Lipopolysaccharide Stimulation Research Articles

H3K27 tri-demethylase JMJD3 inhibits macrophage apoptosis by promoting ADORA2A in lipopolysaccharide-induced acute lung injury

Acute lung injury (ALI) is a common critical disease, which is characterized by an uncontrolled, acute inflammatory response, diffuse lung damage and ultimately directly deteriorates into acute respiratory distress syndrome. The number of pro-inflammatory macrophages is related to the severity of ALI. Up-regulation of lipopolysaccharide (LPS)-activated macrophage apoptosis can reduce the pro-inflammatory reactions. Jumonji domain-containing protein D3 (JMJD3)-mediated histone 3 lysine 27 trimethylation (H3K27me3) demethylation may promote the pro-inflammatory response of macrophages under LPS stimulation. However, the mechanism of JMJD3 affecting macrophage apoptosis is still not clear. To explore this gap in knowledge, the ALI mice model with intratracheal administration of LPS and RAW264.7 cells with LPS stimulation were used as in vivo and in vitro experiments. The expression of JMJD3 and H3K27me3 and their cellular localization were analysed in lung tissue. Apoptosis was evaluated using TUNEL staining and flow cytometry. Expression of H3K27me3, ADORA2A and C/EBPβ were compared among different treatments and chromatin immunoprecipitation was performed to investigate the regulatory relationship. Our study showed that JMJD3 expression was upregulated in LPS-induced ALI mice and RAW264.7 cells. JMJD3-indued H3K27me3 demethylation inhibited caspase-3 cleavage by upregulating ADORA2A to decrease LPS-stimulated macrophage apoptosis and promoted the inflammatory reaction. This H3K27me3 demethylation also increased C/EBPβ expression, which may enhance ADORA2A expression further. Besides, inhibiting ADORA2A can also promote LPS-limited macrophage apoptosis. Moreover, the inhibition of JMJD3 in vivo and in vitro relieved the inhibition of macrophage apoptosis thus leading to the resolution of the inflammation. JMJD3 might inhibit macrophage apoptosis by promoting ADORA2A expression in LPS-induced ALI.

Acetylcholine suppresses LPS-induced endothelial cell activation by inhibiting the MAPK and NF-κB pathways.

Endothelial cell activation plays a critical role in leukocyte recruitment during inflammation and infection. We previously found that cholinergic stimulation (via vagus nerve stimulation) attenuates vascular endothelial impairment and reduces the inflammatory profile in ovariectomized rats. However, the specific molecular mechanism is unclear. This study was designed to explore the effects and molecular mechanisms of cholinergic agonists (acetylcholine [ACh]) on lipopolysaccharide (LPS)-induced endothelial cell activation in vitro. Human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of LPS (10/100/1000ng/mL) to activate endothelial cells. HUVECs were untreated, treated with ACh (10-5 M) alone, treated with 100ng/mL LPS alone, or treated with different concentrations of ACh (10-9/10-8/10-7/10-6/10-5 M) before LPS stimulation. HUVECs were also pre-treated with 10-6 M ACh with or without mecamylamine (an nAChR blocker) (10μΜ) and methyllycaconitine (a specific α7 nAChR blocker) (10μΜ) and incubated with or without LPS. ELISA, western blotting, cell immunofluorescence, and cell adhesion assays were used to examine inflammatory cytokine production, adhesion molecule expression, monocyte-endothelial cell adhesion and activation of the MAPK/NF-κB pathways. LPS (at 10ng/mL, 100ng/mL and 1,000ng/mL) increased VCAM-1 expression in HUVECs in a dose-dependent manner (with no significant difference between LPS at 100ng/mL and 1,000ng/mL). ACh (10-9 M-10-5 M) blocked adhesion molecule expression (VCAM-1, ICAM-1, and E-selectin) and inflammatory cytokine production (TNF-α, IL-6, MCP-1, IL-8) in response to LPS in a dose-dependent manner (with no significant difference between 10-5 and 10-6 M Ach). LPS was also shown to significantly enhance monocyte-endothelial cell adhesion, which was largely abrogated by treatment with ACh (10-6M). VCAM-1 expression was blocked by mecamylamine rather than methyllycaconitine. Lastly, ACh (10-6 M) significantly reduced LPS-induced phosphorylation of NF-κB/p65, IκBα, ERK, JNK and p38MAPK in HUVECs, which was blocked by mecamylamine. ACh protects against LPS-induced endothelial cell activation by inhibiting the MAPK and NF-κB pathways, which are mediated by nAChR, rather than α7 nAChR. Our results may provide novel insight into the anti-inflammatory effects and mechanisms of ACh.

