Cytokines In Supernatants Research Articles

Efficacy of Qingre Huashi decoction on infection of : inhibiting adhesion, antioxidant, and anti-inflammation.

To investigate the phytochemical profile of Qingre Huashi decoction (, QHD) and evaluate the mechanisms rationalizing the use of QHD against ()-associated gastritis. QHD is composed of 11 herbs, which was prepared by a fixed Pharmacy and concentrated into clear paste. High-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (HPLC-QTOF/MS) was used to detect the phytochemical profile of QHD. The toxicity of QHD against and human gastric epithelial cells was evaluated by the toxicology test and cell counting kit-8 assay. The adhesion model was constructed by incubating and gastric mucosal epithelial cells for 2 h. The urease assay was used to examine the anti-adhesion effects of QHD, and gene expression of adhesins was evaluated by quantitative polymerase chain reaction. The antioxidant activity was assessed by DCFH-DA labeling. To evaluate the anti-inflammatory effect, the levels of pro-inflammatory cytokines in the culture supernatant were detected by enzyme-linked immunosorbent assay. HPLC-QTOF/MS profiling indicated the presence of primary compounds 1-20 in QHD. Drug concentration was determined as 1, 2, and 5 mg/mL by the toxic concentration of QHD against and human gastric epithelial cells. QHD prevented adhesion to the human gastric epithelial cells and reduced levels of reactive oxygen species. QHD also reduced the level of interleukin-8 and other pro-inflammatory cytokines that were upregulated by infection. QHD could inhibit adhesion, and exert antioxidant and anti-inflammatory effects .

Establishment of local allergic rhinitis tolerance in mouse model

Objective:To establish a mouse model of local allergic rhinitis tolerance by intranasal infusion of allergen, and study its immunological indexes and characteristics. Methods:The mice were given ovalbumin（OVA） and phosphate buffer solution（PBS） daily, and their allergic symptoms were recorded. OVA-specific antibodies（IgE, IgG1, IgG2a） in serum and cytokine（IL-4, IL-10, IFN-γ） in splenic culture supernatant were detected. The infiltration of eosinophils and goblet cells in nasal mucosa were observed, and the changes in local nasal mucosa genes were analyzed by RNA-seq technique. Results:Mice first showed aggravation of allergic symptoms when stimulated with OVA. The serum OVA-specific antibodies IgE, IgG1, IgG2a and the cytokine（IL-4, IL-10, IFN-γ） Iin splenic culture supernatant were increased, the eosinophils and goblet cells in nasal mucosa were significantly increased. The expression of encoding IL-10, TGF-β gene and eosinophils activation gene in nasal mucosa were up-regulated. As the duration of nasal dripping increased, the allergic symptoms gradually decreased, serum OVA-specific antibodies IgE, IgG1, IgG2a continued to increase. Splenic culture supernatant IL-4 decreased, IL-10 increased, IFN-γ had a downward trend. In nasal mucosa, goblet cells decreased significantly. Genes involved in eosinophils activation were significantly down-regulated. The encoding tolerance genes such as IL-10 and TGF-β genes remained highly expressed. Ten core genes associated with immune tolerance in localized allergic rhinitis were screened, Rps27a, Uba52, Kng2, Gnal, C3, Rtp4, Reep1, Rtp2, Rtp1, Reep5. Conclusion:The mice first develop an allergy and then develop immune tolerance by continuous local drops of the allergen. The generation of immune tolerance may be induced by the continuous action of allergens, which induced systemic and local immune tolerance effects.

NONO regulates multiple cytokine production in sepsis via the ERK1/2 signaling pathway

The massive release of pro-inflammatory cytokines is a crucial step in triggering the inflammatory cascade in sepsis. Exploring the key molecules regulating the expression and release of multiple cytokines has important value for revealing the mechanism of the cytokine storm in sepsis. This study aimed to investigate the role of multifunctional nuclear protein non-POU domain containing octamer-binding protein (NONO) in the sepsis cytokine storm and to elucidate the underlying mechanism. We found that NONO expression in tissues and cells of sepsis mice was significantly upregulated. Downregulation of NONO expression inhibited the mRNA expression of multiple cytokines, including IL-6, IL-1β, MCP-1, MIP-1α, and MIP-1β in inflammatory cells from mice and human leukemic monocyte-THP1 cells challenged with lipopolysaccharide (LPS), and significantly decreased the level of these cytokines and TNF-α in the supernatant of THP1 cells challenged by LPS. Nono knockout also reduced the levels of TNF-α, IL-6, MIP-1α, and MIP-1β in serum, alleviated hepatocyte edema, and improved the survival rate of sepsis mice. Reduced NONO expression decreased the phospho-ERK1/2 level in inflammatory cells from sepsis mice or THP1 cells challenged by LPS. Phospho-ERK1/2 inhibitor decreased the mRNA expression and concentration of cytokines in the culture supernatant of LPS-induced THP1 cells, similar to the effect of NONO knockdown. After LPS challenge, the levels of phospho-ERK1/2 and NONO were increased, with obvious colocalization in the nucleus and vesicular-like organelles in macrophages. NONO knockdown decreased nuclear translocation of phospho-ERK1/2 in LPS-challenged THP1 cells. These results suggest that NONO is a potentially critical molecule involved in multiple cytokine production in sepsis. Upregulated NONO in sepsis may promote the expression and release of multiple cytokines to participate in a sepsis cytokine storm by promoting ERK1/2 phosphorylation.

