LPS-induced Cytokine Production Research Articles

Butyrate and tributyrin reduce LPS-induced inflammatory cytokine production from human visceral fat.

The current obesity crisis has resulted in many people with excess adipose tissue suffering from chronic inflammation. This inflammation is largely due to the release of cytokines and chemokines from visceral fat. The aim of this study was to identify potential anti-inflammatory agents that might alleviate obesity-induced chronic inflammation. To identify agents that might alleviate this obesity-induced chronic inflammation we have developed a simple protocol for incubating intact pieces of human visceral adipose tissue in 35 mm tissue culture plates, in the presence of low-dose lipopolysaccharide (LPS) and co-incubating these samples with potential anti-inflammatory agents. RNA-Seq analysis was performed to identify enriched gene expression signatures among the most significantly differentially expressed genes. From this screen, we have identified the short-chain fatty acid (SCFA) sodium butyrate and its triacylglyceride form, tributyrin, as effective agents, significantly reducing the production of LPS-induced inflammatory cytokines and chemokines from all adipose tissue samples tested. As well, these agents appear to be non-toxic at the concentrations tested. RNA-Seq analysis has revealed that IL36γ is one of the most upregulated genes in response to LPS and one of the most downregulated when sodium butyrate is added to human fat samples stimulated with LPS. IL-36γ ELISAs confirmed this holds true at the protein level as well. These studies suggest that the short-chain fatty acid, sodium butyrate, and its triacylglyceride form, tributyrin, might alleviate the chronic inflammation that is associated with many individuals with obesity.

Prevotella histicola ameliorates DSS-induced colitis by inhibiting IRE1α-JNK pathway of ER stress and NF-κB signaling

Inflammatory bowel disease (IBD) is a chronic and recurrent gastrointestinal inflammation regulated by intricate mechanisms. Recently, prebiotics is considered as promising nutritional strategy for the prevention and treatment of IBD. Prevotella histicola (P. histicola), an emerging probiotic, possesses apparently anti-inflammatory bioactivity. However, the role and underlying mechanism of P. histicola on IBD remain unclear. Hence, we probe into the effect of P. histicola on dextran sulfate sodium (DSS)-induced colitis and clarified the potential mechanism. Our results revealed that DSS-induced colonic inflammatory response and damaged epithelial barrier in mice were attenuated by oral administration of P. histicola. Moreover, supplementary P. histicola significantly enriched short-chain fatty acid (SCFA)-producing bacteria (Lactobacillus, and Bacillus) and reduced pathogenic bacteria (Erysipelotrichaceae, Clostridium, Bacteroides) in DSS-induced colitis. Notably, In DSS-treated mice, endoplasmic reticulum stress (ERS) was persistently activated in colonic tissue. Conversely, P. histicola gavage suppressed expansion of endoplasmic reticulum, downregulated PERK-ATF4-CHOP and IRE1α-JNK pathway. In vitro, the P. histicola supernatant eliminated LPS-induced higher production of pro-inflammatory cytokines regulated by NF-κB and impairment of epithelial barrier by inhibiting IRE1α-JNK signaling in Caco-2 cell. In summary, our study indicated that P. histicola mitigated DSS-induced chronic colitis via inhibiting IRE1α-JNK pathway and NF-κB signaling. These findings provide the new insights into the promotion of gut homeostasis and the application potential of P. histicola as a prebiotic for IBD in the future.

Pomegranate polyphenol punicalin ameliorates lipopolysaccharide-induced memory impairment, behavioral disorders, oxidative stress, and neuroinflammation via inhibition of TLR4-NF-кB pathway.

