Lipopolysaccharide Activation Research Articles

The Modulatory Effect of Selol (Se IV) on Pro-Inflammatory Pathways in RAW 264.7 Macrophages.

Selol is a semi-synthetic mixture of selenized triglycerides. The results of biological studies revealed that Selol exhibits several anticancer effects. However, studies on its potential anti-inflammatory activity are scarce, and underlying signaling pathways are unknown. The aim of our study was to investigate the ability of Selol to exert anti-inflammatory effects in a RAW 264.7 cell line model of LPS (lipopolysaccharide)-induced inflammation. Cells were treated either with Selol 5% (4 or 8 µg Se/mL) or LPS (1 µg/mL) alone or with Selol given concomitantly with LPS. The parameters studied were reactive oxygen species (ROS) production, glutathione and thioredoxin (Txn) levels, and nuclear factor kappa B (NF-κB) activation, as well as nitric oxide/prostaglandin E2 (NO/PGE2) production. The presented research also included the effect of Selol and/or LPS on glucose (Glc) catabolism; for this purpose, the levels of key enzymes of the glycolysis pathway were determined. The results showed that Selol exhibited pro-oxidative properties. It induced ROS generation with a significant increase in the level of Txn; however, it did not affect the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio. Selol moderately activated NF-κB but failed to affect NO/PGE2 production. The effect of Selol on glucose catabolism was not significant. However, the simultaneous administration of Selol with LPS exerted a statistically significant anti-inflammatory effect via a decrease in the production of pro-inflammatory mediators and NF-κB activation. Our study also showed that as a result of LPS action in cells, the anaerobic glycolysis activity was increased, and incubation with Selol caused a partial reprogramming of Glc metabolism towards aerobic metabolism. This may indicate different pharmacological and molecular effects of Selol action in physiological and pathological conditions.

Defective Mitophagy Impairs Response to Inflammatory Activation of Macrophage-Like Cells.

The role of mitophagy in atherosclerosis has been extensively studied during the last few years. It was shown that mitophagy is involved in the regulation of macrophages, which are important players as immune cells in atherosclerosis development. In this study, we investigated the relationship between mitophagy and response to inflammatory stimulation of macrophage-like cells. Six cybrid cell lines with normal mitophagy, that is, increasing in response to stimulation, and 7 lines with defective mitophagy not responding to stimulation were obtained. The objective of the study was to compare the nature of the inflammatory response in normal and defective mitophagy in order to elucidate the role of mitophagy defects in inflammation. We used cytoplasmic hybrids (cybrids) as cellular models, created using mitochondrial DNA from different atherosclerosis patients. Mitophagy was stimulated by carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and assessed as the degree of colocalization of mitochondria with lysosomes using confocal microscopy. Western blotting methods were used for the determination of proteins involved in the exact mechanism of mitophagy. Experiments with stimulation of mitophagy show a high correlation between these two approaches (microscopy and blotting). The pro-inflammatory response of cybrids was stimulated with bacterial lipopolysaccharide (LPS). The extent of the inflammatory response was assessed by the secretion of cytokines CCL2, IL8, IL6, IL1β, and TNF measured by ELISA. Basal level of secretion of cytokines CCL2, IL8 and TNF was 1.5-2 times higher in cultures of cybrids with defective mitophagy compared to cells with normal mitophagy. This suggests a persistently elevated inflammatory response in cells with defective mitophagy, even in the absence of an inflammatory stimulus. Such cells in the tissue will constantly recruit other immune cells, which is characteristic of macrophages derived from monocytes circulating in the blood of patients with atherosclerosis. We observed significant differences in the degree and type of response to inflammatory activation in cybrids with defective mitophagy. These differences were not so much quantitative as they were dramatically qualitative. Compared with cells with normal mitophagy, in cells with defective mitophagy, the relative (to basal) secretion of IL8, IL6 and IL1b increased after the second LPS activation. This indicates a possible lack of tolerance to inflammatory activation in cells with defective mitophagy, since typically, re-activation reveals a smaller pro-inflammatory cytokine response, allowing the inflammatory process to resolve. In cells with normal mitophagy, exactly this normal (tolerant) inflammatory reaction was observed. Data on the involvement of mitophagy, including defective mitophagy, in disturbances of the inflammatory response in sepsis, viral infections, autoimmune diseases and other pathologies have previously been reported. In this work, we studied the role of defective mitophagy in non-infectious chronic inflammatory diseases using the example of atherosclerosis. We showed a dramatic disruption of the inflammatory response associated with defective mitophagy. Compared with cybrids with normal mitophagy, in cybrids with defective mitophagy, the secretion of all studied cytokines changed significantly both quantitatively and qualitatively. In particular, the secretion of 3 of 5 cytokines demonstrated an intolerant inflammatory response manifested by increased secretion after repeated inflammatory stimulation. Such an intolerant reaction likely indicates a significant disruption of the pro-inflammatory response of macrophages, which can contribute to the chronification of inflammation. Elucidating the mechanisms of chronification of inflammation is extremely important for the search for fundamentally new pharmacological targets and the development of drugs for the prevention and treatment of chronic inflammatory diseases, including atherosclerosis and diseases characteristic of inflammation. Such diseases account for up to 80% of morbidity and mortality.