Preclinical Assessment of Immunogenicity and Protectivity of Novel ROR1 Fusion Proteins in a Mouse Tumor Model.

The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a new tumor associated antigen (TAA) which is overexpressed in several hematopoietic and solid malignancies. The present study aimed to produce and evaluate different fusion proteins of mouse ROR1 (mROR1) to enhance immunogenicity and protective efficacy of ROR1. Four ROR1 fusion proteins composed of extracellular region of mROR1, immunogenic fragments of TT as well as Fc region of mouse IgG2a were produced and employed to immunize Balb/C mice. Humoral and cellular immune responses and anti-tumor effects of these fusion proteins were evaluated using two different syngeneic murine ROR1+ tumor models. ROR1-specific antibodies were induced in all groups of mice. The levels of IFN-γ, IL-17 and IL-22 cytokines in culture supernatants of stimulated splenocytes were increased in all groups of immunized mice, particularly mice immunized with TT-mROR1-Fc fusion proteins. The frequency of ROR1-specific CTLs was higher in mice immunized with TT-mROR1-Fc fusion proteins. Finally, results of tumor challenge in immunized mice showed that immunization with TT-mROR1-Fc fusion proteins completely inhibited ROR1+ tumor cells growth in two different syngeneic tumor models until day 120 post tumor challenge. Our preclinical findings, for the first time, showed that our fusion proteins could be considered as a potential candidate vaccine for active immunotherapy of ROR1-expressing malignancies.

Artesunate ameliorates osteoarthritis cartilage damage by updating MTA1 expression and promoting the transcriptional activation of LXA4 to suppress the JAK2/STAT3 signaling pathway.

The objective of this study was to discuss the mechanism of artesunate (ART) in improving cartilage damage in osteoarthritis (OA) by regulating the expression levels of metastatic tumor antigen 1 (MTA1), lipoxin A4 (LXA4) and the downstream JAK2/STAT3 signaling pathway. The OA model in vitro was constructed by stimulating chondrocytes for 24h with 10ng/mL interleukin (IL)-1β, and cell proliferation and apoptosis, expression levels of Aggrecan, MTA1, LXA4, MMP3, MMP13 and Collagen II, and inflammatory cytokines in the culture supernatants were examined. Histopathological changes, inflammatory response and chondrocyte apoptosis of the cartilage tissues of OA mice were performed. In vitro cell experiments, ART enhanced cell proliferation capacity, accompanied by decreased apoptosis rate, decreased expression of MMP-3 and MMP-13, elevated expression of Collagen II and Aggrecan, as well as reduced levels of IL-6 and TNF-α in the cell supernatant. ART also ameliorated IL-1β-induced chondrocyte damage by upregulating MTA1. The LXA4 promoter region had two potential binding sites for MTA1. There was a positive correlation between MTA1 and LXA4. MTA1 enhanced the expression of LXA4 through transcription and blocked the activation of the JAK2/STAT3 signaling pathway. In vivo animal model experiments further showed that ART treatment alleviated cartilage tissue damage in OA model mice by upregulating MTA1. Our study demonstrates that ART improves the cartilage damage of OA by upregulating MTA1 expression and promoting the transcriptional activation of LXA4, and further blocking the JAK2/STAT3 signaling pathway.

AIM2 and NLRC4-driven inflammasome activation in adult-onset Still's disease and the preliminary therapeutic effect exploration of carboxyamidotriazole.

This study aimed to explore the changes of four major inflammasomes in adult-onset Still's disease (AOSD) and preliminarily evaluate the therapeutic effect of carboxyamidotriazole (CAI), which has previously been reported to have the significant anti-inflammatory activity. The mRNA expressions of proinflammatory cytokines and inflammasome components in peripheral blood mononuclear cells (PBMCs) from AOSD patients and healthy controls (HC) were determined by reverse transcription-quantitative PCR. Poly(dA:dT)-induced AIM2 inflammasome and flagellin-induced NLRC4 inflammasome activation models were established in bone marrow-derived macrophages (BMDMs). The levels of cytokines in serum and culture supernatants were measured by ELISA method. The serum levels of IL-1β, IL-6, and TNF-α in AOSD patients were significantly higher than those in HC. However, the mRNA expressions of IL-1β, IL-6, IL-18, and TNF-α in PBMCs did not differ markedly in AOSD patients in comparison with HC. Significantly increased mRNA levels of AIM2, NLRC4, ASC, and caspase-1 were observed in patients with AOSD when compared with HC, while NLRP1 and NLRP3 showed no change in AOSD samples. In addition, CAI treatment could significantly reduce the levels of IL-1β, IL-6, and TNF-α secreted by AOSD PBMCs and inhibit AIM2 and NLRC4 inflammasomes activation in BMDMs. Increased levels of proinflammatory cytokines in AOSD might be associated with NLRC4 and AIM2 inflammasomes activation. CAI is likely to have the therapeutic potential for AOSD by inhibiting NLRC4 and AIM2 inflammasomes activation and reducing the proinflammatory cytokines and worthy of further investigation. These results provide new ideas for elucidating the pathogenesis of AOSD and providing specific targeted therapy. Key points • Significantly higher mRNA levels of AIM2 and NLRC4 inflammsome signaling were observed in AOSD patients compared with health controls, indicating that AIM2 and NLRC4 inflammsome activation might be related to the increased proinflammatory cytokines in AOSD. • CAI treatment markedly reduced the secretion levels of cytokines IL-1β, IL-6, and TNF-α in AOSD PBMCs and inhibited AIM2 and NLRC4 inflammasome activation.

