Induce Cytokine Production Research Articles

Variable Induction of Pro-Inflammatory Cytokines by Commercial SARS CoV-2 Spike Protein Reagents: Potential Impacts of LPS on In Vitro Modeling and Pathogenic Mechanisms In Vivo.

Proinflammatory cytokine production following infection with severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) is associated with poor clinical outcomes. Like SARS CoV-1, SARS CoV-2 enters host cells via its spike protein, which attaches to angiotensin-converting enzyme 2 (ACE2). As SARS CoV-1 spike protein is reported to induce cytokine production, we hypothesized that this pathway could be a shared mechanism underlying pathogenic immune responses. We herein compared the capabilities of Middle East Respiratory Syndrome (MERS), SARS CoV-1 and SARS CoV-2 spike proteins to induce cytokine expression in human peripheral blood mononuclear cells (PBMC). We observed that only specific commercial lots of SARS CoV-2 induce cytokine production. Surprisingly, recombinant SARS CoV-2 spike proteins from different vendors and batches exhibited different patterns of cytokine induction, and these activities were not inhibited by blockade of spike protein-ACE2 binding using either soluble ACE2 or neutralizing anti-S1 antibody. Moreover, commercial spike protein reagents contained varying levels of lipopolysaccharide (LPS), which correlated directly with their abilities to induce cytokine production. The LPS inhibitor, polymyxin B, blocked this cytokine induction activity. In addition, SARS CoV-2 spike protein avidly bound soluble LPS in vitro, rendering it a cytokine inducer. These results not only suggest caution in monitoring the purity of SARS CoV-2 spike protein reagents, but they indicate the possibility that interactions of SARS CoV-2 spike protein with LPS from commensal bacteria in virally infected mucosal tissues could promote pathogenic inflammatory cytokine production.

Features of interleukin-19 production in patients with atherosclerosis

Objective. To study the features of local and systemic production of interleukin-19 in patients with atherosclerosis.Material and Methods. The study comprised a total of 46 patients (26 women and 20 men) treated for arterial hypertension in the therapeutic department of Republican Clinical Hospital named after G.Y. Remishevskaya. The mean age of subjects was 63.4 ± 3.2 years. The control group included 40 patients (23 women and 17 men aged 44.7 ± 5.5 years) who did not have atherosclerosis. Samples of atherosclerotic plaques and venous blood were examined. Atherosclerotic plaques were obtained by endarterectomy and then subjected to homogenization followed by enzymatic hydrolysis for 1 h at 37 °C with collagenase IV in the presence of proteinase III inhibitors. The serum levels of cytokines (in the control group and in patients with atherosclerosis) and in the atherosclerotic plaque homogenate (in patients with atherosclerosis) were determined by ELISA. To assess the cytokine-producing capacity of blood leukocytes and white blood cells isolated from atherosclerotic plaques, spontaneous and phytohemagglutinin (PHA)-induced cytokine production was determined when the cells were cultured in RPMI-1640.Results. The serum levels of IL-19 did not significantly differ between the patients with atherosclerosis and the control group. A statistically significant two-fold increase in the spontaneous expression of IL-19 by blood leukocytes was observed in the group of patients with atherosclerosis in comparison with the control group. When comparing the contents of IL-19 in blood serum and atherosclerotic plaque homogenate in patients with atherosclerosis, no statistically significant differences were found (p = 0.182). The level of PHA-induced IL-19 production by the atherosclerotic plaque white blood cells was significantly lower than that of blood leucocytes.Conclusion. The study showed that the reserve capacity for IL-19 synthesis in the atherosclerotic plaque white blood cells decreases leading to the progression of inflammation. The obtained results suggest that IL-19 plays the anti-atherogenic role and its production is involved in the maintaining the mechanisms for down-regulation of inflammation in atherosclerotic plaques.

Plasma extracellular vesicle delivery of miR-210-3p by targeting ATG7 to promote sepsis-induced acute lung injury by regulating autophagy and activating inflammation

Extracellular vesicles (EVs) can be used for intercellular communication by facilitating the transfer of miRNAs from one cell to a recipient cell. MicroRNA (miR)-210-3p is released into the blood during sepsis, inducing cytokine production and promoting leukocyte migration. Thus, the current study aimed to elucidate the role of plasma EVs in delivering miR-210-3p in sepsis-induced acute lung injury (ALI). Plasma EVs were isolated from septic patients, after which the expression of various inflammatory factors was measured using enzyme-linked immunosorbent assay. Cell viability and apoptosis were measured via cell counting kit-8 and flow cytometry. Transendothelial resistance and fluorescein isothiocyanate fluorescence were used to measure endothelial cell permeability. Matrigel was used to examine the tubulogenesis of endothelial cells. The targeting relationship between miR-210-3p and ATG7 was assessed by dual-luciferase reporter assays. The expression of ATG7 and autophagy-related genes was determined to examine autophagic activation. A sepsis mouse model was established by cecal ligation and puncture (CLP)-induced surgery. The level of miR-210-3p was highly enriched in septic EVs. MiR-210-3p enhanced THP-1 macrophage inflammation, BEAS-2B cell apoptosis, and HLMVEC permeability while inhibiting angiogenesis and cellular activity. MiR-210-3p overexpression reduced ATG7 and LC3II/LC3I expression and increased P62 expression. Improvements in vascular density and autophagosome formation, increased ATG7 expression, and changes in the ratio of LC3II/LC3I were detected, as well as reduced P62 expression, in adenovirus-anti-miR-210-3p treated mice after CLP injury. Taken together, the key findings of the current study demonstrate that plasma EVs carrying miR-210-3p target ATG7 to regulate autophagy and inflammatory activation in a sepsis-induced ALI model.