Impaired postprandial GLP-2 response enhances endotoxemia, systemic inflammation, and kidney injury in metabolic dysfunction-associated steatohepatitis (MASH): effect of phospholipid curcumin meriva

ABSTRACT We investigate the role of homeostatic mechanisms involved in acute, postprandial nutrient metabolism and nutrient-induced systemic inflammation in CKD presence and progression in Metabolic dysfunction-associated steatohepatitis (MASH). We assessed postprandial incretins (GLP-1 and GIP), intestinotropic hormone GLP-2, endotoxin LPS, Zonulin (a marker of intestinal permeability), hepatokines, adipokines and NF-kB activation in circulating MNCs during a meal tolerance test in 52 biopsy proven MASH patients randomized to curcumin Meriva or placebo and 26 matched controls. At baseline, MASH-CKD had a lower GLP-2 response and a 2-fold higher postprandial LPS and NF-kB activation in MNCs than MASH patients without CKD, but similar remaining postprandial or fasting parameters. Postprandial IAUC GLP-2 predicted the presence of CKD in MASH (OR = 0.43, 95%CI:0.32-0.80, p = 0.008) independently of liver histology and traditional risk factors. After 72 weeks, changes in IAUC GLP-2 independently predicted the presence of CKD (OR = 0.49, 95%CI:0.21-0.73, p = 0.010) and eGFR changes [β(SE) = 0.510(0.007, p = 0.006] at end-of-treatment, In MASH, an impaired GLP-2 response to meals is associated with intestinal barrier dysfunction, endotoxemia and NF-kB-mediated systemic inflammation and may promote renal dysfunction and CKD. These data provide the rationale for evaluating GLP-2 analogues in MASH-related CKD.

The physicochemical properties of lipopolysaccharide chemotypes regulate activation of the contact pathway of blood coagulation.

Lipopolysaccharide (LPS) is the primary pathogenic factor in Gram-negative sepsis. While the presence of LPS in the bloodstream during infection is associated with disseminated intravascular coagulation, the mechanistic link between LPS and blood coagulation activation remains ill-defined. The contact pathway of coagulation-a series of biochemical reactions that initiates blood clotting when plasma factors XII (FXII) and XI (FXI), prekallikrein (PK) and high molecular weight kininogen (HK) interact with anionic surfaces-has been shown to be activated in Gram-negative septic patients. In this study, using an in vivo baboon model of Gram-negative Escherichia coli sepsis, we observed activation of the contact pathway including FXII, FXI and PK. We examined whether E.coli LPS molecules could binding and activate contact pathway members by quantifying the interaction and activation of either FXII, FXI, or PK with each of three chemotypes of LPS: O111:B4, O26:B6, or Rd2. The LPS chemotypes exhibited distinct physicochemical properties as aggregates and formed complexes with FXII, FXI and PK. The LPS chemotype O26:B6 uniquely promoted the autoactivation of FXII to FXIIa, and in complex with FXIIa, promoted the cleavage of FXI and prekallikrein to generate FXIa and plasma kallikrein, respectively. Furthermore, in complex with the active forms of FXI or prekallikrein, LPS chemotypes were able to regulate the catalytic activity of FXIa and plasma kallikrein, respectively, despite the inability to promote the autoactivation of either zymogen. These data suggest that the procoagulant phenotype of E.coli is influenced by bacterial strain and the physicochemical properties of the LPS chemotypes.

Unique N-glycosylation signatures in Aβ oligomer-and lipopolysaccharide-activated human iPSC-derived microglia.

