Ginsenoside Rh2 Alleviates LPS-Induced Inflammatory Responses by Binding to TLR4/MD-2 and Blocking TLR4 Dimerization.

Lipopolysaccharide (LPS) triggers a severe systemic inflammatory reaction in mammals, with the dimerization of TLR4/MD-2 upon LPS stimulation serving as the pivotal mechanism in the transmission of inflammatory signals. Ginsenoside Rh2 (G-Rh2), one of the active constituents of red ginseng, exerts potent anti-inflammatory activity. However, whether G-Rh2 can block the TLR4 dimerization to exert anti-inflammatory effects remains unclear. Here, we first investigated the non-cytotoxic concentration of G-Rh2 on RAW 264.7 cells, and detected the releases of pro-inflammatory cytokines in LPS-treated RAW 264.7 cells, and then uncovered the mechanisms involved in the anti-inflammatory activity of G-Rh2 through flow cytometry, fluorescent membrane localization, Western blotting, co-immunoprecipitation (Co-IP), molecular docking and surface plasmon resonance (SPR) analysis in LPS-stimulated macrophages. Our results show that G-Rh2 stimulation markedly inhibited the secretion of LPS-induced interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and nitric oxide (NO). Additionally, G-Rh2 blocked the binding of LPS with the membrane of RAW 264.7 cells through direct interaction with TLR4 and MD-2 proteins, leading to the disruption of the dimerization of TLR4 and MD-2, followed by suppression of the TLR4/NF-κB signaling pathway. Our results suggest that G-Rh2 acts as a new inhibitor of TLR4 dimerization and may serve as a promising therapeutic agent against inflammation.

Cyclic-di-GMP induces inflammation and acute lung injury through direct binding to MD2.

BACKGROUND: Severe bacterial infections can trigger acute lung injury (ALI) and acute respiratory distress syndrome, with bacterial pathogen-associated molecular patterns (PAMPs) exacerbating the inflammatory response, particularly in COVID-19 patients. Cyclic-di-GMP (CDG), one of the PAMPs, is synthesized by various Gram-positve and Gram-negative bacteria. Previous studies mainly focused on the inflammatory responses triggered by intracellular bacteria-released CDG. However, how extracellular CDG, which is released by bacterial autolysis or rupture, activates the inflammatory response remains unclear. METHODS: The interaction between extracellular CDG and myeloid differentiation protein 2 (MD2) was investigated using in vivo and in vitro models. MD2 blockade was achieved using specific inhibitor and genetic knockout mice. Site-directed mutagenesis, co-immunoprecipitation, SPR and Bis-ANS displacement assays were used to identify the potential binding sites of MD2 on CDG. RESULTS: Our data show that extracellular CDG directly interacts with MD2, leading to activation of the TLR4 signalling pathway and lung injury. Specific inhibitors or genetic knockout of MD2 in mice significantly alleviated CDG-induced lung injury. Moreover, isoleucine residues at positions 80 and 94, along with phenylalanine at position 121, are essential for the binding of MD2 to CDG. CONCLUSION: These results reveal that extracellular CDG induces lung injury through direct interaction with MD2 and activation of the TLR4 signalling pathway, providing valuable insights into bacteria-induced ALI mechanisms and new therapeutic approaches for the treatment of bacterial co-infection in COVID-19 patients.

Nicotine Enantioselectively Targets Myeloid Differentiation Protein 2 and Inhibits the Toll-like Receptor 4 Signaling.

Psychoactive substances, including morphine and methamphetamine, have been shown to interact with the classic innate immune receptor Toll-like receptor 4 (TLR4) and its partner protein myeloid differentiation protein 2 (MD2) in a nonenantioselective manner. (-)-Nicotine, the primary alkaloid in tobacco and a key component of highly addictive cigarettes, targets the TLR4/MD2, influencing TLR4 signaling pathways. Existing as two enantiomers, the stereoselective recognition of nicotine by TLR4/MD2 in the context of the innate immune response remains unclear. In this study, we synthesized (+)-nicotine and investigated its effects alongside (-)-nicotine on lipopolysaccharide (LPS)-induced TLR4 signaling. (-)-Nicotine dose-dependently inhibited proinflammatory factors such as tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and cyclooxygenase-2 (COX-2). In contrast, (+)-nicotine showed no such inhibitory effects. Molecular dynamics simulations revealed that (-)-nicotine exhibited a stronger affinity with the TLR4 coreceptor MD2 than (+)-nicotine. Additionally, in silico simulations revealed that both nicotine enantiomers initially attach to the entrance of the MD2 cavity, creating a metastable state before they fully enter the cavity. In the metastable state, (-)-nicotine established more stable interactions with the surrounding residues at the entrance of the MD2 cavity compared to those of (+)-nicotine. This highlights the crucial role of the MD2 cavity entrance in the chiral recognition of nicotine. These findings provide valuable insights into the distinct interactions between nicotine enantiomers and the TLR4 coreceptor MD2, underscoring the enantioselective effect of nicotine on modulating TLR4 signaling.

