IL-12 P40 Research Articles

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

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

Higher expression of Th1/Th2‐related cytokines in the intestine of Wistar rats with ligature‐induced periodontitis

This study aimed to investigate the impact of ligature-induced periodontitis (LIP) on histopathological and immunological outcomes in the colon of Wistar rats. It has been repeatedly shown that inflammatory bowel disease (IBD) patients are at higher risk of developing periodontitis and presenting worse oral health than non-IBD patients. However, whether the chronic inflammatory process around teeth contributes to the pathophysiology of IBD needs to be further explored. Thirteen Wistar rats were allocated into LIP (n = 7) and controls (n = 6). Half of the colon was processed for histopathological analyses and immunohistochemical (CD45); the other half was homogenized for immunological analyses. Periodontal destruction was confirmed by measuring the distance from the cementum-enamel junction to the mandible's apical position of the mesial interproximal bone. The immunological analyses were performed with the Bio-Plex Th1/Th2 assay. There was a significantly higher interproximal bone loss in LIP compared to controls. The LIP group showed a moderate infiltrate of inflammatory cells, predominantly mononucleated cells in the intestinal tissues. There was significantly higher expression of GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-10, IL-12 (p70), IL-13, and TNF-α in the intestinal tissues of LIP group compared to controls. Ligature-induced periodontitis was associated with an overexpression of Th1/Th2-related cytokines in the colon of Wistar rats.

Ketoprofen-Based Polymer-Drug Nanoparticles Provide Anti-Inflammatory Properties to HA/Collagen Hydrogels.

Current limitations of wound dressings for treating chronic wounds require the development of novel approaches. One of these is the immune-centered approach, which aims to restore the pro-regenerative and anti-inflammatory properties of macrophages. Under inflammatory conditions, ketoprofen nanoparticles (KT NPs) can reduce pro-inflammatory markers of macrophages and increase anti-inflammatory cytokines. To assess their suitability as part of wound dressings, these NPs were combined with hyaluronan (HA)/collagen-based hydro- (HGs) and cryogels (CGs). Different HA and NP concentrations and loading techniques for NP incorporation were used. The NP release, gel morphology, and mechanical properties were studied. Generally, colonialization of the gels with macrophages resulted in high cell viability and proliferation. Furthermore, direct contact of the NPs to the cells reduced the level of nitric oxide (NO). The formation of multinucleated cells on the gels was low and further decreased by the NPs. For the HGs that produced the highest reduction in NO, extended ELISA studies showed reduced levels of the pro-inflammatory markers PGE2, IL-12 p40, TNF-α, and IL-6. Thus, HA/collagen-based gels containing KT NPs may represent a novel therapeutic approach for treating chronic wounds. Whether effects observed in vitro translate into a favorable profile on skin regeneration in vivo will require rigorous testing.

The Interaction between Circulating Cell-Free Mitochondrial DNA and Inflammatory Cytokines in Predicting Human Mental Health Issue Risk in Adolescents: An Explorative Study.

Adolescence is often a challenging time in which psychiatric issues have a strong connection to mental health disorders later in life. The early identification of the problems can reduce the burden of disease. To date, the effective identification of adolescents at risk of developing mental health problems remains understudied. Altogether, the interaction between circulating cell-free mtDNA (ccf-mtDNA) and inflammatory cytokines in adolescents is insufficiently understood regarding experienced mental health difficulties. Our study selected the participants based on the Strength and Difficulty Questionnaire (SDQ) score using the cut-off points of 3 and 18 for the low and the high score groups, respectively. The answers of the SDQ at the age of 12.2-15.7 years contributed to the investigation of (i) whether ccf-mtDNA units are associated with cytokines, and (ii) if an interaction model for predicting risk of mental health issues is observed. We discovered a sex-specific correlation between the screened markers associated with mental health problems in the low and high SDQ score groups among the male participants and in the low SDQ score group among the female participants. The mitochondrial MT-ND4 and MT-CO1 genes correlated significantly with interleukin-12p70 (IL-12p70) in males and with monocyte chemoattractant protein-1 (MCP-1) in females. Due to the nature of the explorative study, the studied markers alone did not indicate statistical significance for the prediction of mental health problems. Our analysis provided new insight into potential plasma-based biomarkers to predict mental health issues.

