LPS-induced Pro-inflammatory Responses Research Articles

Radix Sophorae Flavescentis of Sophora flavescens Aiton inhibits LPS-induced macrophage pro-inflammatory response via regulating CFHR2 expression.

Long-term chronic inflammation often leads to chronic diseases. Although Sophora flavescens has been shown to have anti-inflammatory properties, its detailed molecular mechanism is still unknown. This study investigated the effect of Radix Sophorae Flavescentis on the LPS-induced inflammatory response in macrophages. LPS was used to induce the peritoneal macrophages to simulate the inflammatory environment in vitro. Different concentrations of Radix Sophorae Flavescentis-containing (medicated) serum were used for intervention. The peritoneal macrophages were identified by using hematoxylin-eosin and immunofluorescence staining. ELISA was used to measure the TNF-α and IL-6 expression to determine the concentration of LPS. ELISA and Western blot (WB) were used to detect the PGE2 and CFHR2 expression in each group, respectively. The lentiviral vector for interference and overexpression of the CFHR2 gene was constructed, packaged, and transfected into LPS-induced macrophages. The transfection efficiency was verified by WB. Then, ELISA was used to detect the TNF-α, PGE2, and IL-6 expression. WB was used to detect the CFHR2, iNOS, COX-2, TLR2, TLR4, IFN-γ, STAT1, and p-STAT1 expression. The primary isolated cells were identified as macrophages. The LPS-treated macrophages exhibited significantly higher expression of PGE2 and CFHR2 and the inflammatory factors TNF-α and IL-6, as well as iNOS, COX-2, TLR2, TLR4, IFN-γ, STAT1, and p-STAT1 expression compared with the control group (P<0.05). The TNF-α, PGE2, and IL-6 levels, as well as CFHR2, iNOS, COX-2, TLR2, TLR4, IFN-γ, STAT1, and p-STAT1 expression were considerably lower in the LPS-induced+10% medicated-serum group, LPS-induced+20% medicated-serum group, and shCFHR interference group compared with the LPS group (P<0.05). Radix Sophorae Flavescentis might mediate CFHR2 expression and play an important role in inhibiting the LPS-induced pro-inflammatory response of macrophages. Radix Sophorae Flavescentis could be a potential treatment for LPS-induced related inflammatory diseases.

Inhibition of voltage-gated Hv1 alleviates LPS-induced neuroinflammation via regulation of microglial metabolic reprogramming

A growing body of evidence highlights the crucial role of metabolic reprogramming in activated immune cells, significantly contributing to both the initiation and progression of neuroinflammation and neurodegenerative diseases. The voltage-gated H channel (Hv1) has been reported to be involved in microglial activation and acts as a key driver of neuroinflammation. This study aimed to explore how Hv1-mediated metabolic reprogramming contributes to neuroinflammation and to assess the therapeutic potential of the Hv1 inhibitor 2-GBI in a model of lipopolysaccharide (LPS)-induced neuroinflammation. We investigated the influence of 2-GBI on the generation of ROS, metabolic reprogramming, and pro-inflammatory mediator production in vitro and examined the therapeutic effect of 2-GBI on microglial activation and hippocampal neuroinflammation in vivo. The results indicated that 2-GBI attenuated the LPS-induced pro-inflammatory response and aerobic glycolysis in microglia, specifically mitigating HIF1α-mediated upregulation of glycolysis. 2-GBI exerted a protective effect against LPS-induced neuroinflammation through HIF1α pathway-regulated aerobic glycolysis. Using a transwell coculture system, we demonstrated that 2-GBI reversed PC12 cell death caused by BV2-mediated neuroinflammation. In vivo experiments further suggested that 2-GBI mitigated neuroinflammatory processes and cognitive dysfunction via microglial metabolic reprogramming. Collectively, our results highlight the potential of Hv1 inhibition as a therapeutic strategy for alleviating LPS-induced neuroinflammation by modulating microglial metabolic reprogramming.

Polar Lipids of Marine Microalgae Nannochloropsis oceanica and Chlorococcum amblystomatis Mitigate the LPS-Induced Pro-Inflammatory Response in Macrophages.

Microalgae are recognized as a relevant source of bioactive compounds. Among these bioactive products, lipids, mainly glycolipids, have been shown to present immunomodulatory properties with the potential to mitigate chronic inflammation. This study aimed to evaluate the anti-inflammatory effect of polar lipids isolated from Nannochloropsis oceanica and Chlorococcum amblystomatis. Three fractions enriched in (1) digalactosyldiacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol (SQDG), (2) monogalactosyldiacylglycerol (MGDG), and (3) diacylglyceryl-trimethylhomoserine (DGTS) and phospholipids (PL) were obtained from the total lipid extracts (TE) of N. oceanica and C. amblystomatis, and their anti-inflammatory effect was assessed by analyzing their capacity to counteract nitric oxide (NO) production and transcription of pro-inflammatory genes Nos2, Ptgs2, Tnfa, and Il1b in lipopolysaccharide (LPS)-activated macrophages. For both microalgae, TE and Fractions 1 and 3 strongly inhibited NO production, although to different extents. A strong reduction in the LPS-induced transcription of Nos2, Ptgs2, Tnfa, and Il1b was observed for N. oceanica and C. amblystomatis lipids. The most active fractions were the DGTS-and-PL-enriched fraction from N. oceanica and the DGDG-and-SQDG-enriched fraction from C. amblystomatis. Our results reveal that microalgae lipids have strong anti-inflammatory capacity and may be explored as functional ingredients or nutraceuticals, offering a natural solution to tackle chronic inflammation-associated diseases.