RBM3 Induced Immune Resistance through Modulating Tumor Microenvironment in Nasopharyngeal Carcinoma

<h3>Purpose/Objective(s)</h3> We hypothesize RBM3 could lead to immune resistance in nasopharyngeal carcinoma (NPC). In the present study, we aim to explore the role and mechanisms of RBM3 inducing immune resistance by modulating tumor microenvironment (TME) in nasopharyngeal carcinoma. <h3>Materials/Methods</h3> Bioinformatic prediction and analysis were used to explore the correlation between RBM3 expression and NPC patients' survival, TME cell characters and related cytokines level. Immunohistochemistry (IHC) were used to examine RBM3 and PD-L1 expression, as well as to quantify Treg and M2 macrophages at the tissue level. Flow cytometry (FCM) were used to quantify immune cells in different treatment settings. Multiple cytokines were detected by ELISA Assays. Gene overexpression and knockdown were used to regulate RBM3 expression. Western-blot was used to detect related proteins. <h3>Results</h3> TCGA data analysis showed NPC patients with higher RBM3 expression and more Tegs had worse prognosis than those with lower RBM3 expression and fewer Tegs. IHC assays revealed that RBM3 expression was higher in immune resistant patients than in immune sensitive patients, with more Treg and M2 macrophages in TME. However, there was no significant difference in PD-L1 expression between the two groups. CNE1/IR cell line with high RBM3 expression and the parental CNE1 cell line with low RBM3 expression were cultured and cytokines in supernatant were detected. IL-2 and IL-10 levels were significantly higher in CNE1/IR cells and IL-4 and TGF-β were slightly higher. CNE1/IR and CNE1 cell lines were co-cultured with Treg and RAW264.7 cells. Flow cytometry showed there were more Treg cells and higher M2/M1 ratio in RBM3 highly expressed CNE1/IR group than the RBM3 low expression group. Decrease RBM3 expression in CNE1/IR cell could reduce IL-2/IL-10 level in supernatant and attenuate cytokines induced Treg recruit and macrophages transformation. Increase RBM3 expression in CNE1 cell could increase IL-2/IL-10 level and boost Treg recruit and macrophages transformation. Higher p-AKT level was detected in CNE1/IR cells and was correlated with IL-10 mRNA level. P-AKT and MEK1 inhibitor suppressed IL-10 mRNA and protein in RBM3 high expressing CNE1/IR cells. These results suggested RBM3 could induce immune resistant TME through regulating cytokines secretion. We further co-cultured RBM3 low expressing CNE1 cells with Treg, M2 and Treg+M2 cells. CNE1 cells co-cultured with Treg+M2 cells had higher RBM3 level than other groups, suggesting the immune resistant TME could inversely affect tumor cells and preserve immune resistant state. <h3>Conclusion</h3> RBM3 could increase NPC cells IL-10 secretion, recruiting more Tregs and increasing M1/M2 transformation in TME, thus induce immune resistance in nasopharyngeal carcinoma.

Impact of interleukin-32α on T helper cell-related cytokines, transcription factors, and proliferation in patients with type 2 diabetes mellitus

Objective The ability of interleukin (IL)-32α to induce T helper (Th) 1, Th17, and Treg cytokines (interferon gamma [IFN-γ], IL-17, and IL-10, respectively), and transcription factors ([signal transducer and activator of transcription (STAT) 1 and T-box (T-bet) for Th1, STAT3 and retinoid-related orphan receptor (ROR)-γt for Th17, and STAT5 and forkhead box P3 (Foxp3) for Treg]) were investigated in type 2 diabetes mellitus (T2DM). IL-32α effects on Th cell proliferation and related factors including IL-2 and NF-κB were also explored. Methods Serum levels of IL-32α in 31 patients and 31 healthy controls (HCs) were determined by ELISA assay. CD4+ T cells cultured with polyclonal activators in the presence and absence of recombinant IL-32α (rIL-32α). Gene expressions in cultured Th cells were assessed with real-time PCR. Cytokines in supernatants were measured with ELISA. Proliferation experiments were assessed by flow cytometry. Results The patients showed significant increase in IL-32α levels compared with HCs and its levels were positively correlated with fasting plasma glucose (FPG) and hemoglobin A1c (HbA1c). rIL-32α enhanced IL-17 and IL-2 production, increased ROR-γt and NF-κB expression, and enhanced Th proliferation in both patients and HCs. In patients, IL-17, ROR-γt, NF-κB, and proliferation levels were higher than those in HCs, in cultures with and without rIL-32α (rIL-32α+ and rIL-32α-). IL-2 levels in rIL-32α+cultures of patients were significantly higher than the HCs, and it was positively correlated with proliferation rate and NF-κB expression. Conclusions Aberrant IL-32α levels are participated in T2DM pathogenesis. IL-32α potently induces Th17-related factors and amplifies the proliferative function of T cells. Highlights Enhanced serum levels of IL-32α in T2DM patients was correlated with FPG and HbA1c. IL-32α increases ROR-γt expression and IL-17 production, and induces Th17 cells. IL-32α enhances NF-κB expression and IL-2 production, and promotes Th proliferation. IL-32α is more effective for inducing Th17 cells and proliferation in the patients. IL-32α axis could be mentioned as a future therapeutic goal for the T2DM.