Microglia are the immune cells in the central nervous system (CNS) and become pro-inflammatory/activated in Alzheimer's disease (AD). Cell surface glycosylation plays an important role in immune cells; however, the N-glycosylation and glycosphingolipid (GSL) signatures of activated microglia are poorly understood. Here, we study comprehensive combined transcriptomic and glycomic profiles using human induced pluripotent stem cells-derived microglia (hiMG). Distinct changes in N-glycosylation patterns in amyloid-β oligomer (AβO) and LPS-treated hiMG were observed. In AβO-treated cells, the relative abundance of bisecting N-acetylglucosamine (GlcNAc) N-glycans decreased, corresponding with a downregulation of MGAT3. The sialylation of N-glycans increased in response to AβO, accompanied by an upregulation of genes involved in N-glycan sialylation (ST3GAL4 and 6). Unlike AβO-induced hiMG, LPS-induced hiMG exhibited a decreased abundance of complex-type N-glycans, aligned with downregulation of mannosidase genes (MAN1A1, MAN2A2, and MAN1C1) and upregulation of ER degradation related-mannosidases (EDEM1-3). Fucosylation increased in LPS-induced hiMG, aligned with upregulated fucosyltransferase 4 (FUT4) and downregulated alpha-L-fucosidase 1 (FUCA1) gene expression, while sialofucosylation decreased, aligned with upregulated neuraminidase 4 (NEU4). Inhibition of sialyation and fucosylation in AβO- and LPS-induced hiMG alleviated pro-inflammatory responses. However, the GSL profile did not exhibit significant changes in response to AβO or LPS activation. AβO- and LPS- specific glycosylation changes could contribute to impaired microglia function, highlighting glycosylation pathways as potential therapeutic targets for AD.

Study the effect of lactoperoxidase activation in combination with heat treatment or nisin on the microbiological quality of raw milk

The aim of the current study was to improve the microbiological quality of raw cow milk by activating the lactoperoxidase system using either nisin or heat treatment. A central composite experimental design with three factors was used to evaluate the effect of thiocyanate (SCN), hydrogen peroxide (H202) and nisin on the microbiological quality of raw milk. The experimental response was measured in terms of the logarithmic reduction in total microbial, total coliforms count, yeasts and molds. Our results indicate that the combination of both treatments was highly effective in destroying various microorganisms. In fact, the optimal values of each parameter (SCN, H202 and nisin) for a 6log, 5log and 4log reductions of the initial number of total aerobic mesophilic bacteria, coliforms, yeasts and molds were around 7 mg/L of SCN, 15 mg/L of H202 and 50 IU/mL of nisin, obtained after 72 h of incubation at 25 °C. Concerning the LPS activation at moderate temperature, our data suggest that this combination was very effective in destroying different microorganisms. Certainly, the total destruction of coliforms, yeasts and molds was noted after 96 h incubation at 25 °C after LPS activation for 4 h and 6 h followed by heat treatment (63 °C for 30 min). These results highlight a synergistic effect between the LPS activation and nisin or heat treatment in preserving microbiological quality of raw milk for 72 h or 96 h at 25 °C, respectively. Such treatments could be implemented by the dairy industry to enhance the quality of fresh milk and consequently of dairy products.

LL37-mtDNA regulates viability, apoptosis, inflammation, and autophagy in lipopolysaccharide-treated RLE-6TN cells by targeting Hsp90aa1.

Sepsis-induced acute lung injury is associated with lung epithelial cell injury. This study analyzed the role of the antimicrobial peptide LL37 with mitochondrial DNA (LL37-mtDNA) and its potential mechanism of action in lipopolysaccharide (LPS)-treated rat type II alveolar epithelial cells (RLE-6TN cells). RLE-6TN cells were treated with LPS alone or with LL37-mtDNA, followed by transcriptome sequencing. Differentially expressed and pivotal genes were screened using bioinformatics tools. The effects of LL37-mtDNA on cell viability, inflammation, apoptosis, reactive oxygen species (ROS) production, and autophagy-related hallmark expression were evaluated in LPS-treated RLE-6TN cells. Additionally, the effects of Hsp90aa1 silencing following LL37-mtDNA treatment were investigated in vitro. LL37-mtDNA further suppressed cell viability, augmented apoptosis, promoted the release of inflammatory cytokines, increased ROS production, and elevated LC3B expression in LPS-treated RLE-6TN cells. Using transcriptome sequencing and bioinformatics, ten candidate genes were identified, of which three core genes were verified to be upregulated in the LPS + LL37-mtDNA group. Additionally, Hsp90aa1 downregulation attenuated the effects of LL37-mtDNA on LPS-treated RLE-6TN cells. Hsp90aa1 silencing possibly acted as a crucial target to counteract the effects of LL37-mtDNA on viability, apoptosis, inflammation, and autophagy activation in LPS-treated RLE-6TN cells.