Dissecting the Role of the Hydroxyl Moiety at C14 in (+)-Opioid-Based TLR4 Antagonists via Wet-Lab Experiments and Molecular Dynamics Simulations.

Toll-like receptor 4 (TLR4) is pivotal as an innate immune receptor, playing a critical role in mediating neuropathic pain and drug addiction through its regulation of the neuroinflammatory response. The nonclassical (+)-opioid isomers represent a unique subset of TLR4 antagonists known for their effective blood-brain barrier permeability. Despite growing interest in the structure-activity relationship of these (+)-opioid-based TLR4 antagonists, the specific impact of heteroatoms on their TLR4 antagonistic activities has not been fully explored. This study investigated the influence of the hydroxyl group at C14 in six (+)-opioid TLR4 antagonists (1-6) using wet-lab experiments and in silico simulations. The corresponding C14-deoxy derivatives (7-12) were synthesized, and upon comparison with their corresponding counterparts (1-6), it was discovered that their TLR4 antagonistic activities were significantly diminished. Molecular dynamics simulations showed that the (+)-opioid TLR4 antagonists (1-6) possessed more negative binding free energies to the TLR4 coreceptor MD2, which was responsible for ligand recognition. This was primarily attributed to the formation of a hydrogen bond between the hydroxyl group at the C-14 position of the antagonists (1-6) and the R90 residue of MD2 during the binding process. Such an interaction facilitated the entry and subsequent binding of these molecules within the MD2 cavity. In contrast, the C14-deoxy derivatives (7-12), lacking the hydroxyl group at the C-14 position, missed this crucial hydrogen bond interaction with the R90 residue of MD2, leading to their egression from the MD2 cavity during simulations. This study underscores the significant role of the C14 hydroxyl moiety in enhancing the effectiveness of (+)-opioid TLR4 antagonists, which provides insightful guidance for designing future (+)-isomer opioid-derived TLR4 antagonists.

Screening of Poria cocos polysaccharide with immunomodulatory activity and its activation effects on TLR4/MD2/NF-κB pathway.

PCP-W1, the Poria cocos polysaccharide with the strong immunomodulatory activity, was isolated through column chromatography and screened for in vitro immune activity in RAW 264.7 cells in this study. The structure analysis results revealed that the PCP-W1 were composed of galactose, glucose, fucose and mannose in a molar percentage of 35.87: 28.56: 21.77: 13.64. And it exhibited a random coil and branched conformational features with a molecular weight of 18.38 kDa. The main chain consisted of residues→3)-ß-D-Glcp-(1 â†’ 3,6)-ß-D-Glcp-(1 â†’ 3)-ß-D-Glcp-(1 â†’ 6)-ß-D-Glcp-(1 â†’ 6)-α-D-Galp-(1 â†’ 6)-α-D-Galp-(1 â†’ 2,6)-α-D-Galp-(1→6)-α-D-Galp-(1 â†’ 6)-α-D-Galp-(1 â†’ , while branching occurred at ß-D-Glcp-(1→, α-D-Manp-(1→, and α-L-Fucp-(1 â†’ 3)- α-L-Fucp-(1→. The pharmacodynamic studies demonstrated that PCP-W1 activated the release of NO, IL-6, IL-ß, TNF-α, CD86, and ROS to induce polarization of RAW 264.7 murine macrophages towards M1-type through modulation of the TLR4/MD2/NF-κB pathway. The molecular docking results showed that PCP-W1 could primarily dock onto the hydrophobic binding site of TLR4/MD2 complex via its galactose chain. Furthermore, molecular dynamics simulation displayed stable modeling for TLR4-MD2-PCP-W1 complex. Overall, we screened the most immunoactive components of the polysaccharide, analyzed its structure, demonstrated its impact on TLR4/MD2/NF-kB pathway, and studied the interaction between TLR4/MD2 and the polysaccharide fragments. These results provide further support for the structure-activity relationship study of the immunomodulatory effects of Poria cocos polysaccharide.