Oral administration of Forsythiasides-rich extract from Forsythiae fructus decreases the IgE production through suppressing Th2 response in shrimp protein-sensitized BALB/c mice

BackgroundIgE-mediated allergic diseases are characterized by an overreaction of type 2 response which promotes B cell differentiation and IgE production via Th2 cytokines. Forsythiae Fructus, a well-known traditional Chinese medicine, has been clinically used for relieving IgE-mediated type I allergy by continuous treatment for several days. PurposeWe aimed to investigate the effect of continuous oral administration with Forsythiasides-rich extract (FRE) from Forsythiae Fructus on type 2 response and the underlying mechanism in shrimp protein (SP)-sensitized mice. Study design and methodsWe established a SP-sensitized mice model and investigated effect of FRE both in vivo and ex vivo. Serum total IgE (tIgE), specific IgE (sIgE), mouse mast cell protease 1 (mMCP-1) and supernatant Th2 cytokines (IL-4, IL-5 and IL-13) were determined by ELISA. The cell viability was examined by MTS assay. ResultsContinuous oral administration of FRE could significantly lower the levels of serum tIgE, sIgE and mMCP-1 in SP-sensitized mice. Ex vivo study indicated that a single treatment of FRE could potently suppress the release of Th2 cytokines (IL-4, IL-5 and IL-13) in isolated mesenteric lymph nodes cells and splenocytes of SP-sensitized mice in a concentration-dependent manner. ConclusionFRE can suppress Th2 cytokines (IL-4, IL-5 and IL-13) to decrease the production of IgE in SP-sensitized mice. In view of its another ability to stabilize mast cells, Forsythiae Fructus is reasonably suitable for treating IgE-mediated allergic diseases.

IL-37 improves mice myocardial infarction via inhibiting YAP-NLRP3 signaling mediated macrophage programming

ObjectiveMyocardial infarction is the highest cause of cardiovascular death. Previous studies found that patients with myocardial infarction have elevated serum IL-37 and IL-37 treatment significantly alleviates adverse remodeling in myocardial infarction mice. However, the underlying mechanism of IL-37 in myocardial infarction is still unknown. Here we explored the underlying mechanism of IL-37 in attenuating myocardial infarction. MethodsThe myocardial infarction mice model was constructed by left anterior descending ligation and then submitted to recombinant IL-37 administration. The histology and cardiac function were detected by HE & Masson staining and echocardiography, respectively. The macrophage phenotypes were analyzed by flow cytometry and real-time PCR. The cytokines in serum and cell culture supernatant were determined by ELISA. In addition, THP-1 cells were used in vitro to investigate the underlying mechanisms. ResultsInfarcted mice showed increased inflammatory cell infiltration and impaired cardiac function. IL-37 treatment alleviated pro-inflammatory macrophage infiltration, tissue injury, and collagen deposition in hearts on day 3 and 7 after infarction in mice. In addition, IL-37 application modulated the balance between M1 and M2 macrophages in infarcted hearts. In vitro, THP-1 cell line polarization was also regulated by IL-37, companied by YAP phosphorylation and NLRP3 inactivation. Verteporfin, a YAP inhibitor, could abolish IL-37-induced NLRP3 inhibition and M2 macrophage polarization. ConclusionOur results demonstrated that IL-37 achieves a favorable therapeutical function on myocardial infarction by modulating YAP-NLRP3 mediated macrophage programming, providing a promising drug for the treatment of myocardial infarction.

Association of gestational diabetes mellitus and negative modulation of the specific humoral and cellular immune response against Toxoplasma gondii.