Anti-Inflammatory Effect of Cinnamomum japonicum Siebold's Leaf through the Inhibition of p38/JNK/AP-1 Signaling.

Cinnamomum japonicum Siebold (CJ) branch bark, commonly known as Japanese cinnamon, has been used for various culinary and medicinal applications for many centuries. Although the efficacy of CJ branch bark's anti-inflammatory and antioxidant activity for the treatment of various diseases has been confirmed, the efficacy of CJ leaves (CJLs) has not been examined. We therefore investigated whether CJL3, an ethyl acetate extract of a 70% ethanol CJL extract, exerts anti-inflammatory effects on lipopolysaccharide (LPS)-activated Kupffer cells, specialized macrophages found in the liver. Liver inflammation can activate Kupffer cells, inducing the release of pro-inflammatory molecules that contribute to tissue damage. We found that CJL3 has high 2,2-diphenyl-1-picrylhydrazyl and 2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) radical-scavenging activity. Among the CJL extracts, CJL3 exhibited the greatest polyphenol content, with protocatechuic acid and 4-hydroxybenzoic acid being the most abundant. In addition, we verified that CJL3, which has strong antioxidant properties, ameliorates LPS-induced pro-inflammatory responses by inhibiting p38/JNK/AP-1 signaling. CJL3 therefore has potential for treating liver disease, including hepatitis.

Lomerizine inhibits LPS-mediated neuroinflammation and tau hyperphosphorylation by modulating NLRP3, DYRK1A, and GSK3α/β.

Lomerizine is a calcium channel blocker that crosses the blood-brain barrier and is used clinically in the treatment of migraines. However, whether lomerizine is beneficial in modulating neuroinflammatory responses has not been tested yet. To assess the potential of lomerizine for repurposing as a treatment for neuroinflammation, we investigated the effects of lomerizine on LPS-induced proinflammatory responses in BV2 microglial cells, Alzheimer's disease (AD) excitatory neurons differentiated from induced pluripotent stem cells (iPSCs), and in LPS-treated wild type mice. In BV2 microglial cells, lomerizine pretreatment significantly reduced LPS-evoked proinflammatory cytokine and NLRP3 mRNA levels. Similarly, lomerizine pretreatment significantly suppressed the increases in Iba-1, GFAP, proinflammatory cytokine and NLRP3 expression induced by LPS in wild-type mice. In addition, lomerizine posttreatment significantly decreased LPS-stimulated proinflammatory cytokine and SOD2 mRNA levels in BV2 microglial cells and/or wild-type mice. In LPS-treated wild-type mice and AD excitatory neurons differentiated from iPSCs, lomerizine pretreatment ameliorated tau hyperphosphorylation. Finally, lomerizine abolished the LPS-mediated activation of GSK3α/β and upregulation of DYRK1A, which is responsible for tau hyperphosphorylation, in wild-type mice. These data suggest that lomerizine attenuates LPS-mediated neuroinflammatory responses and tau hyperphosphorylation and is a potential drug for neuroinflammation- or tauopathy-associated diseases.

ACAT1/SOAT1 Blockade Suppresses LPS-Mediated Neuroinflammation by Modulating the Fate of Toll-like Receptor 4 in Microglia.

Cholesterol is stored as cholesteryl esters by the enzymes acyl-CoA:cholesterol acyltransferases/sterol O:acyltransferases (ACATs/SOATs). ACAT1 blockade (A1B) ameliorates the pro-inflammatory responses of macrophages to lipopolysaccharides (LPS) and cholesterol loading. However, the mediators involved in transmitting the effects of A1B in immune cells is unknown. Microglial Acat1/Soat1 expression is elevated in many neurodegenerative diseases and in acute neuroinflammation. We evaluated LPS-induced neuroinflammation experiments in control vs. myeloid-specific Acat1/Soat1 knockout mice. We also evaluated LPS-induced neuroinflammation in microglial N9 cells with and without pre-treatment with K-604, a selective ACAT1 inhibitor. Biochemical and microscopy assays were used to monitor the fate of Toll-Like Receptor 4 (TLR4), the receptor at the plasma membrane and the endosomal membrane that mediates pro-inflammatory signaling cascades. In the hippocampus and cortex, results revealed that Acat1/Soat1 inactivation in myeloid cell lineage markedly attenuated LPS-induced activation of pro-inflammatory response genes. Studies in microglial N9 cells showed that pre-incubation with K-604 significantly reduced the LPS-induced pro-inflammatory responses. Further studies showed that K-604 decreased the total TLR4 protein content by increasing TLR4 endocytosis, thus enhancing the trafficking of TLR4 to the lysosomes for degradation. We concluded that A1B alters the intracellular fate of TLR4 and suppresses its pro-inflammatory signaling cascade in response to LPS.

Precision-cut rat placental slices as a model to study sex-dependent inflammatory response to LPS and Poly I:C.

