Cytokine Levels In Supernatants Research Articles

Elucidating the effect of pro and anti-inflammatory recombinant cytokines TNF-α and TGF-β in tuberculosis

Tuberculosis (TB) is a leading cause of death caused by Mycobacterium tuberculosis (M tb) and about one-third of the world’s population is infected with TB. The household contacts of TB patients are at higher risk towards TB than general population. During the initial stages of infection, pro and anti-inflammatory cytokines are induced by innate immune cells, and the course of infection is influenced by general cytokine environment. These cytokines play an important role in the regulation of host immune responses against M tb. Therefore, it is necessary to understand the cytokines role in the immune mechanism to evaluate the correlation between the disease and the immune responses involved in TB. Our current study has focused on recombinant cytokines to understand their effects on cell proliferation and cytokine levels in culture supernatants. We observed that the mean proliferative responses to recombinant rhTNF-α were high and TNF-α levels were significantly low in APTB patients compared to their HHC and HC with p < 0.0375 and p < 0.0051 respectively. The mean proliferative responses to recombinant rhTGF-β were significantly low in APTB when compared to HHC and HC with p < 0.0376, p < 0.0247 respectively, and TGF-β levels were also significantly low in APTB and HHC compared to HC with p < 0.0468 and p < 0.0001 respectively. The lower cytokine secretions in culture supernatants might be due the autocrine signaling by recombinant cytokines towards the inflammatory response. Further, to validate these recombinant cytokines, a larger sample size could aid in identifying individuals at high risk for TB.

The Cytokine Levels of Cord Blood- and Wharton's Jelly-Derived Mesenchymal Stem Cells from Early to Late Passages.

Mesenchymal stem cells (MSCs) are promising for clinical studies owing to their self-renewal, multipotency, trophic, and immunomodulatory properties. This study aimed to investigate the cytokine levels of human umbilical cord blood (CB) and Wharton's Jelly-(WJ) derived MSCs relevant to immune modulation on different passage levels in vitro. Umbilical CB MSCs were isolated using the ficoll-paque gradient method, and WJ-MSCs were isolated by the explant method. After isolation, the MSCs were characterized using flow cytometry. The supernatant cytokine levels (interferon-gamma (IFN-γ), interleukin 4 (IL-4), interleukin 17 (IL-17)) of MSCs at each passage were evaluated using the ELISA assay. MSCs exhibited different cytokine levels with each passage number. In WJ-MSC culture supernatants, IL-17 levels significantly increased at P4 and P5 compared to the first passage (p < 0.005), while the other passages showed a decrease. IFN-γ levels increased at passage P1 and P4 and decreased at other passages (p < 0.005). IL-4 levels significantly increased only at passage P3 (p < 0.005). In CB-MSC culture supernatants, IL-17 and IL-4 cytokines decreased compared to P0, while IFN-γ cytokine increased from P0 (p < 0.005). The changing ratio of cytokine levelsfor both CB-MSCs and WJ-MSCs were similarly maintained from early to late passages. More research is needed to understand the immunomodulatory functions of MSCs.

Inflammatory Response to Wear Particles: Comparisons Between Zirconia and Titanium In Vitro.

To compare the inflammatory responses of peripheral blood mononuclear cells (PBMCs) subjected to titanium (Ti) and/or zirconia (Zr) particles while growing on Ti or Zr discs. In total, 240 discs were fabricated at a size of 2 mm in height and 5 mm in diameter. Of the 240 discs, 120 discs were made of Ti (commercially pure [CP] grade 4), and 120 discs were made of Zr (3 mol% yttria-stabilized zirconia polycrystals [3Y-TZP]). The PBMCs were cultured on the two-disc materials, and particles with a size up to 20 mm Ti (99.5% Ti) and 0.1 to 0.2 mm Zr (3Y-TZP) were added to the cultures. The concentration levels of inflammatory cytokines in culture supernatants were measured through Bio-Plex assay (Bio-Rad Laboratories), and light microscopic analysis was performed to detect cell attachment and characterize particle shape and cell-particle interaction. The inflammatory responses of PBMCs were generally higher when cells were cultured on a Ti surface compared to a Zr surface. In addition, higher cytokine levels were seen when cells were cultured in the presence of Ti particles compared to Zr particles when no discs were used. However, there were only significantly increased levels for three cytokines (MCP-1, IFN-γ, and TNF-α) when particles were added to Ti discs. Higher release of neutrophil extracellular traps (NETs) from neutrophils were seen in presence of Zr particles compared to Ti particles. A reduction in cell death was observed in the presence of Zr particles compared to Ti particles and unstimulated control samples. The type of growth material and presence of particle affects PBMCs in vitro. Cells seeded on Ti discs and together with Ti particles generated higher levels of inflammatory cytokines compared to the Zr counterparts.