APOE genotype alters the lipid droplet proteome and modulates droplet dynamics

AbstractBackgroundThe microglial immune response is a significant contributor to Alzheimer’s disease (AD) pathophysiology and neurodegeneration. Aging microglia accumulate lipid droplets (LDs), have high levels of reactive oxygen species, secrete pro‐inflammatory cytokines, and are defective in phagocytosis. The E4 allele of Apolipoprotein E (APOE) is the strongest genetic risk factor for late‐onset AD, and is associated with heightened neuroinflammation and increased LD formation. We hypothesize E4 microglia have increased LD formation under basal conditions and a higher capacity to form LDs under stress, resulting in greater pro‐inflammatory cytokine production. We characterized LD development in microglia in the context of APOE genotype and analyzed LD surface proteins and lipid content from control and lipopolysaccharide (LPS) stimulated ApoE3 and ApoE4 mice.MethodPrimary microglia were isolated from mice expressing human ApoE3 and ApoE4. Microglia were exposed to 250uM oleic acid (OA), 10ug/mL LPS, OA+LPS, dead N2A cells, or dead N2As+LPS. ApoE3 and ApoE4 expressing mice were injected with saline (control) or LPS (5mg/kg) and perfused at 24h. Livers were extracted, the LD enriched supernatant fraction was collected after centrifugation, and proteomic and lipidomic analyses were performed.ResultPrimary microglia from ApoE4 mice accumulated more LDs at baseline, with exogenous OA, LPS stimulation, and N2As as a percentage of E3 control across multiple experiments (E3 v E4 p‐values: baseline, 0.0317; LPS, 0.0032; OA, 0.0277; N2A, 0.0192). Western blots on LD fractions confirm LD enrichment by surface protein, PLIN2, along with increased expression of PLIN2 (i.e. more LDs) in E4 LPS mice. Proteomics reveal LD fractions from E4 mice are enriched for proteins involved in innate immunity, while E3 LDs are enriched for proteins involved in lipid b‐oxidation.ConclusionE4 microglia accumulate more LDs compared to E3 microglia under all conditions tested. The proteomic profile of E4 liver LDs support the hypothesis that E4 expression increases inflammation under basal conditions, and upon stimulation, causes a more robust response. Increased LD formation is present in non‐aged, non‐diseased E4 cells, suggesting preclinical dysfunction associated with the highest risk APOE genotype. A better understanding of LD dynamics within these cells and their functional implications can provide targets to improve E4‐related outcomes.

Pathogenic variant c.61G > C in NLRP3 lowers NLRP3 inflammasome activation threshold in patients with keratitis Fugax Hereditaria

AbstractPurpose: To elucidate the effect of NLRP3 variant c.61G > C on interleukin‐1β (IL‐1β) secretion in keratitis fugax hereditaria (KFH), a cryopyrin‐associated periodic syndrome limited to the eye, and to probe the potential modifying role of prednisolone.Methods: Peripheral blood mononuclear cells (PBMCs) isolated from whole blood of patients with KFH and healthy controls were grown under steady‐state conditions or primed with lipopolysaccharide (LPS) with or without prednisolone, and subsequently activated with adenosine triphosphate (ATP). Cell lysates and proteins precipitated from the cell culture media were separated by SDS‐PAGE. NLRP3, procaspase‐1, and IL‐1β were visualized by western blotting. The concentration of secreted IL‐1β in the culture media was quantified by ELISA.Results: A lower threshold for IL‐1β secretion following priming of the NLRP3 inflammasome with LPS was observed in patients‐derived PBMCs, compared to healthy controls (median, 124 vs. 16 pg/ml respectively). Interestingly, in PBMCs derived from patients with frequent KFH symptoms, LPS priming alone was able to trigger substantial IL‐1β secretion (median, 522 pg/ml), whereas those of patients experiencing occasional KFH attacks showed a subtler release of IL‐1β (median, 85 pg/ml). LPS and ATP treated PBMCs from patients with KFH showed significantly diminished IL‐1β secretion with prednisolone treatment (p = 0.04). NLRP3 expression was significantly enhanced with LPS stimulation (p = 0.03) whereas procaspase‐1 expression was not affected.Conclusions: PBMCs from patients with KFH are more prone to secrete IL‐1β, confirming the presumption that the c.61G > C is a gain‐of‐function variant. Furthermore, prednisolone is confirmed as a potent drug to reduce IL‐1β secretion in KFH.

