Lipopolysaccharide Stimulation Research Articles

Impacts of plasma microbial lipopolysaccharide translocation on B cell perturbations and anti-CD4 autoantibody production in people with HIV on suppressive antiretroviral therapy

Background. Up to 20% of people with HIV (PWH) who undergo virologically suppressed antiretroviral therapy (ART) fail to experience complete immune restoration. We recently reported that plasma anti-CD4 IgG (antiCD4IgG) autoantibodies from immune non-responders specifically deplete CD4 + T cells via antibody-dependent cytotoxicity. However, the mechanism of antiCD4IgG production remains unclear.Methods. Blood samples were collected from 16 healthy individuals and 25 PWH on suppressive ART. IgG subclass, plasma lipopolysaccharide (LPS), and antiCD4IgG levels were measured by ELISA. Gene profiles in B cells were analyzed by microarray and quantitative PCR. Furthermore, a patient-derived antiCD4IgG–producing B cell line was generated and stimulated with LPS in vitro. B cell IgG class switch recombination (CSR) was evaluated in response to LPS in splenic B cells from C57/B6 mice in vitro.Results. Increased plasma anti-CD4 IgGs in PWH were predominantly IgG1 and associated with increased plasma LPS levels as well as B cell expression of TLR2, TLR4, and MyD88 mRNA in vivo. Furthermore, LPS stimulation induced antiCD4IgG production in the antiCD4IgG B cell line in vitro. Finally, LPS promoted CSR in vitro.Conclusion. Our findings suggest that persistent LPS translocation may promote anti-CD4 autoreactive B cell activation and antiCD4IgG production in PWH on ART, which may contribute to gradual CD4 + T cell depletion. This study suggests that reversing a compromised mucosal barrier could improve ART outcomes in PWH who fail to experience complete immune restoration.

PKM2/STAT1-mediated PD-L1 upregulation on neutrophils during sepsis promotes neutrophil organ accumulation by serving an anti-apoptotic role

BackgroundDelayed neutrophil apoptosis during sepsis may impact neutrophil organ accumulation and tissue immune homeostasis. Elucidating the mechanisms underlying neutrophil apoptosis may help identify potential therapeutic targets. Glycolysis is critical to neutrophil activities during sepsis. However, the precise mechanisms through which glycolysis regulates neutrophil physiology remain under-explored, especially those involving the non-metabolic functions of glycolytic enzymes. In the present study, the impact of programmed death ligand-1 (PD-L1) on neutrophil apoptosis was explored. The regulatory effect of the glycolytic enzyme, pyruvate kinase M2 (PKM2), whose role in septic neutrophils remains unaddressed, on neutrophil PD-L1 expression was also explored.MethodsPeripheral blood neutrophils were isolated from patients with sepsis and healthy controls. PD-L1 and PKM2 levels were determined by flow cytometry and Western blotting, respectively. Dimethyl sulfoxide (DMSO)-differentiated HL-60 cells were stimulated with lipopolysaccharide (LPS) as an in vitro simulation of septic neutrophils. Cell apoptosis was assessed by annexin V/propidium iodide (annexin V/PI) staining, as well as determination of protein levels of cleaved caspase-3 and myeloid cell leukemia-1 (Mcl-1) by Western blotting. An in vivo model of sepsis was constructed by intraperitoneal injection of LPS (5 mg/kg) for 16 h. Pulmonary and hepatic neutrophil infiltration was assessed by flow cytometry or immunohistochemistry.ResultsPD-L1 level was elevated on neutrophils under septic conditions. Administration of neutralizing antibodies against PD-L1 partially reversed the inhibitory effect of LPS on neutrophil apoptosis. Neutrophil infiltration into the lung and liver was also reduced in PD-L1−/− mice 16 h after sepsis induction. PKM2 was upregulated in septic neutrophils and promoted neutrophil PD-L1 expression both in vitro and in vivo. In addition, PKM2 nuclear translocation was increased after LPS stimulation, which promoted PD-L1 expression by directly interacting with and activating signal transducer and activator of transcription 1 (STAT1). Inhibition of PKM2 activity or STAT1 activation also led to increased neutrophil apoptosis.ConclusionIn this study, a PKM2/STAT1-mediated upregulation of PD-L1 on neutrophils and the anti-apoptotic effect of upregulated PD-L1 on neutrophils during sepsis were identified, which may result in increased pulmonary and hepatic neutrophil accumulation. These findings suggest that PKM2 and PD-L1 could serve as potential therapeutic targets.