Serum cytokine profiles predict outcomes of chronic hepatitis B patients discontinuing entecavir or tenofovir therapy

Distinct hepatitis relapse has been observed after discontinuing entecavir (ETV) or tenofovir disoproxil fumarate (TDF) therapy in chronic hepatitis B (CHB) patients. End-of-therapy (EOT) serum cytokines were compared and used for outcome prediction. A total of 80 non-cirrhotic CHB patients in a tertiary medical center in Taiwan who discontinued ETV (n=51) or TDF (n=29) therapy after fulfilling the APASL guidelines were prospectively enrolled. Serum cytokines were measured at EOT and 3rd month afterwards. Multivariable analysis was performed to predict virological relapse (VR, HBV DNA >2000 IU/mL), clinical relapse (CR, VR and alanine aminotransferase > 2-fold upper limit of normal) and hepatitis B surface antigen (HBsAg) seroclearance. Compared with TDF group, ETV stoppers had greater interleukin 5 (IL-5), IL-12 p70, IL-13, IL-17A and tumor necrosis factor alpha (TNF-alpha) (all P<0.05) at EOT. Older age, TDF use, higher EOT HBsAg and IL-18 (Hazard ratio [HR], 1.01; 95% CI, 1.00-1.02) levels at EOT predicted VR, while older age, higher EOT HBsAg and IL-7 (HR, 1.25; 95% CI, 1.00-1.56) levels predicted CR. In TDF stoppers, higher IL-7 (HR, 1.29; 95% CI, 1.05-1.60) and IL-18 (HR, 1.02; 95% CI, 1.00-1.04) levels predicted VR, while IL-7 (HR, 1.34; 95% CI, 1.08-1.65) and interferon-gamma (IFN-gamma) (HR, 1.08; 95% CI, 1.02-1.14) levels predicted CR. A lower EOT HBsAg level was associated with HBsAg seroclearance. Distinct cytokine profiles were observed after stopping ETV or TDF. Higher EOT IL-7, IL-18, and IFN-gamma could be probable predictors for VR and CR in patients discontinuing NA therapies.

Over supplementation with vitamin B12 alters microbe-host interactions in the gut leading to accelerated Citrobacter rodentium colonization and pathogenesis in mice

BackgroundVitamin B12 supplements typically contain doses that far exceed the recommended daily amount, and high exposures are generally considered safe. Competitive and syntrophic interactions for B12 exist between microbes in the gut. Yet, to what extent excessive levels contribute to the activities of the gut microbiota remains unclear. The objective of this study was to evaluate the effect of B12 on microbial ecology using a B12 supplemented mouse model with Citrobacter rodentium, a mouse-specific pathogen. Mice were fed a standard chow diet and received either water or water supplemented with B12 (cyanocobalamin: ~120 μg/day), which equates to approximately 25 mg in humans. Infection severity was determined by body weight, pathogen load, and histopathologic scoring. Host biomarkers of inflammation were assessed in the colon before and after the pathogen challenge.ResultsCyanocobalamin supplementation enhanced pathogen colonization at day 1 (P < 0.05) and day 3 (P < 0.01) postinfection. The impact of B12 on gut microbial communities, although minor, was distinct and attributed to the changes in the Lachnospiraceae populations and reduced alpha diversity. Cyanocobalamin treatment disrupted the activity of the low-abundance community members of the gut microbiota. It enhanced the amount of interleukin-12 p40 subunit protein (IL12/23p40; P < 0.001) and interleukin-17a (IL-17A; P < 0.05) in the colon of naïve mice. This immune phenotype was microbe dependent, and the response varied based on the baseline microbiota. The cecal metatranscriptome revealed that excessive cyanocobalamin decreased the expression of glucose utilizing genes by C. rodentium, a metabolic attribute previously associated with pathogen virulence.ConclusionsOral vitamin B12 supplementation promoted C. rodentium colonization in mice by altering the activities of the Lachnospiraceae populations in the gut. A lower abundance of select Lachnospiraceae species correlated to higher p40 subunit levels, while the detection of Parasutterella exacerbated inflammatory markers in the colon of naïve mice. The B12-induced change in gut ecology enhanced the ability of C. rodentium colonization by impacting key microbe-host interactions that help with pathogen exclusion. This research provides insight into how B12 impacts the gut microbiota and highlights potential consequences of disrupting microbial B12 competition/sharing through over-supplementation.Graphical 5vuKG727Y2QSKPsq8nr33cVideo

Roles and correlations of TIM-3 and LAG-3 with cytokines and chemokines in alcoholic liver disease