Maternal inflammation in pregnancy represents a major hallmark of several pregnancy complications and a significant risk factor for neurodevelopmental and neuropsychiatric disorders in the offspring. As the interface between the mother and the fetus, the placenta plays a crucial role in fetal development and programming. Moreover, studies have suggested that the placenta responds to an inflammatory environment in a sex-biased fashion. However, placenta-mediated immunoregulatory mechanisms are still poorly understood. Therefore, we have developed a model of ex vivo precision-cut placental slices from the rat term placenta to study acute inflammatory response. Rat placental slices with a precise thickness of 200 µm were generated separately from male and female placentas. Inflammation was stimulated by exposing the slices to various concentrations of LPS or Poly I:C for 4 and 18 hours. Treatment of placental slices with LPS significantly induced the expression and release of proinflammatory cytokines TNF-α, IL-6, and IL-1β. In contrast, Poly I:C treatment resulted in a less-pronounced inflammatory response. Interestingly, the female placenta showed higher sensitivity to LPS than male placenta. Anti-inflammatory agents, curcumin, 1α,25- dihydroxyvitamin D3, and progesterone attenuated the LPS-induced proinflammatory cytokine response at both mRNA and protein levels. We conclude that rat placental slices represent a novel alternative model to study the role of sexual dimorphism in the acute inflammatory response and immune activation in pregnancy.

10-Residue MyD88-Peptide Adopts β-Sheet Structure, Self-Assembles, Binds to Lipopolysaccharides, and Rescues Mice from Endotoxin-Mediated Lung-Infection and Death.

Naturally occurring cationic antimicrobial peptides (AMPs) mostly adopt α-helical structures in bacterial membrane mimetic environments. To explore the design of novel β-sheet AMPs, we identified two short cationic amphipathic β-strand segments from the crystal structure of the innate immune protein, MyD88. Interestingly, of these, the 10-residue arginine-valine-rich synthetic MyD88-segment, KRCRRMVVVV (M3), exhibited β-sheet structure when bound to the outer membrane Gram-negative bacterial component, LPS. Isothermal titration calorimetric data showed that M3 bound to LPS with high affinity, and the interaction was hydrophobic in nature. Supporting these observations, computational studies indicated strong interactions of multiple and consecutive valine residues of M3 with the acyl chain of LPS. Moreover, M3 adopted nanosheet and nanofibrillar structure in 25% acetonitrile/water and isopropanol, respectively. M3 showed substantial antibacterial activities against both Gram-positive and Gram-negative bacteria which it appreciably retained in the presence of human serum and physiological salts. M3 was non-hemolytic against human red blood cells and non-cytotoxic to 3T3 cells up to 200 μM and to mice in vivo at a dose of 40 mg/kg. Furthermore, M3 neutralized LPS-induced pro-inflammatory responses in THP-1 cells and rat bone marrow-derived macrophages. Consequently, M3 attenuated LPS-mediated lung inflammation in mice and rescued them (80% survival at 10 mg/kg dose) against a lethal dose of LPS. The results demonstrate the identification of a 10-mer LPS-interacting, β-sheet peptide from MyD88 with the ability to form nanostructures and in vivo activity against LPS challenge in mice. The identified M3-template provides scope for designing novel bioactive peptides with β-sheet structures and self-assembling properties.

Single cell analysis of docosahexaenoic acid suppression of sequential LPS-induced proinflammatory and interferon-regulated gene expression in the macrophage.

Preclinical and clinical studies suggest that consumption of long chain omega-3 polyunsaturated fatty acids (PUFAs) reduces severity of chronic inflammatory and autoimmune diseases. While these ameliorative effects are conventionally associated with downregulated expression of proinflammatory cytokine and chemokine genes, our laboratory has recently identified Type 1 interferon (IFN1)-regulated gene expression to be another key target of omega-3 PUFAs. Here we used single cell RNA sequencing (scRNAseq) to gain new mechanistic perspectives on how the omega-3 PUFA docosahexaenoic acid (DHA) influences TLR4-driven proinflammatory and IFN1-regulated gene expression in a novel self-renewing murine fetal liver-derived macrophage (FLM) model. FLMs were cultured with 25 µM DHA or vehicle for 24h, treated with modest concentration of LPS (20 ng/ml) for 1 and 4h, and then subjected to scRNAseq using the 10X Chromium System. At 0h (i.e., in the absence of LPS), DHA increased expression of genes associated with the NRF2 antioxidant response (e.g. Sqstm1, Hmox1, Chchd10) and metal homeostasis (e.g.Mt1, Mt2, Ftl1, Fth1), both of which are consistent with DHA-induced polarization of FLMs to a more anti-inflammatory phenotype. At 1h post-LPS treatment, DHA inhibited LPS-induced cholesterol synthesis genes (e.g. Scd1, Scd2, Pmvk, Cyp51, Hmgcs1, and Fdps) which potentially could contribute to interference with TLR4-mediated inflammatory signaling. At 4h post-LPS treatment, LPS-treated FLMs reflected a more robust inflammatory response including upregulation of proinflammatory cytokine (e.g. Il1a, Il1b, Tnf) and chemokine (e.g.Ccl2, Ccl3, Ccl4, Ccl7) genes as well as IFN1-regulated genes (e.g. Irf7, Mx1, Oasl1, Ifit1), many of which were suppressed by DHA. Using single-cell regulatory network inference and clustering (SCENIC) to identify gene expression networks, we found DHA modestly downregulated LPS-induced expression of NF-κB-target genes. Importantly, LPS induced a subset of FLMs simultaneously expressing NF-κB- and IRF7/STAT1/STAT2-target genes that were conspicuously absent in DHA-pretreated FLMs. Thus, DHA potently targeted both the NF-κB and the IFN1 responses. Altogether, scRNAseq generated a valuable dataset that provides new insights into multiple overlapping mechanisms by which DHA may transcriptionally or post-transcriptionally regulate LPS-induced proinflammatory and IFN1-driven responses in macrophages.