Gut microbiota indole-3-propionic acid mediates neuroprotective effect of probiotic consumption in healthy elderly: A randomized, double-blind, placebo-controlled, multicenter trial and in vitro study

The beneficial effects of probiotic consumption on age-related decline in cerebral function have been previously reported in the literature; however, the mechanistic link between gut and brain interactions has not yet been fully elucidated. Therefore, this study aimed to identify the role of gut microbiota-derived metabolites in gut-brain interactions via blood metabolomic profiling analysis in clinical trials and invitro mechanistic studies. A randomized, double-blind, placebo-controlled, multicenter clinical trial was conducted in 63 healthy elderly individuals (≥65 years of age). Participants were administered either placebo (placebo group, N=31) or probiotic capsules (Bifidobacterium bifidum BGN4 and Bifidobacterium longum BORI; probiotics group, N=32) for 12 weeks. Global and targeted metabolomic profiling analyses of their blood samples were then performed using 1H nuclear magnetic resonance and liquid chromatography-mass spectrometry methods, both at baseline and at the end of the trial. Gut microbial analysis was conducted using the 16S ribosomal ribonucleic acid gene sequencing method. Subsequently, microglial BV2 cells were treated invitro with indole-3-propionic acid (IPA) following lipopolysaccharide stimulation, and neuronal SH-SY5Y cells were treated with conditioned media from the BV2 cells. Finally, the levels of pro-inflammatory cytokines in BV2 cells and neurotrophins in SH-SY5Y cells were quantified using a real-time polymerase chain reaction or enzyme-linked immunosorbent assay. The metabolomic profiling analyses showed that probiotic consumption significantly altered the levels of metabolites involved in tryptophan metabolism (P<0.01). Among these metabolites, gut microbiota-produced IPA had a 1.91-fold increase in the probiotics group (P<0.05) and showed a significant relation to gut bacterial profiles (P<0.01). Elevated IPA levels were also positively associated with the level of serum brain-derived neurotropic factor (BDNF) in the probiotics group (r=0.28, P<0.05), showing an inverse trend compared to the placebo group. In addition, invitro treatment with IPA (5μM) significantly reduced the concentration of proinflammatory TNF-α in activated microglia (P<0.05), and neuronal cells cultured with conditioned media from IPA-treated microglia showed a significant increase in BDNF and nerve growth factor production (P<0.05). These results show that gut microbiota-produced IPA plays a role in protecting the microglia from inflammation, thus promoting neuronal function. Therefore, this suggests that IPA is a significant mediator linking the interaction between the gut and the brain in the elderly with probiotic supplementation.

Inhibition of the mevalonate pathway causes accumulation of CD14 in the endoplasmic reticulum of macrophages

Abstract Mevalonate kinase deficiency (MKD) is an autoinflammatory disease caused by mutations in the mevalonate kinase enzyme, which acts early in the mevalonate pathway to produce sterol and non-sterol isoprenoid compounds. Patients with MKD have episodes of inflammatory symptoms accompanied by increased plasma levels of pro-inflammatory cytokines. CD14 is a receptor that binds to lipopolysaccharide (LPS) and induces the release of cytokines. Isoprenoid depletion was previously shown to increase cellular and decrease extracellular levels of CD14 in macrophages after LPS stimulation. The objective of this study is to characterize the cellular localization of CD14 following isoprenoid depletion. RAW 264.7 macrophages were treated with lovastatin to deplete isoprenoids and CD14 localization was examined using immunofluorescence and western blotting after LPS stimulation. CD14 co-localized with the endoplasmic reticulum (ER) protein calnexin following lovastatin treatment. To quantitate ER CD14, cell lysates were digested with endoglycosidase (Endo) H, which removes high mannose glycans found on ER proteins, but not complex glycans added in the Golgi apparatus. Western blot results showed that the level of Endo H sensitive CD14 was higher in lovastatin as compared to carrier-treated cells. Co-incubation of lovastatin-treated cells with mevalonate or geranylgeranyl pyrophosphate (GGPP), but not cholesterol, prevented the ER accumulation of CD14 indicating that GGPP depletion may cause this effect. Understanding how mevalonate pathway products contribute to protein trafficking in macrophages will expand our understanding of inflammatory diseases.

AMPK reduces macrophage endotoxin tolerance through inhibition of TGF-β1 production and its signaling pathway.

Adenosine monophosphate-activated protein kinase (AMPK) is involved in suppression of the development of endotoxin tolerance, which is a driver of the immunosuppression induced by sepsis. However, the mechanism by which AMPK inhibits the development of endotoxin tolerance has not been clearly elucidated. Therefore, the present study was performed to investigate the mechanism by which the AMPK activator, metformin, inhibits the development of endotoxin tolerance. Lipopolysaccharide (LPS) increased the production of transforming growth factor (TGF)-β1 in macrophages, which was inhibited by metformin and resveratrol. Knockdown of AMPKα1 inhibited the suppressive effect of metformin on LPS-induced TGF-β1 production. TGF-β neutralizing antibody and TGF-β type I receptor inhibitor increased the production of TNF-α and IL-6 via LPS restimulation in tolerized macrophages. LPS increased Smad2 phosphorylation, but this was inhibited in cells treated with TGF-β neutralizing antibody or metformin. Smad2 knockdown inhibited the development of endotoxin tolerance, as evidenced by increased TNF-α production in response to LPS restimulation in tolerized macrophages. TGF-β1 expression was increased, and the levels of TNF-α and IL-6 production induced by LPS stimulation were decreased, in splenocytes of cecal ligation and puncture (CLP) model mice compared to sham-operated controls. However, metformin treatment suppressed the production of TGF-β1, and enhanced the production of TNF-α and IL-6 induced by LPS stimulation in splenocytes of CLP mice. These results indicated that AMPK activation inhibits LPS-induced TGF-β1 production and its signaling pathway, thus suppressing the development of endotoxin tolerance in macrophages.

