Lymphocyte antigen 96: A new potential biomarker and immune target in Parkinson's disease.

BACKGROUND: Lymphocyte antigen 96 (LY96) plays an important role in innate immunity and has been reported to be associated with various neurological diseases. However, its role in Parkinson's disease (PD) remains unclear. METHODS: Transcriptome data from a total of 49 patients with PD and 34 healthy controls were downloaded from the Gene Expression Omnibus (GEO) database to analyse the expression pattern of LY96 and its relationship with gene function and immune-related markers. In addition, peripheral blood samples were collected from clinical patients to validate LY96 mRNA expression levels. Finally, an in vitro cell model of PD based on highly differentiated SH-SY5Y cells was constructed, with small interfering RNA-silenced LY96 expression, and LY96 mRNA level, cell viability, flow cytometry, and mitochondrial membrane potential assays were performed. RESULTS: The results of the analyses of the GEO database and clinical samples revealed significantly abnormally high LY96 expression in patients with PD compared with healthy controls. The results of cell experiments showed that inhibiting LY96 expression alleviated adverse cellular effects by increasing cell viability, reducing apoptosis, and reducing oxidative stress. Gene set enrichment analysis showed that LY96 was positively correlated with T1 helper cells, T2 helper cells, neutrophils, natural killer T cells, myeloid-derived suppressor cells, macrophages, and activated CD4 cells, and may participate in PD through natural killer cell-mediated cytotoxicity pathways and extracellular matrix receptor interaction pathways. CONCLUSION: These findings suggested that LY96 might be a novel potential biomarker for PD, and offer insights into its immunoregulatory role.

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.

Mice Plasmacytoid Dendritic Cells Were Activated by Lipopolysaccharides Through Toll-Like Receptor 4/Myeloid Differentiation Factor 2.

Plasmacytoid dendritic cells (pDCs) are known to respond to viral infections. However, the activation of pDCs by bacterial components such as lipopolysaccharides (LPS) has not been well studied. Here, we found that pDCs, conventional dendritic cells (cDCs), and B cells express high levels of toll-like receptor 4 (TLR4), a receptor for LPS. Moreover, LPS could effectively bind to not only cDCs but also pDCs and B cells. Intraperitoneal administration of LPS promoted activation of splenic pDCs and cDCs. LPS treatment led to upregulation of interferon regulatory factor 7 (IRF7) and induced production of interferon-alpha (IFN-α) in splenic pDCs. Furthermore, LPS-dependent upregulation of co-stimulatory molecules in pDCs did not require the assistance of other immune cells, such as cDCs. However, the production levels of IFN-α were decreased in cDC-depleted splenocytes, indicating that cDCs may contribute to the enhancement of IFN-α production in pDCs. Finally, we showed that activation of pDCs by LPS requires the TLR4 and myeloid differentiation factor 2 (MD2) signaling pathways. Thus, these results demonstrate that the gram-negative component LPS can directly stimulate pDCs via TLR4/MD2 stimulation in mice.

Sulfatides are endogenous ligands for the TLR4-MD-2 complex.

Many endogenous molecules, mostly proteins, purportedly activate the Toll-like receptor 4 (TLR4)-myeloid differentiation factor-2 (MD-2) complex, the innate immune receptor for lipopolysaccharide (LPS) derived from gram-negative bacteria. However, there is no structural evidence supporting direct TLR4-MD-2 activation by endogenous ligands. Sulfatides (3-O-sulfogalactosylceramides) are natural, abundant sulfated glycolipids that have variously been shown to initiate or suppress inflammatory responses. We show here that short fatty acid (FA) chain sulfatides directly activate mouse TLR4-MD-2 independent of CD14, trigger MyD88- and TRIF-dependent signaling, and stimulate tumor necrosis factor α (TNFα) and type I interferon (IFN) production in mouse macrophages. In contrast to the agonist activity toward the mouse receptor, the tested sulfatides antagonize TLR4-MD-2 activation by LPS in human macrophage-like cells. The agonistic and antagonistic activities of sulfatides require the presence of the sulfate group and are inversely related to the FA chain length. The crystal structure of mouse TLR4-MD-2 in complex with C16-sulfatide revealed that three C16-sulfatide molecules bound to the MD-2 hydrophobic pocket and induced an active dimer conformation of the receptor complex similar to that induced by LPS or lipid A. The three C16-sulfatide molecules partially mimicked the detailed interactions of lipid A to achieve receptor activation. Our results suggest that sulfatides may mediate sterile inflammation or suppress LPS-stimulated inflammation, and that additional endogenous negatively charged lipids with up to six lipid chains of limited length might also bind to TLR4-MD-2 and activate or inhibit this complex.