Neuroinflammation may play an important role in the development of Alzheimer's disease (AD). Previous studies have reported that lipopolysaccharide (LPS)-induced neuroinflammation causes memory impairments and behavioral disorders. We investigated the potential preventive effects of punicalin (PUN), a polyphenolic component of pomegranate, on LPS-induced memory deficiency and anxiety- and depression-like behaviors, along with the underlying mechanisms. LPS-treated cultured microglial BV2 cells and BV2 cell/Neuro-2a (N2a) cell coculture system were investigated for anti-neuroinflammatory effects of PUN in vitro. The in vivo experiments involved mice administered a 4-week course of oral gavage with 1500 mg/kg/d PUN before intraperitoneal LPS (250 mg/kg daily 7 times) injections. The in vitro results demonstrated that PUN inhibited the LPS-induced inflammatory cytokine (IL-18, IL-1β, TNF-ɑ, and IL-6) production in BV2 cells and protected N2a cells from synaptic damage mediated by BV2 microglia-induced neuroinflammation. In in vivo studies, it was observed that PUN improved memory impairment and anxiety- and depression-like behaviors caused by LPS and reduced the expression of inflammatory proteins such as iNOS, COX-2, IL-1β, IL-2, IL-6, and TNF-α. Furthermore, PUN inhibited the LPS-induced production of MDA; increased the activities of CAT, SOD, and GSH-Px, and inhibited LPS-induced Aβ1-42 generation through down-regulation of APP and BACE1 expression. Moreover, PUN also suppressed the expression of TLR4, IRAK4, TRAF6, IKK-β, NF-κB, p65, and HMGB1 in LPS-treated mouse brain and cultured microglial BV-2 cells. These results suggest that PUN inhibits LPS-induced memory impairment via anti-inflammatory and anti-amylogenic mechanisms through inhibition of TLR4-NF-kB activation.

The gut lactic acid bacteria metabolite, 10-oxo-cis-6,trans-11-octadecadienoic acid, suppresses inflammatory bowel disease in mice by modulating the NRF2 pathway and GPCR-signaling.

Various gut bacteria, including Lactobacillus plantarum, possess several enzymes that produce hydroxy fatty acids (FAs), oxo FAs, conjugated FAs, and partially saturated FAs from polyunsaturated FAs as secondary metabolites. Among these derivatives, we identified 10-oxo-cis-6,trans-11-octadecadienoic acid (γKetoC), a γ-linolenic acid (GLA)-derived enon FA, as the most effective immunomodulator, which inhibited the antigen-induced immunoactivation and LPS-induced production of inflammatory cytokines. The treatment with γKetoC significantly suppressed proliferation of CD4+ T cells, LPS-induced activation of bone marrow-derived dendritic cells (BMDCs), and LPS-induced IL-6 release from peritoneal cells, splenocytes, and CD11c+ cells isolated from the spleen. γKetoC also inhibited the release of inflammatory cytokines from BMDCs stimulated with poly-I:C, R-848, or CpG. Further in vitro experiments using an agonist of GPR40/120 suggested the involvement of these GPCRs in the effects of γKetoC on DCs. We also found that γKetoC stimulated the NRF2 pathway in DCs, and the suppressive effects of γKetoC and agonist of GPR40/120 on the release of IL-6 and IL-12 were reduced in Nrf2-/- BMDCs. We evaluated the role of NRF2 in the anti-inflammatory effects of γKetoC in a dextran sodium sulfate-induced colitis model. The oral administration of γKetoC significantly reduced body weight loss, improved stool scores, and attenuated atrophy of the colon, in wild-type C57BL/6 and Nrf2+/- mice with colitis. In contrast, the pathology of colitis was deteriorated in Nrf2-/- mice even with the administration of γKetoC. Collectively, the present results demonstrated the involvement of the NRF2 pathway and GPCRs in γKetoC-mediated anti-inflammatory responses.

Exploring the Anti-Inflammatory Potential of Cyperus pangorei Rhizome Extracts An In Vitro and In Vivo Study

Background and aim: Inflammation is a pivotal process implicated in various physiological and pathological conditions, necessitating the exploration of alternative anti-inflammatory agents with minimal side effects. This study aimed to investigate the anti-inflammatory potential of the standardized ethyl acetate (EtAc) fraction derived from Cyperus pangorei rhizomes. Methods: The rhizomes of C. pangorei were collected, processed, and subjected to extraction and fractionation to obtain the EtAc fraction. RP-HPLC analysis was employed to standardized the EtAc fraction against standard quercetin, luteolin, and apigenin. In vitro studies utilized peritoneal macrophages isolated from male Swiss albino rats to assess NO production and cytokine levels (IL-1β, IL-6, TNF-α) upon treatment with the EtAc fraction. In vivo evaluation was conducted using a carrageenan-induced rat paw edema model. Results: RP-HPLC analysis revealed the presence of quercetin, luteolin, and apigenin in the EtAc fraction. In vitro studies demonstrated dose-dependent inhibition of LPS-induced NO production and suppression of inflammatory cytokines (IL-1β, IL-6, TNF-α) by the EtAc fraction. Furthermore, in the carrageenan-induced rat paw edema model, the EtAc fraction exhibited dose-dependent inhibition of paw edema. Conclusion: The findings of this study highlight the significant anti-inflammatory potential of C. pangorei rhizome extracts, particularly the EtAc fraction. The identified compounds, quercetin, luteolin, and apigenin, contribute to its anti-inflammatory activity by modulating key inflammatory mediators. These results support the potential therapeutic use of C. pangorei in managing inflammation-related disorders. Further research is warranted to elucidate the underlying mechanisms and evaluate the long-term efficacy and safety of C. pangorei extracts as anti-inflammatory agents.