BackgroundDysregulation of immune checkpoint regulators has been reported in alcoholic liver disease (ALD). This study was designed to assess the serum levels of cytokines and chemokines associated with ALD and uncover the possible disease correlations with the soluble TIM-3 and LAG-3. MethodsThe soluble TIM-3 and LAG-3 levels were measured by enzyme-linked immunoassay, and 14 cytokines and chemokines were measured using Luminex-based multiplex assay in 111 male ALD patients and 45 healthy controls (HCs). ResultsOur results showed that soluble TIM-3 was significantly increased (p < 0.001) while soluble LAG-3 was significantly decreased (p < 0.001) in ALD group compared to HCs. Among the 14 cytokines and chemokines assessed, granulocyte-colony stimulating factor (G-CSF) (p = 0.003) and interferon γ-induced protein (IP)-10 (p < 0.001) were significantly increased, while interleukin (IL)-4 (p = 0.005) and IL-12 (p40) (p = 0.001) were significantly decreased in the ALD group. Kaplan-Meier analysis showed that overall survival decreased in higher TIM-3 level individuals. ConclusionsOur results showed that TIM-3, LAG-3, and IP-10 appear to be important for clinical diagnosis of ALD and ALD severity and may represent potential therapeutic targets in ALD.

Blocking interleukin-23 ameliorates neuromuscular and thymic defects in myasthenia gravis

Acetylcholine receptor (AChR) myasthenia gravis (MG) is a chronic autoimmune disease characterized by muscle weakness. The AChR+ autoantibodies are produced by B-cells located in thymic ectopic germinal centers (eGC). No therapeutic approach is curative. The inflammatory IL-23/Th17 pathway is activated in the thymus as well as in the blood and the muscle, contributing to the MG pathogenic events. We aimed to study a potential new therapeutic approach that targets IL-23p19 (IL-23) in the two complementary preclinical MG models: the classical experimental MG mouse model (EAMG) based on active immunization and the humanized mouse model featuring human MG thymuses engrafted in NSG mice (NSG-MG). In both preclinical models, the anti-IL-23 treatment ameliorated MG clinical symptoms. In the EAMG, the treatment reduced IL-17 related inflammation, anti-AChR IgG2b antibody production, activated transduction pathway involved in muscle regeneration and ameliorated the signal transduction at the neuromuscular junction. In the NSG-MG model, the treatment reduced pathogenic Th17 cell population and expression of genes involved in eGC stabilization and B-cell development in human MG thymus biopsies. Altogether, these data suggest that a therapy targeting IL-23p19 may promote significant clinical ameliorations in AChR+ MG disease due to concomitant beneficial effects on the thymus and skeletal muscle defects.

Low circulatory Fe and Se levels with a higher IL-6/IL-10 ratio provide nutritional immunity in tuberculosis.

Tuberculosis (TB) patients show dysregulated immunity, iron metabolism, and anemia. In this study, circulatory cytokines, trace metals, and iron-related proteins (hepcidin, ferroportin, transferrin, Dmt1, Nramp1, ferritin, ceruloplasmin, hemojuvelin, aconitase, and transferrin receptor) were monitored in case (active tuberculosis patients: ATB) and control (non-tuberculosis: NTB and healthy) study populations (n = 72, male: 100%, mean age, 42.94 years; range, 17-83 years). Using serum elemental and cytokine levels, a partial least square discriminate analysis model (PLS-DA) was built, which clustered ATB patients away from NTB and healthy controls. Based on the PLS-DA variable importance in projection (VIP) score and analysis of variance (ANOVA), 13 variables were selected as important biosignatures [IL-18, IL-10, IL-13, IFN-γ, TNF-α, IL-5, IL-12 (p70), IL-1β, copper, zinc, selenium, iron, and aluminum]. Interestingly, low iron and selenium levels and high copper and aluminum levels were observed in ATB subjects. Low circulatory levels of transferrin, ferroportin, and hemojuvelin with higher ferritin and ceruloplasmin levels observed in ATB subjects demonstrate an altered iron metabolism, which partially resolved upon 6 months of anti-TB therapy. The identified biosignature in TB patients demonstrated perturbed iron homeostasis with anemia of inflammation, which could be useful targets for the development of host-directed adjunct therapeutics.

Increased expression of ELOVL7 contributes to production of inflammatory cytokines in THP-1 cell-derived M1-like macrophages.