In order to evaluate and compare the specific immune response of pregnant women (PW) chronically infected with Toxoplasma gondii, with and without gestational diabetes mellitus (GDM), and the humoral response of their respective newborns (NB), the study was carried out on 81 PW (34 GDM and 47 controls) from whose medical records the results of the oral glucose tolerance test (OGTT) were obtained, and blood samples were collected at the third trimester of pregnancy; also, on 45 NBs (20 GDM and 25 controls) from whom umbilical cord blood samples were obtained. Humoral immunity was analyzed by measuring anti-T. gondii total IgG, IgG subclasses and IgG avidity. To evaluate cellular immunity, peripheral blood mononuclear cells (PBMC) from 32 PW (16 GDM and 16 controls) were cultured, supernatant cytokines were determined, and flow cytometry was performed to analyze the expression at lymphocytes of surface molecules, cytokines and transcription factors. All PW and NBs were positive for total IgG, and the prevalent subclass was IgG1. There was a negative correlation between the OGTT glycemia of PW and the levels of total IgG, IgG1 and IgG avidity. The IgG avidity of the GDM group was significantly lower than the control group. Patients from the GDM group had a higher number of T lymphocytes expressing markers of cell activation and exhaustion (CD28 and PD-1). In the presence of T. gondii soluble antigen (STAg) the amount of CD4+ T cells producing IFN-γ, IL-10 and IL-17 was significantly lower in the GDM group, while there was no difference between groups in the number of CD4+ CD25HighFOXP3+LAP+ functional Treg cells. Additionally, under STAg stimulus, the secretion of IL-17, IL-4, TNF and IL-2 cytokines at PBMCs culture supernatant was lower in the GDM group. In conclusion, there was a correlation between the increase in blood glucose and the decrease in levels of anti-T. gondii antibodies, associated with the decreased IgG avidity in patients who develop GDM. Also, the GDM group had decreased immune responses in Th1, Th2 and Th17 profiles, suggesting an association between GDM and the negative modulation of the humoral and cellular immune responses against T. gondii.

ADSCs stimulated by VEGF-C alleviate intestinal inflammation via dual mechanisms of enhancing lymphatic drainage by a VEGF-C/VEGFR-3-dependent mechanism and inhibiting the NF-κB pathway by the secretome

BackgroundAdipose-derived stem cells (ADSCs) have provided promising applications for Crohn’s disease (CD). However, the practical efficacy of ADSCs remains controversial, and their mechanism is still unclear. Based on the pathogenesis of dysregulated immune responses and abnormal lymphatic alterations in CD, vascular endothelial growth factor-C (VEGF-C) is thought to be a favourable growth factor to optimize ADSCs. We aimed to investigate the efficacy of VEGF-C-stimulated ADSCs and their dual mechanisms in both inhibiting inflammation “IN” and promoting inflammation “OUT” in the intestine.MethodsHuman stem cells isolated from adipose tissues were identified, pretreated with or without 100 ng/ml VEGF-C and analysed for the secretion of cell culture supernatants in vitro. Lymphatic endothelial cells (LECs) were treated with ADSCs-conditioned medium or co-cultured with ADSCs and VEGF-C stimulated ADSCs. Changes in LECs transmigration, and VEGF-C/VEGFR-3 mRNA levels were assessed by transwell chamber assay and qRT–PCR. ADSCs and VEGF-C-stimulated ADSCs were intraperitoneally injected into mice with TNBS-induced chronic colitis. ADSCs homing and lymphatic vessel density (LVD) were evaluated by immunofluorescence staining. Lymphatic drainage was assessed using Evans blue. Cytokines and growth factors expression was detected respectively by ELISA and qRT–PCR. The protein levels of VEGF-C/VEGFR-3-mediated downstream signals and the NF-κB pathway were assayed by western blot. Faecal microbiota was measured by 16S rRNA sequencing.ResultsADSCs stimulated with VEGF-C released higher levels of growth factors (VEGF-C, TGF-β1, and FGF-2) and lower expression of cytokines (IFN-γ and IL-6) in cell supernatants than ADSCs in vitro (all P < 0.05). Secretome released by VEGF-C stimulated ADSCs exhibited a stronger LEC migratory capability and led to elevated VEGF-C/VEGFR-3 expression, but these effects were markedly attenuated by VEGFR-3 inhibitor. VEGF-C-stimulated ADSCs homing to the inflamed colon and mesenteric lymph nodes (MLNs) can exert stronger efficacy in improving colitis symptoms, reducing inflammatory cell infiltration, and significantly enhancing lymphatic drainage. The mRNA levels and protein concentrations of anti-inflammatory cytokines and growth factors were markedly increased with decreased proinflammatory cytokines in the mice treated with VEGF-C-stimulated ADSCs. Systemic administration of VEGF-C-stimulated ADSCs upregulated the colonic VEGF-C/VEGFR-3 pathway and activated downstream AKT and ERK phosphorylation signalling, accompanied by decreased NF-κB p65 expression. A higher abundance of faecal p-Bacteroidetes and lower p-Firmicutes were detected in mice treated with VEGF-C-stimulated ADSCs (all P < 0.05).ConclusionVEGF-C-stimulated ADSCs improve chronic intestinal inflammation by promoting lymphatic drainage and enhancing paracrine signalling via activation of VEGF-C/VEGFR-3-mediated signalling and inhibition of the NF-κB pathway. Our study may provide a new insight into optimizing ADSCs treatment and investigating potential mechanisms in CD.

Effect of Porcine Colostral Exosomes on T Cells in the Peripheral Blood of Suckling Piglets.

