S100A8 may govern hyper-inflammation in severe COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic threatens human species with mortality rate of roughly 2%. We can hardly predict the time of herd immunity against and end of COVID-19 with or without success of vaccine. One way to overcome the situation is to define what delineates disease severity and serves as a molecular target. The most successful analogy is found in BCR-ABL in chronic myeloid leukemia, which is the golden biomarker, and simultaneously, the most effective molecular target. We hypothesize that S100 calcium-binding protein A8 (S100A8) is one such molecule. The underlying evidence includes accumulating clinical information that S100A8 is upregulated in severe forms of COVID-19, pathological similarities of the affected lungs between COVID-19 and S100A8-induced acute respiratory distress syndrome (ARDS) model, homeostatic inflammation theory in which S100A8 is an endogenous ligand for endotoxin sensor Toll-like receptor 4/Myeloid differentiation protein-2 (TLR4/MD-2) and mediates hyper-inflammation even after elimination of endotoxin-producing extrinsic pathogens, analogous findings between COVID-19-associated ARDS and pre-metastatic lungs such as S100A8 upregulation, pulmonary recruitment of myeloid cells, increased vascular permeability, and activation coagulation cascade. A successful treatment in an animal COVID-19 model is given with a reagent capable of abrogating interaction between S100A8/S100A9 and TLR4. In this paper, we try to verify our hypothesis that S100A8 governs COVID-19-associated ARDS.

MUC1 Mitigates Renal Injury and Inflammation in Endotoxin-Induced Acute Kidney Injury by Inhibiting the TLR4-MD2 Axis and Reducing Pro-inflammatory Macrophages Infiltration.

ABSTRACT: Sepsis is the leading cause of acute kidney injury (AKI) in critical care patients. A cornerstone of sepsis-associated AKI is dysregulated inflammation driven by excessive activation of Toll-like receptor 4 (TLR4) pathway. MUC1, a membrane-bound mucin expressed in both epithelial tubular cells and renal macrophages, has been shown to be involved in the regulation of TLRs. Therefore, we hypothesized that MUC1 could mitigate the renal inflammatory response to TLR4 activation. To test this hypothesis, we used a murine model of endotoxin-induced AKI by intraperitoneal injection of LPS. We showed that Muc1-/- mice have a more severe renal dysfunction, an increased activation of the tissular NF-kB pathway and secreted more pro inflammatory cytokines compare to Muc1+/+ mice. By flow cytometry, we observed that the proportion of M1 (pro-inflammatory) macrophages in the kidneys of Muc1-/- mice was significantly increased. In human and murine primary macrophages, we showed that MUC1 is only induced in M1 type macrophages and that macrophages derived from Muc1-/- mice secreted more pro-inflammatory cytokines. Eventually, in HEK293 cells, we showed that MUC1 cytosolic domain (CT) seems necessary for the negative regulation of TLR4 by proximity ligation assay, MUC1-CT is in close relationship with TLR4 and acts as a competitive inhibitor of the recruitment of MYD88. Overall our results support that in the context of endotoxin-induced AKI, MUC1 plays a significant role in controlling disease severity by regulating negatively the TLR4-MD2 axis.

Danshen (Salvia miltiorrhiza) restricts MD2/TLR4-MyD88 complex formation and signalling in acute myocardial infarction-induced heart failure.

Heart failure (HF) represents a major public health burden. Inflammation has been shown to be a critical factor in the progression of HF, regardless of the aetiology. Disappointingly, the majority of clinical trials targeting aspects of inflammation in patients with HF have been largely negative. Many clinical researches demonstrate that danshen has a good efficacy on HF, and however, whether danshen exerts anti-inflammatory effects against HF remains unclear. In our study, the employment of a water extracted and alcohol precipitated of danshen extract attenuated cardiac dysfunction and inflammation response in acute myocardial infarction-induced HF rats. Transcriptome technique and validation results revealed that TLR4 signalling pathway was involved in the anti-inflammation effects of danshen. In vitro, danshen reduced the release of inflammatory mediators in LPS-stimulated RAW264.7 macrophage cells. Besides, the LPS-stimulated macrophage conditioned media was applied to induce cardiac H9C2 cells injury, which could be attenuated by danshen. Furtherly, knock-down and overexpression of TLR4 were utilized to confirm that danshen ameliorated inflammatory injury via MyD88-dependent TLR4-TRAF6-NF-κB signalling pathway in cardiomyocytes. Furthermore, by utilizing co-immunoprecipitation, danshen was proved to suppress MD2/TLR4 complex formation and MyD88 recruitment. In conclusion, our results demonstrated that danshen ameliorates inflammatory injury by controlling MD2/TLR4-MyD88 complex formation and TLR4-TRAF6-NF-κB signalling pathway in acute myocardial infarction-induced HF.