Characterization of CD4+ t cell subsets during Chlamydia muridarum genital infection

Characterization of CD4+ T cell subsets in a stressed mouse model during Chlamydia muridarum genital infection. Katrina Davis, Tesfaye Belay. Department of Applied Sciences and Mathematics. Chlamydia genital infection caused by Chlamydia trachomatis is highly prevalent in low socioeconomic status populations. Whether stress is a risk factor for the high incidence of chlamydia remains to be investigated. CD4+ T cells are the major immune cells that fight chlamydia genital infection; however, the dynamics of CD4+ T cells during stressful conditions and chlamydia infection are unclear. The profiles of CD4+ T cell signature cytokines: T helper 1 (IL-12, Interferon-gamma (IFN-γ), Thelper2 (IL-4, IL-5, IL-13), Thelper17(IL-17), and Treg (IL-10). Mice were stressed for 5 minutes daily for 21 days and infected with 1x 105 IFU/ml of Chlamydia muridarum intravaginally. Non-stressed mice were infected as controls. Mice were sacrificed 7 days post-infection, the spleen and lymph nodes were harvested, and T cells were purified and proliferated for 72 h. Determination of the levels of cytokines in culture supernatants of T cells from stressed and non-stressed mice are underway for poster presentation. Supported by the NIH grant # 1R15AI124156-01 awarded to BSU

Neutralizing antibody titers against D8 genotype and persistence of measles humoral and cell-mediated immunity eight years after the first dose of measles, mumps, and rubella vaccine in Brazilian children

Objective: Assess the level of measles vaccine-induced neutralizing antibodies against the D8 genotype and the persistence of humoral and cell-mediated immunity in children who received their first dose of the measles, mumps, and rubella vaccine eight years previously. Methods: Measles-specific IgG and neutralizing antibodies were determined in serum using ELISA and plaque reduction neutralization test, respectively. Cellular response was evaluated from peripheral blood mononuclear cells (PBMC). IFN-γ-secreting cells, memory B and T cells, and immunological mediators were assayed by ELISpot, flow cytometry, and multiplex liquid microarray assay, respectively. Results: Antibody concentrations declined over time; however, the vaccine-induced neutralizing antibodies’ effect against D8 and vaccinal genotypes persisted. PBMC stimulated with the vaccine virus exhibited specific IFN- γ-measles-secreting responses in most participants. Participants with high levels of neutralizing antibodies showed a higher proportion of activated B cells compared to participants with low levels of neutralizing antibodies, while proportions of memory CD4+ and CD8+ T cells were similar between these groups. PBMC supernatant cytokine levels showed a significant difference between stimulated and non-stimulated conditions for IL-2, TNF-α, IL-10, and CXCL10. Conclusion: Despite the decline in antibody concentrations over time, the participants still demonstrated neutralizing capacity against the measles D8 genotype five to eight years after the second dose of the measles, mumps, and rubella vaccine. Additionally, most of the enrolled children exhibited cell-mediated immunity responses to measles virus stimulation.

NETosis Secondary to the Use of Levamisole-Adulterated Cocaine: A Likely Underlying Mechanism of Vasculopathy.