Modulation of vascular endothelial inflammatory response by proprotein convertase subtilisin-kexin type 9

Background and AimsEndotoxins carried within LDL are cleared from the circulation via hepatic LDL receptor (LDLR)-mediated endocytosis. Proprotein convertase subtilisin-kexin type 9 (PCSK9) reduces this clearance by down-regulating LDLR density on hepatocytes. In addition to hepatocytes, vascular endothelial cells also express receptor targets of PCSK9, including LDLR. Therefore, we hypothesized that PCSK9 may regulate vascular endothelial cell uptake of lipopolysaccharide (LPS) and alter the vascular endothelial cell inflammatory response. Methods and resultsWe found that LPS is internalized by human umbilical vein vascular endothelial cells (HUVECs) and LPS uptake dose-dependently increased with increasing LDL concentration. Intracellular LPS co-localized with LDL. PCSK9 and, separately, blocking antibodies against LDLR, dose-dependently decreased the vascular endothelial cell uptake of LPS and, furthermore, inhibition of endocytosis using Dynasore blocked LPS uptake. In contrast, blocking antibodies against TLR4 did not alter LPS uptake. PCSK9 decreased the LPS-induced proinflammatory response (IL-6 and IL-8 gene expression and protein secretion, and VCAM-1/ICAM-1 expression) in vascular endothelial cells. In addition, a decrease in PCSK9 and increase in LDLR, mediated by triciribine or siPCSK9, increased LPS uptake and the LPS-induced proinflammatory response. Similar results were also found in aortic vascular tissue from Pcsk9−/− mice after LPS injection. ConclusionsOur data suggest that, similar to PCSK9 treatment in hepatocytes, PCSK9 reduces vascular endothelial cell uptake of LPS via LDLR-mediated endocytosis. Consequently, PCSK9 decreases the LPS-induced proinflammatory response in vascular endothelial cells. These results raise the possibility that PCSK9 inhibition may have additional effects on vascular endothelial inflammation via this alternative pathway, beyond the known effects of PCSK9 inhibition on LDL lowering and hepatic endotoxin clearance.

MiR-146b-5p/TRAF6 axis is essential for Ginkgo biloba L. extract GBE to attenuate LPS-induced neuroinflammation.

Background: Neuroinflammation plays a crucial role in the pathogenesis and progression of various neurodegenerative diseases, including Alzheimer’s disease. The Ginkgo biloba leaf extract (GBE) has been widely used to treat cerebral and peripheral blood circulation disorders. However, its potential targets and underlying mechanisms regarding neuroinflammation have not yet been characterized. Aims: The purpose of this study was to investigate and validate the anti-neuroinflammatory properties of GBE against lipopolysaccharide (LPS)-mediated inflammation and to determine the underlying molecular mechanisms. Methods: The effect of GBE on LPS-induced release of inflammatory cytokines was examined using ELISA and western blot assay. The effects of GBE on NF-κB binding activity and translocation were determined via luciferase, streptavidin-agarose pulldown, and immunofluorescence assays. The potential targets of GBE were screened from the GEO and microRNA databases and further identified via qPCR, luciferase, gene mutation, and western blot assays. Results: GBE significantly inhibited LPS-induced pro-inflammatory responses in BV-2 and U87 cells, with no obvious cytotoxicity. GBE significantly induced miR-146b-5p expression, which negatively regulated TRAF6 expression by targeting its 3′-UTR. Thus, due to TRAF6 suppression, GBE decreases the transcriptional activity of NF-κB and the expression of pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and cyclooxygenase (COX)-2, and finally reverses LPS-induced neuroinflammation. Conclusion: Our study revealed the anti-neuroinflammatory mechanism of GBE through the miR-146b-5p/TRAF6 axis and provided a theoretical basis for its rational clinical application.

Identification and characterization of two novel functional CD39 splice variants that negatively modulate the proinflammatory response in the Japanese flounder Paralichthys olivaceus

Ecto-nucleoside triphosphate diphosphohydrolase 1 (CD39) is a key regulator of inflammatory responses by scavenging proinflammatory extracellular ATP (eATP) and promoting the generation of immunosuppressive adenosine. In addition to the previously identified CD39 homolog (designated CD39a), here we identified two novel functional CD39 splice variants (termed CD39b and CD39c) from teleost fish Japanese flounder (Paralichthys olivaceus). Transcriptional expression analysis revealed that all three CD39 splicing variants were coexpressed in Japanese flounder head kidney macrophages (HKMs) and a variety of normal tissues but with differential tissue/immune cell expression preferences. Immune challenge experiments showed that the expression of CD39 splice variants was modulated by both in vitro inflammatory stimulation and in vivo bacterium Edwardsiella piscicida infections. However, even though all CD39 splice variants were functional cell surface-expressed proteins and shared generally similar domain structures, their varied cytoplasmic N-terminal sequences resulted in a varied capability for hydrolysis of eATP and attenuation of eATP/P2XR-mediated proinflammatory cytokine expression. Pharmacological inhibition of endogenous CD39 activity in Japanese flounder HKMs or overexpression of CD39 splice variants in Japanese flounder FG-9307 cells further revealed the role of CD39 variants in down-regulating LPS-induced proinflammatory cytokine production. In addition, luciferase reporter analysis demonstrated that the CD39 splice variant promoters had varied transcriptional activities and that Sp1 was a potential transcription factor for CD39c transcription regulation. Taken together, we report for the first time that functional CD39 splice variants provide additional means to downregulate eATP- and LPS-induced proinflammatory response(s) in fish.