Linoleic acid-derived diol 12,13-DiHOME enhances NLRP3 inflammasome activation in macrophages.

12,13-dihydroxy-9z-octadecenoic acid (12,13-DiHOME) is a linoleic acid diol derived from cytochrome P-450 (CYP) epoxygenase and epoxide hydrolase (EH) metabolism. 12,13-DiHOME is associated with inflammation and mitochondrial damage in the innate immune response, but how 12,13-DiHOME contributes to these effects is unclear. We hypothesized that 12,13-DiHOME enhances macrophage inflammation through effects on NOD-like receptor protein 3 (NLRP3) inflammasome activation. To test this hypothesis, we utilized human monocytic THP1 cells differentiated into macrophage-like cells with phorbol myristate acetate (PMA). 12,13-DiHOME present during lipopolysaccharide (LPS)-priming of THP1 macrophages exacerbated nigericin-induced NLRP3 inflammasome activation. Using high-resolution respirometry, we observed that priming with LPS+12,13-DiHOME altered mitochondrial respiratory function. Mitophagy, measured using mito-Keima, was also modulated by 12,13-DiHOME present during priming. These mitochondrial effects were associated with increased sensitivity to nigericin-induced mitochondrial depolarization and reactive oxygen species production in LPS+12,13-DiHOME-primed macrophages. Nigericin-induced mitochondrial damage and NLRP3 inflammasome activation in LPS+12,13-DiHOME-primed macrophages were ablated by the mitochondrial calcium uniporter (MCU) inhibitor, Ru265. 12,13-DiHOME present during LPS-priming also enhanced nigericin-induced NLRP3 inflammasome activation in primary murine bone marrow-derived macrophages. In summary, these data demonstrate a pro-inflammatory role for 12,13-DiHOME by enhancing NLRP3 inflammasome activation in macrophages.

A Whole Blood Method for Assessing the Innate Immune Response in Chickens

Innate immunity is considered the first line of immune defense and is typically an unmeasured response. Here we report a method for evaluating the innate immune response in chickens by using whole blood which has been activated by lipopolysaccharide (LPS) to induce IL-6 release by innate immune cells. It was found that a 24-h LPS activation time interval was the optimum time interval for inducing the IL-6 response. An activation index, defined as the PBS activated control response subtracted from the LPS activated response and then divided by the PBS activated control response and expressed as a percentage, was useful for demonstrating and comparing the magnitude of the innate immune response. Results indicated that there was wide variation between the IL-6 response between individual birds although statistically significant results were obtained for all individual birds at the 24-h activation time interval. The activation indices from all birds were greatest at the 24-h activation time interval. Statistically significant results were achieved when all the data from all birds at the 24-h activation time interval were combined. The cells responsible for the IL-6 response were identified as the peripheral blood monocytes.

A Synthetic Derivative SH 66 of Homoisoflavonoid from Liliaceae Exhibits Anti-Neuroinflammatory Activity against LPS-Induced Microglial Cells.

Naturally occurring homoisoflavonoids isolated from some Liliaceae plants have been reported to have diverse biological activities (e.g., antioxidant, anti-inflammatory, and anti-angiogenic effects). The exact mechanism by which homoisoflavonones exert anti-neuroinflammatory effects against activated microglia-induced inflammatory cascades has not been well studied. Here, we aimed to explore the mechanism of homoisoflavonoid SH66 having a potential anti-inflammatory effect in lipopolysaccharide (LPS)-primed BV2 murine microglial cells. Microglia cells were pre-treated with SH66 followed by LPS (100 ng/mL) activation. SH66 treatment attenuated the production of inflammatory mediators, including nitric oxide and proinflammatory cytokines, by down-regulating mitogen-activated protein kinase signaling in LPS-activated microglia. The SH66-mediated inhibition of the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome complex and the respective inflammatory biomarker-like active interleukin (IL)-1β were noted to be one of the key pathways of the anti-inflammatory effect. In addition, SH66 increased the neurite length in the N2a neuronal cell and the level of nerve growth factor in the C6 astrocyte cell. Our results demonstrated the anti-neuroinflammatory effect of SH66 against LPS-activated microglia-mediated inflammatory events by down-regulating the NLRP3 inflammasome complex, with respect to its neuroprotective effect. SH66 could be an interesting candidate for further research and development regarding prophylactics and therapeutics for inflammation-mediated neurological complications.