The elevation of intracellular very long-chain fatty acids (VLCFAs) augments pro-inflammatory activity of macrophages. VLCFAs are considered to function as regulators in macrophage inflammatory responses; however, the precise mechanism of regulating the production of VLCFAs is unclear. In this study, we focused on elongation of the very‑long‑chain fatty acid protein (ELOVL) family, rate-determining enzymes for VLCFA synthesis, in macrophages. ELOVL7 mRNA was upregulated in human monocytic THP-1 cell-derived M1-like macrophages. Metascape analysis using the RNA-seq data set showed the involvement of NF-κB and STAT1 in transcriptional regulation of ELOVL7 highly correlated genes. Gene ontology (GO) enrichment analysis suggested that ELOVL7 highly correlated genes were closely associated with multiple pro-inflammatory responses, including response to virus and positive regulation of NF-κB signaling. Consistent with RNA-seq analysis, the NF-κB inhibitor BAY11-7082, but not the STAT1 inhibitor fludarabine, canceled ELOVL7 upregulation in M1-like macrophages. ELOVL7 knockdown decreased interleukin (IL)-6 and IL-12/IL-23 p40 production. Moreover, RNA-seq analysis of plasmacytoid dendritic cells (pDCs) revealed that ELOVL7 was upregulated in pDCs treated with TLR7 and TLR9 agonists. In conclusion, we propose that ELOVL7 is a novel pro-inflammatory gene that is upregulated by inflammatory stimuli, and regulates M1-like macrophage and pDC functions.

Epstein-Barr virus-induced gene 3 commits human mesenchymal stem cells to differentiate into chondrocytes via endoplasmic reticulum stress sensor.

Mesenchymal stem cells (MSC) can differentiate into chondrocytes. Epstein-Barr virus-induced gene 3 (EBI3) is differentially expressed during chondrogenic differentiation and can be produced by MSC. EBI3 is also a subunit of interleukin (IL)-27 and IL-35, and it accumulates in the endoplasmic reticulum (ER) when its partners, such as IL-27 p28 and IL-35 p35, are insufficient. ER stress induced by protein accumulation is responsible for chondrogenic differentiation. However, the role of EBI3 and its relevance to the ER stress in chondrogenic differentiation of MSC have never been addressed. Here, we demonstrate that EBI3 protein is expressed in the early stage of chondrogenic differentiation of MSC. Additionally, knockdown, overexpression, or induction of EBI3 through IL-1β inhibits chondrogenesis. We show that EBI3 localizes and accumulates in the ER of MSC after overexpression or induction by IL-1β and TNF-α, whereas ER stress inhibitor 4-phenylbutyric acid decreases its accumulation in MSC. Moreover, EBI3 modulates ER stress sensor inositol-requiring enzyme 1 α (IRE1α) after induced by IL-1β, and MSC-like cells coexpress EBI3 and IRE1α in rheumatoid arthritis (RA) synovial tissue. Altogether, these data demonstrate that intracellular EBI3 commits to chondrogenic differentiation by regulating ER stress sensor IRE1α.

Features of mucosal immunity in the development of periodontal diseases in patients with type II diabetes mellitus

Chronic generalized periodontitis takes one of the leading places in the structure of dental diseases. Inflammatory periodontal disease is one of the most common complications of diabetes. An important role in the progression of periodontitis in type II diabetes mellitus (T2DM) belongs to disorders of the immune system that affect the supportive/retaining complex of the tooth. Inflammation of periodontal tissues which occurs in the patients with carbohydrate metabolism disorders is characterized by a more severe course, which worsens the condition and quality of life of patients with T2DM thus leading to tooth loss. In this regard, the immunological aspects of developing periodontal pathology under conditions of carbohydrate metabolism disorders are of great interest. Purpose of our study included comparative assessment of local TNF, TNF, IFN, IL-1, IL- 12, IL-17A levels in patients with periodontitis with and without carbohydrate metabolism disorders (type II diabetes mellitus), as well as in patients with type II diabetes mellitus without signs of periodontitis.&#x0D; 127 patients were examined, aged 30 to 59 years. The patients were divided into 3 groups, i.e., group I included patients suffering from periodontitis of varying severity without known comorbidities (47 persons); Group II consisted of patients with T2DM and periodontitis of varying severity (49 persons); Group III included patients with T2DM without symptoms of periodontitis (30 persons). The control group consisted of 30 practically healthy volunteers, matched to the patients for age and sex. Saliva specimens were used for laboratory studies. The levels of TNF, TNF, IFN, IL-1, IL-12, IL-17А were determined by ELISA sandwich technique, with specific reagents purchased from RD Diagnostics Inc (USA).&#x0D; A significant increase in the IL-1, TNF, IFN and IL-17A levels was found in patients of all groups compared with controls. At the same time, the increased concentrations of IFN, IL-1, IL-17A correlated with increase in periodontitis severity in the patients of both groups. Decreased TNF levels in saliva samples were revealed in the patients from almost all groups, regardless of the periodontitis severity. Significantly increased levels of IL-12 (p40 subunit) were recorded in saliva of the persons from group II and III when compared with controls and the group without diabetes mellitus.

