Reconstruction of LPS Transfer Cascade Reveals Structural Determinants within LBP, CD14, and TLR4-MD2 for Efficient LPS Recognition and Transfer.

Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, binds Toll-like receptor 4 (TLR4)-MD2 complex and activates innate immune responses. LPS transfer to TLR4-MD2 is catalyzed by both LPS binding protein (LBP) and CD14. To define the sequential molecular interactions underlying this transfer, we reconstituted in vitro the entire LPS transfer process from LPS micelles to TLR4-MD2. Using electron microscopy and single-molecule approaches, we characterized the dynamic intermediate complexes for LPS transfer: LBP-LPS micelles, CD14-LBP-LPS micelle, and CD14-LPS-TLR4-MD2 complex. A single LBP molecule bound longitudinally to LPS micelles catalyzed multi-rounds of LPS transfer to CD14s that rapidly dissociated from LPB-LPS complex upon LPS transfer via electrostatic interactions. Subsequently, the single LPS molecule bound to CD14 was transferred to TLR4-MD2 in a TLR4-dependent manner. The definition of the structural determinants of the LPS transfer cascade to TLR4 may enable the development of targeted therapeutics for intervention in LPS-induced sepsis.

Disulfiram blocks inflammatory TLR4 signaling by targeting MD-2.

Toll-like receptor 4 (TLR4) sensing of lipopolysaccharide (LPS), the most potent pathogen-associated molecular pattern of gram-negative bacteria, activates NF-κB and Irf3, which induces inflammatory cytokines and interferons that trigger an intense inflammatory response, which is critical for host defense but can also cause serious inflammatory pathology, including sepsis. Although TLR4 inhibition is an attractive therapeutic approach for suppressing overexuberant inflammatory signaling, previously identified TLR4 antagonists have not shown any clinical benefit. Here, we identify disulfiram (DSF), an FDA-approved drug for alcoholism, as a specific inhibitor of TLR4-mediated inflammatory signaling. TLR4 cell surface expression, LPS sensing, dimerization and signaling depend on TLR4 binding to MD-2. DSF and other cysteine-reactive drugs, previously shown to block LPS-triggered inflammatory cell death (pyroptosis), inhibit TLR4 signaling by covalently modifying Cys133 of MD-2, a key conserved residue that mediates TLR4 sensing and signaling. DSF blocks LPS-triggered inflammatory cytokine, chemokine, and interferon production by macrophages in vitro. In the aggressive N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease (PD) in which TLR4 plays an important role, DSF markedly suppresses neuroinflammation and dopaminergic neuron loss, and restores motor function. Our findings identify a role for DSF in curbing TLR4-mediated inflammation and suggest that DSF and other drugs that target MD-2 might be useful for treating PD and other diseases in which inflammation contributes importantly to pathogenesis.

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.

SRSF3 Knockdown Inhibits Lipopolysaccharide-Induced Inflammatory Response in Macrophages.

Serine/arginine-rich splicing factor 3 (SRSF3), the smallest member of the SR protein family, serves multiple roles in RNA processing, including splicing, translation, and stability. Recent studies have shown that SRSF3 is implicated in several inflammatory diseases. However, its impact on macrophage inflammation remains unclear. Herein, we determined the expression of SRSF3 in inflammatory macrophages and found that the level of SRSF3 was increased in macrophages within atherosclerotic plaques, as well as in RAW-264.7 macrophages stimulated by lipopolysaccharides. Moreover, the downregulation of SRSF3 suppressed the levels of inflammatory cytokines by deactivating the nuclear factor κB (NFκB) pathway. Furthermore, the alternative splicing of myeloid differentiation protein 2 (MD2), a co-receptor of toll-like receptor 4 (TLR4), is regulated by SRSF3. The depletion of SRSF3 increased the level of the shorter MD2B splicing variants, which contributed to inflammatory inhibition in macrophages. In conclusion, our findings imply that SRSF3 regulates lipopolysaccharide-stimulated inflammation, in part by controlling the alternative splicing of MD2 mRNA in macrophages.

Isoproterenol induces MD2 activation by ß-AR-cAMP-PKA-ROS signalling axis in cardiomyocytes and macrophages drives inflammatory heart failure.