Taurine ameliorates thioacetamide induced liver fibrosis in rats via modulation of toll like receptor 4/nuclear factor kappa B signaling pathway.

Liver fibrosis is a significant health problem that can cause serious illness and death. Unfortunately, a standard treatment for liver fibrosis has not been approved yet due to its complicated pathogenesis. The current study aimed at assessing the anti-fibrotic effect of taurine against thioacetamide induced liver fibrosis in rats through the modulation of toll like receptor 4/nuclear factor kappa B signaling pathway. Both concomitant and late taurine treatment (100 mg/kg, IP, daily) significantly reduced the rise in serum ALT and AST activities and significantly reversed the decrease in serum albumin and total protein. These results were confirmed by histopathological examinations and immunehistochemical inspection of α-SMA, caspase-3 and NF-κB. The antioxidant potential of taurine was verified by a marked increase of GSH content and a reduction of MDA level in liver tissue. The anti-fibrotic effects of taurine were evaluated by investigating the expression of TLR4, NF-κB. The protein levels of IL-6, LPS, MyD88, MD2, CD14, TGF-ß1 and TNF-α were determined. Docking studies were carried out to understand how taurine interacts inside TLR4-MD2 complex and it showed good binding with the hydrophobic binding site of MD2. We concluded that the anti-fibrotic effect of taurine was attributable to the modulation of the TLR4/NF-κB signaling.

Nalmefene non-enantioselectively targets myeloid differentiation protein 2 and inhibits toll-like receptor 4 signaling: wet-lab techniques and in silico simulations.

Nalmefene is an opiate derivative having a similar structure to naltrexone. Recent evidence suggests that nalmefene, acting as the innate immune protein toll-like receptor 4 (TLR4) antagonist, effectively reduces the injury of lung ischemia-reperfusion and prevents neuroinflammation. However, the molecular recognition mechanism, especially the enantioselectivity, of nalmefene by the innate immune receptor is not well understood. Herein in vitro assays and in silico simulations were performed to dissect the innate immune recognition of nalmefene at the atomic, molecular, and cellular levels. Biophysical binding experiments and molecular dynamic simulations provide direct evidence that (-)-nalmefene and (+)-nalmefene bind to the hydrophobic cavity of myeloid differentiation protein 2 (MD-2) and behave similarly, which is primarily driven by hydrophobic interactions. The inhibition activity and the calculated binding free energies show that no enantioselectivity was observed during the interaction of nalmefene with MD-2 as well as the inhibition of TLR4 signaling. Interestingly, nalmefene showed ∼6 times better TLR4 antagonisic activity than naltrexone, indicating that the bioisosteric replacement with the methylene group is critical for the molecular recognition of nalmefene by MD-2. In all, this study provides molecular insight into the innate immune recognition of nalmefene, which demonstrates that nalmefene is non-enantioselectively sensed by MD-2.

Identification and Characterization of Zebrafish Tlr4 Coreceptor Md-2.