Abstract 1280: New generation of STING agonists: Development and characterization of a novel series of systemic immunomodulators with improved potency

Abstract Background. Stimulator of interferon genes, known as STING, is an intracellular sensor of nucleic acids and one of key regulators in activating the innate immune response. Employing synthetic STING agonists has been shown to promote immune-mediated antitumor response in preclinical animal models. Ryvu is developing small-molecule STING agonists suitable for systemic administration. Herein we present unpublished results from characterization of the new generation of our agonist series with significantly improved potency on human immune cells. Methods. Binding to recombinant STING protein was examined using Fluorescence Thermal Shift and Fluorescence Polarisation and was confirmed by X-ray crystallography. Primary screen was performed in THP-1 Dual reporter cells and selectivity was confirmed in THP-1 reporter cells with knocked out STING or expressing varying STING variants. T cell viability and proliferation was assessed by flow cytometry using activated, human T cells exposed to STING agonists. STING pathway activation pattern in cells treated with Ryvu's molecules was confirmed using Western blot analysis. BALB/c mice were injected with compounds and the levels of cytokine release were measured in the plasma. Mice were inoculated with CT26 or EMT6 tumor cells and the compound was administered intravenously followed by the regular monitoring of tumor growth. Results. New generation Ryvu STING agonists are strong binders of human STING protein. Ryvu's compounds show high cellular potency inducing cytokine production in human immune cells at low nM range. Moreover, high activity of developed agonists is maintained irrespective of the natural human STING variant as seen in THP-1 reporter cells as well as in human primary immune cells. High cellular potency of developed compounds also translates into efficacy observed in vivo, where systemic intravenous administration leads to significant tumor growth inhibition and complete tumor regressions in mouse syngeneic models. Conclusion. Ryvu has developed a new generation of potent, direct and selective small-molecule STING agonists. The compounds are characterized by drug-like properties and high in vitro potency on par or outperforming known references. Ryvu agonists are suitable for systemic administration and allow to achieve excellent antitumor efficacy. Taken together, the promising results suggest that the developed series holds high potential for improving immunotherapy in cancer patients. Citation Format: Maciej Krzysztof Rogacki, Stefan Chmielewski, Jolanta Mazurek, Magdalena Zawadzka, Katarzyna Wnuk-Lipińska, Kamil Kuś, Katarzyna Wójcik-Jaszczyńska, Aleksandra Poczkaj, Łukasz Dudek, Wojciech Schonemann, Urszula Głowniak-Kwitek, Marcin Leś, Marek Wronowski, Tushar Mahajan, Urszula Kulesza, Magdalena Zastawna, David Synak, Karol Zuchowicz, Karolina Gluza, Katarzyna Banaszak, Karolina Wiatrowska, Izabela Strojny, Mirosława Gładysz, Justyna Jabłońska, Ewelina Gabor-Worwa, Monika Dobrzańska, Raghuram Tangirala, Peter Littlewood, Krzysztof Brzózka. New generation of STING agonists: Development and characterization of a novel series of systemic immunomodulators with improved potency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1280.

Activation of human skin mast cells by vancomycin via MrgX2: Comparison to the effects of brimonidine

Abstract Vancomycin (VCM) is a glycopeptide antibiotic that inhibits bacterial peptidoglycan biosynthesis and is used for the treatment of certain Gram-positive bacterial infections of the eyes. However, the use of VCM is associated with several potential adverse reactions. We examined the mechanism of activation of human mast cells by vancomycin (VCM) and compared to brimonidine, an alpha2-adrenergic receptor agonist used to treat in glaucoma. VCM induced degranulation and cytokine secretion by SMCs, which were suppressed by pertussis toxin (PTX) (similar to MrgX2 ligands). Brimonidine induced less degranulation than VCM that was partially inhibited by PTX. MrgX2 expression on the surface of SMCs was detected by FACS analysis. VCM enhanced FceRI-mediated degranulation and cytokine secretion in a dose-dependent manner, whereas brimonidine failed to induce cytokine production and suppressed FceRI-mediated cytokine secretion. VCM induced maximal ERK-phosphorylation by 5 min similar to compound 48/80, an MrgX2 ligand and inhibited by PTX. VCM- but not brimonidine-induced degranulation of MCs was significantly inhibited by QWF, an MrgX2 antagonist. This study indicates that VCM but not brimonidine activates human SMCs via MrgX2 and that MCs become more responsive to VCM when co-stimulated by aggregating FceRI, which may occur when sensitive patients exposed to aeroallergen and VCM.