In silico analysis of expression and DNA methylation profiles of NLRP13 inflammasome in tumor cells

BackgroundNOD-like receptor proteins (NLRs) are essential innate pattern recognition receptors that play fundamental roles in inflammation, and they have been associated with many cancers. NLRP13 (NLR Family Pyrin Domain Containing 13) belongs to the NLRP protein family, but its biological function is not as well defined as other related proteins in this family. Methods and resultsThe expression profile and CpG-aggregated methylation status of the NLRP13 gene were determined using GENT2 and TCGA online platforms. Samples from patients with different tumor stages were used to evaluate the promoter methylation status of NLRP13 in cancer versus normal tissues in a tumor stage-dependent manner by using UALCAN server. We further studied NLRP13 expression in response to inflammatory and hypomethylating agents in two distinct cancer cell lines, HT-29 (human colon adenocarcinoma) and C6 (rat glioma cells), following the treatment with LPS and/or a hypomethylating agent, decitabine, to reveal NLRP13 methylation profiles in these cell lines. ConclusionsUsing bioinformatics tools, we found that NLRP13 expression is higher in healthy breast, head and neck, prostate tissues compared to tumor tissues, whereas its expression is significantly higher in colorectal adenocarcinoma tissue than in normal colon tissue. We also found that NLRP13 gene is mostly hypomethylated in cancer tissues compared to normal tissues, and its high expression is linked with worse overall survival depending on cancer type. Based on data from UALCAN database, NLRP13 promoter methylation is similarly decreased in many cancer patients depending on individual cancer stages. Protein-protein interactions (PPIs) data indicated that NLRP13 showed a confidence interaction with MEFV, NLRC4 and CASP1 proteins, and gene ontology terms associated with functional partners of NLRP13 were also identified in the current study. Our results further showed that NLRP13 expression is regulated by DNA methylation, and treatment with decitabine might effect NLRP13-mediated inflammatory responses in cancer since LPS-induced pro-inflammatory responses specified by IL-1β, IL-18 and caspase-1 levels change in certain cases in cancer cell lines following the pretreatment with this epigenetic modulator. Our study is the first to provide evidence for the differential expression and epigenetic regulation of the NLPR13 in multiple cancer types.

Melatonin Attenuates LPS-Induced Proinflammatory Cytokine Response and Lipogenesis in Human Meibomian Gland Epithelial Cells via MAPK/NF-κB Pathway.

PurposeInflammation contributes to the development of meibomian gland dysfunction (MGD) under specific disease conditions, but the underlying mechanisms remain elusive. We examined whether lipopolysaccharide (LPS) induced a proinflammatory cytokine response and lipogenesis in human meibomian gland epithelial cells (HMGECs) and whether melatonin (MLT), a powerful anti-inflammatory regent in the eyes, could protect against LPS-induced disorders.MethodsHuman meibomian gland (MG) tissues and immortalized HMGECs were stained to identify Toll-like receptor (TLR) 4 and MLT receptors (MT1 and MT2). HMGECs were pretreated with or without MLT and then stimulated with LPS. Then, TLR4 activation, cytokine levels, lipid synthesis, apoptosis, autophagy, and MAPK/NF-κB factor phosphorylation in HMGECs were analyzed.ResultsTLR4, MT1, and MT2 were expressed in human MG acini and HMGECs. Pretreatment with MLT inhibited the TLR4/MyD88 signaling and attenuated proinflammatory cytokine response and lipogenesis in LPS-stimulated HMGECs, which manifested as decreased production of cytokines (IL-1β, IL-6, IL-8, and TNF-α), reduced lipid droplet formation, and downregulated expression of meibum lipogenic proteins (ADFP, ELOVL4, and SREBP-1). Phospho-histone H2A.X foci, lysosome accumulation, and cytoplasmic cleaved caspase 3/LC3B-II staining were increased in LPS-stimulated HMGECs, indicating enhanced cell death mediated by apoptosis and autophagy during LPS-induced lipogenesis. MLT downregulated cleaved caspase 3 levels and the Bax/Bcl-2 ratio to alleviate apoptosis and ameliorated the expression of Beclin 1 and LC3B-II to inhibit autophagy. The protective mechanisms of MLT include the inhibition of MAPK and NF-κB phosphorylation.ConclusionsMLT attenuated lipogenesis, apoptosis, and autophagy in HMGECs induced by proinflammatory stimuli, indicating the protective potential of MLT in MGD.

Bronchiectasis enters the inflammation era.