NP-12 peptide functionalized nanoparticles counteract the effect of bacterial lipopolysaccharide on cultured osteoblasts

ObjectiveTo evaluate whether nanoparticles (NPs) functionalized with Tideglusib (TDg, NP-12), and deposited on titanium surfaces, would counteract the effect of bacterial lipopolysaccharide (LPS) on osteoblasts. MethodsExperimental groups were: (a) Titanium discs (TiD), (b) TiD covered with undoped NPs (Un-NPs) and (c) TiD covered with TDg-doped NPs (TDg-NPs). Human primary osteoblasts were cultured onto these discs, in the presence or absence of bacterial LPS. Cell proliferation was assessed by MTT-assay and differentiation by measuring the alkaline phosphatase activity. Mineral nodule formation was assessed by the alizarin red test. Real-time quantitative polymerase chain reaction was used to study the expression of Runx-2, OSX, ALP, OSC, OPG, RANKL, Col-I, BMP-2, BMP-7, TGF-β1, VEGF, TGF-βR1, TGF-βR2, and TGF-βR3 genes. Osteoblasts morphology was studied by Scanning Electron Microscopy. One-way ANOVA or Kruskal-Wallis and Bonferroni multiple comparisons tests were carried out (p < 0.05). ResultsTDg-NPs enhanced osteoblasts proliferation. Similarly, this group increased ALP production and mineral nodules formation. TDg-NPs on titanium discs resulted in overexpression of the proliferative genes, OSC and OSX, regardless of LPS activity. In the absence of LPS, TDg-NPs up-regulated Runx2, COL-I, ALP, BMP2 and BMP7 genes. OPG/RANKL gene ratios were increased about 2500 and 4,000-fold by TDg-NPs, when LPS was added or not, respectively. In contact with the TDg-NPs osteoblasts demonstrated an elongated spindle-shaped morphology with extracellular matrix production. SignificanceTDg-NPs on titanium discs counteracted the detrimental effect of LPS by preventing the decrease on osteoblasts proliferation and mineralization, and produced an overexpression of proliferative and bone-promoting genes on human primary osteoblasts.

Untangling the inflammatory responses of liver x receptor (LXR) activation in human macrophages

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Rembrandt Institute for Cardiovascular Science (grant 2022) The regulation of cholesterol and fatty acid homeostasis is essential and the Liver X Receptor (LXR) plays a crucial role in this process. When activated, LXR promotes cholesterol efflux and suppresses the transcription of pro-inflammatory genes in mouse macrophages, making it a promising therapy against atherosclerosis. Studies indeed have shown that LXR agonism is effective in pre-clinical atherosclerotic mouse models. However, the limited data on human macrophages suggests that LXR activation may have additional pro-inflammatory effects in humans, and the mechanisms contributing to this are not yet fully understood. To investigate the inflammatory properties of LXR activation in human macrophages, monocytes were isolated from buffy coats of healthy donors and differentiated into macrophages using M-CSF. The macrophages were exposed to synthetic and endogenous LXR ligands, such as GW3965, T0901317, and desmosterol, respectively, followed by stimulation with lipopolysaccharide (LPS) for six hours. We used qPCR and ELISA to analyze experiments and plan to supplement our data with RNA-Seq and multiplex assays. Preliminary results show that LXR activation reduces LPS-induced activation of interferon-mediated genes like CXCL9 and CXCL10, similar to mice. However, it promotes the transcription of NF-kB target genes like IL1B and IL12B upon LPS activation. These findings suggest that LXR activation in human macrophages not solely suppresses inflammatory responses in macrophages and that LXR activation has different effects on the regulation of genes mediated by interferon or NF-kB. Our research aims to further uncover the regulatory mechanisms behind LXR activation in human macrophages as central regulators in atherosclerosis.

Cadmium promoted LPS-induced inflammation through TLR4/IκBα/NFκ-B signaling by increasing ROS-mediated incomplete autophagy