Cardiac inflammation contributes to heart failure (HF) induced by isoproterenol (ISO) through activating ß-adrenergic receptors (ß-AR). Recent evidence shows that myeloid differentiation factor 2 (MD2), a key protein in endotoxin-induced inflammation, mediates inflammatory heart diseases. In this study, we investigated the role of MD2 in ISO-ß-AR-induced heart injuries and HF. Mice were infused with ISO (30 mg·kg-1·d-1) via osmotic mini-pumps for 2 weeks. We showed that MD2 in cardiomyocytes and cardiac macrophages was significantly increased and activated in the heart tissues of ISO-challenged mice. Either MD2 knockout or administration of MD2 inhibitor L6H21 (10 mg/kg every 2 days, i.g.) could prevent mouse hearts from ISO-induced inflammation, remodelling and dysfunction. Bone marrow transplantation study revealed that both cardiomyocyte MD2 and bone marrow-derived macrophage MD2 contributed to ISO-induced cardiac inflammation and injuries. In ISO-treated H9c2 cardiomyocyte-like cells, neonatal rat primary cardiomyocytes and primary mouse peritoneal macrophages, MD2 knockout or pre-treatment with L6H21 (10 µM) alleviated ISO-induced inflammatory responses, and the conditioned medium from ISO-challenged macrophages promoted the hypertrophy and fibrosis in cardiomyocytes and fibroblasts. We demonstrated that ISO induced MD2 activation in cardiomyocytes via ß1-AR-cAMP-PKA-ROS signalling axis, and induced inflammatory responses in macrophages via ß2-AR-cAMP-PKA-ROS axis. This study identifies MD2 as a key inflammatory mediator and a promising therapeutic target for ISO-induced heart failure.

Oxypeucedanin hydrate alleviates rheumatoid arthritis by inhibiting the TLR4-MD2/NF-κB/MAPK signaling axis.

Rheumatoid arthritis (RA) is an idiopathic and chronic autoimmune disease for which there are currently no effective treatments. Oxypeucedanin hydrate (OXH) is a natural coumarin known for its potent anti-inflammatory properties. However, further investigations are needed to determine its therapeutic efficacy in treating RA. In this study, we evaluate the anti-inflammatory activity of OXH by treating LPS-induced RAW264.7 macrophages. Our results show that OXH treatment reverses the changes in iNOS, COX-2, IL-1ß, IL-6, and TNF-α levels. Additionally, OXH reduces ROS production. Further analysis reveals that OXH suppresses the activation of the NF-κB/MAPK pathway. CETSA results show that OXH competes with LPS for binding to the TLR4/MD2 complex. MST experiments demonstrate the specific affinity of OXH for the TLR4/MD2 complex, with a Kd value of 33.7 µM. Molecular docking analysis suggests that OXH binds to the pocket of the TLR4/MD2 complex and interacts with specific amino acids, such as GLY-343, LYS-388, and PHE-345. Molecular dynamics simulations further confirm this conclusion. Finally, we investigate the potential of OXH in treating RA using a collagen-induced arthritis (CIA) model in rats. OXH effectively ameliorates the symptoms of CIA, including improving body weight, reducing swelling and redness, increasing talus volume, and decreasing bone erosion. OXH also decreases the mRNA levels of pro-inflammatory factors in synovial tissue. Transcriptome enrichment analysis and western blot analysis confirm that OXH suppresses the NF-κB/MAPK pathway, which is consistent with our in vitro findings.

Toll9 from Bombyx mori functions as a pattern recognition receptor that shares features with Toll-like receptor 4 from mammals.