Transformation of 15-ene steviol by Aspergillus niger, Cunninghamella bainieri, and Mortierella isabellina

Transformation of 15-ene steviol (ent-13-hydroxy-kaur-15-en-19-oic acid) by growth cultures of Aspergillus niger BCRC 32720, Cunninghamella bainieri ATCC 9244, and Mortierella isabellina ATCC 38063 was conducted to generate various derivatives for the development of bioactive compounds. Four previously undescribed compounds along with six known compounds were obtained. The newly identified isolates were characterized using 1D and 2D NMR, IR, and HRESIMS, and three compounds were further confirmed by X-ray crystallographic analyses. Subsequently, the effects of 15-ene steviol and its derivatives on lipopolysaccharide (LPS)-induced cytokine production by THP-1 cells were examined, with dexamethasone used as a positive control. Results indicated that most of the tested compounds showed lower inhibitory effects than those detected in the dexamethasone-treated group, except that 15-ene steviol showed better effects than dexamethasone on the reduction of LPS-induced monocyte chemoattractant protein (MCP)-1, −2, and −3 release. Three specialized products similarly showed better effects than dexamethasone on the inhibition of LPS-induced secretion of regulated on activation, normal T cell expressed and secreted (RANTES). Moreover, none of the tested compounds showed any cytotoxicity or triggered cell apoptosis, and none affected the protein integrity of toll-like receptor 4 (TLR4) or MyD88, suggesting that these compounds may exert the anti-inflammatory activity downstream of membrane-associated TLR4 and MyD88 molecules.

Human Leukemia T-Cell Lines as Alternatives to Animal Use for Detecting Biologically Active Staphylococcal Enterotoxin Type B.

Staphylococcal enterotoxin type B (SEB) is associated with food poisoning. Current methods for the detection of biologically active SEB rely upon its ability to cause emesis when administered to live kittens or monkeys. This technique suffers from poor reproducibility and low sensitivity and is ethically disfavored over concerns for the welfare of laboratory animals. The data presented here show the first successful implementation of an alternative method to live animal testing that utilizes SEB super-antigenic activity to induce cytokine production for specific novel cell-based assays for quantifiable detection of active SEB. Rather than using or sacrificing live animals, we found that SEB can bind to the major histocompatibility complex (MHC) class II molecules on Raji B-cells. We presented this SEB–MHC class II complex to specific Vβ5.3 regions of the human T-cell line HPB-ALL, which led to a dose-dependent secretion of IL-2 that is capable of being quantified and can further detect 10 pg/mL of SEB. This new assay is 100,000 times more sensitive than the ex vivo murine splenocyte method that achieved a detection limit of 1 µg/mL. The data presented here also demonstrate that SEB induced proliferation in a dose-dependent manner for cells obtained by three different selection methods: by splenocyte cells containing 22% of CD4+ T-cells, by CD4+ T-cells enriched to >90% purity by negative selection methods, and by CD4+ T-cells enriched to >95% purity by positive selection methods. The highly enriched and positively isolated CD4+ T-cells with the lowest concentration of antigen-presenting cells (APC) (below 5%) provided higher cell proliferation than the splenocyte cells containing the highest concentration of APC cells.

Impact of heat stress and a feed supplement on hormonal and inflammatory responses of dairy cows

The aim of this trial was to evaluate the effects of an immunomodulatory supplement (OmniGen AF, OG; Phibro Animal Health Corp.) and heat stress on hormonal, inflammatory, and immunological responses of lactating dairy cows. Sixty multiparous Holstein cows were randomly assigned to 4 treatments in a 2 × 2 factorial arrangement using 2 environments: cooled using fans and misters, or noncooled, and 2 top-dressed feed supplements (56 g/d): OG or a placebo (CTL). Temperature-humidity index averaged 78 during the 8-wk trial. Blood was drawn to analyze cortisol, prolactin, and circulating tumor necrosis factor (TNF)-α and IL-10. Peripheral blood mononuclear cells (PBMC) were isolated and stimulated with hydrocortisone, prolactin, or lipopolysaccharide (LPS), individually or in several combinations, to assess induced proliferation and cytokine production. At d 52, 6 cows per treatment were injected i.v. with an LPS bolus (ivLPS) to assess hormone and cytokine responses. For cooled cows, feeding OG increased plasma cortisol concentration relative to CTL. Noncooled cows fed CTL had lower circulating TNF-α concentrations than noncooled-OG and cooled-CTL cows, with cooled-OG intermediate. Hydrocortisone+LPS-stimulated PBMC from OG cows tended to proliferate more than CTL. Relative to cooled cows, PBMC from noncooled cows produced more TNF-α and IL-10 when stimulated with LPS. Following ivLPS, cooled-OG cows had a greater cortisol response than the other treatments. In conclusion, OG supplementation enhanced cortisol release under basal condition and induced inflammation with cooling compared with CTL. This suggests that heat stress inhibits OG-mediated cortisol release. Heat stress seemed to enhance the inflammatory responses of PBMC from lactating cows. However, OG supplementation promoted PBMC proliferation under stress, or in the presence of hydrocortisone.