Overexpression of MD1 ameliorates pathological myocardial remodeling in diabetic cardiomyopathy by TLR4/STAT3 signaling pathway.

Diabetic cardiomyopathy (DCM) is characterized by oxidative damage and inflammatory responses. Myeloid differentiation protein 1 (MD1) exhibits antioxidant and anti-inflammatory properties. However, the specific role of MD1 in DCM has yet to be elucidated. This study aims to investigate the role of MD1 in DCM and to elucidate the underlying mechanisms. We utilized a gain-of-function approach to explore the involvement of MD1 in DCM. Diabetes was induced in MD1-transgenic (MD1-TG) mice and their wild-type (WT) counterparts via streptozotocin (STZ) injection. Additionally, a diabetes cell model was established using H9c2 cells exposed to high glucose levels. We conducted comprehensive evaluations, including pathological analyses, echocardiography, electrocardiography, and molecular assessments, to elucidate the underlying mechanisms of MD1 in DCM. Notably, MD1 expression was reduced in the hearts of STZ-induced diabetic mice. Overexpression of MD1 significantly improved cardiac function and markedly inhibited ventricular pathological hypertrophy and fibrosis in these mice. Furthermore, MD1 overexpression resulted in a substantial decrease in myocardial reactive oxygen species (ROS) accumulation, mitigating myocardial oxidative stress and reducing the levels of inflammation-related markers such as IL-1ß, IL-6, and TNF-α. Mechanistically, MD1 overexpression inhibited the activation of the TLR4/STAT3 signaling pathway, as demonstrated in both in vivo and in vitro experiments. The overexpression of MD1 significantly impeded pathological cardiac remodeling and improved cardiac function in STZ-induced diabetic mice. This effect was primarily attributed to a reduction in ROS accumulation and mitigation of myocardial oxidative stress and inflammation, facilitated by the inhibition of the TLR4/STAT3 signaling pathway.

Retracted: MD-1 Deficiency Accelerates Myocardial Inflammation and Apoptosis in Doxorubicin-Induced Cardiotoxicity by Activating the TLR4/MAPKs/Nuclear Factor kappa B (NF-κB) Signaling Pathway.

This publication has been retracted by the Editor due to the identification of non-original figure images and manuscript content that raise concerns regarding the credibility and originality of the study and the manuscript. Reference: Ying-Jun Zhang, He Huang, Yu Liu, Bin Kong, Guangji Wang. MD-1 Deficiency Accelerates Myocardial Inflammation and Apoptosis in Doxorubicin-Induced Cardiotoxicity by Activating the TLR4/MAPKs/Nuclear Factor kappa B (NF-kappaB) Signaling Pathway. Med Sci Monit, 2019; 25: 7898-7907. DOI: 10.12659/MSM.919861.

In Silico Analyses Indicate a Lower Potency for Dimerization of TLR4/MD-2 as the Reason for the Lower Pathogenicity of Omicron Compared to Wild-Type Virus and Earlier SARS-CoV-2 Variants.

The SARS-CoV-2 Omicron variants have replaced all earlier variants, due to increased infectivity and effective evasion from infection- and vaccination-induced neutralizing antibodies. Compared to earlier variants of concern (VoCs), the Omicron variants show high TMPRSS2-independent replication in the upper airway organs, but lower replication in the lungs and lower mortality rates. The shift in cellular tropism and towards lower pathogenicity of Omicron was hypothesized to correlate with a lower toll-like receptor (TLR) activation, although the underlying molecular mechanisms remained undefined. In silico analyses presented here indicate that the Omicron spike protein has a lower potency to induce dimerization of TLR4/MD-2 compared to wild type virus despite a comparable binding activity to TLR4. A model illustrating the molecular consequences of the different potencies of the Omicron spike protein vs. wild-type spike protein for TLR4 activation is presented. Further analyses indicate a clear tendency for decreasing TLR4 dimerization potential during SARS-CoV-2 evolution via Alpha to Gamma to Delta to Omicron variants.