Effect and mechanism of amphiregulin on acute respiratory distress syndrome in mice

To explore the protective effect of amphiregulin (Areg) on acute respiratory distress syndrome (ARDS) in mice and its underlying mechanism. (1) Male C57BL/6 mice aged 6-8 weeks were selected for animal experiments and divided into 3 groups (n = 10) according to the random number table method, which includes sham-operated group (Sham group), ARDS model group [ARDS model in mice was established by intratracheal instillation of lipopolysaccharide (LPS) 3 mg/kg] and ARDS+Areg intervention group [recombinant mice Areg (rmAreg) 5 μg was injected intraperitoneally 1 hour after LPS modeling]. The mice were sacrificed at 24 h after LPS injection lung histopathological changes were observed under hematoxylin-eosin (HE) staining and scored for lung injury; oxygenation index and wet/dry ratio of lung tissue were measured; the content of protein in bronchoalveolar lavage fluid (BALF) was detected by bicinchoninic acid (BCA) method, the level of inflammatory factors interleukins (IL-1β, IL-6) and tumor necrosis factor-α (TNF-α) in BALF were measured by enzyme-linked immunosorbent assay (ELISA). (2) Mice alveolar epithelial cell line MLE12 cells were obtained and cultured for experiment in vitro. Blank control group (Control group), LPS group (LPS 1 mg/L) and LPS+Areg group (rmAreg 50 μg/L was added 1 hour after LPS stimulation) were set. The cells and culture fluid were collected at 24 hours after LPS stimulation, and the apoptosis level of MLE12 cells was detected by flow cytometry; the activation level of phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) and the expressions of apoptosis-related proteins Bcl-2 and Bax in MLE12 cells were detected by Western blotting. (1) Animal experiments: compared with the Sham group, the lung tissue structure of ARDS model group was destroyed, the lung injury score was significantly increased, the oxygenation index was significantly decreased, the wet/dry weight ratio of lung was significantly increased, and the levels of protein and inflammatory factors in BALF were significantly increased. Compared with ARDS model group, lung tissue structure damage was reduced, pulmonary interstitial congestion, edema and inflammatory cell infiltration were significantly reduced, and lung injury score was significantly decreased (scores: 0.467±0.031 vs. 0.690±0.034) in ARDS+Areg intervention group. In addition, oxygenation index in ARDS+Areg intervention group was significantly increased [mmHg (1 mmHg ≈ 0.133 kPa): 380.00±22.36 vs. 154.00±20.74]. Lung wet/dry weight ratio (5.40±0.26 vs. 6.63±0.25), protein and inflammatory factors levels in BALF [protein (g/L): 0.42±0.04 vs. 0.86±0.05, IL-1β (ng/L): 30.00±2.00 vs. 40.00±3.65, IL-6 (ng/L): 190.00±20.30 vs. 581.30±45.76, TNF-α (ng/L): 30.00±3.65 vs. 77.00±4.16], and the differences were statistically significant (all P < 0.01). (2) Cell experiments: compared with the Control group, the number of apoptotic MLE12 cells was significantly increased in the LPS group, and the levels of PI3K phosphorylation, anti-apoptotic gene Bcl-2 level and pro-apoptotic gene Bax level were increased in MLE12 cells. Compared with the LPS group, the number of apoptosis in MLE12 cells was significantly reduced in the LPS+Areg group after administration of rmAreg treatment [(17.51±2.12)% vs. (36.35±2.84)%], and the levels of PI3K/AKT phosphorylation and Bcl-2 expression in MLE12 cells were significantly increased (p-PI3K/PI3K: 2.400±0.200 vs. 0.550±0.066, p-AKT/AKT: 1.647±0.103 vs. 0.573±0.101, Bcl-2/GAPDH: 0.773±0.061 vs. 0.343±0.071), and Bax expression was significantly suppressed (Bax/GAPDH: 0.810±0.095 vs. 2.400±0.200). The differences were statistically significant (all P < 0.01). Areg could alleviate ARDS in mice by inhibiting the apoptosis of alveolar epithelial cells through activating PI3K/AKT pathway.

Hydrogen regulates mitochondrial quality to protect glial cells and alleviates sepsis-associated encephalopathy by Nrf2/YY1 complex promoting HO-1 expression.