Lovastatin inhibits Toll-like receptor 4 signaling in microglia by targeting its co-receptor myeloid differentiation protein 2 and attenuates neuropathic pain.

There is growing interest in drug repositioning to find new therapeutic indications for drugs already approved for use in people. Lovastatin is an FDA approved drug that has been used clinically for over a decade as a lipid-lowering medication. While lovastatin is classically considered to act as a hydroxymethylglutaryl (HMG)-CoA reductase inhibitor, the present series of studies reveal a novel lovastatin effect, that being as a Toll-like receptor 4 (TLR4) antagonist. Lovastatin selectively inhibits lipopolysaccharide (LPS)-induced TLR4-NF-κB activation without affecting signaling by other homologous TLRs. In vitro biophysical binding and cellular thermal shift assay (CETSA) show that lovastatin is recognized by TLR4's coreceptor myeloid differentiation protein 2 (MD-2). This finding is supported by molecular dynamics simulations that lovastatin targets the LPS binding pocket of MD-2 and lovastatin binding stabilizes the MD-2 conformation. In vitro studies of BV-2 microglial cells revealed that lovastatin inhibits multiple effects of LPS, including activation of NFkB; mRNA expression of tumor necrosis factor-a, interleukin-6 and cyclo-oxygenase 2; production of nitric oxide and reactive oxygen species; as well as phagocytic activity. Furthermore, intrathecal delivery of lovastatin over lumbosacral spinal cord of rats attenuated both neuropathic pain from sciatic nerve injury and expression of the microglial activation marker CD11 in lumbar spinal cord dorsal horn. Given the well-established role of microglia and proinflammatory signaling in neuropathic pain, these data are supportive that lovastatin, as a TLR4 antagonist, may be productively repurposed for treating chronic pain.

sTLR4/MD-2 complex inhibits colorectal cancer migration and invasiveness in vitro and in vivo by lncRNA H19 down-regulation.

Long non-coding RNAs (lncRNAs) have multiple functions in gene regulation and during cellular processes. However, the functional roles of lncRNAs in colorectal cancer (CRC) have not yet been well understood. In our previous study, we demonstrated that sTLR4/MD-2 complex can inhibit CRC in vitro and in vivo by targeting LPS. Therefore, the aim of the present study is to investigate the expression of lncRNA H19 in CRC and to evaluate its effect on the inhibition of sTLR4/MD-2 complex. The expression of H19 is measured in 63 CRC tumor tissues and adjacent normal tissues by quantitative real-time PCR (qRT-PCR). The effects of H19 on migration and invasiveness are evaluated by wound healing assay, migration and invasion assays. Results showed that H19 is significantly overexpressed in cancerous tissues and CRC cell lines compared with adjacent normal tissues and a normal human intestinal epithelial cell line. Moreover, H19 overexpression is closely associated with CRC patients. Our in vitro data indicated that knockdown of H19 inhibits the migration and invasiveness of CRC cells. And in vivo sTLR4/MD-2 complex inhibits tumor growth in mice and the expression of H19 is down-regulated. These results suggest that sTLR4/MD-2 complex inhibits CRC migration and invasiveness in vitro and in vivo by lncRNA H19 down-regulation.

Novel synthetic toll-like receptor 4/MD2 ligands attenuate sterile inflammation.