The zebrafish (Danio rerio) is a powerful model organism for studies of the innate immune system. One apparent difference between human and zebrafish innate immunity is the cellular machinery for LPS sensing. In amniotes, the protein complex formed by TLR4 and myeloid differentiation factor 2 (Tlr4/Md-2) recognizes the bacterial molecule LPS and triggers an inflammatory response. It is believed that zebrafish have neither Md-2 nor Tlr4; Md-2 has not been identified outside of amniotes, whereas the zebrafish tlr4 genes appear to be paralogs, not orthologs, of amniote TLR4s We revisited these conclusions. We identified a zebrafish gene encoding Md-2, ly96 Using single-cell RNA sequencing, we found that ly96 is transcribed in cells that also transcribe genes diagnostic for innate immune cells, including the zebrafish tlr4-like genes. In larval zebrafish, ly96 is expressed in a small number of macrophage-like cells. In a functional assay, zebrafish Md-2 and Tlr4ba form a complex that activates NF-κB signaling in response to LPS. In larval zebrafish ly96 loss-of-function mutations perturbed LPS-induced cytokine production but gave little protection against LPS toxicity. Finally, by analyzing the genomic context of tlr4 genes in 11 jawed vertebrates, we found that tlr4 arose prior to the divergence of teleosts and tetrapods. Thus, an LPS-sensitive Tlr4/Md-2 complex is likely an ancestral feature shared by mammals and zebrafish, rather than a de novo invention on the tetrapod lineage. We hypothesize that zebrafish retain an ancestral, low-sensitivity Tlr4/Md-2 complex that confers LPS responsiveness to a specific subset of innate immune cells.

Myeloid differentiation 2 deficiency attenuates AngII-induced arterial vascular oxidative stress, inflammation, and remodeling.

Vascular remodeling is a pertinent target for cardiovascular therapy. Vascular smooth muscle cell (VSMC) dysfunction plays a key role in vascular remodeling. Myeloid differentiation 2 (MD2), a cofactor of toll-like receptor 4 (TLR4), is involved in atherosclerotic progress and cardiac remodeling via activation of chronic inflammation. In this study, we explored the role of MD2 in vascular remodeling using an Ang II-induced mouse model and cultured human aortic VSMCs. MD2 deficiency suppressed Ang II-induced vascular fibrosis and phenotypic switching of VSMCs without affecting blood pressure in mice. Mechanistically, MD2 deficiency prevented Ang II-induced expression of inflammatory cytokines and oxidative stress in mice and cultured VSMCs. Furthermore, MD2 deficiency reversed Ang II-activated MAPK signaling and Ang II-downregulated SIRT1 expression. Taken together, MD2 plays a significant role in Ang II-induced vascular oxidative stress, inflammation, and remodeling, indicating that MD2 is a potential therapeutic target for the treatment of vascular remodeling-related cardiovascular diseases.

Evidence that angiotensin II does not directly stimulate the MD2-TLR4 innate inflammatory pathway.

The renin-angiotensin system (RAS) plays a critical role in the regulation of blood pressure. Inappropriate activation of the RAS, particularly stimulation of the ACE-Ang II-AT1 receptor axis is a key factor in hypertension and AT1R antagonists (ARBs) are first line therapies in the treatment of cardiovascular disease (CVD). Accumulating evidence suggests that the Ang II-AT1R axis may stimulate both innate and adaptive immune systems. Indeed, recent studies suggest that Ang II stimulates inflammatory events in an AT1R-independent manner by binding the MD2 accessory protein of the TLR4 complex in renal NRK-52E cells. Direct Ang II stimulation of the TLR4 complex is clinically relevant as ARBs increase circulating Ang II levels. Thus, the current study further investigated Ang II stimulation of the TLR4 pathway to release of the pro-inflammatory cytokine CCL2 under identical conditions to the TLR4 ligands LPS and palmitate in the NRK-52E cells. Although LPS (1 ng/mL) and palmitate (100 µM) stimulated CCL2 release 20-fold, Ang II (0.1-10 µM) failed to induce CCL2 release. Both the LPS and palmitate CCL2 responses were abolished by the TLR4 inhibitor Tak242 and significantly reduced by the MD2 inhibitor L48H37. Ang II (1 µM) had no additive effects on LPS (1 ng/mL) or palmitate (100 µM), and the ARB candesartan failed to attenuate CCL2 release to either agent alone. Ang II also failed to induce the release of the putative TLR4 ligand HMBG1. These studies failed to confirm that Ang II directly stimulates the MD2-TLR4 complex to induce cytokine release in NRK-52E cells.

Generation and Characterization of a Novel Anti-Rat TLR4/MD2 Antibody with Potent Neutralizing Activity In Vivo.