Toll/Toll-like receptors (TLRs) are key regulators of the innate immune system in both invertebrates and vertebrates. However, while mammalian TLRs directly recognize pathogen-associated molecular patterns, the insect Toll pathway is thought to be primarily activated by binding Spätzle cytokines that are processed from inactive precursors in response to microbial infection. Phylogenetic and structural data generated in this study supported earlier results showing that Toll9 members differ from other insect Tolls by clustering with the mammalian TLR4 group, which recognizes lipopolysaccharide (LPS) through interaction with myeloid differentiation-2 (MD-2)-like proteins. Functional experiments showed that BmToll9 from the silkmoth Bombyx mori also recognized LPS through interaction with two MD-2-like proteins, previously named BmEsr16 and BmPP, that we refer to in this study as BmMD-2A and BmMD-2B, respectively. A chimeric BmToll9-TLR4 receptor consisting of the BmToll9 ectodomain and mouse TLR4 transmembrane and Toll/interleukin-1 (TIR) domains also activated LPS-induced release of inflammatory factors in murine cells but only in the presence of BmMD-2A or BmMD-2B. Overall, our results indicate that BmToll9 is a pattern recognition receptor for LPS that shares conserved features with the mammalian TLR4-MD-2-LPS pathway.

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.

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.

A non-polar fraction of Saponaria officinalis L. acted as a TLR4/MD2 complex antagonist and inhibited TLR4/MyD88 signaling in vitro and in vivo.

Targeting Toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD2) signaling is regarded as a potential strategy for treating inflammatory diseases. Saponaria officinalis L. is rich in saponin, which include quillaic acid, gypsogenin, saponarin, and hederagenin. We evaluated the pharmacological activity of a Saponaria officinalis extract in THP-1 derived macrophages and RAW264.7 macrophages. TLR4/MyD88 complex formation and downstream signals were investigated by co-immunoprecipitation (Co-IP). In silico docking simulation was conducted to predict binding scores and perform 3D modeling of saponarin-TLR4/MD2 complex. A hexane fraction of Saponaria officinalis (SH) and fr.1 (a sub-fraction 1 of SH) inhibited mitogen-activated protein kinase (MAPK) signaling, nuclear factor kappa b (NF-κB) activity, cytokine production, and the expressions of marker genes specific for M1 polarization. The inhibitory effects of fr.1 and saponarin on TLR4/MyD88 complex formation were observed by western blotting TLR4 co-immunoprecipitated proteins. Saponarin and fr.1 markedly attenuated LPS-induced inflammatory cytokines, thus reducing mortality and morphological abnormality in zebrafish larvae. Finally, docking simulation revealed that saponarin can directly interact with TLR4/MD2 complex to inhibit downstream signalings. Our findings suggest that saponarin reduces downstream inflammatory response by disrupting TLR4/MD2 complex and blocking MyD88-dependent inflammatory signaling.

The antimicrobial peptide LK2(6)A(L) exhibits anti-inflammatory activity by binding to the myeloid differentiation 2 domain and protects against LPS-induced acute lung injury in mice.

Acute lung injury (ALI) is a life-threatening disease that is generally attributable to an uncontrolled inflammatory response in the lung, but there is a lack of effective treatments. At present, regulating the inflammatory response has become an important strategy for treating ALI. In the present study, LK2(6)A(L), a peptide derived from the natural antimicrobial peptide temporin-1CEa, inhibited lipopolysaccharide (LPS)-induced expression of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and NO in RAW264.7 cells. Herein, the anti-inflammatory mechanism of LK2(6)A(L) was investigated. The RNA-sequencing (RNA-seq) results showed that LK2(6)A(L) significantly inhibited the TLR4-mediated NF-κB and MAPK signaling pathways in LPS-induced RAW264.7 cells. The results of co-immunoprecipitation (Co-IP), pull-down experiment, confocal laser scanning microscopy, and surface plasmon resonance (SPR) suggested that MD2 was the direct target of LK2(6)A(L). Chemical inhibition of MD2 and its knockdown abolished the anti-inflammatory effect of LK2(6)A(L). Molecular dynamic simulation indicated that LK2(6)A(L) could bind to the active domain of the MD2 hydrophobic pocket via six hydrogen bonds. The truncated peptides were designed based on analysis of the molecular docking of LK2(6)A(L) to MD2. The truncated peptide IS-7 showed strong affinity to MD2 and a remarkable inhibitory effect on pro-inflammatory factors that was comparable to the effect of LK2(6)A(L). Finally, LK2(6)A(L) and IS-7 relieved inflammatory symptoms and lung tissue destruction in the ALI mouse model. Overall, our study suggested that LK2(6)A(L) showed promising anti-inflammatory activity by targeting MD2, and the amino acid domain 7-13 was an important area that binds with MD2 and also an anti-inflammatory active region. LK2(6)A(L) and IS-7 may be potential new treatments for ALI and other acute inflammatory diseases.