The effect of dietary supplementation with Nigella sativa (black seeds) mediates immunological function in male Wistar rats

This experiment aimed to investigate the effect of dietary Nigella sativa on the cell-mediated immune response. Eighteen male Wistar rats were divided equally into a control group and treated groups that received black seeds at rates of 30 and 50 g/kg in the diet (Sa30 and Sa50 groups, respectively, for 30 days. The weight gain, feed intake, feed conversion ratio (FCR), and cell-mediated immune response were monitored after the injection of 0.1 mL of 10% phytohemagglutinin (PHA). The intumesce index, serum total antioxidant capacity (TAC), catalase (CAT), interleukin-12 (IL-12), gamma interferon (γ-IF) and tumor necrosis factor alpha (TNF-α) were determined. Histopathological examination and an immunohistochemistry analysis of splenic caspase-3 and CD8 were performed. Nigella sativa significantly improved the weight gain and FCR. Intumesce index of Sa50 group was significantly increased. Nigella sativa significantly increased TAC, CAT, IL-12, γ-IF and TNF-α. A histological examination of PHA-stimulated foot pads showed increased leukocyte infiltration and edema in a dose-dependent pattern. Splenic caspase-3 and CD8 showed significant decreases and increases, respectively, in the Sa30 and Sa50 groups. The results indicate that Nigella sativa seeds exhibit immunostimulatory function through their antioxidant potential, induction of cytokine production, promotion of CD8 expression and reduction of splenic apoptosis.

Thrombospondin-1 Restricts Interleukin-36γ-Mediated Neutrophilic Inflammation during Pseudomonas aeruginosa Pulmonary Infection.

Interleukin-36γ (IL-36γ), a member of the IL-1 cytokine superfamily, amplifies lung inflammation and impairs host defense during acute pulmonary Pseudomonas aeruginosa infection. To be fully active, IL-36γ is cleaved at its N-terminal region by proteases such as neutrophil elastase (NE) and cathepsin S (CatS). However, it remains unclear whether limiting extracellular proteolysis restrains the inflammatory cascade triggered by IL-36γ during P. aeruginosa infection. Thrombospondin-1 (TSP-1) is a matricellular protein with inhibitory activity against NE and the pathogen-secreted Pseudomonas elastase LasB-both proteases implicated in amplifying inflammation. We hypothesized that TSP-1 tempers the inflammatory response during lung P. aeruginosa infection by inhibiting the proteolytic environment required for IL-36γ activation. Compared to wild-type (WT) mice, TSP-1-deficient (Thbs1-/-) mice exhibited a hyperinflammatory response in the lungs during P. aeruginosa infection, with increased cytokine production and an unrestrained extracellular proteolytic environment characterized by higher free NE and LasB, but not CatS activity. LasB cleaved IL-36γ proximally to M19 at a cleavage site distinct from those generated by NE and CatS, which cleave IL-36γ proximally to Y16 and S18, respectively. N-terminal truncation experiments in silico predicted that the M19 and the S18 isoforms bind the IL-36R complex almost identically. IL-36γ neutralization ameliorated the hyperinflammatory response and improved lung immunity in Thbs1-/- mice during P. aeruginosa infection. Moreover, administration of cleaved IL-36γ induced cytokine production and neutrophil recruitment and activation that was accentuated in Thbs1-/- mice lungs. Collectively, our data show that TSP-1 regulates lung neutrophilic inflammation and facilitates host defense by restraining the extracellular proteolytic environment required for IL-36γ activation.IMPORTANCEPseudomonas aeruginosa pulmonary infection can lead to exaggerated neutrophilic inflammation and tissue destruction, yet host factors that regulate the neutrophilic response are not fully known. IL-36γ is a proinflammatory cytokine that dramatically increases in bioactivity following N-terminal processing by proteases. Here, we demonstrate that thrombospondin-1, a host matricellular protein, limits N-terminal processing of IL-36γ by neutrophil elastase and the Pseudomonas aeruginosa-secreted protease LasB. Thrombospondin-1-deficient mice (Thbs1-/-) exhibit a hyperinflammatory response following infection. Whereas IL-36γ neutralization reduces inflammatory cytokine production, limits neutrophil activation, and improves host defense in Thbs1-/- mice, cleaved IL-36γ administration amplifies neutrophilic inflammation in Thbs1-/- mice. Our findings indicate that thrombospondin-1 guards against feed-forward neutrophilic inflammation mediated by IL-36γ in the lung by restraining the extracellular proteolytic environment.

Immunomodulatory, Antioxidant Activity and Cytotoxic Effect of Sulfated Polysaccharides from Porphyridium cruentum. (S.F.Gray) Nägeli.