Sepsis-associated encephalopathy (SAE) is a complication of the central nervous system in patients with sepsis. Currently, no effective treatment for sepsis is available. Hydrogen plays a protective role in different diseases; however, the detailed mechanism of hydrogen-treated disease remains unclear. The purpose of this study was to investigate the effect of hydrogen on SAE in vitro and in vivo and the mechanism of hydrogen in mitochondrial dynamics and its function in astrocytes and microglia stimulated by lipopolysaccharides (LPSs). Animal models of SAE were generated by cecal ligation and puncture, and the SAE model was established by in vitro LPS stimulation. MTT, lactate dehydrogenase (LDH), reactive oxygen species (ROS), heme oxygenase-1 (HO-1) activity, mitochondrial membrane potential (MMP), and cell apoptosis assays were used to determine the effect of hydrogen on astrocytes and microglia stimulated by LPSs. The relationships between nuclear factor erythroid 2-related factor 2 (Nrf2), YY1, and HO-1 were examined by chromatin immunoprecipitation and co-immunoprecipitation. Mitochondrial homeostasis-related proteins in LPS-stimulated glial cells and brain tissues of SAE mice were detected by western blotting. The effects of hydrogen treatment in the SAE mouse model were investigated using Morris water maze and Y-maze analyses. After performing experiments with different concentrations of LPSs in vitro, we selected 1000ng/ml for subsequent experiments. Hydrogen attenuated the increase in ROS, LDH, and apoptosis and promoted decreases in cell activity and MMP, further promoting an increase in HO-1 expression induced by LPSs in astrocytes and microglia. Moreover, hydrogen further promoted the expression of Nrf2, HO-1, PGC-1α, TFAM, PARKIN, and PINK1, inhibited LPS-induced OPA1 and MFN2 expression in astrocytes and microglia, and downregulated the expression of DRP1 after LPS induction. Intriguingly, hydrogen treatment enhanced the binding between Nrf2 and YY1. However, silencing Nrf2 or YY1 abolished the protective effects of hydrogen on cell activity, LDH, ROS, and MMP; apoptosis; and regulation of Nrf2, HO-1, PGC-1α, TFAM, OPA1, DRP1, MFN2, PARKIN, and PINK1 in microglia. Finally, hydrogen treatment improved the results of behavioral detection, apoptosis, Nrf2, HO-1, PGC-1α, TFAM, OPA1, DRP1, MFN2, PARKIN, PINK1, and cytokines in SAE in vivo. Hydrogen improved cell injury and mitochondrial quality, which were associated with HO-1 expression promoted by the Nrf2/YY1 complex in vitro. Thus, hydrogen treatment may represent a novel therapeutic method for treating SAE.

Inhibition of DAI refrains dendritic cells from maturation and prolongs murine islet and skin allograft survival.

Central to allograft rejection is the T cell-mediated adaptive immune response initiated by activated dendritic cells (DCs). Previous studies have shown that the DNA-dependent activator of IFN regulatory factors (DAI) is involved in the maturation and activation of DCs. Therefore, we hypothesized that inhibition of DAI could prevent DCs from maturation and prolong murine allograft survival. Donor mouse bone marrow-derived dendritic cells (BMDCs) were transduced with the recombinant adenovirus vector (AdV-DAI-RNAi-GFP) to inhibit DAI expression (DC-DAI-RNAi), and the immune cell phenotype and function of DC-DAI-RNAi upon lipopolysaccharide (LPS) stimulation were evaluated. Then DC-DAI-RNAi was injected into recipient mice before islet transplantation and skin transplantation. The survival times of islet and skin allograft were recorded and the proportions of T cell subsets in spleen and secretion levels of cytokines in serum were measured. We identified that DC-DAI-RNAi inhibited the expression of main co-stimulatory molecules and MHC-II, exhibited strong phagocytic ability, and secreted high levels of immunosuppressive cytokines and low levels of immunostimulating cytokines. Recipient mice treated with DC-DAI-RNAi had longer islet and skin allograft survival times. In the murine islet transplantation model, we observed an increase in Treg cells proportion, a reduction in Th1 and Th17 cells proportions in spleen, and similar trends in their secreted cytokines in serum in the DC-DAI-RNAi group. Inhibition of DAI by adenovirus transduction inhibits the maturation and activation of DCs, affects the differentiation of T cell subsets as well as their secreted cytokines, and prolongs allograft survival.

Detection of tissue factor–positive extracellular vesicles using the ExoView R100 system

BackgroundTissue factor (TF) is essential for hemostasis. TF-expressing extracellular vesicles (TF+ EVs) are released in pathological conditions, such as trauma and cancer, and are linked to thrombosis. Detection of TF+ EV antigenically in plasma is challenging due to their low concentration but may be of clinical utility. ObjectivesWe hypthesised that ExoView can allow for direct measurement of TF+ EV in plasma, antigenically. MethodsWe utilized the anti-TF monoclonal antibody 5G9 to capture TF EV onto specialized ExoView chips. This was combined with fluorescent TF+ EV detection using anti-TF monoclonal antibody IIID8-AF647. We measured tumor cell-derived (BxPC-3) TF+ EV and TF+ EVs from plasma derived from whole blood with or without lipopolysaccharide (LPS) stimulation. We used this system to analyze TF+ EVs in 2 relevant clinical cohorts: trauma and ovarian cancer. We compared ExoView results with an EV TF activity assay. ResultsBxPC-3-derived TF+ EVs were identified with ExoView using 5G9 capture with IIID8-AF647 detection. 5G9 capture with IIID8-AF647 detection was significantly higher in LPS+ samples than in LPS samples and correlated with EV TF activity (R2 = 0.28). Trauma patient samples had higher levels of EV TF activity than healthy controls, but activity did not correlate with TF measurements made by ExoView (R2 = 0.15). Samples from patients with ovarian cancer have higher levels of EV TF activity than those from healthy controls, but activity did not correlate with TF measurement by ExoView (R2 = 0.0063). ConclusionTF+ EV measurement is possible in plasma, but the threshold and potential clinical applicability of ExoView R100, in this context, remain to be established.