Simple SummaryExosomes in porcine colostrum have gained attention as the possible key compounds involved in the growth and/or development of suckling piglets. In this study, peripheral blood mononuclear cells (PBMCs) from suckling piglets were cultured with or without milk-derived exosomes (control) in vitro. Porcine colostral exosomes increased the proportion of cytotoxic T (Tc) cells, while this phenomenon was not observed in PBMC whose endocytosis was inhibited. Moreover, exosome-treated PBMCs had a higher cytokine IL-2 concentration in the culture supernatant than the control. The present study demonstrated that porcine colostral exosomes could increase the Tc cell proportion in the peripheral blood of a suckling piglet, with the underlying mechanism believed to be the stimulation of IL-2 production in PBMCs via endocytosis.Growing evidence indicates that porcine colostral exosomes may contribute to the healthy development of piglets. Here, we evaluated in vitro the effect of porcine milk-derived exosomes, in particular colostral exosomes, on T cells in the peripheral blood of suckling piglets. A total of seven sows and thirteen suckling piglets were used. Peripheral blood mononuclear cells (PBMCs) from suckling piglets were cultured with or without milk-derived exosomes (control). Using flow cytometry, the proportion of each T cell subset in cultured PBMCs was analyzed three days post-incubation. PBMCs cultured with porcine colostral exosomes had a higher proportion of CD3+CD4−CD8+ T cells (cytotoxic T cells; Tc) than the control. However, exosomes induced no increase in the Tc cell population in PBMC whose endocytosis was inhibited. We further measured the concentrations of cytokines in the culture supernatant. Exosome-treated PBMCs had a higher cytokine IL-2 concentration than the control. The present study demonstrated that porcine colostral exosomes could increase the Tc cell proportion in the peripheral blood of suckling piglets, with the underlying mechanism believed to be the stimulation of IL-2 production in PBMCs via endocytosis. Moreover, our results suggested that porcine colostral exosomes were involved in the development of cellular immunity in suckling piglets.

Rapid Synergistic Biofilm Production of Pseudomonas and Candida on the Pulmonary Cell Surface and in Mice, a Possible Cause of Chronic Mixed Organismal Lung Lesions.

Due to the possible co-presence of Pseudomonas aeruginosa and Candida albicans (the most common nosocomial pathogens) in lungs, rapid interkingdom biofilm production is possible. As such, PA+CA produced more dominant biofilms on the pulmonary epithelial surface (NCI-H292) (confocal fluorescent extracellular matrix staining) with dominant psl upregulation, as demonstrated by polymerase chain reaction (PCR), after 8 h of experiments than PA alone. With a proteomic analysis, rhamnosyltransferase RhlB protein (Psl-associated quorum-sensing protein) was found to be among the high-abundance proteins in PA+CA than in PA biofilms, supporting psl-mediated biofilms in PA+CA on the cell surface. Additionally, PA+CA increased supernatant cytokines (IL-8 and IL-13, but not TNF-α, IL-6, and IL-10) with a similar upregulation of TLR-4, TLR-5, and TLR-9 (by PCR) compared with PA-stimulated cells. The intratracheal administration of PA+CA induced a greater severity of sepsis (serum creatinine, alanine transaminase, serum cytokines, and histology score) and prominent biofilms (fluorescent staining) with psl upregulation (PCR). In comparison with PA+CA biofilms on glass slides, PA+CA biofilms on biotic surfaces were more prominent (fluorescent staining). In conclusion, PA+CA induced Psl-predominant biofilms on the pulmonary cell surface and in mice with acute pneumonia, and these biofilms were more prominent than those induced by PA alone, highlighting the impact of Candida on rapid interkingdom biofilm production.

Immune Biomarkers in Blood from Sarcoma Patients: A Pilot Study.

The main role of the host immune system is to identify and eliminate cancer cells, which is a complex process, but it is not a fail-safe mechanism. Many sarcoma patients succumb to this disease despite treatments rendered. The aim of this pilot study was to compare the levels of CD4+ T-cells, T-regulatory (Treg) cells, and cytokines such as tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin-17A (IL-17A), and transforming growth factor-beta-1 (TGF-β1) in peripheral blood leukocytes of sarcoma patients and healthy controls. For gene expression studies, total ribonucleic acid (RNA) was extracted from peripheral blood leukocytes and genes that were differentially regulated in peripheral blood leukocytes of sarcoma patients compared with healthy controls were determined using a commercial T-helper cell differentiation quantitative polymerase chain reaction (qPCR) array. Flow cytometer analysis was performed on blood samples from 26 sarcoma patients and 10 healthy controls to identify the levels of CD4+ T-cells and T-reg cells. The level of cytokines in plasma and culture supernatant were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits. A marked reduction in the percentage of CD4+ T-cells (p = 0.037) and levels of TNF-α (p = 0.004) and IFN-γ (0.010) was observed in sarcoma patients. Gene expression analysis showed five genes (homeobox A10 (HOXA10), GATA binding protein 3 (GATA3), prostaglandin D2 receptor 2 (PTGDR2), thymocyte selection associated high mobility group box (TOX), and C-C motif chemokine receptor 3 (CCR3)) were dysregulated (p < 0.05) in sarcoma patients. This study suggests that T-helper-1 immune responses are reduced in sarcoma patients.