Cadmium (Cd) exposure is considered as non-infectious stressor to human and animal health. Recent studies suggest that the immunotoxicity of low dose Cd is not directly apparent, but disrupts the immune responses when infected with some bacteria or virus. But how Cd alters the adaptive immunity organ and cells remains unclear. In this study, we applied lipopolysaccharide (LPS, infectious stressor) to induced inflammation in spleen tissues and T cells, and investigated the effects after Cd exposure and the underlying mechanism. Cd exposure promoted LPS-induced the expressions of the inflammatory factors, induced abnormal initiation of autophagy, but blocked autophagic flux. The effects Cd exposure under LPS activation were reversed by the autophagy promoter Rapamycin. Under LPS activation conditions, Cd also induced oxidative stress by increasing the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), and reducing total antioxidant capacity (T-AOC) activity. The increased superoxide dismutase (SOD) activity after Cd exposure might be a negative feedback or passive adaptive regulation of oxidative stress. Cd-increased autophagic flux inhibition and TNF-α expression were reversed by ROS scavenger α-tocopherol (TCP). Furthermore, under LPS activation condition, Cd promoted activation of toll-like receptor 4 (TLR4)/IκBα/NFκ-B signaling pathway and increased TLR4 protein stability, which were abolished by the pretreatment of Rapamycin. The present study confirmed that, by increasing ROS-mediated inhibiting autophagic degradation of TLR4, Cd promoted LPS-induced inflammation in spleen T cells. This study identified the mechanism of autophagy in Cd-aggravated immunotoxicity under infectious stress, which could arouse public attention to synergistic toxicity of Cd and bacterial or virus infection.

JAK1/2 Regulates Synergy Between Interferon Gamma and Lipopolysaccharides in Microglia.

Microglia, the resident immune cells of the brain, regulate neuroinflammation which can lead to secondary neuronal damage and cognitive impairment under pathological conditions. Two of the many molecules that can elicit an inflammatory response from microglia are lipopolysaccharide (LPS), a component of gram-negative bacteria, and interferon gamma (IFNγ), an endogenous pro-inflammatory cytokine. We thoroughly examined the concentration-dependent relationship between LPS from multiple bacterial species and IFNγ in cultured microglia and macrophages. We measured the effects that these immunostimulatory molecules have on pro-inflammatory activity of microglia and used a battery of signaling inhibitors to identify the pathways that contribute to the microglial response. We found that LPS and IFNγ interacted synergistically to induce a pro-inflammatory phenotype in microglia, and that inhibition of JAK1/2 completely blunted the response. We determined that this synergistic action of LPS and IFNγ was likely dependent on JNK and Akt signaling rather than typical pro-inflammatory mediators such as NF-κB. Finally, we demonstrated that LPS derived from Escherichia coli, Klebsiella pneumoniae, and Akkermansia muciniphila can elicit different inflammatory responses from microglia and macrophages, but these responses could be consistently prevented using ruxolitinib, a JAK1/2 inhibitor. Collectively, this work reveals a mechanism by which microglia may become hyperactivated in response to the combination of LPS and IFNγ. Given that elevations in circulating LPS and IFNγ occur in a wide variety of pathological conditions, it is critical to understand the pharmacological interactions between these molecules to develop safe and effective treatments to suppress this process.

The LPS-inactivating enzyme acyloxyacyl hydrolase protects the brain from experimental stroke

Blood-brain-barrier (BBB) disruption is a pathological hallmark of ischemic stroke, and inflammation occurring at the BBB contributes to the pathogenesis of ischemic brain injury. Lipopolysaccharide (LPS), a cell wall component of Gram-negative bacteria, is elevated in patients with acute stroke. The activity of LPS is controlled by acyloxyacyl hydrolase (AOAH), a host enzyme that deacylates LPS to inactivated forms. However, whether AOAH influences the pathogenesis of ischemic stroke remain elusive. We performed in vivo experiments to explore the role and mechanism of AOAH on neutrophil extravasation, BBB disruption, and brain infarction. We found that AOAH was upregulated in neutrophils in peri-infarct areas from mice with transient focal cerebral ischemia. AOAH deficiency increased neutrophil extravasation into the brain parenchyma and proinflammatory cytokine production, broke down the BBB and worsened stroke outcomes in mice. These effects require Toll-like receptor 4 (TLR4) because absence of TLR4 or pharmacologic inhibition of TLR4 signaling prevented the exacerbated inflammation and BBB damage in Aoah−/− mice after ischemic stroke. Importantly, neutrophil depletion or inhibition of neutrophil trafficking by blocking LFA-1 integrin dramatically reduced stroke-induced BBB breakdown in Aoah−/− mice. Furthermore, virus-mediated overexpression of AOAH induced a substantial decrease in neutrophil recruitment that was accompanied by reducing BBB damage and stroke volumes. Our findings show the importance of AOAH in regulating neutrophil-dependent BBB breakdown and cerebral infarction. Consequently, strategies that modulate AOAH may be a new therapeutic approach for treatment of ischemic stroke.