Cardiac-specific ITCH overexpression ameliorates septic cardiomyopathy via inhibition of the NF-κB signaling pathway

Septic cardiomyopathy is a common complication of septic shock and organ dysfunction. ITCH is a HECT (homologous to the E6-AP carboxyl-terminus)-type ubiquitin E3 ligase that plays a critical role in inflammatory suppression. Herein, we focused on the interaction between ITCH and key regulators of nuclear factor-κB (NF-κB), such as tumor necrosis factor receptor-associated factor 6 (TRAF6) and transforming growth factor-β activated kinase 1 (TAK1), and examined the impact of ITCH on the development of septic cardiomyopathy. In H9C2 cardiomyocytes, ITCH protein expression decreased in response to lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNFα). The protein interactions of ITCH with TRAF6 and TAK1 were confirmed by immunoprecipitation in vitro and in vivo . Based on overexpression and knockdown studies of ITCH in H9C2 cardiomyocytes, ITCH regulates the phosphorylation of NF-κB and subsequent interleukin 6 ( IL-6 ) expression in response to LPS and TNFα stimulation. LPS was intraperitoneally injected into transgenic mice with cardiac-specific overexpression of ITCH (ITCH-Tg) and wild-type (WT) mice. Compared with WT mice, phosphorylation of NF-κB and subsequent IL-6 expression were inhibited in ITCH-Tg mice. Cardiac systolic dysfunction after LPS administration was ameliorated in ITCH-Tg mice, and the survival rate was higher in ITCH-Tg mice than in WT mice. ITCH interacts with TRAF6 and TAK1 in cardiomyocytes and improves cardiac function and survival rates in septic cardiomyopathy by suppressing the NF-κB pathway. • ITCH interacted with TRAF6 and TAK1 in cardiomyocytes. • ITCH modulates activation of NFκB in cardiomyocytes. • Cardiac overexpression of ITCH preserves cardiac function in septic cardiomyopathy. • Cardiac overexpression of ITCH improves survival rate in septic cardiomyopathy. • ITCH plays a protective role in the development of septic cardiomyopathy.

Quantification of extracellular proteins, protein complexes and mRNAs in single cells by proximity sequencing.

We present proximity sequencing (Prox-seq) for simultaneous measurement of proteins, protein complexes and mRNAs in thousands of single cells. Prox-seq combines proximity ligation assay with single-cell sequencing to measure proteins and their complexes from all pairwise combinations of targeted proteins, providing quadratically scaled multiplexing. We validate Prox-seq and analyze a mixture of T cells and B cells to show that it accurately identifies these cell types and detects well-known protein complexes. Next, by studying human peripheral blood mononuclear cells, we discover that naïve CD8+ T cells display the protein complex CD8-CD9. Finally, we study protein interactions during Toll-like receptor (TLR) signaling in human macrophages. We observe the formation of signal-specific protein complexes, find CD36 co-receptor activity and additive signal integration under lipopolysaccharide (TLR4) and Pam2CSK4 (TLR2) stimulation, and show that quantification of protein complexes identifies signaling inputs received by macrophages. Prox-seq provides access to an untapped measurement modality for single-cell phenotyping and can discover uncharacterized protein interactions in different cell types.

Tripartite motif containing 23 functions as a critical regulator in macrophages to control the pathological feature of diabetic nephropathy.

Diabetic nephropathy (DN) is a kidney disease resulting from diabetes. Macrophages and macrophage-mediated inflammation contributed to the development of DN. Tripartite motif containing 23 (TRIM23) is E3 ligase and has been involved in inflammation. Until now, the precise roles of TRIM23 in DN are not described yet. Therefore, we evaluated the functions of TRIM23 in DN. We generated mice with TRIM23 deficiency in macrophages. We also established diabetic mice model. The expression of TRIM23 was measured in diabetic animals. The DN symptoms were compared between diabetic wild-type (WT) mice and TRIM23 conditional knock out mice. The cytokine expression, ubiquitination of TAB2, and interactions between TAB2 and IKK were compared in oxidized low-density lipoprotein (oxLDL) or lipopolysaccharides (LPS)-treated WT and TRIM23-deficient macrophages. Upregulation of TRIM23 was observed in diabetic mice and LPS or oxLDL-treated macrophages. Diabetic mice with TRIM23 deficiency in macrophages had attenuated DN symptoms. TRIM23-deficient macrophages had decreased pro-inflammatory cytokines production after oxLDL or LPS stimulation. TRIM23 was predicted to interact with TAB2. The ubiquitination of TAB2 was abolished in oxLDL-treated TRIM23-deficient macrophages, which correlated with decreased activation of IKK. TRIM23 regulates inflammation in macrophages and plays important role in DN.

NOS2/miR-493-5p Signaling Regulates in the LPS-Induced Inflammatory Response in the RAW264.7 Cells.