Structure-Based Design and Synthesis of Lipid A Derivatives to Modulate Cytokine Responses.

Agonists of Toll like receptors (TLRs) have attracted interest as adjuvants and immune modulators. A crystal structure of TLR4/MD2 with E. coli LPS indicates that the fatty acid at C-2 of the lipid A component of LPS induces dimerization of two TLR4-MD2 complexes, which in turn initiates cell signaling leading to the production of (pro)inflammatory cytokines. To probe the importance of the (R)-3-hydroxymyristate at C-2 of lipid A, a range of bis- and mono-phosphoryl lipid A derivatives with different modifications at C-2 were prepared by a strategy in which 2-methylnaphthyl ethers were employed as permanent protecting group that could be readily removed by catalytic hydrogenation. The C-2 amine was protected as 9-fluorenylmethyloxycarbamate, which at a later stage could be removed to give a free amine that was modified by different fatty acids. LPS and the synthetic lipid As induced the same cytokines, however, large differences in activity were observed. A compound having a hexanoyl moiety at C-2 still showed agonistic properties, but further shortening to a butanoyl abolished activity. The modifications had a larger influence on monophosphoryl lipid As. The lipid As having a butanoyl moiety at C-2 could selectively antagonize TRIF associated cytokines induced by LPS or lipid A.

Dried tangerine peel polysaccharide (DTPP) alleviates hepatic steatosis by suppressing TLR4/MD-2-mediated inflammation and endoplasmic reticulum stress.

Non-alcoholic fatty liver disease (NAFLD) is a complex pathogenic metabolic syndrome characterized by increased inflammation and endoplasmic reticulum stress. In recent years, natural polysaccharides derived from traditional Chinese medicine have shown significant anti-inflammatory effects, making them an attractive therapeutic option. However, little research has been conducted on the therapeutic potential of dried tangerine peel polysaccharide (DTPP) - one of the most important medicinal resources in China. The results of the present study showed that DTPP substantially reduced macrophage infiltration in vivo and suppressed the expression of pro-inflammatory factors and endoplasmic reticulum stress-related genes. Additionally, surface plasmon resonance analysis revealed that DTPP had a specific affinity to myeloid differentiation factor 2, which consequently suppressed lipopolysaccharide-induced inflammation via interaction with the toll-like receptor 4 signaling pathway. This study provides a potential molecular mechanism underlying the anti-inflammatory effects of DTPP on NAFLD and suggests DTPP as a promising therapeutic strategy for NAFLD treatment.

Gastrodin Attenuates Colitis and Prevents Tumorigenesis in Mice by Interrupting TLR4/MD2/NF-κB Signaling Transduction.

INTRODUCTION: Chronic inflammation is one of the causative factors for tumorigenesis. Gastrodin is a main active ingredient isolated from Gastrodia elata Blume, a famous medicinal herb with a long edible history. AIM: This study aimed to explore the effects of gastrodin on colitis-associated carcinogenesis (CRC) in mice and to elucidate its potential molecular mechanisms. METHODS: Balb/c mice were induced with azoxymethane (AOM) and dextran sulfate sodium (DSS) for 12 weeks. Gastrodin (50 mg/kg) was administered via oral gavage three times per week until the end of the experiment. Disease indexes, including body weight, bloody diarrhea, colon length, histopathological score, and tumor size, were measured. Tumor cell proliferation was evaluated by BrdU incorporation assay and tumor cell cytotoxicity was assessed by cell counting kit (CCK-8). The expression levels of toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) signaling molecules, NF-κB luciferase, and pro-inflammatory cytokines were determined by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), immunoblotting, immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), or reporter gene assays. The binding affinity between gastrodin and myeloid differentiation protein-2 (MD2) was analyzed by molecular docking and cellular thermal shift assay (CETSA). RESULTS: Gastrodin administration was demonstrated to mitigate various CRC-related symptoms in mice, including weight loss, diarrhea, and tissue abnormalities. Notably, gastrodin suppressed tumor cell growth during colitis- associated tumorigenesis, resulting in fewer and smaller adenomas in the colon. Unlike irinotecan, a broadspectrum antitumor drug, gastrodin did not exhibit apparent cytotoxicity in various colorectal adenocarcinoma cell lines. Additionally, gastrodin downregulated TLR4/NF-κB signaling molecules and pro-inflammatory mediators in mice and macrophages. Molecular docking and CETSA experiments suggested that gastrodin binds to the MD2 protein, potentially interfering with the recognition of lipopolysaccharide (LPS) by TLR4, leading to NF-κB pathway inhibition. CONCLUSION: This study provides evidence for the first time that gastrodin attenuated colitis and prevented colitisrelated carcinogenesis in mice, at least partially, by diminishing tumor-promoting cytokines through the interruption of TLR4/MD2/NF-κB signaling transduction.