Modulatory actions of Echinococcus granulosus antigen B on macrophage inflammatory activation.

Cestodes use own lipid-binding proteins to capture and transport hydrophobic ligands, including lipids that they cannot synthesise as fatty acids and cholesterol. In E. granulosus s.l., one of these lipoproteins is antigen B (EgAgB), codified by a multigenic and polymorphic family that gives rise to five gene products (EgAgB8/1-5 subunits) assembled as a 230 kDa macromolecule. EgAgB has a diagnostic value for cystic echinococcosis, but its putative role in the immunobiology of this infection is still poorly understood. Accumulating research suggests that EgAgB has immunomodulatory properties, but previous studies employed denatured antigen preparations that might exert different effects than the native form, thereby limiting data interpretation. This work analysed the modulatory actions on macrophages of native EgAgB (nEgAgB) and the recombinant form of EgAg8/1, which is the most abundant subunit in the larva and was expressed in insect S2 cells (rEgAgB8/1). Both EgAgB preparations were purified to homogeneity by immunoaffinity chromatography using a novel nanobody anti-EgAgB8/1. nEgAgB and rEgAgB8/1 exhibited differences in size and lipid composition. The rEgAgB8/1 generates mildly larger lipoproteins with a less diverse lipid composition than nEgAgB. Assays using human and murine macrophages showed that both nEgAgB and rEgAgB8/1 interfered with in vitro LPS-driven macrophage activation, decreasing cytokine (IL-1β, IL-6, IL-12p40, IFN-β) secretion and ·NO generation. Furthermore, nEgAgB and rEgAgB8/1 modulated in vivo LPS-induced cytokine production (IL-6, IL-10) and activation of large (measured as MHC-II level) and small (measured as CD86 and CD40 levels) macrophages in the peritoneum, although rEgAgB8/1 effects were less robust. Overall, this work reinforced the notion that EgAgB is an immunomodulatory component of E. granulosus s.l. Although nEgAgB lipid's effects cannot be ruled out, our data suggest that the EgAgB8/1 subunit contributes to EgAgB´s ability to regulate the inflammatory activation of macrophages.

Induction of MTHFD2 in Macrophages Inhibits Reactive Oxygen Species-mediated NF-κB Activation and Protects against Inflammatory Responses.

The one-carbon metabolism enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) is critical for cancer cell proliferation and immune cell phenotypes, but whether it can contribute to macrophage inflammatory responses remains unclear. In this study, we show that MTHFD2 was upregulated by LPS in murine macrophages upon activation of the TLR4-MyD88-IKKα/β-NF-κB signaling pathway. MTHFD2 significantly attenuated LPS-induced macrophage proinflammatory cytokine production through its enzymatic activity. Notably, ablation of myeloid MTHFD2 rendered mice more sensitive to septic shock and CCl4-induced acute hepatitis. Mechanistically, MTHFD2 restrained IKKα/β-NF-κB activation and macrophage inflammatory phenotype by scavenging reactive oxygen species through the generation of NADPH. Our study reveals MTHFD2 as a "self-control" mechanism in macrophage-mediated inflammatory responses.

The Effects of Psilocybin on Lipopolysaccharide-Induced Inflammation in THP-1 Human Macrophages

Psilocybin, an innate compound produced by mushrooms belonging to the Psilocybe genus, is primarily known for its agonistic effects on the serotonin 5-HT2A receptor. This receptor’s functioning is involved in many neurological processes. In the context of this research, our primary aim was to comprehensively investigate the influence of psilocybin as a serotonin receptor agonist on the intricate cascade of events involved in THP-1 macrophages stimulated by lipopolysaccharide (LPS). THP-1 monocyte cells were subjected to differentiation into macrophages through a controlled incubation with phorbol 12-myristate 13-acetate (PMA). The next step involved the induction of an inflammatory response by exposing THP-1 macrophages to 500 ng/mL LPS for 4 h. Subsequently, we triggered the activation of the second phase of the NLRP3 inflammasome by introducing adenosine triphosphate (ATP) immediately following LPS stimulation. Our findings have revealed a dose-dependent inverse correlation between psilocybin exposure and the production of LPS-induced proinflammatory cytokines and proteins. Our work indicates that psilocybin likely mediates these responses by influencing key signaling pathways, including NF-κB, IL-6/TYK2/STAT3, and TYK2/STAT1.