Tuberculosis (TB) is a fatal infectious disease; however, the molecular mechanisms underlying the pathogenicity of TB remain elusive. The present study aims to identify potential biomarkers associated with Mycobacterium tuberculosis (M.tb) infection by using integrated bioinformatics and in vitro validation studies. GSE50050, GSE78706, and GSE108844 data from the gene expression omnibus (GEO) database were downloaded to identify differentially expressed genes (DEGs). The functions of DEGs were further subjected to gene ontology (GO) and KEGG pathway analysis. The hub genes from the DEGs were determined based on the protein-protein interaction (PPI) network analysis. Finally, the hub genes were experimentally validated using the in vitro functional studies. A total of 26 common DEGs were identified among GSE50050, GSE78706, and GSE108844. The functional enrichment analysis showed that the common DEGs were associated with cytokines response and TB pathways. The PPI network analysis identified nine hub genes. Further in vitro studies showed that nitric oxide synthase 2 (NOS2) was up-regulated in RAW264.7 cells upon lipopolysaccharides (LPS) stimulation, which was accompanied by increased inflammatory cytokines release. Furthermore, NOS2 was found to be a target of miR-493-5p, which was confirmed by luciferase reporter assay. NOS2 was repressed by miR-493-5p overexpression and was up-regulated after miR-493-5p inhibition in RAW264.7 cells. The rescue experiments showed that LPS-induced increase in the inflammatory cytokines of the RAW264.7 cells was significantly attenuated by NOS2 knockdown and miR-493-5p overexpression. Collectively, our results for the first time demonstrated that NOS2/miR-493-5p signaling pathway may potentially involve in the inflammatory response during bacterial infection such as M. tb infection.

Casein Lactose-Glycation of the Maillard-Type Attenuates the Anti-Inflammatory Potential of Casein Hydrolysate to IEC-6 Cells with Lipopolysaccharide Stimulation

During the thermal processing of dairy products, the Maillard reaction occurs between milk proteins and lactose, resulting in the formation of various products including glycated proteins. In this study, lactose-glycated casein was generated through the Maillard reaction between casein and lactose and then hydrolyzed by a trypsin preparation. The anti-inflammatory effect of the resultant glycated casein hydrolysate (GCH) was investigated using the lipopolysaccharide (LPS)-sitmulated rat intestinal epithelial (IEC-6) cells as a cell model and corresponding casein hydrolysate (CH) as a control. The results indicated that the preformed glycation enabled lactose conjugation to casein, which endowed GCH with a lactose content of 12.61 g/kg protein together with a lower activity than CH to enhance the viability value of the IEC-6 cells. The cells with LPS stimulation showed significant inflammatory responses, while a pre-treatment of the cells with GCH before LPS stimulation consistently led to a decreased secretion of three pro-inflammatory mediators, namely, IL-6, IL-1β and tumor necrosis factor-α (TNF-α) but an increased secretion of two anti-inflammatory mediators, including IL-10 and transforming growth factor-β (TGF-β), demonstrating the anti-inflammatory potential of GCH in LPS-stimulated cells. In addition, GCH up-regulated the expression of TLR4, p-p38, and p-p65 proteins in the stimulated cells, resulting in the suppression of NF-κB and MAPK signaling pathways. Collectively, GCH was mostly less efficient than CH to exert these assessed anti-inflammatory activities in the cells and more importantly, GCH also showed an ability to cause cell inflammation by promoting IL-6 secretion and up-regulating the expression of TLR4 and p-p65. The casein lactose-glycation of the Maillard-type was thereby concluded to attenuate the anti-inflammatory potential of the resultant casein hydrolysate. It is highlighted that the casein lactose-glycation of the Maillard-type might cause a negative impact on the bioactivity of casein in the intestine, because the glycated casein after digestion could release GCH with reduced anti-inflammatory activity.

EFFECT OF STIMULATING ORGANISM WITH BACTERIAL LIPOPOLYSACCHARIDE ON DEVELOPMENT OF OXIDATIVE STRESS IN BICEPS FEMORIS OF RAT UNDER MODELLED METABOLIC SYNDROME