Dehydrocostus lactone alleviates irinotecan-induced intestinal mucositis by blocking TLR4/MD2 complex formation.

BACKGROUND: Irinotecan (CPT-11) is used as chemotherapeutic drug for treatment of colorectal cancer. However, without satisfactory treatments, its gastrointestinal toxicities such as diarrhea and intestinal inflammation severely restrained its clinical application. Roots of Aucklandia lappa Decne. are used as traditional Chinese medicine to relieve gastrointestinal dysfunction and dehydrocostus lactone (DHL) is one of its main active components. Nevertheless, the efficacy and mechanism of DHL against intestinal mucositis remains unclear. PURPOSE: The present study aimed to investigate the protective effects of DHL on CPT-11-induced intestinal mucositis and its underlying mechanisms. METHODS: The protective effect of DHL was investigated in CPT-11-induced mice and lipopolysaccharide (LPS)+CPT-11 induced THP-1 macrophages. Body weight, diarrhea score, survival rate, colon length, and histopathological changes in mice colon and jejunum were analyzed to evaluate the protective effect of DHL in vivo. And DHL on reducing inflammatory response and regulating TLR4/NF-κB/NLRP3 pathway in vivo and in vitro were explored. Moreover, DHL on the interaction between TLR4 and MD2 was investigated. And silencing TLR4 targeted by siRNA was performed to validate the mechanisms of DHL on regulating the inflammation. RESULTS: DHL prevented CPT-11-induced intestinal damage, represented by reducing weight loss, diarrhea score, mortality rate and the shortening of the colon. Histological analysis confirmed that DHL prevented intestinal epithelial injury and improved the intestinal barrier function in CPT-11 induced mice. Besides, DHL significantly downregulated the level of inflammatory cytokines by inhibiting TLR4/NF-κB/NLRP3 signaling pathway in CPT-11-induced mice and LPS+CPT-11-induced THP-1 macrophages. In addition, DHL blocked TLR4/MD2 complex formation. Molecular docking combined with SIP and DARTS assay showed that DHL could bind to TLR4/MD2 and occludes the hydrophobic pocket of MD2. Furthermore, Silencing TLR4 abrogated the effect of DHL on LPS+CPT-11 induced inflammatory response in THP-1 macrophages. Additionally, DHL ameliorate the CPT-11-induced intestinal mucositis without affecting the anti-tumor efficacy of CPT-11 in the tumor xenograft mice. CONCLUSION: This study found that DHL exhibited the anti-inflammatory effects in CPT-11-induced intestinal mucositis by inhibiting the formation of TLR4/MD2 complex and then regulation of NF-κB/NLRP3 signaling pathway. DHL is potentially served as a novel strategy of combined medication with CPT-11.

Insights into the molecular basis and mechanism of heme-triggered TLR4 signalling: The role of heme-binding motifs in TLR4 and MD2.

Haemolytic disorders, such as sickle cell disease, are accompanied by the release of high amounts of labile heme into the intravascular compartment resulting in the induction of proinflammatory and prothrombotic complications in affected patients. In addition to the relevance of heme-regulated proteins from the complement and blood coagulation systems, activation of the TLR4 signalling pathway by heme was ascribed a crucial role in the progression of these pathological processes. Heme binding to the TLR4-MD2 complex has been proposed recently, however, essential mechanistic information of the processes at the molecular level, such as heme-binding kinetics, the heme-binding capacity and the respective heme-binding sites (HBMs) is still missing. We report the interaction of TLR4, MD2 and the TLR4-MD2 complex with heme and the consequences thereof by employing biochemical, spectroscopic, bioinformatic and physiologically relevant approaches. Heme binding occurs transiently through interaction with up to four HBMs in TLR4, two HBMs in MD2 and at least four HBMs in their complex. Functional studies highlight that mutations of individual HBMs in TLR4 preserve full receptor activation by heme, suggesting that heme interacts with TLR4 through different binding sites independently of MD2. Furthermore, we confirm and extend the major role of TLR4 for heme-mediated cytokine responses in human immune cells.