Aquaporins (AQPs) as a marker in the physiology of inflammation and its interaction studies with garcinol.

Aquaporins like AQP1, AQP3, and AQP4 are known to be involved in the pathophysiology of inflammation based on earlier reports. This study aimed to evaluate the involvement of Aquaporins as a potential target of inflammation. The study also investigates the efficacy of methanolic extract of Garcinia (GME) and its potent phytocompound (garcinol) against the Aquaporins involvedin inflammation. siRNA silencing of AQP3 was carried out in RAW264.7 cells followed by LPS stimulation (1µg/ml) and assessment of important markers of inflammation including NO, PGE2, TNF-α, IL-6, IL-1β, CCL20, iNOS and COX-2. To assess the anti-inflammatory potential of Garcinia extract and garcinol, cells were stimulated with 1µg/ml LPS in the absence and presence of increasing concentrations of GME and garcinol. During the experimental period, extract concentrations (115µg/ml and 230µg/ml for RAW264.7; 118µg/ml and 236µg/ml for THP-1) and garcinol concentrations (6µM and 12µM for RAW264.7; 3µM and 6µM for THP-1) were selected based on the IC50. The anti-inflammatory effects were assessed by measuring the levels of TNF-α, IL-1β, IL-6, and CCL20 in LPS-stimulated cells. The AQP expression was studied at transcriptional and translational levels using qPCR and Western blot analysis respectively. AQP3 knockdown significantly decreased the NO, PGE2, TNF-α, IL-1β levels along with iNOS andCOX-2 mRNA expression. LPS stimulation led to a significant increase in the mRNA and protein level expression AQP1, AQP3, and AQP4 in RAW264.7 cells; and AQP1 and AQP3 in THP-1 cells indicating their role as markers of inflammation. GME and garcinol effectively suppressed the LPS-induced proinflammatory cytokine production in both cell lines. The results indicate that AQP1, AQP3, and AQP4 could play a crucial role as markers of inflammation. Anti-inflammatory agents like Garcinia could potentially decrease the expression of such AQPs, thus inhibiting the inflammatory process.

Investigation of an aminothiazole-based scaffold as an anti-inflammatory agent: Potential application in the management of cytokine storm in SARS-CoV-19

Aminothiazole-based structures, including compounds like anakinra, nitazoxanide, amiphenazole, and tizoxanide, along with cytokine-blocking agents such as tocilizumab and siltuximab are currently under extensive investigation for their potential in managing severe COVID-19 by specifically targeting and inhibiting cytokines responsible for cytokine release syndrome (CRS). These targeted therapies have shown promising results in effectively mitigating the effects of severe COVID-19. Herein, we carried out a biological assessment of a novel aminothiazole-based derivative 5-(phenylthio)-4-(2-(trifluoromethyl) phenyl) thiazol-2-amine (X22) to gauge its potential as an anti-inflammatory agent. Utilizing in vitro assays, which included measuring nitric oxide (NO) inhibition and observing the production of LPS-induced pro-inflammatory cytokines (TNF-α and IL-6) in RAW 264.7 macrophages. Furthermore, the X22 was characterized by experimental FT-IR, NMR, and UV–visible spectral analysis. The optimized molecular geometry and vibrational wave numbers were calculated using the DFT and B3LYP/6–311++G (d,p) basis set. The vibrational assignments were calculated using VEDA's Potential Energy Distribution (PED) program. 1H and 13C NMR were calculated by the GIAO method. Additionally, compounds HOMO-LUMO and HF interactions were studied. Subsequently, the MEP map was traced to find the reactive sites of X22. In addition, Mulliken population analysis and thermodynamic properties with respect to various temperatures were also calculated. All the theoretical predictions were carried out by using the DFT-B3LYP method at the level of 6–311++G (d,p). Finally, the molecular docking interactions revealed that ligand (X22) binds to TNF-α and IL-6 via H-bonding with amino acids Tyr 151, Ser60, Gln61, and Arg179. These findings suggest that X22 could be a valuable therapeutic agent for mitigating the progression of severe SARS-CoV-2 infections.