The glycolysis/HIF-1α axis defines the inflammatory role of IL-4-primed macrophages

T helper type 2 (Th2) cytokine-activated M2 macrophages contribute to inflammation resolution and wound healing. This study shows that IL-4-primed macrophages exhibit a stronger response to lipopolysaccharide stimulation while maintaining M2 signature gene expression. Metabolic divergence between canonical M2 and non-canonical proinflammatory-prone M2 (M2INF) macrophages occurs after the IL-4Rα/Stat6 axis. Glycolysis supports Hif-1α stabilization and proinflammatory phenotype of M2INF macrophages. Inhibiting glycolysis blunts Hif-1α accumulation and M2INF phenotype. Wdr5-dependent H3K4me3 mediates the long-lasting effect of IL-4, with Wdr5 knockdown inhibiting M2INF macrophages. Our results also show that the induction of M2INF macrophages by IL-4 intraperitoneal injection and transferring of M2INF macrophages confer a survival advantage against bacterial infection invivo. In conclusion, our findings highlight the previously neglected non-canonical role of M2INF macrophages and broaden our understanding of IL-4-mediated physiological changes. These results have immediate implications for how Th2-skewed infections could redirect disease progression in response to pathogen infection.

Glucose Transporter Glut1-Dependent Metabolic Reprogramming Regulates Lipopolysaccharide-Induced Inflammation in RAW264.7 Macrophages.

This study investigated the critical role of Glut1-mediated glucose metabolism in the inflammatory response of macrophages, which are energy-intensive cells within the innate immune system. Inflammation leads to increased Glut1 expression, ensuring sufficient glucose uptake to support macrophage functions. We demonstrated that using siRNA to knock down Glut1 reduces the expression of various pro-inflammatory cytokines and markers, such as IL-6, iNOS, MHC II/CD40, reactive oxygen species, and the hydrogen sulfide (H2S)-producing enzyme cystathionine γ-lyase (CSE). Glut1 activates a pro-inflammatory profile through a nuclear factor (NF)-κB, while silencing Glut1 can prevent lipopolysaccharide (LPS)-induced IκB degradation, blocking NF-κB activation. Glut1's role in autophagy, an essential process for macrophage functions such as antigen presentation, phagocytosis, and cytokine secretion, was also measured. The findings show that LPS stimulation decreases autophagosome formation, but Glut1 knockdown reverses this effect, increasing autophagy beyond control levels. The study highlights Glut1's importance in macrophage immune responses and its regulation of apoptosis during LPS stimulation. Knocking down Glut1 negatively impacts cell viability and mitochondrial intrinsic pathway signaling. These findings collectively suggest that targeting macrophage glucose metabolism through Glut1 could potentially serve as a target for controlling inflammation.

Hepatocyte CD36 modulates UBQLN1-mediated proteasomal degradation of autophagic SNARE proteins contributing to septic liver injury

ABSTRACT Macroautophagy/autophagy plays a protective role in sepsis-induced liver injury. As a member of class B scavenger receptors, CD36 plays important roles in various disorders, such as atherosclerosis and fatty liver disease. Here we found that the expression of CD36 in hepatocytes was increased in patients and a mouse model with sepsis, accompanied by impaired autophagy flux. Furthermore, hepatocyte cd36 knockout (cd36-HKO) markedly improved liver injury and the impairment of autophagosome-lysosome fusion in lipopolysaccharide (LPS)-induced septic mice. Ubqln1 (ubiquilin 1) overexpression (OE) in hepatocyte blocked the protective effect of cd36-HKO on LPS-induced liver injury in mice. Mechanistically, with LPS stimulation, CD36 on the plasma membrane was depalmitoylated and distributed to the lysosome, where CD36 acted as a bridge molecule linking UBQLN1 to soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and hence promoting the proteasomal degradation of SNARE proteins, resulting in fusion impairment. Overall, our data reveal that CD36 is essential for modulating the proteasomal degradation of autophagic SNARE proteins in a UBQLN1-dependent manner. Targeting CD36 in hepatocytes is effective for improving autophagic flux in sepsis and therefore represents a promising therapeutic strategy for clinical treatment of septic liver injury. Abbreviations: AAV8: adeno-associated virus 8; AOSC: acute obstructive suppurative cholangitis; ATP1A1: ATPase, Na+/K+ transporting, alpha 1 polypeptide; CASP3: caspase 3; CASP8: caspase 8; CCL2: chemokine (C-C motif) ligand 2; cd36-HKO: hepatocyte-specific cd36 knockout; Co-IP: co-immunoprecipitation; CQ: chloroquine; Cys: cysteine; GOT1: glutamic-oxaloacetic transaminase 1, soluble; GPT: glutamic–pyruvic transaminase, soluble; IL1B: interleukin 1 beta; IL6: interleukin 6; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LDH, lactate dehydrogenase; LPS: lipopolysaccharide; LYPLA1: lysophospholipase 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OE: overexpression; qPCR: quantitative polymerase chain reaction; SNAP29: synaptosome associated protein 29; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TNF: tumor necrosis factor; TRIM: tripartite motif-containing; UBA: ubiquitin-associated; UBL: ubiquitin-like; UBQLN: ubiquilin; VAMP8: vesicle associated membrane protein 8; WT: wild-type.