Concentration of anti-inflammatory cytokines in supernatants of peripheral blood cell cultures in children with autoimmune and infectious diseases

Cytokines belong to the class of signaling molecules, being involved into regulation of proliferation, differentiation and effector functions of immunocompetent cells. The ratio of pro- and anti-inflammatory cytokines is important for development of any inflammatory process. IL-1ra, IL-4, IL-10 are among the most important anti-inflammatory cytokines. Their function is to limit and suppress immune response in inflammatory processes of any etiology. In this respect, the aim of this study was to evaluate production of the anti-inflammatory cytokines IL-1ra, IL-4 and IL-10 by peripheral blood cells in children with autoimmune and infectious diseases. Patients and methods: Pediatric parients (2-17 years old) participated in the study including those with juvenile idiopathic arthritis (n = 101); unspecified reactive arthropathy (n = 24); systemic lupus erythematosus (SLE, n = 14); chronic viral hepatitis C (n = 24). 33 healthy children (n = 33) comprised the control group. Heparinized blood samples were diluted with glutamine-containing medium RPMI-1640. Control samples were not treated by any stimulants, and the stimulated samples were supplied with phytohemagglutinin (20 mkg/ml). The samples of diluted blood were incubated for 24 hours (37 C, 5% CO2). The supernates were frozen once. The concentrations of IL-1ra, IL-4 and IL-10 in these cell supernatants were determined by ELISA technique (Vector-Best, Russia). Results: It was found that the spontaneous production of anti-inflammatory cytokines (IL-1ra, IL-4 and IL-10) didnt differ, or was lower in the groups of patients with SLE, juvenile idiopathic arthritis and hepatitis C if compared with control group. SLE is an autoimmune disease, whereas juvenile arthritis is of mixed autoimmune-autoinflammatory etiology. Chronic hepatitis C is a viral disease, but autoimmune responses may manifest at the chronic stage of the disorder. Decreased production of anti-inflammatory cytokines could be an evidence for autoimmune mechanisms of these diseases, despite their different etiology. More intensive spontaneous production of IL-1ra and IL-10 in children with unspecified reactive arthropathy may suggest some compensatory reactions which inhibit development of inflammation in this disorder. IL-1ra, IL-4 and IL-10 production in stimulated cultures didnt differ between all groups of the patients, or it was lower in comparison with healthy children. Decrease cytokine production in groups of children with different diseases suggests exhausted functional reserve of immunocompetent cells caused by their chronic activation.

Evaluation of tuberculosis diagnostic biomarkers in immunocompromised hosts based on cytokine levels in QuantiFERON-TB Gold Plus

Tuberculosis (TB) remains a serious health concern globally. QuantiFERON-TB (QFT) is a diagnostic tool for TB detection, and its sensitivity is reduced in immunocompromised hosts with low T lymphocyte counts or abnormal T cell function. This study aimed to evaluate the correlation between T cell and cytokine levels in patients with active TB using QFT-Plus. Forty-five patients with active TB were enrolled, and the cytokines in QFT-Plus tube supernatants were quantified using the MAGPIX System. CD4+ T cell count negatively correlated with patient age (p < 0.001, r = −0.51). The levels of TB1-responsive interleukin-1 receptor antagonist (IL-1Ra) and IL-2 correlated with CD4+ T cell count, whereas the levels of TB2-responsive IL-1Ra and IFN-γ-induced protein 10 correlated with both CD4+ and CD8+ T cell counts. Cytokines that correlated with CD4+ and CD8+ T cell counts might not be suitable TB diagnostic biomarkers in immunocompromised hosts. Notably, cytokines that did not correlate with the T cell counts, such as monocyte chemoattractant protein-1, might be candidate biomarkers for TB in immunocompromised hosts. Our findings might help improve TB diagnosis, which could enable prompt treatment and minimize poor disease outcomes.

The Role of D2-like Dopaminergic Receptor in Dopamine-mediated Modulation of Th17-cells in Multiple Sclerosis.

Dopamine is one of the main mediators capable regulate the neuroimmune interaction and is involved in multiple sclerosis (MS) pathogenesis. The aim of this study was to clarify the role of dopamine and its receptors in modulation of Th17-cells in MS. 34 relapsing-remitting MS patients and 23 healthy subjects were examined. To assess the effect of dopamine on Th17-cells, CD4+ T-cells were cultured in the presence of dopamine and antagonist or agonist of D1- or D2-like dopaminergic receptors and stimulated with anti-CD3/CD28- microbeads. The levels of cytokines in supernatants were assessed by ELISA. Production of interleukin-17 (IL-17), interferon-γ (IFN-γ), granulocyte-colony stimulating factor (GM-CSF), and IL-21 by CD4+ T-cells as well as dopamine were comparable between the groups. Dopamine suppressed cytokine secretion by activated СD4+ T-cells in both groups. Blockade of D1-like dopaminergic receptor with a specific antagonist SCH23390 did not affect dopaminemediated cytokine suppression. In contrast, blockade of D2-like dopaminergic receptor by sulpiride decreased dopamine's inhibitory effect on IL-17 secretion in both groups and GM-CSF and IL-21 production in MS patients. Blockade of D1-like dopaminergic receptor directly inhibited IL-17, IFN- γ, GM-CSF in both groups and IL-21 production in healthy subjects, while blockade of D2-like dopaminergic receptor had no effect on cytokine secretion. Finally, activation of D2-like dopaminergic receptor with a specific agonist quinpirole decreased cytokine production in both groups. These data suggest an inhibitory role of dopamine on Th17-cells in MS, which could be mediated by the activation of the D2-like dopaminergic receptor.