TLR4 and MD2 variation among horses with differential TNFα baseline concentrations and response to intravenous lipopolysaccharide infusion.

Gram-negative bacterial septicemia is mediated through binding of lipopolysaccharide (LPS) to mammalian toll-like receptor protein 4 (TLR4). TLR4 and its cognate protein, myeloid differentiation factor 2 (MD2) form a heterodimeric complex after binding LPS. This complex induces a cascade of reactions that results in increased proinflammatory cytokine gene expression, including TNFα, which leads to activation of innate immunity. In horses, the immune response to LPS varies widely. To determine if this variation is due to differences in TLR4 or MD2, DNA from 15 healthy adult horses with different TNFα dynamics after experimental intravenous LPS infusion was sequenced across exons of TLR4 and MD2. Haplotypes were constructed for both genes using all identified variants. Four haplotypes were observed for each gene. No significant associations were found between either TNFα baseline concentrations or response to LPS and haplotype; however, there was a significant association (P value = 0.0460) between the baseline TNFα concentration and one MD2 missense variant. Three-dimensional structures of the equine TLR4-MD2-LPS complex were built according to haplotype combinations observed in the study horses, and the implications of missense variants on LPS binding were modeled. Although the sample size was small, there was no evidence that variation in TLR4 or MD2 explains the variability in TNFα response observed after LPS exposure in horses.

Radix Glycyrrhizae extract and licochalcone a exert an anti-inflammatory action by direct suppression of toll like receptor 4.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Glycyrrhizae (GL), a herbal medicine that is widely available, has shown advantages for a variety of inflammatory diseases. Toll like receptor 4 (TLR4) pathway has been shown to play a key role in the progression of inflammation. AIM OF THE STUDY: The purpose of this study was to investigate the involvement of TLR4 in the anti-inflammatory mechanism of GL extract and its active constituent on acute lung injury (ALI). MATERIALS AND METHODS: A model of inflammation produced by lipopolysaccharide (LPS) was established in C57BL/6 mice and macrophages derived from THP-1. To screen the active components of GL, molecular docking was used. Molecular dynamics and surface plasmon resonance imaging (SPRi) were used to study the interaction of a specific drug with the TLR4-MD2 complex. TLR4 was overexpressed by adenovirus to confirm TLR4 involvement in the anti-inflammatory activities of GL and the chosen chemical. RESULTS: We observed that GL extract significantly reduced both LPS-induced ALI and the production of pro-inflammatory factors including TNF-α, IL-6 and IL-1ß. Additionally, GL inhibited the binding of Alexa 488-labeled LPS (LPS-488) to the membrane of THP-1 derived macrophages. GL drastically reduce on the expression of TLR4 and the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-B (NF-κB). Furthermore, molecular docking revealed that Licochalcone A (LicoA) docked into the LPS binding site of TLR4-MD2 complex. MD2-LicoA binding conformation was found to be stable using molecular dynamic simulations. SPRi indicated that LicoA bound to TLR4-MD2 recombinant protein with a KD of 3.87 × 10-7 M. LicoA dose-dependently reduced LPS-488 binding to the cell membrane. LicoA was found to significantly inhibit LPS-induced lung damage and inflammation. Furthermore, LicoA inhibited TLR4 expression, MAPK and NF-κB activation in a dose-dependent manner. The inhibitory effects of GL and LicoA on LPS-induced inflammation and TLR4 signaling activation were partly eliminated by TLR4 overexpression. CONCLUSION: Our findings imply that GL and LicoA exert inhibitory effects on inflammation by targeting the TLR4 directly.

Cisplatin Toxicity Is Mediated by Direct Binding to Toll-Like Receptor 4 through a Mechanism That Is Distinct from Metal Allergens.