Since 2010, several cases of a new vasculopathy induced by the use of levamisole-adulterated cocaine (LAC) have been reported. This vasculopathy is characterized by retiform purpura, earlobe necrosis, multisystem compromise, and multiple autoantibodies. Given its similarity to antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, LAC-associated vasculopathy is postulated to be mediated by pathophysiologic processes resulting from neutrophil cell death by NETosis, a phenomenon previously described in ANCA vasculitis. This study tries to establish the presence of NETosis induced by cocaine, levamisole, or both. Methodology. Neutrophils were isolated from the peripheral blood of healthy controls by Ficoll-Hystopaque density gradient centrifugation followed by dextran sedimentation. Cell viability and purity were evaluated by flow cytometry after staining with PI/DiOC6 and labeling with fluorescent anti-CD45/anti-CD3 monoclonal antibodies (mAbs), respectively. Neutrophils were exposed to levamisole, cocaine, a cocaine-levamisole mixture, and sera pools from healthy controls and patients with LAC-associated vasculopathy. NETosis was then assessed by flow cytometry after staining cells with Sytox Green, Hoechst-33342, and fluorescent antineutrophil elastase (NE) and antimyeloperoxidase (MPO) mAbs. In addition, NETosis was morphologically confirmed by fluorescence microscopy. Proinflammatory cytokine levels in culture supernatants and reactive oxygen species (ROS) synthesis were determined by flow cytometry. The involvement of calcium and muscarinic receptors in cell death induction was evaluated in parallel experiments carried out in the presence of 1,2-bis (o-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid (BAPTA) and hyoscine butylbromide (HBB), their respective inhibitors. Cocaine, levamisole, and a cocaine-levamisole mixture induced neutrophil cell death. DNA/MPO extrusion and cell morphology patterns were consistent with NETosis. Neither proinflammatory cytokines nor ROS behaved as proNETotic factors. Preliminary results suggested that muscarinic receptors and calcium-dependent signals were involved in LAC-induced NETosis. Cocaine, levamisole, and a cocaine-levamisole mixture can induce NETosis through mechanisms involving muscarinic receptors and calcium-dependent pathways.

Cardamonin decreases inflammatory mediator expression in IL-1β-stimulated human periodontal ligament cells.

Cardamonin is classified as a natural chalcone, and has been reported to possess various bioactive effects. However, there have been limited attempts to utilize cardamonin in the treatment of periodontitis. This study aimed to investigate whether cardamonin has anti-inflammatory effects on human periodontal ligament cells (HPDLCs), which are a component cell of periodontal tissue. Specifically, the study seeks to determine whether cardamonin affects the expression of inflammatory mediators, such as cytokines and adhesion molecules, induced by interleukin-1β (IL-1β) in HPDLCs, as well as the signaling pathways activated by IL-1β. Cytokine and chemokine levels in supernatants of HPDLCs were measured by ELISA. Western blot analysis was used to measure protein expression and signal transduction pathway activation in HPDLCs. We found that IL-1β-induced CC chemokine ligand (CCL)2, CCL5, CCL20, CXC-chemokine ligand (CXCL)10, and interleukin (IL)-6 production and intercellular adhesion molecule (ICAM)-1 and cyclooxygenase (COX)-2 expression in HPDLCs were suppressed by cardamonin treatment. We also found that cardamonin suppressed IL-1β-activated nuclear factor (NF)-κB pathway, and the phosphorylation of signal transducer and activator of transcription (STAT)3. Furthermore, cardamonin treatment enhanced the expression of the antioxidant enzymes, heme oxygenase (HO)-1 and NAD(P)H dehydrogenase [quinone] 1 (NQO1), in HPDLCs. In this study, we found that cardamonin could suppress the production of inflammatory mediators in HPDLCs as well as the activation of several signaling pathways induced by IL-1β treatment.