Toll-like receptor (TLR) stimulation has been implicated as a major contributor to chronic inflammation. Among these receptors, TLR4 has been described as a key regulator of endogenous inflammation and has been proposed as a therapeutic target. Previously, we discovered by high-throughput screening a group of substituted pyrimido[5,4-b]indoles that activated a nuclear factor-κB reporter in THP-1 human monocytic cells. A biologically active hit compound was resynthesized, and derivatives were prepared to assess structure-activity relationships. The derived compounds activated cells in a TLR4/myeloid differentiation protein 2 (MD2)-dependent and CD14-independent manner, using the myeloid differentiation primary response 88 and Toll/IL-1 receptor domain-containing adapter-inducing interferon-ß pathways. Two lead compounds, 1Z105 and 1Z88, were selected for further analysis based on favorable biologic properties and lack of toxicity. In vivo pharmacokinetics indicated that 1Z105 was orally bioavailable, whereas 1Z88 was not. Oral or parenteral doses of 1Z105 and 1Z88 induced undetectable or negligible levels of circulating cytokines and did not induce hepatotoxicity when administered to galactosamine-conditioned mice, indicating good safety profiles. Both compounds were very effective in preventing lethal liver damage in lipopolysaccharide treated galatosamine-conditioned mice. Orally administered 1Z105 and parenteral 1Z88 prevented arthritis in an autoantibody-driven murine model. Hence, these low molecular weight molecules that target TLR4/MD2 were well tolerated and effective in reducing target organ damage in two different mouse models of sterile inflammation.

Recombinant human annexin A5 inhibits proinflammatory response and improves cardiac function and survival in mice with endotoxemia.

OBJECTIVES: Annexin A5 is a 35-kDa protein with high affinity binding to negatively charged phospholipids. However, its effects on sepsis are not known. Our aim was to study the effects of annexin A5 on myocardial tumor necrosis factor-α expression, cardiac function, and animal survival in endotoxemia. DESIGN: Prospective experimental study. SETTING: University laboratory. SUBJECTS: Adult male C57BL/6 mice. INTERVENTIONS: Mice were challenged with lipopolysaccharide (4 or 20 mg/kg, i.p.) to induce endotoxemia with and without recombinant human annexin A5 treatment (5 or 10 µg/kg, i.v.). Cytokine expression and cardiac function were assessed, and animal survival was monitored. MEASUREMENTS AND MAIN RESULTS: Treatment with annexin A5 inhibited myocardial mitogen-activated protein kinase, and nuclear factor-κB activation in mice with endotoxemia. Furthermore, annexin A5-treated animals showed significant reductions in myocardial and plasma levels of tumor necrosis factor-α and interleukin-1ß while cardiac function was significantly improved during endotoxemia. Additionally, 5-day animal survival was significantly improved by either an immediate or a 4-hour delayed annexin A5 treatment after lipopolysaccharide challenge. Importantly, annexin A5 dose-dependently inhibited lipopolysaccharide binding to a toll-like receptor-4/myeloid differentiation factor 2 fusion protein. CONCLUSIONS: Annexin A5 treatment decreases cytokine expression and improves cardiac function and survival during endotoxemia. These effects of annexin A5 are mediated by its ability to inhibit lipopolysaccharide binding to toll-like receptor-4, leading to reductions in mitogen-activated protein kinase and Akt signaling. Our study suggests that annexin A5 may have therapeutic potential in the treatment of sepsis.

Antiinflammation effect of human placental multipotent mesenchymal stromal cells is mediated by prostaglandin E2 via a myeloid differentiation primary response gene 88-dependent pathway.

BACKGROUND: We sought to elucidate the antiinflammation effect of human placental multipotent mesenchymal stromal cells (hPMSCs) and the possible molecular mechanisms. METHODS: Immortalized murine macrophages (RAW264.7 cells), with or without hPMSCs coincubation, were treated with endotoxin to induce expression of the relevant molecules. RESULTS: The peak concentrations (means ± SD) of inflammatory molecules in endotoxin-activated macrophages with hPMSCs coincubation were significantly lower than those in endotoxin-activated macrophages without hPMSCs coincubation (tumor necrosis factor-α: 9.4±0.8 vs. 13.0±1.1 ng/ml; interleukin-6: 0.8±0.1 vs. 1.2±0.1 ng/ml; macrophage inflammatory protein-2: 345±30 vs. 666±51 ng/ml; intercellular adhesion molecule 1: 1.4±0.1 vs. 1.7±0.1 ng/ml; prostaglandin E2: 5.7±0.3 vs. 8.5±0.6 ng/ml; all P<0.008). Data of the activation of nuclear factor-κB and mitogen-activated protein kinases as well as the interaction between toll-like receptor 4 and myeloid differentiation primary response gene 88 paralleled those of the inflammatory molecules. In contrast, the endotoxin binding and toll-like receptor 4/myeloid differential-2 complex activation in endotoxin-activated macrophages with hPMSCs coincubation were comparable with those in endotoxin-activated macrophages without hPMSCs coincubation. As our data revealed that hPMSCs could induce low-grade prostaglandin E2 expression in macrophages, we also employed the selective cyclooxygenase-2 inhibitor NS-398 to further elucidate the possible role of prostaglandin E2. Our data revealed that the above-mentioned hPMSC-triggered inhibitory effects were significantly reversed by NS-398. CONCLUSIONS: The antiinflammation effect of human placental multipotent mesenchymal stromal cells is mediated, at least in part, by prostaglandin E2 via a myeloid differentiation primary response gene 88-dependent pathway.