Lumbricus Extract Prevents LPS-Induced Inflammatory Activation of BV2 Microglia and Glutamate-Induced Hippocampal HT22 Cell Death by Suppressing MAPK/NF-κB/NLRP3 Signaling and Oxidative Stress.

Microglia-induced inflammatory signaling and neuronal oxidative stress are mutually reinforcing processes central to the pathogenesis of neurodegenerative diseases. Recent studies have shown that extracts of dried Pheretima aspergillum (Lumbricus) can inhibit tissue fibrosis, mitochondrial damage, and asthma. However, the effects of Lumbricus extracts on neuroinflammation and neuronal damage have not been previously studied. Therefore, to evaluate the therapeutic potential of Lumbricus extract for neurodegenerative diseases, the current study assessed the extract's anti-inflammatory and antioxidant activities in BV2 microglial cultures stimulated with lipopolysaccharide (LPS) along with its neuroprotective efficacy in mouse hippocampal HT22 cell cultures treated with excess glutamate. Lumbricus extract dose-dependently inhibited the LPS-induced production of multiple proinflammatory cytokines (tumor necrosis factor-α, interleukin (IL)-6, and IL-1β) and reversed the upregulation of proinflammatory enzymes (inducible nitric oxide synthase and cyclooxygenase-2). Lumbricus also activated the antioxidative nuclear factor erythroid 2-relayed factor 2/heme oxygenase-1 pathway and inhibited LPS-induced activation of the nuclear factor-κB/mitogen-activated protein kinases/NOD-like receptor family pyrin domain containing 3 inflammatory pathway. In addition, Lumbricus extract suppressed the glutamate-induced necrotic and apoptotic death of HT22 cells, effects associated with upregulated expression of antiapoptotic proteins, downregulation of pro-apoptotic proteins, and reduced accumulation of reactive oxygen species. Chromatography revealed that the Lumbricus extract contained uracil, hypoxanthine, uridine, xanthine, adenosine, inosine, and guanosine. Its effects against microglial activation and excitotoxic neuronal death reported herein support the therapeutic potential of Lumbricus for neurodegenerative diseases.

Traditional Chinese medicine Pien-Tze-Huang ameliorates LPS-induced sepsis through bile acid-mediated activation of TGR5-STAT3-A20 signalling

Pien Tze Huang (PZH), a class-1 nationally protected traditional Chinese medicine (TCM), has been used to treat liver diseases such as hepatitis; however, the effect of PZH on the progression of sepsis is unknown. Here, we reported that PZH attenuated lipopolysaccharide (LPS)-induced sepsis in mice and reduced LPS-induced production of proinflammatory cytokines in macrophages by inhibiting the activation of mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signalling. Mechanistically, PZH stimulated signal transducer and activator of transcription 3 (STAT3) phosphorylation to induce the expression of A20, which could inhibit the activation of NF-κB and MAPK signalling. Knockdown of the bile acid (BA) receptor G protein-coupled bile acid receptor 1 (TGR5) in macrophages abolished the effects of PZH on STAT3 phosphorylation and A20 induction, as well as the LPS-induced inflammatory response, suggesting that BAs in PZH may mediate its anti-inflammatory effects by activating TGR5. Consistently, deprivation of BAs in PZH by cholestyramine resin reduced the effects of PZH on the expression of phosphorylated-STAT3 and A20, the activation of NF-κB and MAPK signalling, and the production of proinflammatory cytokines, whereas the addition of BAs to cholestyramine resin-treated PZH partially restored the inhibitory effects on the production of proinflammatory cytokines. Overall, our study identifies BAs as the effective components in PZH that activate TGR5-STAT3-A20 signalling to ameliorate LPS-induced sepsis.

Strontium Attenuates LPS-Induced Inflammation via TLR4/MyD88/NF-κB Pathway in Bovine Ruminal Epithelial Cells.