Mahuang Xixin Fuzi decoction protects the BALB/c-nude mice infected with influenza A virus by reducing inflammatory cytokines storm and weakly regulating SIgA immune response

Mahuang Xixin Fuzi Decoction (MXF), as a classical prescription of traditional Chinese medicine (TCM), has been used to treat the immunocompromised individuals infected with influenza A virus (IAV). The study aims to explore the regulatory of MXF on inflammation and secretory immunoglobulin A (SIgA) antibodies immune response in BALB/c-nude mice infected with IAV. The BALB/c-nude mice were infected with IAV, then different dosages of MXF were orally administrated to the mice. The weight, rectal temperature, spontaneous activity, spleen index, lung index, pathological changes of lung tissues, and the relative mRNA expression level of H1N1 M gene were measured for the purpose of valuing the antiviral effect of MXF. The expression levels of cytokines in lungs and immunoglobulin A (IgA) in serum of BALB/c-nude mice were determined with Cytometric Bead Array System (CBA). SIgA in bronchoalveolar lavage fluids (BALF) was detected with Enzyme-linked Immunosorbent Assay (ELISA). The mRNA and protein expression levels of B cell activating factor (BAFF), chemokine receptors 10 (CCR10), and polymeric immunoglobulin receptor (pIgR) in the lung tissues, which are related to the secretion of SIgA, were determined by using RT-PCR and Western blot. MXF could alleviate the clinical features and reduce the severity of viral lung lesions, including improving the body weight, rectal temperature and spontaneous activity of nude mice infected with IAV, increasing spleen index, decreasing lung index, alleviating pathological damage, and decreasing the relative expression level of H1N1 M gene. Levels of pro-inflammatory cytokines, including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2), interleukin-12p70 (IL-12p70), and interleukin-17A (IL-17A) were also significantly decreased after treatment with MXF. Interferon-γ (IFN-γ), an antiviral cytokine, was significantly up-regulated in high dose MXF (3.12g/kg) group. Moreover, after MXF treatment, the expressions of SIgA in BALF and IgA in serum were both at relatively low levels. And the mRNA and protein expressions of BAFF, CCR10, and pIgR were significantly decreased after treatment with MXF. MXF has obviously protective effects on BALB/c-nude mice infected with IAV by inhibiting virus replication, calming inflammatory cytokine storm, and regulating SIgA immune response weakly.

444 Lactate and its induced EGR1 are novel key factors that turn hot tumors into cold tumors

Tumors with an inflamed tumor microenvironment are considered “hot tumors” and show good responses to immune checkpoint inhibitors (ICIs). Conversely, non-inflamed tumors, so-called “cold tumors,” have no tumor-suppressive cytokines and rarely respond to ICIs. Therefore, turning non-inflamed (cold) tumors into inflamed (hot) tumors is important for maximizing the effect of ICIs. We showed that lactate, a product of the Warburg effect, inhibited the efficacy of ICIs and suppressed IL-12 p40 expression in dendritic cells (DCs) through reducing NF-κB p65, p50 and c-Rel DNA-binding activity to the IL-12 p40 promoter.

Dietary squalene supplementation decreases triglyceride species and modifies phospholipid lipidomic profile in the liver of a porcine model of non-alcoholic steatohepatitis