Cisplatin is an effective chemotherapeutic agent, yet its use is limited by several adverse drug reactions, known as cisplatin-induced toxicities (CITs). We recently demonstrated that cisplatin could elicit proinflammatory responses associated with CITs through Toll-like receptor 4 (TLR4). TLR4 is best recognized for binding bacterial lipopolysaccharide (LPS) via its coreceptor, MD-2. TLR4 is also proposed to directly bind transition metals, such as nickel. Little is known about the nature of the cisplatin-TLR4 interaction. Here, we show that soluble TLR4 was capable of blocking cisplatin-induced, but not LPS-induced, TLR4 activation. Cisplatin and nickel, but not LPS, were able to directly bind soluble TLR4 in a microscale thermophoresis binding assay. Interestingly, TLR4 histidine variants that abolish nickel binding reduced, but did not eliminate, cisplatin-induced TLR4 activation. This was corroborated by binding data that showed cisplatin, but not nickel, could directly bind mouse TLR4 that lacks these histidine residues. Altogether, our findings suggest that TLR4 can directly bind cisplatin in a manner that is enhanced by, but not dependent on, histidine residues that facilitate binding to transition metals. SIGNIFICANCE STATEMENT: This work describes how the xenobiotic cisplatin interacts with Toll-like receptor 4 (TLR4) to initiate proinflammatory signaling that underlies cisplatin toxicities, which are severe adverse outcomes in cisplatin treatment. Here, this study provides a mechanistic bridge between cisplatin extracellular interactions with TLR4 and previous observations that genetic and chemical inhibition of TLR4 mitigates cisplatin-induced toxicity.

Human MD2 deficiency-an inborn error of immunity with pleiotropic features.

BACKGROUND: Toll-like receptors (TLRs) are important pattern recognition receptors that sense microbes and control host defense. Myeloid differentiation protein 2 (MD2) is the indispensable coreceptor for TLR4, facilitating the binding to the gram-negative bacterial cell wall component LPS and activation of downstream signaling. OBJECTIVE: We sought to provide phenotypic and mechanistic insights into human MD2 deficiency. METHODS: To elucidate the genetic cause in a patient with very early onset inflammatory bowel disease, we performed whole-exome sequencing and studied the functional consequences of the identified mutation in LY96 (encoding for MD2) in genetically engineered induced pluripotent stem cell-derived macrophages with knockout of MD2 or knockin of the patient-specific mutation, including TLR4-mediated signaling, cytokine production, and bacterial handling. RESULTS: Whole-exome sequencing identified a homozygous in-frame deletion in the LY96 gene (c.347_349delCAA; p.Thr116del) in a patient with very early onset inflammatory bowel disease and a sibling presenting with pneumonia and otitis media. Induced pluripotent stem cell-derived macrophages with knockout of MD2 or expression of the Thr116del mutation showed impaired activation of nuclear factor kappa B and mitogen-activated protein kinase signaling as well as TLR4 endocytosis on challenge with LPS or bacteria. In addition, MD2-deficient macrophages showed decreased cytokine expression (eg, IL-6, TNF, and IL-10) in response to LPS or gram-negative but not gram-positive bacteria. CONCLUSIONS: Human MD2 deficiency causes defective TLR4 signaling in response to LPS or gram-negative bacteria. The clinical manifestations and expressivity might be variable due to unknown secondary risk factors. Because TLR4 represents a therapeutic target for multiple inflammatory conditions, our study may provide insights into potential side effects of pharmacological TLR4 targeting.

Heme induces inflammatory injury by directly binding to the complex of myeloid differentiation protein 2 and toll-like receptor 4.

Heme, as an essential component of hemoproteins, is a prosthetic co-factor found in many cells, which is essential for physiologically vital oxygen transport. However, extracellular or circulatory heme is cytotoxic and triggers inflammation. Although the proinflammatory role of heme has been reported to be associated with Toll-like receptor 4 (TLR4) signaling, the exact mechanism remains unknown. Here, we show that heme promotes TLR4 signaling and inflammation via directly physically interacting with TLR4 and its adaptor protein myeloid differentiation protein 2 (MD2). Genetic loss of MD2 ameliorates heme-induced inflammation and inflammatory cytokine production in the spleen of MD2 knockout (MD2-/-) mice. Using mouse macrophage RAW 264.7 cell line, we show that heme induces TLR4 dimerization and MD2/TLR4/MyD88 activation by physically interacting with TLR4 and MD2 in vitro. Genetic loss of MD2 inhibits heme-induced inflammation and MAPK/NF-κB pathway in mouse primary macrophages extracted from MD2-/- mice. Furthermore, pharmacological inhibition of MD2 by L6H9 ameliorates heme-induced inflammation in macrophages. These findings demonstrate that heme causes inflammation by directly binding to MD2/TLR4 complex, leading to activation of TLR4/MAPK/NF-κB signaling pathway and production of downstream effectors of inflammation.