Subacute ruminal acidosis (SARA) is a common nutritional metabolic disease in ruminants that causes significant economic losses to dairy farming. Strontium (Sr) is known to be involved in bone metabolism and exhibits potent anti-inflammatory effects. To evaluate the effect of Sr on inflammation in bovine ruminal epithelial cells, a model of LPS-induced inflammation was established in this study, and the cell viability of bovine ruminal epithelial cells was measured using CCK-8. The production of pro-inflammatory cytokines was measured by ELISA and real-time PCR, respectively. The related proteins of the TLR4/MyD88/NF-κB pathway were assayed through Western blotting, and the fluorescence of p-p65 and p-IκB were assayed by immunofluorescence. Molecular docking of Sr and TLR4/MyD88/NF-κB pathway-related proteins was performed using MIB2 ( http://bioinfo.cmu.edu.tw/MIB2/ ). Results showed that after treatment for 24 h, the cell viability was decreased at the high concentration of Sr (≥ 10 mmol/L). Sr significantly decreased the production of TNF-α, IL-1β, and IL-6, downregulated the related proteins expression of the TLR4/MyD88/NF-κB pathway, and reduced the fluorescence levels of p-p65 and p-IκB. The NF-κB pathway inhibitor PDTC and molecular docking further revealed that Sr reduced LPS-induced pro-inflammatory cytokines production via the TLR4/MyD88/NF-κB pathway. These results suggest that Sr reduces LPS-induced pro-inflammatory cytokines production via the TLR4/MyD88/NF-κB pathway, thereby exerting an anti-inflammatory effect in bovine ruminal epithelial cells, providing a basis for Sr in the treatment of bovine rumen acidosis disease.

Modulation of In Vitro Macrophage Responses via Primary and Secondary Bile Acids in Dogs

Bile acids (BA) are important metabolites secreted into the intestinal lumen and impacted by luminal microbes and dietary intake. Prior studies in humans and rodents have shown that BAs are immunologically active and that primary and secondary BAs have distinct immune properties. Therefore, the composition of the gut BA pool may influence GI inflammatory responses. The current study investigated the relative immune modulatory properties of primary (cholic acid, CA) and secondary BAs (lithocholic acid, LCA) by assessing their effects on canine macrophage cytokine secretion and BA receptor (TGR5) expression. In addition, RNA sequencing was used to further interrogate how CA and LCA differentially modulated macrophage responses to LPS (lipopolysaccharide). We found that exposure to either CA or LCA influenced LPS-induced cytokine production via macrophages similarly, with suppression of TNF-α secretion and enhancement of IL-10 secretion. Neither BA altered the expression of the BA receptor TGR5. Transcriptomic analysis revealed that CA activated inflammatory signaling pathways in macrophages involving type II interferon signaling and the aryl hydrocarbon receptor, whereas LCA activated pathways related to nitric oxide signaling and cell cycle regulation. Thus, we concluded that both primary and secondary BAs are active modulators of macrophage responses in dogs, with differential and shared effects evident with sequencing analysis.

6-Pentyl-α-Pyrone from Trichoderma gamsii Exert Antioxidant and Anti-Inflammatory Properties in Lipopolysaccharide-Stimulated Mouse Macrophages.

Filamentous fungi produce several beneficial secondary metabolites, including bioactive compounds, food additives, and biofuels. Trichoderma, which is a teleomorphic Hypocrea that falls under the taxonomic groups Ascomycota and Dikarya, is an extensively studied fungal genus. In an ongoing study that seeks to discover bioactive natural products, we investigated potential bioactive metabolites from the methanolic extract of cultured Trichoderma gamsii. Using liquid chromatography-mass spectrometry (LC-MS), one major compound was isolated and structurally identified as 6-pentyl-α-pyrone (6PP) based on nuclear magnetic resonance data and LC-MS analysis. To determine its antioxidant and anti-inflammatory activity, as well as the underlying mechanisms, we treated lipopolysaccharide (LPS)-stimulated Raw264.7 mouse macrophages with 6PP. We found that 6PP suppresses LPS-induced increase in the levels of nitric oxide, a mediator of oxidative stress and inflammation, and restores LPS-mediated depletion of total glutathione by stabilizing nuclear factor erythroid 2-related factor 2 (Nrf2), an antioxidative factor, and elevating heme oxygenase-1 levels. Furthermore, 6PP inhibited LPS-induced production of proinflammatory cytokines, which are, at least in part, regulated by heme oxygenase-1 (HO-1). 6PP suppressed proinflammatory responses by inhibiting the nuclear localization of nuclear factor kappa B (NF-κB), as well as by dephosphorylating the mitogen-activated protein kinases (MAPKs). These results indicate that 6PP can protect macrophages against oxidative stress and LPS-induced excessive inflammatory responses by activating the Nrf2/HO-1 pathway while inhibiting the proinflammatory, NF-κB, and MAPK pathways.