Porphyridium cruentum is a unicellular microalga that can synthesize and secrete to the culture medium-high amounts of polysaccharides. In this study, the immunomodulatory, cytotoxic effect and antioxidant activity of the sulfated polysaccharides (PcSPs) were determinate. The PcSPs were precipitated with 2% Cetylpyridinium bromide hydrate and ethanol and purified by dialysis. The extract was lyophilized for its characterization by Fourier transform-Infrared (FT-IR) spectroscopy and gas chromatography–mass spectrometry (GC-MS). The antioxidant activity of PcSPs were examined with assay 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) and compared with that of the biomass, observing significant differences between the results obtained from the PcSPs and biomass. To determine their ability to induce cytokine production Tumor Necrosis Factor alpha (TNF-α) and interleukina-6 (IL-6), the immunomodulatory activity of the PcSPs has been evaluated. In the mouse macrophage cell line (RAW 264.7), PcSPs are potent inducers of IL-6 cytokines but mainly of TNF-α. The cytotoxic capacity of PcSPs was measured by the MTT colorimetric assay in colorectal carcinoma (HTC-116), human leukemia (U-937 and HL-60), breast cancer (MCF-7), lung cancer (NCI-H460) and human gingival fibroblasts (HGF-1) cell lines. The IC50 value of 2311.20 µg mL−1, 1676.74 µg mL−1, 1089.63 µg mL−1, 5498.14 µg mL−1 and 2861.49 µg mL−1 respectively in the tumor lines and 5022.55 µg mL−1 in gingival fibroblasts were obtained. Our study suggested that PcSPs from P. cruentum have a moderate immunomodulatory and cytotoxic effect. The results obtained indicate that the polysaccharides from P. cruentum are potent inducers of IL-6 cytokines and, most importantly, of TNF-α. PcSPs showed no evidence of antigenic activity or hypersensitivity when administered intraperitoneally in mice. Furthermore, the in vivo study revealed an improvement of local inflammatory response against stress in the peritoneum. These findings suggest that the PcSPs from P. cruentum might have potential as a valuable ingredient in nutraceutical products.

Protective effect of Bacteroides fragilis LPS on Escherichia coli LPS-induced inflammatory changes in human monocytic cells and in a rheumatoid arthritis mouse model

It has been reported that patients with rheumatoid arthritis (RA) have significantly less bacteria belonging to the Bacteroides group in their microbiota. We speculate that inhibition of cytokine production is impaired in patients with RA owing to their low levels of intestinal bacteria belonging to the Bacteroidetes group. Here we investigated the effect of Bacteroides fragilis lipopolysaccharide (B-LPS) on cytokine production in vitro and on the development of collagen antibody-induced arthritis (CAIA) in DBA/1 mice, an animal model of RA. in vitro culture experiments showed that Escherichia coli LPS (E-LPS)-induced cytokine production from THP-1 monocytic cells and peripheral blood mononuclear cells was significantly suppressed by B-LPS in a dose-dependent manner. A decrease in TNF-α and IL-1β production was also observed in LPS-tolerized macrophages induced by B-LPS at concentrations equal to and higher than that of E-LPS. Similar results were obtained when autoclaved feces were used to induce cytokine production instead of E-LPS. In in vivo experiments using CAIA models, B-LPS had no adverse effects even when administered at 10 times the concentration of E-LPS, which elicits severe arthritis. In addition, simultaneous administration of high dose B-LPS with E-LPS or administration of B-LPS prior to E-LPS significantly suppressed arthritis development in CAIA model animals when compared with administration of E-LPS alone. These results suggest that increasing certain bacterial groups such as Bacteroides is an effective strategy for preventing arthritis development in patients with RA.

5-aza-2'-deoxycytidine may regulate the inflammatory response of human odontoblast-like cells through the NF-κB pathway.

To explore the role of DNA methylation in the innate immunity of the dental pulp, this study investigated the effect of 5-aza-2'-deoxycytidine (AZA) on lipoteichoic acid (LTA)-induced cytokine production and related intracellular signalling pathways in human odontoblast-like cells (hOBs). hOBs were cultured and differentiated from human dental pulp tissue, and the odontoblastic phenotype of the cells was detected using immunofluorescence, qRT-PCR and Western blotting. hOBs were pretreated with AZA and then stimulated with 10μgmL-1 LTA. The levels of 42 cytokines related to immunity and inflammation were examined using a cytokine antibody array and verified using qRT-PCR and ELISA. The effect of AZA on the LTA-induced NF-κB and MAPK signalling pathways was explored using Western blotting. The cells were treated with the specific NF-κB inhibitor PDTC and MAPK inhibitors (the ERK inhibitor U0126, the p38 inhibitor SB203580, and the JNK inhibitor SP600125) to further confirm the role of the signalling pathways in LTA-treated hOBs. DNA immunoprecipitation-PCR was used to examine the dynamic methylation status of the gene promoters of myeloid differentiation primary response 88 (MyD88) and tumour necrosis factor receptor-associated factor 6 (TRAF6) in the LTA-induced hOBs. Statistical analyses of the differences between two groups were performed using Student's t-test. One-way analysis of variance (anova) or repeated-measures anova with a post hoc Dunnett's test was used to assess the differences between multiple sets of data. P<0.05 was considered to be statistically significant. The odontoblastic markers were significantly higher in hOBs than those in human dental pulp cells (hDPCs) (P<0.05). According to the cytokine antibody array results, hOBs pretreated with AZA had significantly increased production of several inflammatory cytokines (P<0.05), in which the expression levels of IL-6 and IL-8 were the most dramatically increased upon LTA stimulation (P<0.01). Furthermore, AZA resulted in the significant upregulation of p-IKKα/β, p-IκBα, p-p65, p-p38 and p-ERK in LTA-stimulated hOBs (P<0.01). Treatment with the NF-κB pathway inhibitor suppressed both IL-6 and IL-8 expression (P<0.05), whereas inhibitors of the MAPK pathway (SB203580 and SP600125) did not. In LTA-treated hOBs, AZA significantly increased the expression levels of TRAF6 and MyD88 (P<0.05). AZA induced MyD88 promoter hypomethylation but did not affect TRAF6 methylation. AZA regulated the LTA-induced inflammatory response through the NF-κB signal pathway in hOBs. This study highlights the important role of DNA methylation in the immunity defence of odontoblasts during the dental pulp immunity response to caries.