Toll-like receptor 4 (TLR4) plays a critical role in the innate immune system and is involved in the pathogenesis of multiple diseases. Here, we report the antagonistic and ratized antibody, 52-1H4 e2 (e2), which completely inhibited lipopolysaccharide-induced interleukin-6 secretion in vitro. The average serum drug concentration was above 10 µg/mL for 28 days in rats injected with e2. The novel anti-rat TLR4/myeloid differentiation factor 2 antibody, e2, may be a useful tool for investigating the role of TLR4 in rat disease models.

Inhibition of myeloid differentiation protein 2 attenuates renal ischemia/reperfusion-induced oxidative stress and inflammation via suppressing TLR4/TRAF6/NF-kB pathway.

As a major risk factor of acute kidney injury, renal ischemia/reperfusion (I/R) has a high mortality rate. Myeloid differentiation protein 2 (MD-2) is a secretory glycoprotein that plays an important role in inflammation. Our study aimed to explore the roles of MD-2 in I/R-induced inflammation and oxidative stress in vivo and in vitro. For the in vivo studies, male C57BL/6 mice were randomly divided into four groups: 1) sham, 2) I/R, 3) negative control for siRNA (siNC) and I/R treatment, or 4) MD-2 siRNA (siMD-2) and I/R. Levels of blood urea nitrogen and creatinine in the plasma were tested, and hematoxylin and eosin staining was performed at 24 h after I/R injury. The inflammatory cytokines TNF-α, IL-6, and MCP-1 were measured using ELISA and Real-time qPCR (RT-qPCR). Malondialdehyde (MDA) content and superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activity were estimated. For the in vitro studies, HK-2 cells were transfected with siMD-2 and then exposed to hypoxia/reoxygenation (H/R). Inflammatory cytokine expression and oxidative stress then were evaluated. We found decreased levels of blood urea nitrogen and creatinine levels after MD-2 silencing. MD-2 deficiency improved histological damage. MD-2 downregulation attenuated levels of inflammatory cytokines. Inhibition of MD-2 resulted in reduced MDA content and increased SOD, CAT, and GPx activity. Loss of function of MD-2 inhibited the H/R-induced production and expression of inflammatory cytokines. MD-2 silencing reduced MDA content after H/R, and MD-2 suppression enhanced SOD, CAT, and GPx activity. MD-2 deficiency also blocked H/R-mediated activation of the TLR4/TRAF6/NF-κB pathway, and pyrrolidinedithiocarbamate (PDTC) pretreatment strengthened the anti-inflammatory and antioxidant damage effects of MD-2 silencing. Taken together, our study revealed that MD-2 deficiency ameliorated renal I/R-induced inflammation and oxidative stress via inhibition of TLR4/TRAF6/NF-κB pathway.

HIV-1 Tat - TLR4/MD2 interaction drives the expression of IDO-1 in monocytes derived dendritic cells through NF-κB dependent pathway.

In the present study we showed that HIV-1 Tat protein stimulated the expression of Indoleamine 2,3 dioxygenase (IDO) -1 in human monocytes derived dendritic cells (MoDC) but not IDO-2 by acting directly at the cell membrane level. This induction of IDO-1 is dependent on the secondary structure of Tat protein, since stimulation with a chemically oxidized Tat protein loses its capacity to induce the production of IDO-1. Among the variety of candidate receptors described for Tat, we demonstrated that Tat protein interacted physically with TLR4/MD2 complex. Strikingly, blockade of Tat-TLR4 interaction by anti-TLR4 antibodies (clone HTA125), LPS-RS, a known TLR4 antagonist, or by soluble recombinant TLR4/MD2 complex inhibited strongly or totally the capacity of Tat to induce IDO-1 in MoDC while such treatments had no effect on IFN-γ-induced IDO-1. Furthermore, we showed that the activation of the transcription factor NF-κB by Tat is essential for the production of IDO-1 by human MoDC. Indeed, Tat activated NF-κB pathway in MoDC as demonstrated by the phosphorylation of p65 in Tat-treated MoDC. Further, we demonstrate that the stimulation of IDO-1 by Tat or by IFN-γ was totally or partially inhibited in the presence of NF-κB inhibitor respectively. These results suggest that Tat and IFN-γ act probably by two distinct mechanisms to induce the production of IDO-1. Our results clearly demonstrated that, although TLR4 pathway is necessary for Tat-induced IDO-1 in MoDC, it seems not to be sufficient since stable transfection of a functional TLR4/MD2 pathway in HEK or HeLa cell lines which are endogenously defectives for TLR4, did not restore the capacity of Tat to induce IDO-1 while IFN-γ treatment induces IDO-1 in HeLa cells independently of TLR4 pathway. These results suggest the involvement of additional stimuli in addition to TLR4 pathway which remain to be identified. Altogether our results demonstrated that, in human MoDC, HIV-1 Tat protein induced IDO-1 expression and activity in a NF-κB dependent-manner by recruiting TLR4 pathway.