ACT001 Inhibits TLR4 Signaling by Targeting Co-Receptor MD2 and Attenuates Neuropathic Pain.

Neuropathic pain is a common and challenging neurological disease, which renders an unmet need for safe and effective new therapies. Toll-like receptor 4 (TLR4) expressed on immune cells in the central nervous system arises as a novel target for treating neuropathic pain. In this study, ACT001, an orphan drug currently in clinical trials for the treatment of glioblastoma, was identified as a TLR4 antagonist. In vitro quenching titrations of intrinsic protein fluorescence and saturation transfer difference (STD)-NMR showed the direct binding of ACT001 to TLR4 co-receptor MD2. Cellular thermal shift assay (CETSA) showed that ACT001 binding affected the MD2 stability, which implies that MD2 is the endogenous target of ACT001. In silico simulations showed that ACT001 binding decreased the percentage of hydrophobic area in the buried solvent-accessible surface areas (SASA) of MD2 and rendered most regions of MD2 to be more flexible, which is consistent with experimental data that ACT001 binding decreased MD2 stability. In keeping with targeting MD2, ACT001 was found to restrain the formation of TLR4/MD2/MyD88 complex and the activation of TLR4 signaling axes of NF-κB and MAPKs, therefore blocking LPS-induced TLR4 signaling downstream pro-inflammatory factors NO, IL-6, TNF-α, and IL-1ß. Furthermore, systemic administration of ACT001 attenuated allodynia induced by peripheral nerve injury and activation of microglia and astrocyte in vivo. Given the well-established role of neuroinflammation in neuropathic pain, these data imply that ACT001 could be a potential drug candidate for the treatment of chronic neuropathic pain.

Myeloid differentiation factor-2 activates monocytes in patients with dilated cardiomyopathy.

Plasma levels of myeloid differentiation factor-2 (MD-2), a co-receptor of toll-like-receptor 4 (TLR4), independently predict mortality in patients with dilated cardiomyopathy (DCM). We tested whether monocyte activation by MD-2 contributes to immune activation and inflammatory status in DCM patients. We found increased MD-2 plasma levels in 25 patients with recent-onset DCM (1250 ± 80.7 ng/ml) compared to 25 age- and gender-matched healthy controls (793.4 ± 52.0 ng/ml; p < 0.001). Monocytes isolated from DCM patients showed a higher expression (141.7 ± 12.4%; p = 0.006 vs. controls) of the MD-2 encoding gene, LY96 and an increased NF-κB-activation. Further, the TLR4-activator lipopolysaccharide (LPS) caused a higher increase in interleukin (IL)-6 in monocytes from DCM patients compared to controls (mean fluorescence intensity: 938.7 ± 151.0 vs. 466.9 ± 51.1; p = 0.005). MD-2 increased IL-6 secretion in a TLR4/NF-κB-dependent manner in monocyte-like THP-1-cells as demonstrated by TLR4-siRNA and NF-κB-inhibition. Since endothelial cells (ECs) are responsible for recruiting monocytes to the site of inflammation, ECs were treated with MD-2 leading to an activation of Akt and increased secretion of monocyte-chemoattractant-protein-1 (MCP-1). Activation of ECs by MD-2 was accompanied by an increased expression of the adhesion molecules CD54, CD106 and CD62E, resulting in an increased monocyte recruitment, which was attenuated by CD54 inhibition. In addition, in murine WT but not LY96-KO bone marrow-derived macrophages LPS increased the amount of CD54 and CD49d/CD29. MD-2 facilitates a pro-inflammatory status of monocytes and EC-mediated monocyte recruitment via TLR4/NF-κB. Elevated MD-2 plasma levels are possibly involved in monocyte-related inflammation-promoting disease progression in DCM. Our results suggest that MD-2 contributes to increasing monocytic inflammatory activity and triggers the recruitment of monocytes to ECs in DCM.