Impact of sphingomyelin synthase 1 deficiency on sphingolipid metabolism and atherosclerosis in mice.

OBJECTIVE: Sphingomyelin synthase (SMS) catalyzes the conversion of ceramide to sphingomyelin and sits at the crossroads of sphingolipid biosynthesis. SMS has 2 isoforms: SMS1 and SMS2. Although they have the same SMS activity, they are different enzymes with distinguishable subcellular localizations and cell expression patterns. It is conceivable that these differences could yield different consequences, in terms of sphingolipid metabolism and its related atherogenesis. METHODS AND RESULTS: We created Sms1 gene knockout mice and found that Sms1 deficiency significantly decreased plasma, liver, and macrophage sphingomyelin (59%, 45%, and 54%, respectively), but only had a marginal effect on ceramide levels. Surprisingly, we found that Sms1 deficiency dramatically increased glucosylceramide and GM3 levels in plasma, liver, and macrophages (4- to 12-fold), whereas Sms2 deficiency had no such effect. We evaluated the total SMS activity in tissues and found that Sms1 deficiency causes 77% reduction in SMS activity in macrophages, indicating SMS1 is the major SMS in macrophages. Moreover, Sms1-deficient macrophages have a significantly higher glucosylceramide synthase activity. We also found that Sms1 deficiency significantly attenuated toll-like 4 receptor-mediated nuclear factor-κB and mitogen-activated protein kinase activation after lipopolysaccharide treatment. To evaluate atherogenicity, we transplanted Sms1 knockout mouse bone marrow into low-density lipoprotein receptor knockout mice (Sms1(-/-)→Ldlr(-/-)). After 3 months on a western diet, these animals showed a significant decrease of atherosclerotic lesions in the root and the entire aorta (35% and 44%, P<0.01, respectively) and macrophage content in lesions (51%, P<0.05), compared with wild-type→Ldlr(-/-) mice. CONCLUSIONS: Sms1 deficiency decreases sphingomyelin, but dramatically increases the levels of glycosphingolipids. Atherosclerosis in Sms1(-/-)→Ldlr(-/-) mice is significantly decreased.

Prolonged hepatomegaly in mice that cannot inactivate bacterial endotoxin.

UNLABELLED: Transient hepatomegaly often accompanies acute bacterial infections. Reversible, dose-dependent hepatomegaly also occurs when animals are given intravenous infusions of bacterial lipopolysaccharide (LPS). We found that recovery from LPS-induced hepatomegaly requires a host enzyme, acyloxyacyl hydrolase (AOAH), that inactivates LPS. When we challenged Aoah(-/-) mice with low doses of LPS or gram-negative bacteria, their livers remained enlarged (as much as 80% above normal) many weeks longer than did the livers of Aoah(+/+) animals. When compared with livers from LPS-primed Aoah(+/+) mice, LPS-primed Aoah(-/-) livers had (1) more numerous and larger Kupffer cells, (2) intrasinusoidal leukocyte aggregates and activated sinusoidal endothelial cells, and (3) sustained production of interleukin (IL)-10 and messenger RNAs (mRNAs) for tumor necrosis factor (TNF), IL-10, and IRAK-M. Depleting Kupffer cells decreased the liver enlargement by ≈40%, whereas depletion of neutrophils, dendritic cells, natural killer (NK) cells, NK-T cells, or B cells had no effect. Pretreatment with dexamethasone almost completely prevented prolonged hepatomegaly in Aoah(-/-) mice, whereas neutralizing TNF or interleukin-1ß was only partially effective. In contrast, an antagonistic antibody to the IL-10 receptor increased LPS-induced hepatomegaly by as much as 50%. CONCLUSION: our findings suggest that persistently active LPS induces Kupffer cells to elaborate mediators that promote the accumulation of leukocytes within enlarged sinusoids. Large increases in IL-10 and several other modulatory molecules are unable to prevent prolonged hepatomegaly in mice that cannot inactivate LPS. The striking findings in this mouse model should encourage studies to find out how AOAH contributes to human liver physiology and disease.