Macrophage-derived myeloid differentiation protein 2 plays an essential role in ox-LDL-induced inflammation and atherosclerosis.

BACKGROUND: Atherosclerosis is a chronic inflammatory disease. Although Toll-like receptor 4 (TLR4) has been involved in inflammatory atherosclerosis, the exact mechanisms by which oxidized-low-density lipoproteins (ox-LDL) activates TLR4 and elicits inflammatory genesis are not fully known. Myeloid differentiation factor 2 (MD2) is an extracellular molecule indispensable for lipopolysaccharide recognition of TLR4. METHOD: Apoe-/-Md2-/- mice and pharmacological inhibitor of MD2 were used in this study. We also reconstituted Apoe-/- mice with either Apoe-/- or Apoe-/-Md2-/- marrow-derived cells. Mechanistic studies were performed in primary macrophages, HEK-293T cells, and cell-free system. FINDING: MD2 levels are elevated in atherosclerotic lesion macrophages, and MD2 deficiency or pharmacological inhibition in mice reduces the inflammation and stunts the development of atherosclerotic lesions in Apoe-/- mice fed with high-fat diet. Transfer of marrow-derived cells from Apoe-Md2 double knockout mice to Apoe knockout mice confirmed the critical role of bone marrow-derived MD2 in inflammatory factor induction and atherosclerosis development. Mechanistically, we show that MD2 does not alter ox-LDL uptake by macrophages but is required for TLR4 activation and inflammation via directly binding to ox-LDL, which triggers MD2/TLR4 complex formation and TLR4-MyD88-NFκB pro-inflammatory cascade. INTERPRETATION: We provide a mechanistic basis of ox-LDL-induced macrophage inflammation, illustrate the role of macrophage-derived MD2 in atherosclerosis, and support the therapeutic potential of MD2 targeting in atherosclerosis-driven cardiovascular diseases. FUNDING: This work was supported by the National Key Research Project of China (2017YFA0506000), National Natural Science Foundation of China (21961142009, 81930108, 81670244, and 81700402), and Natural Science Foundation of Zhejiang Province (LY19H020004).

Role of TLR4 in Persistent Leptospira interrogans Infection: A Comparative In Vivo Study in Mice.

Toll-Like Receptor (TLR) 4, the LPS receptor, plays a central role in the control of leptospirosis and absence of TLR4 results in lethal infection in mice. Because human TLR4 does not sense the atypical leptospiral-LPS, we hypothesized that TLR4/MD-2 humanized transgenic mice (huTLR4) may be more susceptible to leptospirosis than wild-type mice, and thus may constitute a model of acute human leptospirosis. We infected huTLR4 mice, which express human TLR4 but not murine TLR4, with a high dose of L. interrogans serovar Copenhageni FioCruz (Leptospira) in comparison to C57BL/6J wild-type (WT) and, as a control, a congenic strain in which the tlr4 coding sequences are deleted (muTLR4Lps-del). We show that the huTLR4 gene is fully functional in the murine background. We found that dissemination of Leptospira in blood, shedding in urine, colonization of the kidney and overall kinetics of leptospirosis progression is equivalent between WT and huTLR4 C57BL/6J mice. Furthermore, inflammation of the kidney appeared to be subdued in huTLR4 compared to WT mice in that we observed less infiltrates of mononuclear lymphocytes, less innate immune markers and no relevant differences in fibrosis markers. Thus, huTLR4 mice showed less inflammation and kidney pathology, and are not more susceptible to leptospirosis than WT mice. This study is significant as it indicates that one intact TLR4 gene, be it mouse or human, is necessary to control acute leptospirosis.