Therapeutic effect of Echinococcus granulosus cyst fluid on bacterial sepsis in mice

BackgroundThe primary pathophysiological process of sepsis is to stimulate a massive release of inflammatory mediators to trigger systemic inflammatory response syndrome (SIRS), the major cause of multi-organ dysfunction and death. Like other helminths, Echinococcus granulosus induces host immunomodulation. We sought to determine whether E. granulosus cyst fluid (EgCF) displays a therapeutic effect on sepsis-induced inflammation and tissue damage in a mouse model.MethodsThe anti-inflammatory effects of EgCF were determined by in vitro culture with bone marrow-derived macrophages (BMDMs) and in vivo treatment of BALB/C mice with cecal ligation and puncture (CLP)-induced sepsis. The macrophage phenotypes were determined by flow cytometry, and the levels of cytokines in cell supernatants or in sera of mice were measured (ELISA). The therapeutic effect of EgCF on sepsis was evaluated by observing the survival rates of mice for 72 h after CLP, and the pathological injury to the liver, kidney, and lung was measured under a microscope. The expression of TLR-2/MyD88 in tissues was measured by western blot to determine whether TLR-2/MyD88 is involved in the sepsis-induced inflammatory signaling pathway.ResultsIn vitro culture with BMDMs showed that EgCF promoted macrophage polarization to M2 type and inhibited lipopolysaccharide (LPS)-induced M1 macrophages. EgCF treatment provided significant therapeutic effects on CLP-induced sepsis in mice, with increased survival rates and alleviation of tissue injury. The EgCF conferred therapeutic efficacy was associated with upregulated anti-inflammatory cytokines (IL-10 and TGF-β) and reduced pro-inflammatory cytokines (TNF-α and INF-γ). Treatment with EgCF induced Arg-1-expressed M2, and inhibited iNOS-expressed M1 macrophages. The expression of TLR-2 and MyD88 in EgCF-treated mice was reduced.ConclusionsThe results demonstrated that EgCF confers a therapeutic effect on sepsis by inhibiting the production of pro-inflammatory cytokines and inducing regulatory cytokines. The anti-inflammatory effect of EgCF is carried out possibly through inducing macrophage polarization from pro-inflammatory M1 to regulatory M2 phenotype to reduce excessive inflammation of sepsis and subsequent multi-organ damage. The role of EgCF in regulating macrophage polarization may be achieved by inhibiting the TLR2/MyD88 signaling pathway.Graphical

Neonatal immune cells have heightened responses following in-utero exposure to chorioamnionitis or COVID-19.

Chorioamnionitis alters neonatal immune responses. Gestational COVID-19 infection is associated with adverse pregnancy outcomes, but its impact on neonatal immunity is unclear. We hypothesized that gestational COVID-19 exposure would result in exaggerated neonatal immune responses, similar to chorioamnionitis-exposed neonates. Term umbilical cord blood mononuclear cells (CBMCs) were isolated from neonates exposed to chorioamnionitis, gestational COVID-19 or unexposed controls. CBMCs were cultured and stimulated with heat-killed Escherichia coli, Streptococcus agalactiae or Staphylococcus epidermidis. A multiplexed protein assay was used to measure cytokine levels in cell culture supernatants and flow cytometry was used to evaluate cellular-level cytokine expression. Both chorioamnionitis-exposed and COVID-19 exposed CBMCs demonstrated upregulation of IL-1β and IL-6 compared to unexposed CBMCs, while only COVID-19 exposure resulted in IL-8 upregulation. There were no differences between chorioamnionitis-exposed and COVID-19 exposed CBMCs when these groups were directly compared. Flow cytometry demonstrated immune cell subset specific differences in cytokine expression between the exposure groups. The fetal/neonatal response to maternal inflammation differed based on immune cell subset and etiology of inflammation, but the global neonatal cytokine responses were similar between exposure groups. This suggests that targeting perinatal inflammation rather than the specific etiology may be a possible therapeutic approach. Neonatal immune cells have similar pathogen-associated global cytokine responses, but different cell-level immune responses, following in-utero exposure to chorioamnionitis or COVID-19. This is the first study to directly compare immune responses between neonates exposed to chorioamnionitis and COVID-19. This suggests that the fetal/neonatal cellular response to perinatal inflammation differs based on the etiology and severity of maternal inflammation, but still results in a similar overall inflammatory profile regardless of the cause of perinatal inflammation.