Abstract 6639: Mechanisms of action of a killed, bacteria-based, multiple immune receptor agonist in development for pulsed anti-tumor immunotherapy

Abstract Activation of immune receptors, such as Toll-like (TLR), NOD-like (NLR) and Stimulator of Interferon Genes (STING) is required for efficient innate and adaptive immunity. Gram-negative bacteria (G-NB) contain multiple TLR, NOD and STING agonists. Potential utility of G-NB for cancer immunotherapy is supported by observations of tumor regression in the setting of infection and Coley’s Toxins. Coley reported that intravenous (i.v.) administration was likely most effective but produced toxicity. The discovery of TLRs and their agonists, particularly the potent/multi-functional TLR4 agonist lipopolysaccharide (LPS)-endotoxin, comprising ~75% of the outer membrane of G-NB, suggests that it may be both a critical active ingredient and responsible for dose-limiting toxicity of i.v. G-NB. We have produced killed, intact bacteria products from non-pathogenic G-NB with ~90% reduction of LPS activity. One product, Decoy10, contained TLR2,4,8,9, NOD2 and STING agonist activity and exhibited reduced i.v. toxicity in mice and rabbits relative to unprocessed cells. Decoy10 and closely related Decoy20 produced single agent activity or combination-mediated durable regressions, with immunological memory, in mice with syngeneic or human breast, colorectal, hepatocellular, pancreatic carcinomas or non-Hodgkin’s lymphoma. Regressions were observed in combination with chemotherapy, a non-steroidal anti-inflammatory drug, anti-PD-1, or rituximab, were associated with induction of 18-26 plasma cytokines/chemokines, activation of innate and adaptive immune pathways in tumors, and were dependent on NK, CD4+ and CD8+ T cells (Newman, Cancer Res 2023;83(7 Suppl):Abstract nr 4165). These and preliminary clinical results demonstrating rapid clearance of Decoy20 with transient induction of &amp;gt;50 plasma cytokines/chemokines (Newman et al., J Immunother Cancer 2023;11(Suppl 2)782-E:A1770) are supportive of a “pulse-prime” mechanism, whereby Decoy bacteria produce transient, but broad innate and adaptive immune activation. We have now analyzed immune activation by Decoy bacteria in vitro using human peripheral blood mononuclear cells (PBMCs). Decoy10 enhanced polarization of CD14+ monocytes to M1 macrophages and inhibited Treg polarization. Decoy10 enhanced monocyte-derived dendritic cell maturation and enhanced Th1, Th2 and Th17 CD4+ polarization, with Th1 at 10-100-fold lower Decoy10 concentrations than Th2 or Th17. Decoy10 also activated NKT and T cells, and enhanced PBMC-mediated tumor cell killing. The results demonstrate that Decoy bacteria stimulate polarization, maturation, or activation of cellular mediators of innate and adaptive anti-tumor immune responses, inhibit an immune-suppressive mechanism, and enhance immune cell killing of tumor cells. The results are consistent with and significantly extend our previously reported in vivo data. Citation Format: Michael J. Newman, Deepak Singh. Mechanisms of action of a killed, bacteria-based, multiple immune receptor agonist in development for pulsed anti-tumor immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6639.

Polysaccharide of Atractylodes macrocephala Koidz alleviates LPS-induced proliferation, differentiation inhibition and excessive apoptosis in chicken embryonic myogenic cells.

Lipopolysaccharide (LPS) can induce systemic inflammation and affect the growth and development of poultry. As a kind of traditional Chinese medicine, polysaccharide of Atractylodes macrocephala Koidz (PAMK) can effectively improve the growth performance of animals and improve the immunity of animal bodies. The purpose of this study was to investigate the effects of PAMK on LPS-induced inflammatory response, proliferation, differentiation and apoptosis of chicken embryonic myogenic cells. We used chicken embryonic myogenic cells as a model by detecting EdU/MYHC immunofluorescence, the expression of inflammation, proliferation, differentiation-related genes and proteins and the number of apoptotic cells in the condition of adding LPS, PAMK, belnacasan (an inhibitor of Caspase1) or their combinations. The results showed that LPS stimulation increased the expression of inflammatory factors, inhibited proliferation and differentiation, and excessive apoptosis in chicken embryonic myogenic cells, and PAMK alleviated these adverse effects induced by LPS. After the addition of belnacasan (inhibitor of Caspase1), apoptosis in myogenic cells was inhibited, and therefore, the number of apoptotic cells and the expression of pro-apoptotic genes Caspase1 and Caspase3 were increased. In addition, belnacasan inhibited the increased expression of inflammatory factors, inhibited proliferation, differentiation and excessive apoptosis in chicken embryonic myogenic cells induced by LPS. This study provides a theoretical basis for further exploring the mechanism of action of PAMK and exogenous LPS on chicken embryonic myogenic cells and lays the foundation for the development and application of green feed additives in animal husbandry industry.