BRISC is required for optimal activation of NF-κB in Kupffer cells induced by LPS and contributes to acute liver injury

BRISC (BRCC3 isopeptidase complex) is a deubiquitinating enzyme that has been linked with inflammatory processes, but its role in liver diseases and the underlying mechanism are unknown. Here, we investigated the pathophysiological role of BRISC in acute liver failure using a mice model induced by D-galactosamine (D-GalN) plus lipopolysaccharide (LPS). We found that the expression of BRISC components was dramatically increased in kupffer cells (KCs) upon LPS treatment in vitro or by the injection of LPS in D-GalN-sensitized mice. D-GalN plus LPS-induced liver damage and mortality in global BRISC-null mice were markedly attenuated, which was accompanied by impaired hepatocyte death and hepatic inflammation response. Constantly, treatment with thiolutin, a potent BRISC inhibitor, remarkably alleviated D-GalN/LPS-induced liver injury in mice. By using bone marrow-reconstituted chimeric mice and cell-specific BRISC-deficient mice, we demonstrated that KCs are the key effector cells responsible for protection against D-GalN/LPS-induced liver injury in BRISC-deficient mice. Mechanistically, we found that hepatic and circulating levels of TNF-α, IL-6, MCP-1, and IL-1β, as well as TNF-α- and MCP-1-producing KCs, in BRISC-deleted mice were dramatically decreased as early as 1 h after D-GalN/LPS challenge, which occurred prior to the elevation of the liver injury markers. Moreover, LPS-induced proinflammatory cytokines production in KCs was significantly diminished by BRISC deficiency in vitro, which was accompanied by potently attenuated NF-κB activation. Restoration of NF-κB activation by two small molecular activators of NF-κB p65 effectively reversed the suppression of cytokines production in ABRO1-deficient KCs by LPS. In conclusion, BRISC is required for optimal activation of NF-κB-mediated proinflammatory cytokines production in LPS-treated KCs and contributes to acute liver injury. This study opens the possibility to develop new strategies for the inhibition of KCs-driven inflammation in liver diseases.

Galacto-Oligosaccharides Alleviate LPS-Induced Immune Imbalance in Small Intestine through Regulating Gut Microbe Composition and Bile Acid Pool.

Recent evidence suggests that the protective effect of gut microbiota on intestinal inflammation can be achieved through a microbe-bile acids (BAs) mechanism. Galacto-oligosaccharides (GOS) are a kind of prebiotic that alter gut microbiota composition. To verify whether GOS has a protective effect on intestinal inflammation through a microbe-BAs mechanism, this research was performed in a lipopolysaccharide (LPS) porcine model with the presence or absence of GOS. GOS prevented LPS-induced production of pro-inflammatory cytokines, the decrease of bacterial bile salt hydrolase-containing bacteria abundance, and the decrease of chendoxycholic acid (CDCA) level in piglets. Additionally, CDCA decreased LPS-induced production of pro-inflammatory cytokines, induced the expression of the takeda G-protein receptor 5 (TGR5), and its downstream cyclic adenosine monophosphate (cAMP) production in lamina propria-derived CD11b+ cells. The cAMP inhibitor eliminated the protective effect of CDCA on lamina propria-derived CD11b+ cells. These results suggested that GOS reduced the production of pro-inflammatory cytokines and inhibited NF-κB activation via microbe-BA-dependent TGR5-cAMP signaling in LPS-challenged piglets.

In-depth biological characterization of two black soldier fly anti-Pseudomonas peptides reveals LPS-binding and immunomodulating effects.