Squalene is a key minor component of virgin olive oil, the main source of fat in the Mediterranean diet, and had shown to improve the liver metabolism in rabbits and mice. The present research was carried out to find out whether this effect was conserved in a porcine model of hepatic steatohepatitis and to search for the lipidomic changes involved. The current study revealed that a 0.5% squalene supplementation to a steatotic diet for a month led to hepatic accumulation of squalene and decreased triglyceride content as well as area of hepatic lipid droplets without influencing cholesterol content or fiber areas. However, ballooning score was increased and associated with the hepatic squalene content. Of forty hepatic transcripts related to lipid metabolism and hepatic steatosis, only citrate synthase and a non-coding RNA showed decreased expressions. The hepatic lipidome, assessed by liquid chromatography-mass spectrometry in a platform able to analyze 467 lipids, revealed that squalene supplementation increased ceramide, Cer(36:2), and phosphatidylcholine (PC[32:0], PC[33:0] and PC[34:0]) species and decreased cardiolipin, CL(69:5), and triglyceride (TG[54:2], TG[55:0] and TG[55:2]) species. Plasma levels of interleukin 12p40 increased in pigs receiving the squalene diet. The latter also modified plasma lipidome by increasing TG(58:12) and decreasing non-esterified fatty acid (FA 14:0, FA 16:1 and FA 18:0) species without changes in total NEFA levels. Together this shows that squalene-induced changes in hepatic and plasma lipidomic profiles, non-coding RNA and anti-inflammatory interleukin are suggestive of an alleviation of the disease despite the increase in the ballooning score.

Cytokine profile in &lt;i&gt;in vitro&lt;/i&gt; mouse macrophage culture infected with &lt;i&gt;Bacillus anthracis&lt;/i&gt; spores with varying plasmid composition

Bacillus anthracis, the causative agent of anthrax, is able to exist both in environmental conditions (soil) and in the macroorganism. The manifestation of pathogenic properties of B. anthracis strains is determined by relevant plasmid composition, because the main toxin and the capsule-related virulence factors are located in bacterial plasmid. Modeling anthrax infection in vitro in macrophage culture might reveal an influence of individual B. anthracis strain characteristics on infection and development of infectious process. The aim of this study was to analyze cytokine level during infection of in vitro macrophage cell cultures with spores of anthrax microbe strains bearing varying plasmid composition. The dependence of the macrophage cell cytokine profile on the plasmid composition of B. anthracis strains was revealed while modeling anthrax infection in vitro. The presence of the toxin-producing plasmid pXO1 in anthrax microbe strains has a powerful stimulating effect on the production of macrophages J774A cell line cytokines. B. anthracis strains lacking the pXO1 plasmid virtually stimulated no production of IL-1, caused very low secretion of IL-1, IL-6, MCP-1, MIP-1, MIP-1, IL-12 (p70) and active G-CSF products. The low cytokine response of macrophage cells infected with monoplasmid strains bearing only the capsule-forming plasmid was due not only to the absence of a binary toxin, but also to disturbed regulation of capsule production associated with the absence of the atxA gene. The capsule, along with lethal and edematous toxins, belongs to the main virulence factors of B. anthracis, but strains lacking the pXO1 virulence plasmid, had its production impaired, because the main regulator of capsule synthesis is the atxA gene localized on the pXO1 plasmid being positively regulated by the acpA and acpB genes, so that strains lacking the toxin-forming plasmid, even in the presence of the encapsulation plasmid, elicit a weak cytokine response in infected cells. Diplasmid strains of B. anthracis, due to produced main virulence factors a two-component toxin and a capsule, enforce macrophages (in the experiment) to actively produce IL-1, IL-6, MCP-1, G-CSF, MIP-1; MIP-1, IL-12 (p70). Strains with moderate virulence and capable of capsulation in air virtually did not differ from highly virulent strains in terms of their effect on in vitro macrophage culture.

Chronic Inflammation Drives p53 Mutant Clonal Hematopoiesis Via Activating the NLRP1 Inflammasome