Bronchiectasis (BE) is an increasing global problem affecting children and adults. Historically regarded as an orphan condition, there has been an increase in both clinical and translational research in recent years. Pathophysiology of BE is understood in terms of the vicious vortex which has three key components leading to airway structural damage: impaired mucociliary clearance, airway infection and inflammation.1 While airway clearance through physiotherapy and antibiotic treatment has been the cornerstone of BE treatment for decades, there is a lack of effective therapies targeting inflammation.1, 2 Neutrophils are the most abundant inflammatory cells identified in the airways of patients with BE.1 Neutrophilic inflammation is a double-edged sword, leading on one hand to the release of harmful proteases and tissue damage, and on the other hand, contributing to immune defence. Previous clinical trials aiming to reduce neutrophil recruitment to the airway have shown similar patterns of increased infections. Understanding the mechanisms of neutrophilic inflammation is the key to unlocking new therapeutic targets. We recently conducted a series of studies with international collaborators in Spain, Italy and Australia to better understand the nature of neutrophilic inflammation in BE.1 Utilizing proteomic analysis of sputum samples, we identified that severe disease in BE was characterized by an upregulation of neutrophil and neutrophil extracellular trap (NET)-associated proteins.1 We validated these findings in two large cohort studies and found that increased sputum NET complexes were associated with disease severity, reduced quality of life, severe exacerbations and mortality. Azithromycin treatment has been proven to reduce exacerbation frequency, including in patients with Pseudomonas aeruginosa infection which is intrinsically resistant to the antibiotic effect of macrolides. Analysis of two long-term studies of azithromycin, one in BE and the AMAZES trial in asthma, demonstrated that NETs could be reduced, and clinical outcomes improved with macrolide treatment.1 This provides a potential mechanism for the beneficial effects of macrolides, particularly in patients with neutrophilic disease. Our results suggest that NETs may contribute to symptoms and exacerbations in BE and NETs are likely to be the primary mechanism by which harmful neutrophil serine proteases such as elastase are released and damage the airway. We recently reported the results of a phase 2 study of a DPP1 inhibitor in BE patients with a history of exacerbations. A total of 256 patients with BE were randomized to either two doses (10 and 25 mg) of the DPP1 inhibitor brensocatib or placebo and followed up for 6 months.2 DPP1 inhibition prevents the activation of the neutrophil proteases elastase, cathepsin-G and proteinase-3 in the bone marrow. Brensocatib treatment at both doses prolonged the time to first exacerbation and was associated with marked reductions in sputum neutrophil elastase. There were small numerical increases in skin and dental adverse effects in the phase 2 trial but no increase in infection risk. While the encouraging results need to be confirmed in a larger phase 3 trial, these data suggest the potential to directly target neutrophilic inflammation in patients with BE.2 BE is a heterogeneous disease and although neutrophilic inflammation and infection represent the most common ‘endotype’, clinical experience and clinical trial results suggest that there are other important contributors to disease burden and exacerbation.3, 4 Eosinophils are well-established drivers of exacerbation in asthma and chronic obstructive pulmonary disease, but until recently their role in BE was not established. Shoemark et al. recently reported the results of a pan-European cohort of 1007 patients with BE. This study showed that based on sputum eosinophil counts >3%, the gold standard for defining eosinophilic inflammation in sputum, approximately 20% of BE patients have eosinophilic inflammation after the exclusion of patients with asthma and allergic bronchopulmonary aspergillosis. Eosinophil counts in blood >300 cells/μl were shown to be a valid surrogate marker of sputum counts. In a sub-study of 144 patients, eosinophilic inflammation in blood was associated with increased risk of exacerbation.3 Oriano et al. extended these observations by also incorporating measurement of FeNO showing that a Th2-high endotype of BE was present in 31% of patients when considering a high eosinophil count (>300 cells/μl) and a raised FeNO ≥25 dpp. Patients with T2-high inflammation had more severe disease and worse symptoms.4 These studies demonstrate that BE is not exclusively a neutrophilic disease. Eosinophilic inflammation may contribute to a subset of BE exacerbations and biomarkers may be useful to target anti-eosinophil strategies such as inhaled corticosteroids or anti-IL5 therapies in future. Observational studies and post hoc analyses of randomized trials already suggest that eosinophil counts in blood may predict treatment responses in BE, although these results need to be confirmed in prospective trials.5 BE is therefore entering a new era where patients can be potentially classified into inflammatory endotypes based on the presence of neutrophilic or eosinophilic (or mixed) profiles which may require different treatments. Ongoing phase 3 trials of both anti-neutrophil (NCT04594369) and anti-eosinophil (NCT05006573) anti-inflammatory strategies represent important opportunities to transform the management of BE. Infection remains a crucial component of the vicious vortex, and targeting inflammation cannot ignore the important contribution of the airway microbiome. While much research has focused on the roles of conventional pathogens such as P. aeruginosa, which are consistently associated with poor clinical outcomes, recent research using next-generation sequencing has given us a more nuanced view of the role of bacteria in BE.6, 7 The concept of ‘good bacteria’ in the microbiome is well accepted in the gut but poorly defined in the airways. Commensals such as Rothia may have a beneficial effect on inflammation, as demonstrated in a recent study by Rigauts et al. Using murine models, Rothia mucilaginosa was shown to have an inhibitory effect on pathogen and LPS-induced proinflammatory responses through inhibiting nuclear factor kappa B (NF-kB) pathway activation. In sputum samples obtained from BE patients, Rothia spp. were negatively correlated with pro-inflammatory mediators, demonstrating that the presence of this organism in the lower airways may reduce inflammation.6 These studies add to evidence that the interactions between bacteria are important in determining patient outcomes7 and demonstrate that modulation of the microbiome may be a powerful tool in reducing inflammation. How to modulate the microbiome remains a challenging question. Our studies using proteomics found that antibiotic treatment in patients with dominance of Gram-negative organisms such as Pseudomonas and Haemophilus reduces neutrophilic inflammation and upregulates antiprotease defence.1 Whether the microbiome can be modified using non-antibiotic approaches such as immune modulators or probiotics requires investigation. BE therapy is entering a new era where immune-modulating therapy has a key role alongside airway clearance and antibiotics. Classifying patients into endotypes, based on neutrophilic, eosinophilic or mixed inflammatory profiles, offers a range of treatment targets under a personalized medicine approach. Targeting inflammation directly, through treatments such as DPP1-inhibition, and indirectly through modulation of the microbiome may both lead to improvements in patient outcomes in the future. Holly R. Keir and James D. Chalmers are funded by Asthma and Lung UK. Holly R. Keir declares no conflicts of interests. James D. Chalmers reports research grants and consultancy from Astrazeneca, Boehringer Ingelheim, Chiesi, Gilead Sciences, Glaxosmithkline, Grifols, Insmed, Novartis, Pfizer and Zambon.