Evidence that tricyclic small molecules may possess toll-like receptor and myeloid differentiation protein 2 activity.

Opioids have been discovered to have Toll-like receptor (TLR) activity, beyond actions at classical opioid receptors. This raises the question whether other pharmacotherapies for pain control may also possess TLR activity, contributing to or opposing their clinical effects. We document that tricyclics can alter TLR4 and TLR2 signaling. In silico simulations revealed that several tricyclics docked to the same binding pocket on the TLR accessory protein, myeloid differentiation protein 2 (MD-2), as do opioids. Eight tricyclics were tested for effects on TLR4 signaling in HEK293 cells over-expressing human TLR4. Six exhibited mild (desipramine), moderate (mianserin, cyclobenzaprine, imiprimine, ketotifen) or strong (amitriptyline) TLR4 inhibition, and no TLR4 activation. In contrast, carbamazepine and oxcarbazepine exhibited mild and strong TLR4 activation, respectively, and no TLR4 inhibition. Amitriptyline but not carbamazepine also significantly inhibited TLR2 signaling in a comparable cell line. Live imaging of TLR4 activation in RAW264.7 cells and TLR4-dependent interleukin-1 release from BV-2 microglia revealed that amitriptyline blocked TLR4 signaling. Lastly, tricyclics with no (carbamazepine), moderate (cyclobenzeprine), and strong (amitriptyline) TLR4 inhibition were tested intrathecally (rats) and amitriptyline tested systemically in wildtype and knockout mice (TLR4 or MyD88). While tricyclics had no effect on basal pain responsivity, they potentiated morphine analgesia in rank-order with their potency as TLR4 inhibitors. This occurred in a TLR4/MyD88-dependent manner as no potentiation of morphine analgesia by amitriptyline occurred in these knockout mice. This suggests that TLR2 and TLR4 inhibition, possibly by interactions with MD2, contributes to effects of tricyclics in vivo. These studies provide converging lines of evidence that several tricyclics or their active metabolites may exert their biological actions, in part, via modulation of TLR4 and TLR2 signaling and suggest that inhibition of TLR4 and TLR2 signaling may potentially contribute to the efficacy of tricyclics in treating chronic pain and enhancing the analgesic efficacy of opioids.

Mutational analysis of Cys(88) of Toll-like receptor 4 highlights the critical role of MD-2 in cell surface receptor expression.

The role of MD-2 in cell surface expression of Toll-like receptor (TLR) 4 has been controversial. The purposes of this study were to characterize the N-glycan of TLR4 and to investigate the roles of MD-2 in N-linked glycosylation and cell surface expression of TLR4. Lectin blot and cell surface biotinylation revealed that TLR4 exhibited the 110 kDa protein with high mannose type N-glycans and the 130 kDa protein with complex type N-glycans and that only the 130 kDa TLR4 with complex type N-glycans was expressed on the cell surface. The cells transfected with a mutant TLR4(C88A) alone expressed only the 110 kDa TLR4 with a high mannose type N-glycan, which did not appear on the cell surface. However, TLR4(C88A) acquired complex type N-glycans and was expressed on the cell surface when MD-2 was co-transfected. The amount of the 130 kDa TLR4(C88A) with complex type N-glycans expressed on the cell surface depended on that of MD-2 transfected. alpha-Mannosidase II inhibitor blocked the processing N-glycans to complex type, but TLR4 with high mannose type appeared on the cell surface, suggesting that TLR4 is destined to locate on the cell surface before processing N-glycans from a high mannose type to a complex type. From these results, we conclude that MD-2 is critical for cell surface expression of TLR4(C88A). This study provides evidence that MD-2 possesses potential ability to play an essential role in cell surface expression of TLR4.