Individuals with somatic TP53 mutations manifest clonal hematopoiesis and are at high risk of developing hematological malignancies. However, how environmental factors influence the expansion of p53 mutant hematopoietic stem and progenitor cells (HSPCs) is unclear. We found that pI:pC or LPS treatment promotes the clonal expansion of p53 mutant HSPCs in transplantation assays. To determine the impact of LPS on HSPC function in vivo, we treated p53+/+ and p53R248W/+ mice with LPS or PBS and then performed competitive transplantation assays. LPS-treated WT BM cells show decreased engraftment in both primary and secondary transplantation assays, whereas LPS-treated p53 mutant BM cells continued to show increased engraftment. To understand how LPS-induced chronic inflammation promotes p53 mutant HSPC expansion, we examined the impact of mutant p53 on cytokine secretion from BM derived macrophages. We found that p53 mutant macrophages show increased secretion of multiple pro-inflammatory cytokines, including IL-1β, IL-1α, IL-12 (p70), KC, VEGF, IP-10, CXCL-9, LIX, and MCP-1. To further investigate how chronic inflammation promotes p53 mutant HSPC expansion, we performed ATAC-seq assays in LSKs to identify differential regions of chromatin accessibility. Notably, we found that mutant p53 increases the chromatin accessibility to inflammasome genes, including Nlrp1a and Nlrp1b. Inflammasomes are multiprotein complexes that activate Caspase-1 and increase the release of pro-inflammatory cytokines such as IL-1β, leading to caspase-1-dependent death, known as pyroptosis. We found that loss of NLRP1 significantly decreased the secretion of multiple pro-inflammatory cytokines, including IL-1β, IL-1α, IL-6, MCP-1, KC, M-CSF, RANTES, LIX, GM-CSF G-CSF, TNF-α and IL-12 (p40), from macrophages expressing mutant p53. Loss of NLRP1 significantly decreases the repopulating potential of HSPCs expressing mutant p53 in vivo. To further understand how chronic inflammation may confer a competitive advantage to p53 mutant HSPCs, we performed in vitro cell competition assays. We found that WT CD45.1+ cells co-cultured with p53 mutant cells show decreased replating potential compared to WT CD45.1+ cells co-cultured with WT CD45.2+ cells. We measured the levels of cytokines in the condition media utilizing cytokine arrays and found increased levels of IL-1β, IL-6, CXCL-9, and IL-4 in mutant cell cultures compared to that of the WT cells. We found that WT CD45.1+ cells co-cultured with mutant cells show increased pyroptosis. Importantly, we found that the enhanced pyroptosis of WT CD45.1+ cells seen in the mutant group is IL-1β dependent. We found that IL-1β treatment decreases the replating potential of WT BM cells, but not that of p53 mutant cells. To determine the mechanism by which p53 mutant HSPCs are resistant to IL-1β treatment, we performed RNA sequencing in p53+/+ and p53R248W/+ LSKs treated with IL-1β (10 ng/ml) or PBS in vitro for 90 minutes. GSEA analysis revealed that hematopoietic stem and progenitor cell differentiation genes are significantly upregulated in p53 mutant LSKs compared to WT LSKs following IL-1β stimulation. Notably, we found that p53 mutant LSKs treated with IL-1β are enriched with MYC target genes. Furthermore, blocking of IL-1β using IL-1β neutralizing antibody decreases the fitness of p53 mutant HSPCs. Our studies will likely establish both the NLRP1 inflammasome and the pro-inflammatory cytokine IL-1β as novel potential therapeutic targets for preventing clonal hematopoiesis development and treating hematological malignancies with TP53 mutations.

BIOM-40. COMPARATIVE STUDY OF INFLAMMATORY MARKERS IN THE CEREBROSPINAL FLUID OF PATIENTS WITH GLIOMA AND CENTRAL NERVOUS SYSTEM MALIGNANT LYMPHOMA

Abstract INTRODUCTION Cytokines in the cerebrospinal fluid (CSF) are useful as markers for primary central nervous system malignant lymphoma (PCBSL). However, there are few reports of CSF markers in glioma. We analyzed and examined glioma and PCNSL CSF inflammation-related mediators. MATERIALS AND METHODS The patients with glioma and PCNSL who were operated on at Kobe University between 2006 and 2017 and whose histology was diagnosed, and whose CSF was stored at -80 °C before treatment. Using Bio-Plex Pro Human Inflammation Assays (Bio-Rad Laboratories, Inc.), 37 types of inflammation-related molecules such as TNF superfamilies, interferonss, and Interleukines were measured and compared with glioma and PCNSL. RESULTS The subjects were 53 glioma patients and 24 PCNSL patients. There were 22 types of significant differences between glioma and PCNSL, 18 with high PCNSL and 4 with high glioma. In addition to IL-10, which has been reported so far, the significant higher concentrations in PCNSL was found in Osteopontin, TNFSF8, TNFSF12, TNFSF13, TNFSF13B, TNFSF14, sTNF-R1, sTNF-R2, IL-6Ra, IL-8, IL-12(p40), IL-20, IL-27, sCD163, MMP2, MMP3, CHI3L1. On the other hand, IL-2, Il-12(p70), IL-22, and MMP-1 levels were significant higher in CSF in glioma. Although CD163 is an important molecule as a marker for M2 macrophages, immunostaining of tissues showed no significant difference in infiltration between Glioblastoma and PCNSL. This suggests that M2 macrophages infiltrating PCNSL and Glioblastoma have different activation states. CONCLUSION PCNSL had higher levels of inflammatory mediators such as TNF-related molecules. PCNSL was significantly higher in sCD163 than in glioblastoma, suggesting different activation of intratumoral M2 macrophages.