Jararhagin, a snake venom metalloproteinase, induces mechanical hyperalgesia in mice with the neuroinflammatory contribution of spinal cord microglia and astrocytes

Jararhagin is a hyperalgesic metalloproteinase from Bothrops jararaca venom. In rodents, jararhagin induces nociceptive behaviors that correlate with an increase in peripheral cytokine levels. However, the role of the spinal cord glia in pain processing after peripheral stimulus of jararhagin has not been investigated. Aiming to explore this proposal, mice received intraplantar (i.pl.) injection of jararhagin and the following parameters were evaluated: hyperalgesia, spinal cord TNF-α, IL-1β levels, and CX3CR1, GFAP and p-NFκB activation. The effects of intrathecal (i.t.) injection of TNF-α soluble receptor (etanercept), IL-1 receptor antagonist (IL-1Ra), and inhibitors of NFκB (PDTC), microglia (minocycline) and astrocytes (α-aminoadipate) were investigated. Jararhagin inoculation induced cytokine production (TNF-α and IL-1β) in the spinal cord, which was reduced by treatment with PDTC (40% and 50%, respectively). Jararhagin mechanical hyperalgesia and cytokine production were inhibited by treatment with etanercept (67%), IL-1Ra (60%), PDTC (70%), minocycline (60%) and α-aminoadipate (45%). Furthermore, jararhagin induced an increase in p-NFκB, CX3CR1 and GFAP detection in the spinal cord indicating activation of NFκB, microglia and astrocytes. These results demonstrate for the first time that jararhagin-induced mechanical hyperalgesia is dependent on spinal cord activation of glial cells, consequent NFκB activation, and cytokine production in mice.

Apigetrin inhibits cytotoxicity and dysregulation of ACE2, IL1α and TGFβ expression induced by recombinant spike protein of SARS-CoV-2

SARS-CoV-2 virus is a novel coronavirus that was first identified in Wuhan, China in December 2019 and has caused an ongoing global health crisis. It has been a worldwide focus in research to understand viral pathogenesis and discover effective therapies. SARS-CoV-2 belongs to the same genus as the viruses responsible for Severe Acute Respiratory Syndrome (SARS) and the Middle East Respiratory Syndrome (MERS). Spike protein (SP) on SARS-CoV-2 plays a key role in the pathogenesis of SARS-CoV-2. The virus enters human cells via the binding of SP to the angiotensin-converting enzyme 2 (ACE2) on human cells. The binding of SP inhibits ACE2 function by reducing formation of angiotensin-(1-7), a compound that has inhibitory effects on inflammation. In addition, SARS-CoV-2 induces excessive proinflammatory cytokine production through various other signaling pathways such as the NFκB and NLRP3 inflammasome pathways. Previous evidence showed that apigenin (APG), a plant phenolic compound, can bind to SP. However, whether or not apigetrin (APT), the glucoside conjugate of APG, can protect human cells against cell injury caused by SARS-CoV-2 is still unknown. Studies have shown that SARS-CoV-2 induced dysregulation of host cell ACE2 expression is one of the major pathophysiological factors of COVID-19 infection. Our current study demonstrated that recombinant SP significantly reduced ACE2 level in human neuronal cells in a dose dependent manner using ELISA assay. Interestingly, APT reversed the SP induced ACE2 downregulation in these cells. In this study, the effect of SP on cell proliferation and immune regulation was also investigated. Using MTT and LDH assays, I discovered that SP had a cytotoxic effect on these cells and significantly inhibited cell proliferation. This cytotoxic effect was mitigated by adding APT treatment. Furthermore, APT reduced SP induced cytokine production such as IL1α and TGFβ . In sum, my study demonstrated that APT inhibited SARS-CoV-2 SP induced dysregulation of human cells and reduced its cytotoxic effects on cells. APT significantly upregulated ACE2 expression and inhibited the production of cytokines IL1α and TGFβ in the cells treated with SP. My study indicated that APT has potential to be a novel therapy for COVID-19 infection. More experiments to further elucidate molecular mechanisms of how APT modulates pathological effect of SP with different in vitro models including other human cell lines and in vivo animal models are currently being performed.