Discovery of novel TLR4/MD-2 inhibitors: Receptor structure-based virtual screening studies and anti-inflammatory evaluation.

In this study, a receptor structure-based virtual screening strategy was constructed using a computer-aided drug design. First, the compounds were filtered based on the Lipinski pentad and adsorption, distribution, metabolism, excretion, and toxicity profiles. Then, receptor structure-based pharmacophore models were constructed and screened. Finally, the in vitro toxicity and anti-inflammatory activities of hit compounds were initially evaluated to investigate their in vitro anti-inflammatory effects and mechanisms of action. The results revealed that hit 94 had the best anti-inflammatory activity and low toxicity while inhibiting the activation of Toll-like receptor (TLR) 4/myeloid differentiation factor 2 (MD2)-associated signaling pathways of nuclear factor-κB and mitogen-activated protein kinase. In vivo adjuvant arthritis results also revealed that hit 94 ameliorated foot swelling to a greater extent in rats compared with the positive control drug indomethacin. These results suggest that hit 94 can be used as a potential TLR/MD2 inhibitor for inflammatory diseases.

Drug Investigation to Dampen the Comorbidity of Rheumatoid Arthritis and Osteoporosis via Molecular Docking Test.

At present, most rheumatoid arthritis (RA) patients are at risk of osteoporosis (OP), which is increased by 1.5 times compared to non-RA individuals. Hence, we investigated overlapping targets related directly to the occurrence and development of RA and OP through public databases (DisGeNET, and OMIM) and literature. A total of 678 overlapping targets were considered as comorbid factors, and 604 out of 678 were correlated with one another. Interleukin 6 (IL-6), with the highest degree of value in terms of protein−protein interaction (PPI), was considered to be a core target against comorbidity. We identified 31 existing small molecules (< 1000 g/mol) as IL-6 inhibitors, and 19 ligands were selected by the 3 primary criteria (Lipinski's rule, TPSA, and binding energy). We postulated that MD2-TLR4-IN-1 (PubChem ID: 138454798), as confirmed by the three criteria, was the key ligand to alleviate comorbidity between RA and OP. In conclusion, we described a promising active ligand (MD2-TLR4-IN-1), and a potential target (IL-6) against comorbidity of RA and OP, providing scientific evidence for a further clinical trial.

The Bursaphelenchus xylophilus effector BxML1 targets the cyclophilin protein (CyP) to promote parasitism and virulence in pine.

BACKGROUND: Bursaphelenchus xylophilus is the causal agent of pine wilt disease (PWD) that has caused enormous ecological and economic losses in China. The mechanism in the interaction between nematodes and pine remains unclear. Plant parasitic nematodes (PPNs) secrete effectors into host plant tissues. However, it is poorly studied that role of effector in the infection of pine wood nematode (PWN). RESULTS: We cloned, characterized and functionally validated the B. xylophilus effector BxML1, containing an MD-2-related lipid-recognition (ML) domain. This protein inhibits immune responses triggered by the molecular pattern BxCDP1 of B. xylophilus. An insitu hybridization assay demonstrated that BxML1 was expressed mainly in the dorsal glands and intestine of B. xylophilus. Subcellular localization analysis showed the presence of BxML1 in the cytoplasm and nucleus. Furthermore, number of B. xylophilus and morbidity of pine were significantly reduced in Pinus thunbergii infected with B. xylophilus when BxML was silenced. Using yeast two-hybrid (Y2H) and coimmunoprecipitation (CoIP) assays, we found that the BxML1 interacts with cyclophilin protein PtCyP1 in P. thunbergii. CONCLUSIONS: This study illustrated that BxML1 plays a critical role in the B. xylophilus-plant interaction and virulence of B. xylophilus.

Myeloid differentiation protein-2 has a protective role in house dust mite-mediated asthmatic characteristics with the proinflammatory regulation of airway epithelial cells and dendritic cells.

