in culture supernatants from these cells. When animals were experimentally infected with a virulent isolate (E-75), enhanced TNF-α expression in mainly affected organs correlated with viral protein expression. Finally, elevated levels of TNF-α were detected in serum, corresponding to the onset of clinical signs. TNF-α has been reported to be critically involved in the pathogenesis of major clinical events in ASF, such as intravascular coagulation, tissue injury, apoptosis, and shock. In the present study, TNF-α containing supernatants from ASFV-infected cultures induced apoptosis in uninfected lymphocytes; this effect was partially abrogated by preincubation with an anti-TNF-α specific antibody. These results suggest a relevant role for TNF-α in the pathogenesis of ASF.     

Flavonoid Apigenin Inhibits Lipopolysaccharide-Induced Inflammatory Response through Multiple Mechanisms in Macrophages

Background

Apigenin is a non-toxic natural flavonoid that is abundantly present in common fruits and vegetables. It has been reported that apigenin has various beneficial health effects such as anti-inflammation and chemoprevention. Multiple studies have shown that inflammation is an important risk factor for atherosclerosis, diabetes, sepsis, various liver diseases, and other metabolic diseases. Although it has been long realized that apigenin has anti-inflammatory activities, the underlying functional mechanisms are still not fully understood.

Methodology and Principal Findings

In the present study, we examined the effect of apigenin on LPS-induced inflammatory response and further elucidated the potential underlying mechanisms in human THP-1-induced macrophages and mouse J774A.1 macrophages. By using the PrimePCR array, we were able to identify the major target genes regulated by apigenin in LPS-mediated immune response. The results indicated that apigenin significantly inhibited LPS-induced production of pro-inflammatory cytokines, such as IL-6, IL-1β, and TNF-α through modulating multiple intracellular signaling pathways in macrophages. Apigenin inhibited LPS-induced IL-1β production by inhibiting caspase-1 activation through the disruption of the NLRP3 inflammasome assembly. Apigenin also prevented LPS-induced IL-6 and IL-1β production by reducing the mRNA stability via inhibiting ERK1/2 activation. In addition, apigenin significantly inhibited TNF-α and IL-1β-induced activation of NF-κB.

Conclusion and Significance

Apigenin Inhibits LPS-induced Inflammatory Response through multiple mechanisms in macrophages. These results provided important scientific evidences for the potential application of apigenin as a therapeutic agent for inflammatory diseases.     

IRAK-4 in macrophages contributes to inflammatory osteolysis of wear particles around loosened hip implants

TLRs recognizing PAMPS play a role in local immunity and participate in implant-associated loosening. TLR-mediated signaling is primarily regulated by IL-1 receptor associated kinase-M (IRAK-M) negatively and IRAK-4 positively. Our previous studies have proved that wear particles promote endotoxin tolerance in macrophages by inducing IRAK-M. However, whether IRAK-4 is involved in inflammatory osteolysis of wear particles basically, and the specific mechanism of IRAK-4 around loosened hip implants, is still unclear. IRAK-4 was studied in the interface membranes from patients in vivo and in particle-stimulated macrophages to clarify its role. Also, IL-1β and TNF-α levels were measured after particle and LPS stimulation in macrophages with or without IRAK-4 silenced by siRNA. Our results showed that the interface membranes around aseptic and septic loosened prosthesis expressed more IRAK-4 compared with membranes from osteoarthritic patients. IRAK-4 in macrophages increased upon particle and LPS stimulation. In the former, IL-1β and TNF-α levels were lower compared with those of LPS stimulation, and IRAK-4 siRNA could suppress production of pro-inflammatory cytokines. These findings suggest that besides IRAK-M, IRAK-4 also plays an important role in the local inflammatory reaction and contributes to prosthesis loosening.     

Chitohexaose Activates Macrophages by Alternate Pathway through TLR4 and Blocks Endotoxemia