Pathogenetic rationale for usage of recombinant interleukins in the patients with mandibular fractures to prevent post-traumatic osteomyelitis

The paper presents the results on spontaneous and induced production of IFNγ, IL-4, IL-13 cytokines in blood cells of patients with mandibular fractures and post-traumatic osteomyelitis. Osteomyelitis of the jaw represents one of the urgent challenges in modern medicine. There are many reasons for development of purulent necrotic processes of the jaw bones, including disorders of innate and adaptive immune response. Currently, the immunological aspects of post-traumatic complications of maxillofacial region remain poorly understood. There are no unambiguous and systematic studies of immune mechanisms in pathogenesis of post-traumatic osteomyelitis of the lower jaw. The aim of our study was to theoretically confirm application of recombinant interleukins (IL-1β, IL-2, IFNγ) in the patients with jaw fractures, in order to prevent osteomyelitis, as based on the studies of spontaneous and induced cytokine production (IFNγ, IL-4, IL-13). Spontaneous and stimulated production of IFNγ, IL-4, and IL-13 cytokines by blood cells was determined using specific reagent kits from RD Diagnostic Inc. (USA). The results were recorded using an ELISA Multiscan analyzer (Finland). Statistical significance of intergroup differences was determined by the Mann–Whitney method. The level of statistical significance at which the null hypotheses were rejected was 0.05. To stratify the cases of post-traumatic osteomyelitis and uncomplicated mandibular fracture, the models were developed with a singlelayer neural network, using the nnet R-studio package. To assess quality of the models, the areas under the ROC curves (AUC), Akaike criterion (AIC), and the relative classification accuracy (OTC) were used, which was determined as the ratio of correctly established model diagnosis numbers to the total number of patients. The study results demonstrate that the patients with mandibular fractures exhibit a moderate impairment of LPS-induced IL-4 and IFNγ production by leukocytes with IL-13 activation. In presence of osteomyelitis, this imbalance is promoted, thus suggesting an impaired ability of the cells to produce IL-4 and IFNγ. An opportunity for usage immunomodulation when treating the patients with mandibular fractures is represented by P = 1/(1+e-z) where z is defined via IL-1β, IL-2 and IFNγ levels on the first day of observation. In vitro supplementation with recombinant IL-1β and IFNγ modulates cell function, improving the cytokine profile. The prognostic models, imitating binary logistic regression, were used to demonstrate that recombinant IL-1β could be used to prevent patients with mandibular fractures from developing post-traumatic osteomyelitis (AIC = 13.2, AUC = 0.96, р = 0.026).

New Biomarker in Chagas Disease: Extracellular Vesicles Isolated from Peripheral Blood in Chronic Chagas Disease Patients Modulate the Human Immune Response.

Chagas disease, a neglected tropical disease (NTD) caused by the flagellated protozoan Trypanosoma cruzi (T. cruzi), is a major public health problem. It was initially restricted to Latin America, but it is now expanding globally. Host and pathogen interactions are crucial in the establishment of disease, and since 1970, it has been known that eukaryotic cells release extracellular vesicles (EVs), which in turn have an important role in intercellular communication in physiological and pathological conditions. Our study proposed to characterize and compare circulating EVs isolated from the plasma of chronic Chagas disease (CCD) patients and controls. For this, peripheral blood was collected from patients and controls, and mononuclear cells (PBMCs) were isolated and stimulated with parasite EVs, showing that patient cells released fewer EVs than control cells. Then, after plasma separation followed by EV total shedding enrichment, the samples were subjected to ultracentrifugation to isolate the circulating EVs, which then had their size and concentration characterized by nanoparticle tracking analysis (NTA). This showed that patients had a lower concentration of circulating EVs while there were no differences in size, corroborating the in vitro data. Additionally, circulating EVs were incubated with THP-1 cells (macrophages) that, after the interaction, had their supernatant analyzed by ELISA for cytokine detection. In relation to their ability to induce cytokine production, the CCD patient EVs were able to induce a differential production of IFN-γ and IL-17 in relation to controls, with differences being more evident in earlier/less severe stages of the disease. In summary, a decreased concentration of circulating EVs associated with differential activation of the immunological system in patients with CCD is related to parasite persistence and the establishment of chronic disease. It is also a potential biomarker for monitoring disease progression.

Inhibiting Protein Kinase D Promotes Airway Epithelial Barrier Integrity in Mouse Models of Influenza A Virus Infection

RationaleProtein kinase D (PKD) is a serine/threonine kinase family that is involved in a wide array of signaling pathways. Although PKD has been implicated in immune responses, relatively little is known about the function of PKD in the lung or during viral infections.ObjectivesWe investigated the hypothesis that PKD is involved in multiple aspects of host response to viral infection.MethodsThe selective PKD inhibitor CRT0010166 was administered to C57BL/6 mice prior to and during challenge with either inhaled double-stranded RNA or Influenza A Virus. PKD signaling pathways were investigated in human bronchial epithelial cells treated with CRT0010166, double-stranded RNA, and/or infected with Influenza A Virus.MeasurementsTotal protein and albumin accumulation in the bronchoalveolar fluid was used to asses inside/out leak. Clearance of inhaled FITC-dextran out of the airspace was used to assess outside/in leak. Cytokines and neutrophils in bronchoalveolar lavage were assayed with ELISAs and cytospins respectively. Viral RNA level was assessed with RT-PCR and protein level assessed by ELISA.Main ResultsPKD inhibition prevented airway barrier dysfunction and pro-inflammatory cytokine release. Epithelial cells express PKD3, and PKD3 siRNA knock-down inhibited polyI:C induced cytokine production. Lung epithelial-specific deletion of PKD3 (CC10-Cre x PKD3-floxed mice) partially attenuated polyI:C-induced barrier disruption in vivo. Mechanistically, we found that PKD promoted cytokine mRNA transcription, not secretion, likely through activating the transcription factor Sp1. Finally, prophylactic CRT treatment of mice promoted barrier integrity during influenza virus infection and reduced viral burden.ConclusionsInhibiting PKD promotes barrier integrity, limit pathogenic cytokine levels, and restrict Influenza A Virus infection. Therefore, PKD is an attractive target for novel antiviral therapeutics.