BACKGROUND: Myeloid differentiation protein-2 (MD-2) is a lipopolysaccharide-binding protein involved in lipopolysaccharide signalling via Toll-like receptor 4 (TLR4). TLR4 plays an essential role in HDM-mediated allergic airway inflammation. Moreover, MD-2 is structurally similar to Der f 2, a major allergen from house dust mite (HDM). OBJECTIVES: We aimed to clarify the role of MD-2 in the pathogenesis of HDM-mediated allergic airway inflammation. METHODS: Wild-type (WT), TLR4 knockout and MD-2 knockout mice were subjected to intranasal instillation of HDM extract, and asthmatic features were evaluated. We also evaluated gene sets regulated by MD-2 in HDM-treated airway epithelial cells and examined the function of dendritic cells from lymph nodes and from lungs. RESULTS: Aggravated allergic airway inflammation with increased airway hyperresponsiveness was observed in MD-2 knockout mice compared with WT and TLR4 knockout mice. Global gene expression analysis revealed an MD-2 regulated proinflammatory response and reconstituted TLR4 signalling in airway epithelial cells. The ability of dendritic cells to evoke an allergic immune response was enhanced in MD-2 knockout mice. CONCLUSIONS & CLINICAL RELEVANCE: MD-2 plays a protective role in HDM-induced airway allergy with the proinflammatory regulation of airway epithelial cells and dendritic cells. MD-2 may serve as a therapeutic target in the treatment of asthma.

Flavokawain B alleviates LPS-induced acute lung injury via targeting myeloid differentiation factor 2.

Acute lung injury (ALI) is a sudden onset systemic inflammatory response. ALI causes severe morbidity and death and currently no effective pharmacological therapies exist. Natural products represent an excellent resource for discovering new drugs. Screening anti-inflammatory compounds from the natural product bank may offer viable candidates for molecular-based therapies for ALI. In this study, 165 natural compounds were screened for anti-inflammatory activity in lipopolysaccharide (LPS)-challenged macrophages. Among the screened compounds, flavokawain B (FKB) significantly reduced LPS-induced pro-inflammatory IL-6 secretion in macrophages. FKB also reduced the formation of LPS/TLR4/MD2 complex by competitively binding to MD2, suppressing downstream MAPK and NF-κB signaling activation. Finally, FKB treatment of mice reduced LPS-induced lung injury, systemic and local inflammatory cytokine production, and macrophage infiltration in lungs. These protective activities manifested as increased survival in the ALI model, and reduced mortality upon bacterial infection. In summary, we demonstrate that the natural product FKB protects against LPS-induced lung injury and sepsis by interacting with MD2 and inhibiting inflammatory responses. FKB may potentially serve as a therapeutic option for the treatment of ALI.

First Report of Bacterial Pineapple Heart Rot Caused by Dickeya zeae in Puerto Rico.