Somatostatin limits intestinal ischemia-reperfusion injury in macaques via suppression of TLR4-NF-kappaB cytokine pathway.

OBJECTIVE: Intestinal ischemia-reperfusion (IIR)-induced gut injury remains a challenge for critically ill patients despite the oxidative stress theory that has been elaborated. This study aimed to test whether Toll-like receptor 4 (TLR4) is involved in gut injury during IIR and whether somatostatin (SST) affects TLR4-nuclear factor-kappaB (NF-kappaB) cytokine pathway in the intestinal mucosa of macaques. DESIGN: Fifteen macaques were randomized into control, IIR, and SST + IIR groups. Pieces of isolated ileal epithelium from each animal were incubated with lipopolysaccharide (LPS), interferon-gamma, or SST. Expression of TLR4 and NF-kappaBp65 was evaluated by immunohistochemical staining, Western blot analysis and reverse transcription polymerase chain reaction. Cytokine levels were measured by ELISA. Radioimmunoassay was used to determine of SST levels. MEASUREMENTS AND MAIN RESULTS: Significant overexpression (IIR vs control) of ileal TLR4 (0.17 +/- 0.03 vs 0.05 +/- 0.02), NF-kappaBp65 (0.55 +/- 0.11 vs 0.15 +/- 0.05), and TNF-alpha (213.2 +/- 29.2 vs 56.0 +/- 10.04) after IIR was greatly decreased (p < 0.05) by prophylactic use of SST (TLR4: 0.06 +/- 0.02; NF-kappaBp65: 0.26 +/- 0.09; TNF-alpha: 97.1 +/- 32.3) in vivo. TLR4 expression in the ileal epithelium treated with LPS and SST (1,330 +/- 93) was significantly lower than that in the ileal epithelium treated with LPS alone (2,088 +/- 126) in vitro. SST levels in plasma (3.67 +/- 0.41 ng/ml) and ileal mucosa (1,402.3 +/- 160 ng/mg protein) of the IIR group were significantly lower than those (6.09 +/- 1.29 ng/ml, 2,234. 8 +/- 301.8 ng/mg protein) in the control group (p < 0.05). CONCLUSIONS: Endogenous SST is a crucial inhibitor of massive inflammatory injury in the intestinal mucosa via direct suppression of the TLR4-NF-kappaB cytokine pathway induced by LPS in ileal epithelium. IIR attacks caused shortages of endogenous SST in the plasma and intestinal mucosa of macaques in this study. Therefore, preventive supplements of SST may limit intestinal injury of macaques by IIR.

MD-2 controls bacterial lipopolysaccharide hyporesponsiveness in human intestinal epithelial cells.

Intestinal epithelial cells (IEC) have adapted to the presence of commensal bacteria through a state of tolerance that involves a limited response to lipopolysaccharide (LPS). Low or absent expression of two LPS receptor molecules, the myeloid differentiation (MD)-2 receptor, and toll-like receptor (TLR)4 was suggested to underlie LPS tolerance in IEC. In the present study we performed transfections of TLR4 and MD-2 alone or combined in different IEC lines derived from intestinal cancer (Caco-2, HT-29, and SW837). We found that LPS responsiveness increased more than 100-fold when IEC were transfected with MD-2 alone, but not TLR4. The release of interleukin (IL)-8, but also the expression of cyclooxygenase (Cox-)2 and the related secretion of prostaglandin (PG)E2 were coordinately stimulated by LPS in IEC transfected with MD-2 alone. Supernatants collected from MD-2-transfected IEC supported LPS activation of naïve HT-29, providing additional support to the concept that MD-2 alone endows IEC with LPS responsiveness. LPS responsiveness detected at concentrations as low as 110 pg/ml, and maximal values obtained by 10 ng/ml were clearly beyond those evoked by classical stimuli as IL-1beta. In polarized cells, apical LPS stimulation was markedly more efficient than basolateral. Our data contradict previous opinion that both TLR4 and MD-2 limit IEC response to LPS, and emphasize the prominent role of MD-2 in intestinal immune responses to Gram-negative bacteria.

Recognition of hyaluronan released in sterile injury involves a unique receptor complex dependent on Toll-like receptor 4, CD44, and MD-2.