Asymptomatic malaria and hepatitis B do not influence cytokine responses of persons involved in chronic sedentary activities

BackgroundChronic Sedentary lifestyles have been linked to increased odds of stress, elevated anxiety and diminished wellbeing, inducing cytokine production and predispose to hypertension and other cardiovascular diseases. In endemic areas, Plasmodium falciparum and hepatitis B virus (HBV) infections can trigger pro-inflammatory cytokine responses. However, the impact of these infections on cytokine response profiles in individuals engaged in chronic sedentary activities is unknown. This study was aimed at addressing these concerns using a predominantly sedentary population of traders in the Tamale metropolis of Ghana.MethodFour hundred respondents were categorized, based on their number of working years (< or ≥ 5 years) and number of working hours per day (< or ≥ 10 h), into sedentary (≥5 years + ≥ 10 h) and non-sedentary (≥ 5 years + < 10 h, < 5 years + ≥ 10 h and < 5 years + < 10 h) groups. The participants were tested for P. falciparum and HBV infections using polymerase chain reaction. Blood pressure and cytokines responses were measured. Associations and comparison analysis between variables were determined, and test statistics with p < 0.05 were considered statistically significant.ResultsInfection status included: un-infected (93.5%), P. falciparum mono-infected (1.0%), HBV mono-infected (3.0%) or P. falciparum /HBV co-infected (2.5%). Majority of the participants, 57.0% (n = 228) were involved in chronic sedentary life style. That notwithstanding, sedentary lifestyle was independent of the infection groups (χ2 = 7.08, p = 0.629). Hypertension was diagnosed in 53.8% of respondents and was independent of infection status (X2 = 6.33, p = 0.097). Pro-inflammatory (TNF-α, IL-1β, IL-6, IL-8 and IL-12) and anti-inflammatory (IL-10, IL-7 and IL-13) cytokine responses were similar among individuals with different sedentary working time and between hypertensive and non-hypertensive individuals (p > 0.05 for all comparisons). Among individuals with different infection status, pro-inflammatory (TNF-α; p = 0.290, IL-1β; p = 0.442, IL-6; p = 0.686, IFN-γ; p = 0.801, IL-8; p = 0.546, IL-12; p = 0.154) and anti-inflammatory (IL-10; p = 0.201, IL-7; p = 0.190, IL-13; p = 0.763) cytokine responses were similar.ConclusionOur data suggest that asymptomatic infections of P. falciparum and HBV together with a high prevalence of hypertension did not have any significant impact on cytokine response profiles among predominantly sedentary traders in the Tamale metropolis of Ghana.

D-Lactate Increases Cytokine Production in Bovine Fibroblast-Like Synoviocytes via MCT1 Uptake and the MAPK, PI3K/Akt, and NFκB Pathways.

Simple SummaryLameness is commonly observed in dairy cattle, affecting animal welfare and farm production. Ruminal acidosis is a metabolic disorder that frequently has been associated with lameness. Has been observed that during acute ruminal acidosis increase the presence of D-lactate in synovial fluid and induces the appearance of a joint inflammation, characterized by an aseptic synovitis. The fibroblast-like synoviocytes is one the main synovium cells involved in the onset of joint inflammation. The aim of this study was to assess the proinflammatory effect of D-lactate on bovine fibroblast-like synoviocytes. We demonstrated that D-lactate increase the release of proinflammatory cytokines, activating intracellular signaling pathways in bovine synoviocytes. Our results support the role of D-lactate in the onset of synovitis and lameness in cattle.Acute ruminal acidosis (ARA) is caused by the excessive intake of highly fermentable carbohydrates, followed by the massive production of D-lactate and the appearance of neutrophilic aseptic polysynovitis. Bovines with ARA develop different lesions, such as ruminitis, polioencephalomalacia (calves), liver abscess and lameness. Lameness in cattle with ARA is closely associated with the presence of laminitis and polysynovitis. However, despite decades of research in bovine lameness as consequence of ruminal acidosis, the aetiology and pathogenesis remain unclear. Fibroblast-like synoviocytes (FLSs) are components of synovial tissue, and under pathological conditions, FLSs increase cytokine production, aggravating inflammatory responses. We hypothesized that D-lactate could induce cytokine production in bovine FLSs. Analysis by qRT-PCR and ELISA revealed that D-lactate, but not L-lactate, increased the expression of IL-6 and IL-8 in a monocarboxylate transporter-1-dependent manner. In addition, we observed that the inhibition of the p38, ERK1/2, PI3K/Akt, and NF-κB pathways reduced the production of IL-8 and IL-6. In conclusion, our results suggest that D-lactate induces an inflammatory response; this study contributes to the literature by revealing a potential key role of D-lactate in the polysynovitis of cattle with ARA.