As effector molecules of the innate immune system, antimicrobial peptides (AMPs) have gathered substantial interest as a potential future generation of antibiotics. Here, we demonstrate the anti-Pseudomonas activity and lipopolysaccharide (LPS)-binding ability of HC1 and HC10, two cecropin peptides from the black soldier fly (Hermetia Illucens). Both peptides are active against a wide range of Pseudomonas aeruginosa strains, including drug-resistant clinical isolates. Moreover, HC1 and HC10 can bind to lipid A, the toxic center of LPS and reduce the LPS-induced nitric oxide and cytokine production in murine macrophage cells. This suggests that the peptide-LPS binding can also lower the strong inflammatory response associated with P. aeruginosa infections. As the activity of AMPs is often influenced by the presence of salts, we studied the LPS-binding activity of HC1 and HC10 in physiological salt concentrations, revealing a strong decrease in activity. Our research confirmed the early potential of HC1 and HC10 as starting points for anti-Pseudomonas drugs, as well as the need for structural or formulation optimization before further preclinical development can be considered. IMPORTANCE The high mortality and morbidity associated with Pseudomonas aeruginosa infections remain an ongoing challenge in clinical practice that requires urgent action. P. aeruginosa mostly infects immunocompromised individuals, and its prevalence is especially high in urgent care hospital settings. Lipopolysaccharides (LPSs) are outer membrane structures that are responsible for inducing the innate immune cascade upon infection. P. aeruginosa LPS can cause local excessive inflammation, or spread systemically throughout the body, leading to multi-organ failure and septic shock. As antimicrobial resistance rates in P. aeruginosa infections are rising, the research and development of new antimicrobial agents remain indispensable. Especially, antimicrobials that can both kill the bacteria themselves and neutralize their toxins are of great interest in P. aeruginosa research to develop as the next generation of drugs.

Molecular binding mechanism and novel antidiabetic and anti-hypertensive bioactive peptides from fermented camel milk with anti-inflammatory activity in raw macrophages cell lines.

The investigation was to determine the effect of camel milk fermented with Limosilactobacillus fermentum KGL4 (MTCC 25515) on ACE-inhibiting, anti-inflammatory, and diabetes-preventing properties and also to release the novel peptides with antidiabetic and anti-hypertensive attributes with molecular interaction studies. Growth conditions were optimised on the basis of total peptide production by inoculating the culture in camel milk at different rates (1.5, 2.0, and 2.5%) along with different incubation periods (12, 24, 36, and 48h). However, after 48h of fermentation with a 2.5% rate of inoculum, the highest proteolytic activity was obtained. Reverse phase high-pressure liquid chromatography (RP-HPLC) was used to calculate the % Rpa from permeates of 3kDa and 10kDa fractions. Molecular weight distributions of fermented and unfermented camel milk protein fractions were compared using SDS-PAGE. Spots obtained from 2D gel electrophoresis were separated on the basis of pH and molecular weight. Spots obtained from 2D gel were digested with trypsin, and the digested samples were subjected to RP-LC/MS for the generation of peptide sequences. The inhibition of tumour necrosis factor alpha, interleukin-6, and interleukin-1 during fermentation was studied using RAW 264.7 macrophages. In the study, fermented camel milk with KGL4 (CMKGL4) inhibited LPS-induced nitric oxide (NO) production and pro-inflammatory cytokine production (TNF-α, IL-6, and IL-1β) by the murine macrophages. The results showed that the peptide structures (YLEELHRLNK and YLQELYPHSSLKVRPILK) exhibited considerable binding affinity against hPAM and hMGA during molecular interaction studies.

Inhibitory Effects of Ehretia tinifolia Extract on the Excessive Oxidative and Inflammatory Responses in Lipopolysaccharide-Stimulated Mouse Kupffer Cells.

Ehretia tinifolia (E. tinifolia) L., an evergreen tree with substantial biological activity, including antioxidant and anti-inflammatory effects, has been used in many herbal and traditional medicines. To elucidate its antioxidant and anti-inflammatory activity and the underlying mechanisms, we applied a methanol extract of E. tinifolia (ETME) to lipopolysaccharide (LPS)-stimulated mouse immortalized Kupffer cells. ETME suppressed the LPS-induced increase in nitric oxide, a mediator for oxidative stress and inflammation, and restored LPS-mediated depletion of total glutathione level by stabilizing antioxidative nuclear factor erythroid 2-related factor 2 (Nrf2) and the subsequent increase in heme oxygenase-1 levels. Furthermore, ETME inhibited the LPS-induced production of pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-1β, and IL-6. The inhibitory effects of ETME on pro-inflammatory responses were regulated by ETME-mediated dephosphorylation of mitogen-activated protein kinases (MAPKs: p38, p44/p42, and stress-associated protein kinase/c-Jun N-terminal kinase) and inhibition of nuclear localization of nuclear factor kappa B (NF-κB). These results suggest that ETME is a possible candidate for protecting Kupffer cells from LPS-mediated oxidative stress and excessive inflammatory responses by activating antioxidant Nrf2/HO-1 and inhibiting pro-inflammatory NF-κB and MAPKs, respectively.