Sepsis is a consequence of systemic bacterial infections leading to hyper activation of immune cells by bacterial products resulting in enhanced release of mediators of inflammation. Endotoxin (LPS) is a major component of the outer membrane of Gram negative bacteria and a critical factor in pathogenesis of sepsis. Development of antagonists that inhibit the storm of inflammatory molecules by blocking Toll like receptors (TLR) has been the main stay of research efforts. We report here that a filarial glycoprotein binds to murine macrophages and human monocytes through TLR4 and activates them through alternate pathway and in the process inhibits LPS mediated classical activation which leads to inflammation associated with endotoxemia. The active component of the nematode glycoprotein mediating alternate activation of macrophages was found to be a carbohydrate residue, Chitohexaose. Murine macrophages and human monocytes up regulated Arginase-1 and released high levels of IL-10 when incubated with chitohexaose. Macrophages of C3H/HeJ mice (non-responsive to LPS) failed to get activated by chitohexaose suggesting that a functional TLR4 is critical for alternate activation of macrophages also. Chitohexaose inhibited LPS induced production of inflammatory molecules TNF-α, IL-1β and IL-6 by macropahges in vitro and in vivo in mice. Intraperitoneal injection of chitohexaose completely protected mice against endotoxemia when challenged with a lethal dose of LPS. Furthermore, Chitohexaose was found to reverse LPS induced endotoxemia in mice even 6/24/48 hrs after its onset. Monocytes of subjects with active filarial infection displayed characteristic alternate activation markers and were refractory to LPS mediated inflammatory activation suggesting an interesting possibility of subjects with filarial infections being less prone to develop of endotoxemia. These observations that innate activation of alternate pathway of macrophages by chtx through TLR4 has offered novel opportunities to cell biologists to study two mutually exclusive activation pathways of macrophages being mediated through a single receptor.     

RAGE and TGF-β1 Cross-Talk Regulate Extracellular Matrix Turnover and Cytokine Synthesis in AGEs Exposed Fibroblast Cells

AGEs accumulation in the skin affects extracellular matrix (ECM) turnover and triggers diabetes associated skin conditions and accelerated skin aging. The receptor of AGEs (RAGE) has an essential contribution to cellular dysfunction driven by chronic inflammatory responses while TGF-β1 is critical in both dermal homeostasis and inflammation. We investigated the contribution of RAGE and TGF-β1 to the modulation of inflammatory response and ECM turnover in AGEs milieu, using a normal fibroblast cell line. RAGE, TGF-β1, collagen I and III gene and protein expression were upregulated after exposure to AGEs-BSA, and MMP-2 was activated. AGEs-RAGE was pivotal in NF-κB dependent collagen I expression and joined with TGF-β1 to stimulate collagen III expression, probably via ERK1/2 signaling. AGEs-RAGE axis induced upregulation of TGF-β1, TNF-α and IL-8 cytokines. TNF-α and IL-8 were subjected to TGF-β1 negative regulation. RAGE’s proinflammatory signaling also antagonized AGEs-TGF-β1 induced fibroblast contraction, suggesting the existence of an inhibitory cross-talk mechanism between TGF-β1 and RAGE signaling. RAGE and TGF-β1 stimulated anti-inflammatory cytokines IL-2 and IL-4 expression. GM-CSF and IL-6 expression appeared to be dependent only on TGF-β1 signaling. Our data also indicated that IFN-γ upregulated in AGEs-BSA milieu in a RAGE and TGF-β1 independent mechanism. Our findings raise the possibility that RAGE and TGF-β1 are both involved in fibrosis development in a complex cross-talk mechanism, while also acting on their own individual targets. This study contributes to the understanding of impaired wound healing associated with diabetes complications.     

High-Mobility Group Box-1 Induces Proinflammatory Cytokines Production of Kupffer Cells through TLRs-Dependent Signaling Pathway after Burn Injury

Kupffer cells (KCs) were a significant source of cytokine release during the early stage of severe burns. High mobility group box protein 1 (HMGB1) was recently identified as a new type of proinflammatory cytokine. The ability of HMGB1 to generate inflammatory responses after burn trauma has not been well characterized. KCs were isolated from sham animals and rats with a 30% full-thickness burn, and then were stimulated with increasing concentrations of HMGB1. The levels of Tumor necrosis factor (TNF)-α and interleukin (IL)-1β in culture supernatant were measured by enzyme-linked immunosorbent assay. Northern blot analysis was performed to detect the expressions of TNF-α and IL-1β mRNAs. The activities of p38 MAPK and JNK (by Western blot analysis) as well as NF-κB (by EMSA) in KCs were also examined. As a result, HMGB1 in vitro upregulated expressions of TNF-α and IL-1β of KCs in a dose-dependent manner, and HMGB1 promoted KCs from burn rats to produce significantly more TNF-α and IL-1β proteins than those from sham animals. After harvested from burn rats, KCs were pre-incubated with anti-TLR2 or anti-TLR4 antibody prior to HMGB1 administration. HMGB1 exposure not only significantly increased expressions of TNF-α and IL-1β mRNAs in KCs from burn rats, but also enhanced activities of p38 MAPK, JNK and NF-κB. However, these upregulation events were all reduced by pre-incubation with anti-TLR2 or anti-TLR4 antibody. These results indicate that HMGB1 induces proinflammatory cytokines production of KCs after sever burn injury, and this process might be largely dependent on TLRs-dependent MAPKs/NF-κB signal pathway.