Metabolic syndrome is one of the most widespread non-infectious pathologies in the world. Metabolic syndrome is quite often accompanied by an increase in body weight and can lead to the development of type 2 diabetes. Between 1990 and 2015, global mortality associated with high body mass index increased by 28.3%.
 The purpose of this work is to determine the activity of antioxidant enzymes, the production of superoxide anion radical, the content of oxidatively modified proteins and the concentration of malondialdehyde in the biceps femoris of rats under experimental metabolic syndrome and stimulation of the organism with bacterial lipopolysaccharide.
 The study was conducted on 24 sexually mature male Wistar rats weighing 200-260 g. The animals were divided into 4 groups of 6 animals each. The first group was the control group. The second group was the test metabolic syndrome group. Metabolic syndrome was modelled by using a 20% fructose solution as the only source of water for 60 days. The third group received S. typhi bacterial lipopolysaccharide in the first week in a dose of 0.4 μg/kg intraperitonealy three times a week, then once a week throughout the experiment. The fourth group underwent the combined exposure to lipopolysaccharide and metabolic syndrome. The development of oxidative stress was observed in the three experimental groups. During modelling of the metabolic syndrome, oxidative stress was characterized by an increase in the production of the superoxide anion radical whilst the activity of the studied antioxidant enzymes decreased. During bacterial lipopolysaccharide stimulation, oxidative stress was characterized by an increase in the production of superoxide anion radical under an increase in superoxide dismutase activity and a decrease in catalase activity. Combined stimulation of the organism by bacterial lipopolysaccharide and modelled metabolic syndrome on the oxidative stress was characterized by an increase in the production of the superoxide anion radical under an increase in the activity of the studied antioxidant enzymes.
 The combined stimulation by bacterial lipopolysaccharide and modelled metabolic syndrome leads to the development of oxidative damage to the lipid and protein components of biceps femoris that is a consequence of an increase in the production of reactive oxygen species under a compensatory increase in the activity of antioxidant enzymes.

Anti-Inflammatory and Anti-oxidative Effects of Puerarin in Postmenopausal Cardioprotection: Roles of Akt and Heme Oxygenase-1.

During menopause, the sharp decline in estrogen levels leads to an increased risk of cardiovascular disease in women. The inflammatory response and oxidative stress are reportedly involved in the development of cardiovascular disorders postmenopause. In this study, we evaluated the cardioprotective effects of puerarin, a phytoestrogen derived from the root of Pueraria lobate, and investigated its underlying molecular mechanisms. Puerarin alleviated cytotoxicity and the production of reactive oxygen species (ROS) in lipopolysaccharide (LPS)- and hydrogen peroxide-stimulated H9c2 cardiomyoblasts. Puerarin scavenges free radicals and reduces apoptosis, thereby suppressing NADPH oxidase-1 and Bax activation to attenuate the production of ROS and restore Bcl-2 expression. Additionally, puerarin inhibited the expression of inducible nitric oxide synthase, cyclooxygenase-2, and nitric oxide production and decreased the hypertrophic phenotype under LPS stimulation. Treatment with puerarin reduced the levels of malondialdehyde and restored glutathione levels when facing oxidative stress. Mechanistically, puerarin inhibited both the LPS-induced Toll-like receptor 4/NF-[Formula: see text]B and mitogen-activated protein kinase signaling pathways. Furthermore, it reversed both the LPS-mediated downregulation of Akt activation and heme oxygenase-1 (HO-1) expression. The cardioprotective effects of puerarin were abolished by inhibitors of Akt and HO-1 and the estrogen receptor antagonist fulvestrant (ICI). This indicated that the estrogen receptor mediated by these two molecules plays important roles in conferring the anti-inflammatory and anti-oxidative functions of puerarin. These results demonstrate the therapeutic potential of puerarin for treating heart disease in postmenopausal women through Akt and HO-1 activation.

Molecular Characterization of a B Cell Adaptor for Phosphoinositide 3-Kinase Homolog in Lamprey (Lampetra japonica) and Its Function in the Immune Response.

Human B cell adaptor for phosphoinositide 3-kinase (BCAP) is identified as an adaptor protein expressed in B cells and plays a critical immunomodulatory role in B cell receptor signaling and humoral immune response. In the current study, a homolog of BCAP (Lja-BCAP) was identified in Lampetra japonica. The open reading frame of Lja-BCAP contains 2181bp nucleotides and encodes a protein of 726 amino acids. After being stimulated by mixed bacteria, the mRNA and protein expression levels of Lja-BCAP and the activation levels of tyrosine kinases increased significantly in peripheral blood lymphocytes, gills and supraneural myeloid bodies, respectively. However, after the knockdown of Lja-BCAP by RNAi in vivo, the activation of tyrosine kinases was inhibited in the above tissues, which indicated that Lja-BCAP participated in the anti-bacterial immune response of lampreys. After lipopolysaccharide (LPS) stimulation, the expression of Lja-BCAP in peripheral blood lymphocytes, gills and supraneural myeloid bodies were significantly up-regulated 2.5, 2.2, and 11.1 times (p < 0.05) compared to the control group, respectively; while after phytohemagglutinin (PHA) stimulation, the up-regulation of Lja-BCAP was only detected in peripheral blood lymphocytes. The above results show that Lja-BCAP mainly participates in the LPS-mediated immune response of lampreys.

Morusin Protected Ruminal Epithelial Cells against Lipopolysaccharide-Induced Inflammation through Inhibiting EGFR-AKT/NF-κB Signaling and Improving Barrier Functions.