In Puerto Rico, the agricultural production of pineapple (Ananas comosus (L.) Merr.) comprises nearly 5,000 tons harvested annually from over 250 ha (USDA 2018). With an annual income of approximately $3 million USD, pineapple ranks fourth in importance among Puerto Rican crops (USDA 2018). Recently, the pineapple industry on the island underwent a change from growing a local cultivar known as "Cabezona" to cultivar MD2, introduced from Hawaii around 1996 (SEA 2015), because this cultivar produces fruit more than once during a single growing season. In August 2018 (when the rainy season normally starts in Puerto Rico), soft rot symptoms appeared at commercial fields in Manatí (WGS 84 Lat 18.42694, Lng -66.44779) and persisted through 2019. Symptoms observed in the field included foliar water-soaked lesions with gas-filled blisters, especially at the base of the leaf. Leaves exhibited brown discoloration and a fetid odor (rot) at the basal portion of the plant. Finally, leaves collapsed at the center of the pineapple crown, effectively killing the apex and preventing the fruit from developing. Disease incidence ranged from 25% to 40% depending on the weather and season; when there was more rain, there was higher disease incidence. Symptomatic leaves were collected in February 2019, disinfected with 70% ethanol, and rinsed with sterile distilled water. Tissue sections (5mm2) were placed in nutrient agar. Bacterial colony-forming units (CFU) were a translucent cream color, circular, with a flat convex surface and wavy edge. Biochemical analysis showed that bacteria were Gram-negative, oxidase positive, catalase positive, and facultatively anaerobic. Pathogenicity was tested on leaves of one-and-a-half-year-old pineapple seedlings in humid chambers. Bacteria were grown on sterile nutrient agar for 3 days at 25 ± 2°C. Inoculation assays (three replications) were performed using 1X108 CFU/ml of bacteria suspended in sterile water and applied with a cotton swab to leaves wounded with a needle. The inoculated tissue was incubated at 28°C and kept in a dark environment. Negative controls were inoculated with sterile water. Five days after inoculation, foliar water-soaked lesions were observed, followed by the formation of brown leaf tissue and gas-filled blisters, the same symptoms observed in the field. A partial DNA sequence of the 16S rRNA gene of the bacterial isolate and the re-isolated bacteria were amplified using primers 27F and 1492R (Lane et al. 1985) and sequenced. The isolate was determined to the genus Dickeya through a BLAST® search against sequences available in the database of the National Center for Biotechnology Information (NCBI). This partial 16S rRNA sequence of the bacterial isolate was deposited in GenBank® at NCBI (Accession no. MT672704). To determine the identity of the Dickeya species, we sequenced the genes dnaA, gyrB, dnaX, and recN (Marrero et al. 2013) for the bacterial isolate (GenBank accession nos. OM276852, OM276853, OM276854, and OM276855) and conducted a Multilocus Sequence Analysis including reference Dickeya sequences of Marrero et al., 2013. The Phylogenetic analysis (using WinClada) resolved the Puerto Rican isolate as belonging to a clade broadly ascribable to D. zeae, most closely related to strains isolated from earlier Hawaiian pineapple bacterial heart rot outbreaks. Dickeya zeae was responsible for bacterial heart rot of pineapple in Malaysia and was later reported as the causal agent for outbreaks in Costa Rica and Hawaii (Kaneshiro et al. 2008; Sueno et al. 2014; Ramachandran et al. 2015). D. zeae had not previously been reported as causing bacterial heart rot in pineapples in Puerto Rico and this study points to a close relationship with strains first detected in Hawaii and which should further be explored to determine the precise nature of this relationship. This information should facilitate the adoption of effective control measures for this disease on the island, promote more effective methods of preventing future introductions of pathogens, and encourage further investigations into the occurrence of D. zeae on the island.

HMGB1 augments cognitive impairment in sepsis-associated encephalopathy by binding to MD-2 and promoting NLRP3-induced neuroinflammation.

BACKGROUND: Sepsis-associated encephalopathy (SAE) always manifests with severe inflammatory symptoms and cognitive impairment. High mobility group box 1 (HMGB1) is a pro-inflammatory cytokine. In this study we investigated the role of HMGB1 in SAE. METHODS: An SAE mouse model was established through cecal ligation and puncture surgery and then injected with adenovirus short hairpin RNA (Ad-sh)-HMGB1 or Ad-sh-myeloid differentiation protein (MD-2). The cognitive impairment and pathological injury in mice of different groups were evaluated using the Morris water maze experiment, Y-maze test, tail suspension test, fear conditioning test, and haematoxylin-eosin staining. The expressions of HMGB1 (fully reduced and disulfide (ds)HMGB1), MD-2, and NLRP3 in SAE mice were determined. Then, levels of inflammatory cytokines were measured. The binding relation between HMGB1 and MD-2 was predicted and certified. Additionally, MD-2 was downregulated to verify the role of the binding of HMGB1 and MD-2 in neuroinflammation and cognitive impairment in SAE. RESULTS: Expressions of HMGB1, MD-2, NLRP3, and inflammatory cytokines were enhanced in the SAE mouse model, which were in parallel with impaired cognitive function. HMGB1 silencing resulted in downregulated NLRP3 expression and alleviated neuroinflammation and cognitive impairment in SAE mice. Mechanically, dsHMGB1 bound to MD-2 to activate NLRP3, thereby exacerbating neuroinflammation and cognitive impairment in SAE mice. The limited binding of HMGB1 and MD-2 downregulated NLRP3 expression to alleviate neuroinflammation and cognitive impairment in SAE mice. CONCLUSION: HMGB1 was overexpressed in SAE, and dsHMGB1 bound to MD-2 to activate NLRP3 inflammasome, inducing neuroinflammation and cognitive impairment in SAE.