Inflammation under sterile conditions is not well understood despite its importance in trauma and autoimmune disease. To investigate this process we established mouse models of sterile injury and explored the role of hyaluronan in mediating inflammation following injury. The response of cultured monocytes to hyaluronan was different than the response to lipopolysaccharide (LPS) despite both being dependent on Toll-like receptor 4 (TLR4). Cultured cells exposed to hyaluronan showed a pattern of gene induction that mimics the response seen in mouse skin after sterile injury with an increase in molecules such as transforming growth factor-beta2 and matrix metalloproteinase-13. These factors were not induced by LPS despite the mutual dependence of both hyaluronan and LPS on TLR4. Explanation for the unique response to hyaluronan was provided by observations that a lack of TLR4 or CD44 in mice diminished the response to sterile injury, and together with MD-2, was required for responsiveness to hyaluronan in vitro. Thus, a unique complex of TLR4, MD-2, and CD44 recognizes hyaluronan. Immunoprecipitation experiments confirmed the physical association of TLR4 and CD44. Taken together, our results define a previously unknown mechanism for initiation of sterile inflammation that involves recognition of released hyaluronan fragments as an endogenous signal of tissue injury.

Analysis of TLR4 polymorphic variants: new insights into TLR4/MD-2/CD14 stoichiometry, structure, and signaling.

TLR4 is the signal-transducing receptor for structurally diverse microbial molecules such as bacterial LPS, respiratory syncytial virus fusion (F) protein, and chlamydial heat shock protein 60. Previous studies associated two polymorphic mutations in the extracellular domain of TLR4 (Asp(299)Gly and Thr(399)Ile) with decreased LPS responsiveness. To analyze the molecular basis for diminished responsiveness, site-specific mutations (singly or coexpressed) were introduced into untagged and epitope (Flag)-tagged wild-type (WT) TLR4 expression vectors to permit a direct comparison of WT and mutant signal transduction. Coexpression of WT TLR4, CD14, and MD-2 expression vectors in HEK293T cells was first optimized to achieve optimal LPS-induced NF-kappaB reporter gene expression. Surprisingly, transfection of cells with MD-2 at high input levels often used in the literature suppressed LPS-induced signaling, whereas supraoptimal CD14 levels did not. Under conditions where WT and polymorphic variants were comparably expressed, significant differences in NF-kappaB activation were observed in response to LPS and two structurally unrelated TLR4 agonists, chlamydial heat shock protein 60 and RSV F protein, with the double, cosegregating mutant TLR4 exhibiting the greatest deficiency. Overexpression of Flag-tagged WT and mutant vectors at input levels resulting in agonist-independent signaling led to equivalent NF-kappaB signaling, suggesting that these mutations in TLR4 affect appropriate interaction with agonist or coreceptor. These data provide new insights into the importance of stoichiometry among the components of the TLR4/MD-2/CD14 complex. A structural model that accounts for the diminished responsiveness of mutant TLR4 polymorphisms to structurally unrelated TLR4 agonists is proposed.

[Induction of apoptosis in human head and neck cancer cell lines by an active component of OK-432 through p53-independent pathway via toll-like receptor (TLR) 4 signaling].

OK-PSA, an active component of OK-432, induces anti-tumor immunity via Toll-like receptor (TLR) 4/MD-2 complex. In the current study, we evaluated the effect of the OK-PSA on human head and neck cancer cell lines. Twelve cancer cell lines including 7 squamous cell carcinoma (SCC) cell lines and 5 salivary gland cancer (SGC) cell lines were examined. The quantitative real-time PCR analysis revealed that TLR4 mRNA was expressed in all 12 cell lines, and that MD-2 mRNA was expressed in 5 cell lines. OK-PSA stimulation resulted in the activation of NF-kappaB in the 4 SCC cell lines which express both TLR4 and MD-2 genes, and in 5 SGC cell lines which express at least TLR4 gene independently of MD-2 expression. In these OK-PSA-responsive cell lines, OK-PSA activated caspase-1, caspase-3 and caspase-8, and induced apoptosis. OK-PSA-induced apoptosis were observed even in a SGC cell line in which p53 is mutated and its function is impaired. These findings strongly suggest that OK-PSA induces apoptosis by the activation of caspases through p53-independent pathway via TLR4 signaling in head and neck cancer cells.