3H-1,2-dithiole-3-thione suppresses LPS-induced proinflammatory responses in macrophages: potential involvement of antioxidant induction, NF-κB, and Nrf2.

Previously, we reported that 3H-1,2-dithiole-3-thione (D3T), an Nrf2 activator, acted as a potential chemoprotectant against lipopolysaccharide (LPS)-induced mortality in mice. In view of the critical involvement of macrophages in the pathogenesis of LPS-induced endotoxemia, in the present study, we investigated the protective effects of D3T on LPS-induced proinflammatory responses in cultured murine RAW 264.7 macrophage cell line and primary peritoneal macrophages and the potential involvement of antioxidant induction, NF-κB, and Nrf2. We showed that treatment with D3T resulted in increased levels of a series of antioxidants in RAW 264.7 cells in a concentration-dependent manner. These included the reduced form of glutathione, glutathione peroxidase, glutathione reductase, glutathione S-transferase, and NADPH:quinone oxidoreductase 1. Catalase was also potently induced by D3T which, however, did not show a concentration dependency. Concurrent with the ability to induce the above cellular antioxidants, D3T pretreatment of RAW 264.7 cells also led to a concentration-dependent suppression of LPS-induced interleukin-1beta (IL-1β) production and nitric oxide release. LPS-stimulated tumor necrosis factor-alpha (TNF-α) production was also suppressed by D3T, but to a much lesser extent. Using NF-κB reporter gene-expressing RAW 264.7 cells, we further showed that D3T pretreatment also suppressed LPS-induced NF-κB activation. To investigate the potential involvement of Nrf2, a chief regulator of cellular antioxidant genes, we used peritoneal macrophages isolated from Nrf2+/+ and Nrf2-/- mice. Our results showed that D3T pretreatment suppressed LPS-induced proinflammatory responses in Nrf2+/+ macrophages, and this inhibitory effect of D3T was completely lost in Nrf2-/- macrophages. Collectively, the results of the present study demonstrated that D3T acted as a potent suppressor of LPS-induced proinflammatory responses in macrophages. Antioxidant induction, NF-κB suppression, and Nrf2 activation appeared to contribute to the anti-proinflammatory activity of D3T in macrophages.

OLFM4 Regulates Lung Epithelial Cell Function in Sepsis-Associated ARDS/ALI via LDHA-Mediated NF-κB Signaling.

BackgroundAcute respiratory distress syndrome (ARDS) is one of the leading causes of death in patients with sepsis. As such, early and accurate identification of sepsis-related ARDS is critical.MethodsBioinformatic analysis was used to explore the GEO datasets. ELISA method was used to detect the plasma or cellular supernatant of relevant proteins. Quantitative real-time PCR was used for mRNA measurements and Western blot was applied for protein measurements. Immunohistochemistry staining and Immunofluorescence staining were used to identify the localization of OLFM4. Cecal ligation and puncture (CLP) model was used to establish sepsis model.ResultsThe bioinformatic analysis results identified ten genes (CAMP, LTF, RETN, LCN2, ELANE, PGLYRP1, BPI, DEFA4, MPO, and OLFM4) as critical in sepsis and sepsis-related ARDS. OLFM4, LCN2, and BPI were further demonstrated to have diagnostic values in sepsis-related ARDS. Plasma expression of OLFM4 and LCN2 was also upregulated in sepsis-related ARDS patients compared to septic patients alone. OLFM4 expression was significantly increased in the lung tissues of septic mice and was co-localized with Ly6G+ neutrophils, F4/80+ macrophages and pro-surfactant C+ lung epithelial cells. In vitro data showed that OLFM4 expression in lung epithelial cells was downregulated upon LPS stimulation, whereas neutrophil media induced OLFM4 expression in lung epithelial cells. Overexpression of OLFM4 and treatment with recombinant OLFM4 effectively suppressed LPS-induced pro-inflammatory responses in lung epithelial cells. Furthermore, the increased levels of LDHA phosphorylation and the downstream NF-κB activation induced by LPS in epithelial cells were effectively diminished by OLFM4 overexpression and recombinant OLFM4 treatment via a reduction in ROS production and HIF1α expression.ConclusionOLFM4 may regulate the pro-inflammatory response of lung epithelial cells in sepsis-related ARDS by modulating metabolic disorders; this result could provide new insights into the treatment of sepsis-induced ARDS.