A novel ML domain-containing protein (SpMD2) functions as a potential LPS receptor involved in anti-Vibrio immune response.

The myeloid differentiation protein 2 (MD2)-related lipid-recognition (ML) proteins display diverse biological functions in host immunity and lipid metabolism by interacting with different lipids. Human MD2, an indispensable accessory protein in TLR4 signaling pathway, specifically recognizes lipopolysaccharides (LPS), thereby leading to the activation of TLR4 signaling pathway to produce many effectors that participate in inflammatory and immuneresponses against Gram-negative bacteria. Toll and immune deficiency (IMD) pathways are first characterized in Drosophila and are reportedly present in crustaceans, but the recognition and activation mechanism of these signaling pathways in crustaceans remains unclear. In the present study, a novel ML protein was characterized in mud crab (Scylla paramamosain) and designated as SpMD2. The complete SpMD2 cDNA sequence is 1114 bp long with a 465 bp open reading frame; it encodes a protein that contains 154 amino acids (aa). In the deduced protein, a signal peptide (1-21 aa residues) and a ML domain (43-151 aa residues) were predicted. SpMD2 shared a similar three-dimensional structure and a close evolutionary relationship with human MD2. SpMD2 was highly expressed in gills, hemocytes, intestine, and hepatopancreas and was upregulated in gills and hemocytes after challenges with bacteria, thereby suggesting its involvement in antibacterial defense. Western blot assay showed that SpMD2 possesses strong binding activities to different bacteria and two fungi. ELISA demonstrated that SpMD2 exhibits binding abilities to LPS, lipid A, peptidoglycan (PGN), and lipoteichoic acid (LTA). Its binding ability to LPS and lipid A were stronger than to PGN or LTA, implying that SpMD2 was an important LPS-binding protein in mud crab. Bacterial clearance assay revealed that the pre-incubation of Vibrio parahemolyticus with SpMD2 facilitates bacterial clearance in vivo and that knockdown of SpMD2 dramatically suppresses the bacterial clearance and decreases the expression of several antimicrobial peptides (AMPs). Furthermore, SpMD2 overexpression could enhance the promoter activity of SpALF2. These results revealed that SpMD2 affects bacterial clearance by regulating AMPs. Thus, by binding to LPS and by regulating AMPs, SpMD2 may function as a potential receptor, which is involved in the recognition and activation of a certain immune signaling pathway against Gram-negative bacteria. This study provides new insights into the diverse functions of ML proteins and into the antibacterial mechanisms of crustaceans.

Myeloid Differentiation Protein 2 Mediates Angiotensin II-Induced Liver Inflammation and Fibrosis in Mice.

Angiotensin II (Ang II) participates in the pathogenesis of liver injury. Our previous publications reported that myeloid differentiation protein 2 (MD2) mediates Ang II-induced cardiac and kidney inflammation by directly binding to Ang II. Thus, we hypothesize that MD2 is critical to Ang II-induced liver injury. Subcutaneous injections of Ang II for 8 weeks were adopted to build the liver injury model. With a specific MD2 inhibitor L6H21 and MD2 knockout mice, we reported that MD2 inhibition and knockout significantly mitigate liver inflammation and fibrosis in mice injected with Ang II. To be more specific, the functional and pathological damages induced by Ang II were mitigated by L6H21 or MD2 knockout. MD2 knockout or L6H21 administration inhibited the Ang II-induced upregulation of fibrosis markers, inflammatory cytokines, and adhesion molecules in gene or protein levels. The activation of NF-κB and Extracellular signal-regulated kinases (ERK) induced by Ang II was also reversed by L6H21 treatment or MD2 deficiency. Note that the co-immunoprecipitation study showed that L6H21 downregulated the ANG II-induced toll-like receptor 4 (TLR4)/MD2 complex in liver tissues while having no effects on MD2 expression. Our results reported the critical role of MD2 in the progress of liver injury and suggested that MD2 is a potential therapeutic target for liver injury.