Using phytogenic extracts for preventing or treating rumen epithelial inflammatory injury is a potential alternative to antibiotic use due to their residue-free characteristics. In this study, the efficacy of Morus root bark extract Morusin on ruminal epithelial cells (RECs) against pathogenic stimulus was investigated for the first time. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and quantitative real-time polymerase chain reaction (qPCR) results showed that the Morusin did not affect the cell viability of RECs and exerted anti-inflammatory effects in a concentration-dependent manner. Transcriptome analysis further revealed that the Morusin significantly downregulated the inflammatory-response-related cell signaling, while it upregulated the cell-proliferation-inhibition- and barrier-function-related processes in RECs upon lipopolysaccharide (LPS) stimulation. The epidermal growth factor receptor (EGFR) blocking and immunoblotting analysis further confirmed that the Morusin suppressed LPS-induced inflammation in RECs by downregulating the phosphorylation of protein kinase B (AKT) and nuclear factor-kappaB (NF-κB) p65 protein via inhibiting the EGFR signaling. These findings demonstrate the protective roles of Morusin in LPS-induced inflammation in RECs.

Phenolic compounds from Chaenomeles speciosa alleviate inflammation in lipopolysaccharide-treated RAW264.7 macrophages via the NF-κB and MAPK pathways

Chaenomeles speciosa (Sweet) Nakai cultivated widely in temperate regions possesses anti-inflammatory properties, however, the underlying molecular mechanisms remain not fully understood. In this study, a purified phenolic extract of C. speciosa rich in chlorogenic acid, procyanidin B1 and catechin (determined by HPLC-Q-TOF-MS/MS) exhibited dose-dependent anti-inflammatory effects on lipopolysaccharide (LPS)-treated RAW264.7 macrophages. The extract at 30 μg/mL was most potent and enabled most cells in normal morphology under LPS stimulation without causing cytotoxicity. The extract suppressed the levels of nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β), and the mRNA and protein expressions of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). The mechanisms underlying such anti-inflammatory actions included the regulation of phosphorylation of related proteins to monitor the expressions of inflammatory mediators and genes in the nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. Therefore, the phenolic extract from C. speciosa is a desirable anti-inflammatory agent for inflammatory conditions to meet the rising demand for natural and cost-effective therapeutics.

Glucose metabolism is upregulated in the mononuclear cell proteome during sepsis and supports endotoxin-tolerant cell function.

Metabolic adaptations shape immune cell function. In the acute response, a metabolic switch towards glycolysis is necessary for mounting a proinflammatory response. During the clinical course of sepsis, both suppression and activation of immune responses take place simultaneously. Leukocytes from septic patients present inhibition of cytokine production while other functions such as phagocytosis and production of reactive oxygen species (ROS) are preserved, similarly to the in vitro endotoxin tolerance model, where a first stimulation with lipopolysaccharide (LPS) affects the response to a second stimulus. Here, we sought to investigate how cellular metabolism is related to the modulation of immune responses in sepsis and endotoxin tolerance. Proteomic analysis in peripheral blood mononuclear cells (PBMCs) from septic patients obtained at intensive care unit admission showed an upregulation of proteins related to glycolysis, the pentose phosphate pathway (PPP), production of ROS and nitric oxide, and downregulation of proteins in the tricarboxylic acid cycle and oxidative phosphorylation compared to healthy volunteers. Using the endotoxin-tolerance model in PBMCs from healthy subjects, we observed increased lactate production in control cells upon LPS stimulation, while endotoxin-tolerant cells presented inhibited tumor necrosis factor-α and lactate production along with preserved phagocytic capacity. Inhibition of glycolysis and PPP led to impairment of phagocytosis and cytokine production both in control and in endotoxin-tolerant cells. These data indicate that glucose metabolism supports leukocyte functions even in a condition of endotoxin tolerance.

Functional consequences of pathogenic variant c.61G>C in the inflammasome gene NLRP3 underlying keratitis fugax hereditaria

AimsTo elucidate the effect of NLRP3 variant c.61G>C on interleukin-1β (IL-1β) secretion in keratitis fugax hereditaria (KFH), a cryopyrin-associated periodic syndrome limited to the eye, and to probe the potential...