Surfactant Protein A Enhances the Degradation of LPS-Induced TLR4 in Primary Alveolar Macrophages Involving Rab7, β-arrestin2, and mTORC1.

Respiratory infections by Gram-negative bacteria are a major cause of global morbidity and mortality. Alveolar macrophages (AMs) play a central role in maintaining lung immune homeostasis and host defense by sensing pathogens via pattern recognition receptors (PRR). The PRR Toll-like receptor (TLR) 4 is a key sensor of lipopolysaccharide (LPS) from Gram-negative bacteria. Pulmonary surfactant is the natural microenvironment of AMs. Surfactant protein A (SP-A), a multifunctional host defense collectin, controls LPS-induced pro-inflammatory immune responses at the organismal and cellular level via distinct mechanisms. We found that SP-A post-transcriptionally restricts LPS-induced TLR4 protein expression in primary AMs from healthy humans, rats, wild-type and SP-A-/- mice by further decreasing cycloheximide-reduced TLR4 protein translation and enhances the co-localization of TLR4 with the late endosome/lysosome. Both effects as well as the SP-A-mediated inhibition of LPS-induced TNF-α release are counteracted by pharmacological inhibition of the small GTPase Rab7. SP-A-enhanced Rab7 expression requires β-arrestin2 and, in β-arrestin2-/- AMs and after intratracheal LPS challenge of β-arrestin2-/- mice, SP-A fails to enhance TLR4/lysosome co-localization and degradation of LPS-induced TLR4. In SP-A-/- mice, TLR4 levels are increased after pulmonary LPS challenge. SP-A-induced activation of mechanistic target of rapamycin complex 1 (mTORC1) kinase requires β-arrestin2 and is critically involved in degradation of LPS-induced TLR4. The data suggest that SP-A post-translationally limits LPS-induced TLR4 expression in primary AMs by lysosomal degradation comprising Rab7, β-arrestin2, and mTORC1. This study may indicate a potential role of SP-A-based therapeutic interventions in unrestricted TLR4-driven immune responses to lower respiratory tract infections caused by Gram-negative bacteria.

Neuron-Derived Extracellular Vesicles Modulate Microglia Activation and Function.

Simple SummaryIn this study we investigated how neuron-derived extracellular vesicles (NDEVs) mediate neuroimmune regulation in primary cell culture systems. Rat cortical neurons released EVs that improved microglial survival and inhibited the expression of activation markers on microglia. Furthermore, NDEVs reduced the LPS-induced proinflammatory response and promoted an anti-inflammatory response. Thus, neurons critically regulate microglia activity and control inflammation via EV-mediated neuron–glia communication.Microglia act as the immune cells of the central nervous system (CNS). They play an important role in maintaining brain homeostasis but also in mediating neuroimmune responses to insult. The interactions between neurons and microglia represent a key process for neuroimmune regulation and subsequent effects on CNS integrity. However, the molecular mechanisms of neuron-glia communication in regulating microglia function are not fully understood. One recently described means of this intercellular communication is via nano-sized extracellular vesicles (EVs) that transfer a large diversity of molecules between neurons and microglia, such as proteins, lipids, and nucleic acids. To determine the effects of neuron-derived EVs (NDEVs) on microglia, NDEVs were isolated from the culture supernatant of rat cortical neurons. When NDEVs were added to primary cultured rat microglia, we found significantly improved microglia viability via inhibition of apoptosis. Additionally, application of NDEVs to cultured microglia also inhibited the expression of activation surface markers on microglia. Furthermore, NDEVs reduced the LPS-induced proinflammatory response in microglia according to reduced gene expression of proinflammatory cytokines (TNF-α, IL-6, MCP-1) and iNOS, but increased expression of the anti-inflammatory cytokine, IL-10. These findings support that neurons critically regulate microglia activity and control inflammation via EV-mediated neuron–glia communication. (Supported by R21AA025563 and R01AA025591).

Modification and enhanced anti-inflammatory activity by Bifidobacterial fermentation of an exopolysaccharide from a medicinal fungus Cs-HK1

The exopolysaccharide (EPS) from the mycelial fermentation of a medicinal fungus Cordyceps sinensis Cs-HK1 had shown significant anti-inflammatory activity previously, and EPS-LM was a highly active fraction with a relatively low molecular weight (MW) isolated from the Cs-HK1 EPS. This study was to assess the effects of Bifidobacterial fermentation in anaerobic conditions on the molecular properties and anti-inflammatory activity of EPS-LM. In both Bifidobacterial cultures (B. breve and B. longum), EPS-LM was fractionally consumed as a carbon source, increasing the bacterial growth and acetic acid production. Analytical results from the fermentation digesta (supernatant) suggested that EPS-LM was partially degraded to lower molecular weight (MW) products with modified structures during the Bifidobacterial fermentation. More interestingly, the higher MW digesta fraction containing the partially degraded EPS-LM showed even stronger inhibiting activity than the original EPS-LM on the LPS-induced pro-inflammatory responses in THP-1 cell culture, including NF-κB activation, release of NO, TNF-α and IL-8. The study has shown that the fermentation by selected Bifidobacterial strains is effective to modify natural polysaccharides with enhanced bioactivities.