LPS-TLR4/MD-2-TNF-α signaling mediates alcohol-induced liver fibrosis in rats.

Liver fibrosis results from liver inflammation and progresses to liver cirrhosis or liver cancer. It is known that nonalcoholic liver disease is mediated by the Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD-2)-tumor necrosis factor-alpha (TNF-α) signaling pathway. This study aimed to investigate whether alcoholic liver disease is also mediated by this pathway. To this end, we first established rat models of liver fibrosis by administering alcohol. Next, the rats were injected with anti-TLR4 and anti-MD-2 antibodies. Real Time Quantitative PCR (RT-qPCR) and Western blotting were used to detect the activation of the TLR4/MD-2-TNF-α signaling pathway and hepatic stellate cells (HSCs). Moreover, the expression of molecules related to liver fibrosis was estimated. The morphology of rat liver tissue was observed through hematoxylin-eosin staining and Masson staining. For in vitro studies, Kupffer cells (KCs) isolated from the liver were transfected with si-TLR4 and si-MD-2 and co-cultured with HSCs to determine the activity of HSCs. It was found that alcohol treatment activated the TLR4/MD-2-TNF-α signaling pathway and upregulated the molecules associated with liver fibrosis. However, inhibition of TLR4 and MD-2 partially reversed this trend. Notably, in vitro studies indicated that knockdown of TLR4 and MD-2 in KCs partially inhibited LPS-induced activation of KCs and HSCs. Overall, this study showed that alcohol induces liver fibrosis via the LPS-TLR4/MD-2-TNF-α signaling pathway.

MD2 contributes to the pathogenesis of perioperative neurocognitive disorder via the regulation of α5GABAA receptors in aged mice.

BACKGROUND: Perioperative neurocognitive disorder (PND) is a long-term postoperative complication in elderly surgical patients. The underlying mechanism of PND is unclear, and no effective therapies are currently available. It is believed that neuroinflammation plays an important role in triggering PND. The secreted glycoprotein myeloid differentiation factor 2 (MD2) functions as an activator of the Toll-like receptor 4 (TLR4) inflammatory pathway, and α5GABAA receptors (α5GABAARs) are known to play a key role in regulating inflammation-induced cognitive deficits. Thus, in this study, we aimed to investigate the role of MD2 in PND and determine whether α5GABAARs are involved in the function of MD2. METHODS: Eighteen-month-old C57BL/6J mice were subjected to laparotomy under isoflurane anesthesia to induce PND. The Barnes maze was used to assess spatial reference learning and memory, and the expression of hippocampal MD2 was assayed by western blotting. MD2 expression was downregulated by bilateral injection of AAV-shMD2 into the hippocampus or tail vein injection of the synthetic MD2 degrading peptide Tat-CIRP-CMA (TCM) to evaluate the effect of MD2. Primary cultured neurons from brain tissue block containing cortices and hippocampus were treated with Tat-CIRP-CMA to investigate whether downregulating MD2 expression affected the expression of α5GABAARs. Electrophysiology was employed to measure tonic currents. For α5GABAARs intervention experiments, L-655,708 and L-838,417 were used to inhibit or activate α5GABAARs, respectively. RESULTS: Surgery under inhaled isoflurane anesthesia induced cognitive impairments and elevated the expression of MD2 in the hippocampus. Downregulation of MD2 expression by AAV-shMD2 or Tat-CIRP-CMA improved the spatial reference learning and memory in animals subjected to anesthesia and surgery. Furthermore, Tat-CIRP-CMA treatment decreased the expression of membrane α5GABAARs and tonic currents in CA1 pyramidal neurons in the hippocampus. Inhibition of α5GABAARs by L-655,708 alleviated cognitive impairments after anesthesia and surgery. More importantly, activation of α5GABAARs by L-838,417 abrogated the protective effects of Tat-CIRP-CMA against anesthesia and surgery-induced spatial reference learning and memory deficits. CONCLUSIONS: MD2 contributes to the occurrence of PND by regulating α5GABAARs in aged mice, and Tat-CIRP-CMA is a promising neuroprotectant against PND.