Bacterial induced miR-144-5p modulates intestinal inflammatory response of Japanese flounder by targeting Hsp90α-dependent NLR signaling pathway

Pathogens infection could induce intestinal inflammatory processes, appropriate intestinal inflammation response had beneficial effects in fish, while excessive inflammation alters enteroendocrine signaling, leading to deregulated immune response. Recently, many researches revealed that microRNAs in teleost serve as vital regulators in innate immunity responses against pathogen infection. However, there are few studies on miRNAs mediated regulatory networks of intestinal inflammatory response in teleost. In our study, miR-144-5p was identified as negative regulators in teleost against bacterial infection. First, we found that the expression of miR-144-5p in flounder both in vivo and in vitro was significantly up-regulated, while the expression of Hsp90α, NLRP3, Caspase1 and IL-1β were significantly down-regulated upon the stimulation of Edwardsiella tarda and lipopolysaccharide (LPS). Furthermore, we confirmed that miR-144-5p modulates intestinal inflammatory responses of flounder by targeting Hsp90α-dependent NLR signaling pathway, in which the miR-144-5p-Hsp90α-NLRP3 signaling pathway was constructed. Besides, the miR-144-5p-Hsp90α-NLRP3 signaling pathway could inhibit antibacterial immune response of flounder and promote the survival of E. tarda. Moreover, the study demonstrated that the miR-144-5p-Hsp90α regulatory network was widely existed in other teleost and human. In conclusion, the expression of miR-144-5p was significantly up-regulated after challenged by E. tarda and LPS, which then down-regulated the expression of inflammation-related cytokines of NLRP3, Caspase1 and IL-1β by targeting Hsp90α, and ultimately promoted bacterial infection. Our findings constructed a novel miR-144-5p-Hsp90α-NLRP3 signaling pathway, which enriched the regulatory network of miRNA on fish intestinal mucosal immunity and would benefit the development of fish disease therapeutic strategies to prevent potentially lethal bacterial infection.

Role of Heparan Sulfate 6-O-Endosulfatases in Hematopoiesis

Heparan sulfate proteoglycans (HSPGs) are expressed at the cell surface and in the extracellular matrix, including within the bone marrow microenvironment. Heparan sulfate (HS) is the glycosaminoglycan side chain of HSPGs, and these side chains bind to a myriad of growth factors, cytokines, chemokines, and extracellular matrix molecules, mediating a diverse range of biological functions. It is well established that HS-protein interactions critically depend on the amount and position of 6-O-sulfate groups, which form binding sites for proteins. The HS 6-O-endosulfatases, the Sulfs (Sulf1 and Sulf2), remove 6-O-sulfates from specific HS intra-chain sites and are the only known enzymes that can modify HS sulfation status extracellularly. A gain or a loss of HS 6-O-sulfation can alter the outcome of HS-ligand interactions, such as with Wnt ligands, fibroblast growth factors (FGF), and transforming growth factor (TGF)-β1. Monocytes and macrophages express the Sulfs, predominantly Sulf2, and their secretion of the Sulfs can influence HS sulfation status in their surrounding microenvironments. The objective of this study was to examine the impact of myeloid lineage Sulf deletion on hematopoiesis. For this investigation, we generated a murine model of Sulf deletion in Lysozyme 2 (Lyz2) positive myeloid lineage cells by crossing Sulf1 and Sulf2 double floxed mice with the LysMcre mouse line (hereinafter LysM-Sulf-/-). At 8-12 weeks of age, the bone marrow of LysM-Sulf-/- male mice displayed myelodysplastic changes with reduced number of megakaryocytes (MK) compared to the control mice (Lyz2-cre+/-). Bone marrow-derived (BMD) macrophages from LysM-Sulf-/- mice exhibited enhanced proliferation but reduced phagocytosis of Escherichia Coli (E. Coli)in vitro. In addition, LysM-Sulf-/- BMD macrophages expressed reduced levels of key inflammatory mediators including inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α and interferon gamma-induced protein 10 (IP-10) in response to lipopolysaccharide stimulation compared to control BMD macrophages. Complete blood cell counts revealed decreases in total white blood cells (4.6 ± 0.6 10E9 Cells/L vs. 8.1 ± 1.010E9Cells/L in control mice, P = 0.0193), red blood cells (RBC, 10.9 ± 0.1 10E12 Cells/L vs. 11.4 ± 0.2 10E12 Cells/L in control mice, P = 0.0385), and hematocrit (43.5 ± 0.3% vs. 46.3 ± 0.7% in control mice, P = 0.0054) in LysM-Sulf-/- males. In addition, LysM-Sulf-/- males (8-12 weeks of age) exhibit extramedullary hematopoiesis with splenomegaly (spleen size increased by 2.6-fold, 162.1 ± 11.5 mg vs. 79.1 ± 1.2 mg in control mice, P < 0.0001). Bone and spleen morphologies are shown in the accompanied Figure. In older LysM-Sulf-/- males (8-12 months of age), the reductions in RBC and hematocrits were further exacerbated with increased reticulocyte counts in the peripheral blood. In addition, thrombocytopenia was observed in 8-12 months old LysM-Sulf-/- males (platelet counts 125.8 ± 41.4 vs. 483.3 ± 7.1 in control mice, P = 0.0007). The above changes were accompanied by amplified splenomegaly (spleen size increased by 9.2-fold, 860.4 ± 157.6 mg vs. 93.2 ± 8.2 mg in control mice, P = 0.0152) and erythropoiesis in the liver. In terms of signaling, enhanced Smad2/3 phosphorylation (primary transducers of TGF-β1 signaling) was observed in both the spleens and the lungs of LysM-Sulf-/- male mice. Finally, in contrast to the male mice, complete blood cell counts were normal in both young and old LysM-Sulf-/- females without obvious extramedullary hematopoiesis. Our investigation revealed the importance of HS 6-O-sulfation in supporting normal hematopoiesis in the bone marrow, and alterations in HS 6-O-sulfation status in the bone marrow microenvironment due to LysM-Sulfdeletion leads to not only impairment in monocyte/macrophage development and function, but also age- and sex-dependent deficiencies in erythropoiesis and thrombopoiesis. Our study identifies TGF-β1 as one of the signaling pathways regulated by the Sulfs. The regulatory roles of the Sulfs in TGF-β1 as well as other signaling pathways in hematopoiesis warrant further investigation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genetic deletion of Cyp4f18 disrupts the omega-3 epoxidation pathway and results in psoriasis-like dermatitis.