Immunopharmacological Properties of VitD3: 1, 25VitD3 Modulates Regulatory T Cells and Th17 Cells and the Cytokine Balance in PBMCs from Women with Unexplained Recurrent Spontaneous Abortion (URSA).

VitD3 may contribute to a successful pregnancy through modulation of immune responses. Therefore, VitD3 deficiency may have a role in the immunopathogenesis of unexplained recurrent spontaneous abortion (URSA). However, the mechanisms of immunomodulatory actions of VitD3 in decreasing the risk of recurrent spontaneous abortion have not been understood well. The purpose of this research was to investigate the influence of 1,25VitD3 on regulatory T cells /Th17 axis, the gene expressions and concentrations of related cytokines including, TGF-β, IL-10, IL-6, IL-23, and IL-17A in peripheral blood mononuclear cells (PBMCs) of healthy women as a control group and women with URSA. Isolation of PBMCs was performed from peripheral blood of the subjects of the studied groups (20 women with URSA as a case group, and 20 control women). The effects of 1,25VitD3 (50 nM, for 24 hours) on the studied parameters were evaluated and were compared to the positive and negative controls in vitro. Flow cytometry analysis was used to determine the percentages of regulatory T cells and Th17 cells. For gene expression measurement and cytokines assay, Realtime PCR and ELISA were carried out. The proportion of regulatory T cells was markedly lower, while the proportion of Th17 cells in women with URSA was considerably higher than in the control group (P=0.01, P=0.01). The ratio of the frequency of Tregs to the baseline (1,25VitD3/Untreated) increased, while the ratio of the frequency of Th17 cells to the baseline decreased in women with URSA relative to the controls (P= 0.01, P=0.04). 1,25VitD3 increased IL-10 expressions at both the protein and mRNA levels in PBMCs in women with URSA relative to the control group (P=0.0001, P=0.04). TGF-β levels in the cultured supernatants decreased significantly in the case group in the presence of 1,25Vit- D3 relative to the controls (P=0.03). 1,25VitD3 treatment also significantly decreased gene expressions of IL-6, IL-17A, and IL-23 in PBMCs of women with URSA (P=0.01, P=0.001, P=0.0005), as well as the levels of those cytokines in cell culture supernatants (P=0.03, P=0.02, P=0.01, respectively) in women with URSA relative to the controls. According to the findings of this research, modulation of immune responses by 1,25VitD3 is accomplished by strengthening Tregs function and inhibiting inflammatory responses of Th17 cells, which may have a positive impact on pregnancy outcome. Thus, as an immunomodulating agent, VitD3 may be effective in reducing the risk of URSA.

YY1 alleviates lupus nephritis-induced renal injury by reducing the Th17/Treg cell ratio via the IFN-γ/Fra2 axis

AbstractLupus nephritis (LN) is associated with extensive injury and nephron loss in the afflicted kidney. Evidence has revealed the involvement of dysregulated Yin Yang 1 (YY1), a reported inflammatory modulator, in LN-induced kidney injury, and our microarray profile identified downregulated YY1 expression. Therefore, this study explored the functional relevance and mechanism of YY1 in LN-induced kidney injury. LN was modeled in mice by intraperitoneal injection of pristane, and Jurkat cells (CD41 human T lymphocytes) were activated with TNF-α to mimic the inflammatory environment found in LN. The expression patterns of YY1 and bioinformatics predictions of the downstream factor IFN-γ were confirmed in renal tissues from the mice with LN using qRT-PCR and Western blot analyses. The contents of proinflammatory cytokines in mouse serum samples and cell supernatants were determined using enzyme-linked immunosorbent assays (ELISAs). Ectopic expression and depletion approaches were subsequently used in vitro and in vivo to examine the effects of the YY1/IFN-γ/Fra2/PARP-1/FOXO1 axis on TNF-α-induced inflammation and LN-induced kidney injury. The results showed downregulated expression of YY1 and FOXO1 in the kidney tissues of the mice with LN. Increased proinflammatory factor production was observed in the mice with LN and TNF-α-treated Jurkat cell supernatant, accompanied by increased cell apoptosis and a high ratio of Th17/Treg cells, and these effects were reversed by YY1 restoration. YY1 was further shown to inhibit IFN-γ expression and thereby downregulate Fra2 expression. Fra2 depletion then inhibited PARP-1 expression and promoted FOXO1 expression to suppress cell apoptosis and the release of inflammatory factors. Collectively, our findings revealed that YY1 may alleviate LN-induced renal injury via the IFN-γ/Fra2/PARP-1/FOXO1 axis.