Comtrifosides A and B, two new triterpenoid saponins from the leaves of Combretum trifoliatum

The phytochemical composition of the Combretum trifoliatum leaves was studied for the first time. Two new triterpenoid saponins, named comtrifoside A (1) and comtrifoside B (2), together with two other saponins (3–4) were purified by variously chromatographic techniques. For the first time, compound 3 was informed from the Combretum genus, as well as all of the isolated compounds (1–4) were reported from C. trifoliatum. The chemical structures of them were clearly characterised using extensive UV-VIS, IR, HRMS-ESI, and NMR experimental data. The in vitro anti-inflammatory activities of 1 & 2 were examined against NO overproduction in LPS activation of RAW264.7.

Microbiome analysis reveals the inducing effect of Pseudomonas on prostatic hyperplasia via activating NF-κB signalling

ABSTRACT Benign prostatic hyperplasia (BPH) is a prevalent disease among middle-aged and elderly males, but its pathogenesis remains unclear. Dysbiosis of the microbiome is increasingly recognized as a significant factor in various human diseases. Prostate tissue also contains a unique microbiome, and its dysbiosis has been proposed to contribute to prostate diseases. Here, we obtained prostate tissues and preoperative catheterized urine from 24 BPH individuals, and 8 normal prostate samples as controls, which followed strict aseptic measures. Using metagenomic next-generation sequencing (mNGS), we found the disparities in the microbiome composition between normal and BPH tissues, with Pseudomonas significantly enriched in BPH tissues, as confirmed by fluorescence in situ hybridization (FISH). Additionally, we showed that the prostate microbiome differed from the urine microbiome. In vitro experiments revealed that lipopolysaccharide (LPS) of Pseudomonas activated NF-κB signalling, leading to inflammation, proliferation, and EMT processes, while inhibiting apoptosis in prostatic cells. Overall, our research determines the presence of microbiome dysbiosis in BPH, and suggests that Pseudomonas, as the dominant microflora, may promote the progression of BPH through LPS activation of NF-κB signalling.

Green Robusta Coffee Bean Extract (GRCBE) inhibits bone loss in wistar rat models of Lps P. gingivalis and NiTi wire-induced experimental periodontitis

Oral exposure to alloys frequently leads to negative reactions, both in the immediate area and across the body. The usage of coffee extracts is anticipated to enhance cellular function by counteracting the effects of pro-inflammatory signals that are involved in chronic inflammatory responses triggered by inflammatory agents like bacteria and metal ions. The objective is to develop a new approach using an extract obtained from Robusta coffee beans as an anti-inflammatory substance, which may have therapeutic benefits in preventing bone loss in Periodontitis Rat. The main cause is primarily attributed to the existence of metals and LPS P. gingivalis in the oral cavity. Methods. A randomized controlled trial was conducted, enroling 35 male Wistar rats that were divided into 7 groups and used as the experimental animals. Groups 1–4 consisted of rats that did not receive any therapy, while groups 5–7 comprised rats that were treated with GRCBE. The rats were subjected to decapitation after 14 days. The Green Robusta Coffee bean extract (GRCBE) was derived from the unprocessed green beans of Javanese Robusta coffee. We discovered in prior experimental experiments that GRCBE at a concentration of 500 mg/ml can inhibit the progression of periodontitis in rats. The substances used to cause inflammation and immune system impairment were NiTi wire and LPS P. gingivalis. Immunohistochemistry was used to assess the expressions of OCN, BMP2, and COX-2. Results. The simultaneous exposure of P. gingivalis lipopolysaccharide (LPS) and NiTi wire in rats led to an upregulation of OCN and BMP2 expression, while the expression of COX-2 reduced. The administration of GRCBE resulted in a steady drop in pro-inflammatory biomarkers, specifically OCN and BMP2, while the anti-inflammatory biomarker COX2 rose. In conclusion. In the field of dentistry, the coexistence of P. gingivalis LPS and metallic biomaterials can influence the occurrence of alveolar bone resorption in a rat model of periodontitis. GRCBE exhibits potential as an innovative approach for reducing periodontitis induced by the simultaneous activation of metal and LPS P. gingivalis. In this setting, it has the potential to be employed as an anti-inflammatory remedy.