Dolphin leukocytes exhibit an attenuated cytokine response and increase heme oxygenase activity upon exposure to lipopolysaccharides

Cetaceans exhibit physiological adaptations that allowed the transition to aquatic life, including a robust antioxidant defense system that prevents injury from repeated exposure to ischemia/reperfusion events associated with breath-hold diving. The signaling cascades that characterize ischemic inflammation in humans are well characterized. In contrast, cetaceans' molecular and biochemical mechanisms that confer tolerance to inflammatory events are poorly understood. Heme oxygenase (HO) is a cytoprotective protein with anti-inflammatory properties. HO catalyzes the first step in the oxidative degradation of heme. The inducible HO-1 isoform is regulated by various stimuli, including hypoxia, oxidant stress, and inflammatory cytokines. The objective of this study was to compare the response of HO-1 and cytokines to a proinflammatory challenge in leukocytes isolated from humans and bottlenose dolphins (Tursiops truncatus). We measured changes in HO activity, and abundance and expression of interleukin 1 beta (IL-1β), interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), and heme oxygenase 1 (HMOX1) in leukocytes treated with lipopolysaccharide (LPS) for 24 and 48 h. HO activity increased (p < 0.05) in dolphin (48 h) but not human cells. TNF-α expression increased in human (24 h, 48 h), but not dolphin cells following LPS stimulation. LPS-induced cytokine expression was lower in dolphin than in human leukocytes, suggesting a blunted cytokine response in bottlenose dolphin leukocytes treated with LPS. Results suggest species-specific regulation of inflammatory cytokines in leukocytes treated with LPS, which may lead to differential responses to a pro-inflammatory challenge between marine and terrestrial mammals.

The pluripotent factor OCT4A enhances the self-renewal of human dental pulp stem cells by targeting lncRNA FTX in an LPS-induced inflammatory microenvironment

BackgroundRegulating the pluripotency of human dental pulp stem cells (hDPSCs) is key for the self-repair of injured dental pulp. We previously found that OCT4A promotes the proliferation and odontogenic differentiation of human dental pulp cells (hDPCs). Recent studies have shown the interaction between OCT4A and lncRNAs in pluripotency maintenance of various stem cells. The aim of this study was to explore the underlying roles and mechanisms of OCT4A and its related lncRNAs in the proliferation and multidirectional differentiation of hDPSCs in an inflammatory microenvironment.MethodsHuman lncRNA microarrays were applied to screen out the differentially expressed lncRNAs in hDPSCs between the OCT4A-overexpressing and vector groups. Lipopolysaccharide (LPS) was used to simulate the inflammatory microenvironment. The effects of OCT4A and the lncRNA FTX on the proliferation and multidifferentiation of hDPSCs were observed by the CCK-8 assay, EdU staining, real-time PCR, western blotting, and Alizarin red and oil red O staining. Bioinformatics analysis and chromatin immunoprecipitation (ChIP) assays were performed to clarify the targeted mechanism of OCT4A on FTX. The regulation by FTX of the expression of OCT4A and its downstream pluripotent transcription factors SOX2 and c-MYC was further detected by real-time PCR and western blotting.ResultsThe microarray results showed that 978 lncRNAs (250 of which were upregulated and 728 downregulated) were potentially differentially expressed genes (fold change ≥ 2, P < 0.05). LPS stimulation attenuated the self-renewal of hDPSCs. OCT4A enhanced the cell proliferation and multidifferentiation capacities of hDPSCs in an inflammatory microenvironment, while FTX exhibited the opposite effects. OCT4A negatively regulated FTX function by binding to specific regions on the FTX promoter, thereby inhibiting the transcription of FTX. Moreover, overexpression of FTX downregulated the expression of OCT4A, SOX2 and c-MYC, whereas knockdown of FTX facilitated their expression.ConclusionsOCT4A was found to be a crucial factor maintaining the self-renewal of hDPSCs by transcriptionally targeting FTX in an inflammatory microenvironment. Moreover, we proposed a novel function of FTX in negatively regulating the pluripotency and multilineage differentiation capacity of hDPSCs. The hierarchical organization between OCT4A and FTX expanded the understanding of the network between transcription factors and lncRNAs in fine-tuning the pluripotency/differentiation balance of adult stem cells, and provided prospective targets for optimizing dental-derived stem cell sources for regenerative endodontics.

Rnf144b alleviates the inflammatory responses and cardiac dysfunction in sepsis.