Cyp4f18 catalyzes the conversion of n-3 polyunsaturated fatty acids (PUFAs) into omega-3 epoxides, such as 17,18-epoxyeicosatetraenoic acid (17,18-EpETE) and 19,20-epoxydocosapentaenoic acid (19,20-EpDPE) from eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), respectively. Cyp4f18-deficient mice spontaneously develop psoriasis-like dermatitis. A significant increase in the number of IL-17A-positive gamma delta (γδ) T cells in the skin and enlargement of draining lymph nodes was observed. These symptoms were drastically suppressed by antibiotic treatment. Cyp4f18 is highly expressed in dendritic cells (DCs), and Cyp4f18-deficient bone marrow-derived dendritic cells (BMDCs) show markedly increased expression levels of cytokines such as IL-23 and IL-1β in response to lipopolysaccharide (LPS) stimulation. Lipidomic analysis of lymph nodes and BMDCs revealed a significant decrease in a series of omega-3 epoxidized metabolites. Among them, 17,18-dihydroxyeicosatetraenoic acid (17,18-diHETE), a vicinal diol derived from EPA omega-3 epoxidation suppressed IL-23 production in LPS-stimulated BMDCs in Cyp4f18-deficient mice. These results demonstrate that Cyp4f18 endogenously produces omega-3-epoxidized metabolites in the draining lymph nodes, and these metabolites contribute to skin homeostasis by suppressing the excessive activation of the IL-23/IL-17 axis initiated by DCs.

Olfactory mucosa tissue-derived mesenchymal stem cells lysate ameliorates LPS-induced acute liver injury in mice

BackgroundAcute liver injury (ALI) induced by sepsis seriously endangers the health of human beings every year. Mesenchymal stem cells (MSCs) lysate containing various regulators had a positive effect on anti-inflammation, hoping to provide a promising strategy in ALI.MethodsOlfactory mucosa-derived mesenchymal stem cells (OM-MSCs) were extracted and identified. The collected OM-MSCs were prepared after repeated freeze–thaw in phosphate buffer solution (PBS). Then, OM-MSCs lysate was filtered for future experiments. To understand the composes of OM-MSCs clearly, we detected the components of OM-MSCs lysate by western blotting. In vitro, OM-MSCs lysate was applied to evaluate the effects on normal human liver cells (LO-2) under stimulation of LPS. Lipopolysaccharide (LPS) was also injected intraperitoneally to build ALI model in mice. We further assessed the anti-inflammatory capacity of OM-MSCs lysate on ALI in vivo by aminotransferase determination, pathology observation, and immunohistochemical staining. Moreover, the immunoblot technique was performed to recognize the changes in inflammatory factors and related proteins.ResultsIn this study, we found that OM-MSCs lysate could protect structure effectively, improve the plasma aminotransferases, diminish inflammation by releasing interleukin-10 (IL-10) and transforming growth factor-beta (TGF-β). A significant decrease in tumor necrosis factor-α (TNF-α) also occurred under the treatment of OM-MSCs lysate. In addition, trophic factors originating from OM-MSCs lysate provided a supportive micro-environment for liver recovery. Especially, up-expression of vascular endothelial growth factor (VEGF) in vivo revealed that OM-MSCs might have a great potential for healing.ConclusionsOur results demonstrated that OM-MSCs lysate could alleviate LPS-induced ALI via decreasing inflammatory cytokines and promoting recovery.