Key residues in TLR4-MD2 tetramer formation identified by free energy simulations.

Toll-like receptors (TLRs) play a central role in both the innate and adaptive immune systems by recognizing pathogen-associated molecular patterns and inducing the release of the effector molecules of the immune system. The dysregulation of the TLR system may cause various autoimmune diseases and septic shock. A series of molecular dynamics simulations and free energy calculations were performed to investigate the ligand-free, lipopolysaccharide (LPS)-bound, and neoseptin3-bound (TLR4-MD2)2 tetramers. Compared to earlier simulations done by others, our simulations showed that TLR4 structure was well maintained with stable interfaces. Free energy decomposition by molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method suggests critical roles that two hydrophobic clusters I85-L87-P88 and I124-L125-P127 of MD2, together with LPS and neoseptin3, may play in TLR4 activation. We propose that 1) direct contacts between TLR4 convex surface and LPS and neoseptin3 at the region around L442 significantly increase the binding and 2) binding of LPS and neoseptin3 in the central hydrophobic cavity of MD2 triggers burial of F126 and exposure of I85-L87-P88 that facilitate formation of (TLR4-MD2)2 tetramer and activation of TLR4 system.

[Effects of MD2 gene silencing on high glucose-induced proliferation inhibition, apoptosis and inflammation in rat cardiomyocytes].

OBJECTIVE: To investigate the effects of myeloid differentiation-2 (MD2) gene silencing on high glucose-induced proliferation inhibition, apoptosis and inflammation in rat cardiomyocytes. METHODS: The immortalized rat cardiomyocyte cell line H9C2 were transfected with MD2 small interfering RNA (si-MD2) and negative control for 24 h, then stimulated with high glucose (HG) for 48 h. RT-qPCR was performed to detect the mRNA levels of MD2 and inflammatory factors TNF-α, IL-1ß and IL-6. MTS and flow cytometry were used to evaluate cell proliferation, cell cycle and apoptosis rate. Western blot was used to detect protein expression levels and phosphorylation levels. RESULTS: The mRNA and protein levels of MD2 in H9C2 cells were dramatically decreased after transfected with si-MD2 (P＜0.01). After stimulation of high glucose, the mRNA levels of inflammatory factors, the cells in G0/G1 phase , the cell apoptosis rate and the protein level of cleaved Caspase-3 were significantly increased, while the cell proliferation ability was decreased (P＜0.01). MD2 gene silencing antagonized the effects of high glucose on cell proliferation, cell cycle, cell apoptosis and the mRNA levels of TNF-α, IL-1ß , IL-6(P＜0.05). Western blot analysis showed that the phosphorylation levels of extracellular signal-regulated kinase(ERK1/2), P38 mitogen-activated protein kinase(P38 MAPK) and C-Jun N-terminal kinase(JNK) protein were increased significantly in H9C2 cells treated with high glucose, which could be reversed by silencing of MD2 (P＜0.01). CONCLUSION: This study demonstrates that MD2 gene silencing reverses high glucose-induced myocardial inflammation, apoptosis and proliferation inhibition via the mechanisms involving suppression of ERK, P38 MAPK, JNK signaling pathway.

Construction of a sensitive pyrogen-testing cell model by site-specific knock-in of multiple genes.

A pyrogen test is crucial for evaluating the safety of drugs and medical equipment, especially those involved in injections. As existing pyrogen tests, including the rabbit pyrogen test, the limulus amoebocyte lysate (LAL) test and the monocyte activation test have limitations, development of new models for pyrogen testing is necessary. Here we develop a sensitive cell model for pyrogen test based on the lipopolysaccharides (LPS) signal pathway. TLR4, MD2, and CD14 play key roles in the LPS-mediated pyrogen reaction. We established a new TLR4/MD2/CD14-specific overexpressing knock-in cell model using the CRISPR/CAS9 technology and homologous recombination to detect LPS. Stimulation of our TLR4/CD14/MD2 knock-in cell line model with LPS leads to the release of the cytokines IL-6 and TNF-alpha, with a detection limit of 0.005 EU/ml, which is greatly lower than the lower limit of 0.015 EU/ml detected by the Tachypleus amebocyte lysate (TAL) assay.