Sepsis is an inflammatory disease with high mortality and morbidity. Inflammation plays an essential role in sepsis, and suppressing inflammation has been shown to ameliorate sepsis. Rnf144b is an ubiquitin E3 ligation with anti-inflammation activities. Its precise roles in sepsis remain unknown. Here, we explored the function of Rnf144b in sepsis. We generated conditional knockout mice with Rnf144b deficiency in the myeloid cells. We monitored the Rnf144b expression in peripheral blood mononuclear cells from healthy donor and patients with sepsis, and in lipopolysaccharides (LPS)-treated bone marrow-derived macrophages (BMDMs). The cytokine expression between wild-type BMDMs and Rnf144b-deficient BMDMs after LPS and CpG treatments was compared. The survival rate and cardiac function were monitored. The activation of TANK binding kinase 1 and nuclear factor kappa-B was examined by Western blot and real-time PCR. Up-regulated expression of Rnf144b was observed in peripheral blood mononuclear cells from patients with sepsis. LPS induced the expression of Rnf144b in BMDMs. Rnf144b-deficient BMDMs produced more inflammatory cytokines after LPS or CpG stimulation. Septic mice with Rnf144b deficiency in myeloid cells had higher mortality and exacerbated cardiac dysfunction. Rnf144b interacted with TANK binding kinase 1 and Rnf144b deficiency resulted in impaired activation of TBK1 but enhanced activation of nuclear factor kappa-B. Rnf144b prevents inflammatory responses and cardiac dysfunction in sepsis.

RNA-Sequencing Characterization of lncRNA and mRNA Functions in Septic Pig Liver Injury.

We assessed differentially expressed (DE) mRNAs and lncRNAs in the liver of septic pigs to explore the key factors regulating lipopolysaccharide (LPS)-induced liver injury. We identified 543 DE lncRNAs and 3642 DE mRNAs responsive to LPS. Functional enrichment analysis revealed the DE mRNAs were involved in liver metabolism and other pathways related to inflammation and apoptosis. We also found significantly upregulated endoplasmic reticulum stress (ERS)-associated genes, including the receptor protein kinase receptor-like endoplasmic reticulum kinase (PERK), the eukaryotic translation initiation factor 2α (EIF2S1), the transcription factor C/EBP homologous protein (CHOP), and activating transcription factor 4 (ATF4). In addition, we predicted 247 differentially expressed target genes (DETG) of DE lncRNAs. The analysis of protein-protein interactions (PPI) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway detected key DETGs that are involved in metabolic pathways, such as N-Acetylgalactosaminyltransferase 2 (GALNT2), argininosuccinate synthetase 1 (ASS1), and fructose 1,6-bisphosphatase 1 (FBP1). LNC_003307 was the most abundant DE lncRNA in the pig liver, with a marked upregulation of >10-fold after LPS stimulation. We identified three transcripts for this gene using the rapid amplification of the cDNA ends (RACE) technique and obtained the shortest transcript sequence. This gene likely derives from the nicotinamide N-methyltransferase (NNMT) gene in pigs. According to the identified DETGs of LNC_003307, we hypothesize that this gene regulates inflammation and endoplasmic reticulum stress in LPS-induced liver damage in pigs. This study provides a transcriptomic reference for further understanding of the regulatory mechanisms underlying septic hepatic injury.

High-Glucose Media Reduced the Viability and Induced Differential Pro-Inflammatory Cytokines in Human Periodontal Ligament Fibroblasts.

Hyperglycemic condition in diabetic patients tends to exacerbate periodontitis severity. Thus, the influence of hyperglycemia on the biological and inflammatory response of periodontal ligament fibroblasts (PDLFs) needs to be elucidated. In this study, PDLFs were seeded in media containing glucose concentrations (5.5, 25, or 50 mM) and stimulated with 1 µg/mL of lipopolysaccharide (LPS). PDLFs' viability, cytotoxicity, and the migration ability were determined. The mRNA expression of Interleukin (IL)-6, IL-10, and IL-23 (p19/p40), and Toll-like receptor (TLR)-4 were analyzed; at 6 and 24 h, protein expression of IL-6 and IL-10 was also determined. PDLFs grown in 50 mM glucose medium showed lower viability. The 5.5 mM glucose led to the highest percentage of wound closure compared to 25 mM and 50 mM glucose with/without LPS. Additionally, 50 mM glucose with LPS exhibited the least migration ability among all groups. The expression of IL-6 was amplified significantly in LPS-stimulated cells in 50 mM glucose medium. IL-10 was constitutively expressed in different glucose concentrations, and LPS stimulation decreased it. IL-23 p40 was up-regulated after LPS stimulation in 50 mM glucose concentration. TLR-4 was highly expressed after LPS stimulation in all glucose concentrations. Hyperglycemic conditions limit PDLF proliferation and migration, and enhance the expression of certain pro-inflammatory cytokines to induce periodontitis.