Matrine Promotes Dendritic Cell Activation and Enhances Anti-tumor Effect of Human Gastric Carcinoma Dendritic Cells Vaccine in Vitro

Objective: The purpose of this study was to investigate whether matrine can affect the phenotypes and functional maturation of dendritic cells (DC), and to secrete many cytokines, as well as the cytotoxic T lymophocyte (CTL) specific killing effect induced by gastric carcinoma dendritic cells vaccine in vitro. Materials and methods: The experimental groups consisted of three concentrations of matrine (0.5, 1.0, and 2.0 mg/mL). The DC maturation was detected by flow cytometry. The proliferation of T cells was detected by cell counting kit-8 (CCK-8) method. The secretions of cytokines by CTLs were detected by enzyme-linked immunosorbent assay (ELISA). The specific killing capacity of CTLs to target MKN45 gastric cancer cells was detected by lactate dehydrogenase (LDH) release assay. Results: The results showed that matrine could increase the expressions of CD86 and CD83 in a dose-dependent manner. Matrine could promote T cell proliferation (P&lt;0.05). Moreover, matrine also significantly increased the levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin-12p70 (IL-12p70), respectively (P&lt;0.05). The therapeutic vaccination with DCs vaccine treated with lipopolysaccharide (LPS) plus matrine resulted in improved killing effect as compared to that observed with DCs treated with LPS alone (P&lt;0.05). Conclusion: These findings define matrine as an immune adjuvant that enhances the DC activation and demonstrate a new pharmacological approach to improve the therapeutic effect of autogenous DC vaccines.

Inflammation and cell-to-cell communication, two related aspects in frailty

BackgroundFrailty is a complex, multi-dimensional age-related syndrome that increases the susceptibility to adverse health outcomes and poor quality of life. A growing consensus supports the contribution of chronic inflammation and immune system alterations to frailty, however a clear role of such alterations remains to be elucidated. Furthermore, pro- and anti-inflammatory cytokines together with other signaling molecules might spread from activated cells to the adjacent ones through extracellular vesicles (EVs), which have also a role in cellular aging. The aim of the present research was to investigate if EVs play a role in the immune function in frailty. ResultsIn 219 older adults aged 76–78 years, selected from the InveCe.Ab study (Abbiategrasso, Italy), we investigated inflammation and EVs-mediated intercellular communication. C-reactive protein (CRP) and pro- (IL-1β, IL-2, IL-6, IL-8, IL-12 p70, TNFα and IFNγ) and anti- (IL-4, IL-10, IL-13) inflammatory cytokines were evaluated on plasma of Frail and non-Frail subjects. We reported a significant increase in CRP, interleukin-1β and -6 (IL-1β, IL-6) and tumor necrosis factor alpha (TNFα) plasma levels in frailty. In female Fr subjects, we also reported an increase in interferon‐gamma (IFN‐γ) and, surprisingly, in IL-13, an anti-inflammatory cytokine, whose increase seems to oppose the inflammaging theory.An inflammatory panel (toll-like receptors 2 and 4 (TLR2 and TLR4), tumor necrosis factor receptors TNFRec5/CD 40 and TNFRec1B/CD120B) and a panel including receptors involved in cellular senescence (insulin-like growth factor 1 receptor (CD221) and interleukin 6 receptor (IL-6R)) were indeed analysed in plasma isolated large EVs (lEVs) from Frail (n = 20) and non-Frail (n = 20) subjects. In lEVs isolated from plasma of Frail subjects we reported an increase in TLR2 and TLR4, TNFRec5/CD 40 and TNFRec1B/CD120B, suggesting a chronic state of inflammation. In addition, CD221 and IL-6R increases in lEVs of Frail individuals.ConclusionsTo conclude, the pro-inflammatory status, notably the increase in circulating cytokines is pivotal to understand the potential mechanisms underlying the frailty syndrome. Moreover, cytokines release from EVs, mainly the large ones, into the extracellular space suggest their contribution to the formation of a pro-inflammatory and pro-senescent microenvironment that, in turn, can contribute to frailty.