Protective effects of an anti-4-HNE monoclonal antibody against liver injury and lethality of endotoxemia in mice

4-hydroxy-2-nonenal (4-HNE) is a lipid peroxidation product that is known to be elevated during oxidative stress. During systemic inflammation and endotoxemia, plasma levels of 4-HNE are elevated in response to lipopolysaccharide (LPS) stimulation. 4-HNE is a highly reactive molecule due to its generation of both Schiff bases and Michael adducts with proteins, which may result in modulation of inflammatory signaling pathways. In this study, we report the production of a 4-HNE adduct-specific monoclonal antibody (mAb) and the effectiveness of the intravenous injection of this mAb (1 mg/kg) in ameliorating LPS (10 mg/kg, i.v.)-induced endotoxemia and liver injury in mice. Endotoxic lethality in control mAb-treated group was suppressed by the administration of anti-4-HNE mAb (75 vs. 27%). After LPS injection, we observed a significant increase in the plasma levels of AST, ALT, IL-6, TNF-α and MCP-1, and elevated expressions of IL-6, IL-10 and TNF-α in the liver. All these elevations were inhibited by anti-4-HNE mAb treatment. As to the underlining mechanism, anti-4-HNE mAb inhibited the elevation of plasma high mobility group box-1 (HMGB1) levels, the translocation and release of HMGB1 in the liver and the formation of 4-HNE adducts themselves, suggesting a functional role of extracellular 4-HNE adducts in hypercytokinemia and liver injury associated with HMGB1 mobilization. In summary, this study reveals a novel therapeutic application of anti-4-HNE mAb for endotoxemia.

Phloroglucinol possesses anti-inflammatory activities by regulating AMPK/Nrf2/HO-1 signaling pathway in LPS-stimulated RAW264.7 murine macrophages

Background Inflammation is closely related to the pathogenesis of chronic illnesses. Secondary metabolites of marine seaweeds are recognized as reliable sources of bioactive compounds due to their health benefits besides their nutritional value. The objective of this study was to determine the potential anti-inflammatory effect of phloroglucinol (Phl) in RAW264.7 murine macrophages after lipopolysaccharides (LPS) stimulation. Methods MTT, nitric oxide (NO), and DCFH-DA assays were conducted to determine cell viability, NO production, and reactive oxygen species (ROS) generation respectively. Pro-inflammatory cytokines and prostaglandin E2 (PGE2) levels were measured using ELISA assay kits. Protein expression levels were determined by western blot analysis. Results Phl treatment showed a promising anti-inflammatory effect by reducing NO production, secretion of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6), PGE2 production, protein expression levels of inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), and ROS generation in LPS-stimulated RAW264.7 murine macrophages. Phl treatment upregulated heme oxygenase-1 (HO-1) expression by inducing nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and activating AMPK. However, Zinc protoporphyrin (ZnPP), an inhibitor of HO-1, partially reversed these effects, including NO production, pro-inflammatory cytokine secretion, iNOS, COX-2 and HO-1 expression, and ROS generation. Conclusion Phl has potential anti-inflammatory activities by regulating AMPK/Nrf2/HO-1 pathway in LPS-stimulated RAW264.7 murine macrophages.

Establishment of specific age-related macular degeneration relevant gene expression panels using porcine retinal pigment epithelium for assessing fucoidan bioactivity

PurposeAge-related macular degeneration (AMD) is the leading cause of severe vision loss in industrialized nations. Important factors in pathogenesis are oxidative stress, inflammation, and, in the wet form of AMD, angiogenesis. Fucoidans, sulfated polysaccharides from brown algae, may have antioxidant, anti-inflammatory, and antiangiogenic effects. In this study, we established specific gene expression panels for inflammation, oxidative stress and angiogenesis in porcine retinal pigment epithelium (RPE), and investigated the effect of fucoidans on gene expression under different noxious agents. MethodsPrimary porcine RPE cells cultured for at least 14 days were used. Using viability assays with tetrazolium bromide and real-time polymerase chain reaction of marker genes, positive controls were established for appropriate concentrations and exposure times of selected noxious agents (lipopolysaccharide (LPS), H2O2, CoCl2). Three different AMD relevant gene panels specific for porcine RPE for inflammation, oxidative stress, and angiogenesis were established, and the influence of fucoidans (mainly Fucus vesiculosus; FV) on gene expression was investigated. ResultsThe following was shown by gene expression analyses:(1) Inflammation panel: Expression of 18 genes was affected under LPS (three days). Among them, LPS increased genes for interleukin 1 receptor 2, interleukin 8, cyclooxygenase-2 and vascular cell adhesion protein 1 expression which were diminished when FV was present.(2) Oxidative stress panel: Under stimulation of H2O2 (one day) and LPS (one day), expression of a total of 15 genes was affected. LPS induced increase in genes for superoxide dismutase-1, C-X-C motif chemokine 10, and CC chemokine ligand-5 expression was not detected when FV was present.(3) Angiogenesis panel: Under stimulation with CoCl2 (three days) expression of six genes was affected, with the increase of genes for angiopoietin 2, vascular endothelial growth factor receptor-1, and follistatin being diminished when FV was present. ConclusionThree specific gene expression panels for porcine RPE that map genes for three of the major pathological factors of AMD, inflammation, oxidative stress, and angiogenesis, were established. Further, we demonstrated that fucoidans can reduce stress related gene activation in all of these three major pathogenic pathways. This study is another indication that fucoidans can act on different pathomechanisms of AMD simultaneously, which provides further evidence for fucoidans as a possible drug for treatment and prevention of AMD.