Boost of innate immunity cytokines as biomarkers of response to extracorporeal photopheresis in patients with leukaemic cutaneous T-cell lymphoma.

Extracorporeal photopheresis (ECP) has emerged as a systemic first-line immunomodulatory therapy in leukaemic cutaneous T-cell lymphoma (L-CTCL) and is now beginning to be utilized in other T-cell-mediated diseases. Although ECP has been used for nearly 30 years, its mechanisms of action are not sufficiently understood, and biomarkers for response are scarce. We aimed to investigate the immunomodulatory effects of ECP on cytokine secretion patterns in patients with L-CTCL, to help elucidate its mechanism of action. A total of 25 patients with L-CTCL and 15 healthy donors (HDs) were enrolled in this retrospective cohort study. Concentrations of 22 cytokines were simultaneously quantified by using multiplex bead-based immunoassays. Neoplastic cells in patients' blood were evaluated by flow cytometry. Firstly, we observed a distinct cytokine profile pattern difference between L-CTCLs and HDs. There was a significant loss of tumour necrosis factor (TNF)-α, and significant increase of interleukins (IL)-9, IL-12 and IL-13 in the sera of patients with L-CTCL compared with HDs. Secondly, patients with L-CTCL who received ECP were classified as treatment responders and nonresponders according to the quantitative reduction of malignant burden in their blood. We evaluated cytokine levels in culture supernatants from patients' peripheral blood mononuclear cells (PBMCs) at baseline and 27 weeks after ECP initiation. Strikingly, PBMCs purified from ECP responders released statistically higher concentrations of innate immune cytokines IL-1α, IL-1β, granulocyte-macrophage colony-stimulating factor (GM-CSF) and TNF-α in comparison with ECP nonresponders. In parallel, responders showed clearance of erythema, reduction of malignant clonal T cells in the blood, and a potent boost of relevant innate immune cytokines in individual patients with L-CTCL. Taken together, our results demonstrate that ECP stimulates the innate immune network, and facilitates redirection of the tumour-biased immunosuppressive microenvironment towards proactive antitumour immune responses. The alterations of IL-1α, IL-1β, GM-CSF and TNF-α can be used as biomarkers of response to ECP in patients with L-CTCL.

Ophiopogonin A attenuates acute lung inflammation in Klebsiella pneumonia mice by regulating RELA (v-rel reticuloendotheliosis viral oncogene homolog A (avian))

Ophiopogonin are natural products from the roots of Ophiopogon japonicas with various pharmacological activities. This study explored the mechanism of ophiopogonin A to alleviate the acute inflammation of the lungs of Klebsiella pneumonia. 64 rats were equally assigned into blank control group, injury model group (intratracheal injection of Klebsiella pneumonia to establish Klebsiella pneumonia model), Ophiopogonin A group and Ophiopogonin A+RELA group (combined group, on the basis of injury model group, received RELA mediated ophiopogonin A), with the dose of 10 mg/kg. The histopathological damage was observed under the microscope after HE staining, as well as inflammatory cells in bronchoalveolar lavage fluid (BALF), cytokine levels, and expression of RELA. After HE staining, compared with injury model group, bronchial epithelium structure in Ophiopogonin A group and combination group were relatively intact, the degree of infiltration of inflammatory cells was reduced, and the thickness of alveolar septum was reduced, especially in combination group. The pathological scores of Ophiopogonin A group and combination group were significantly lower, with a further lower score in combination group. The number of inflammatory cells in BALF of rats in Ophiopogonin A group and combination group were significantly reduced, especially in combination group. Similarly, cytokine levels in BALF supernatant and serum of rats in treatment group were reduced, especially in combination group (P &lt; 0.05). Ophiopogonin A could down-regulate RELA, which was further reduced in combination group. In conclusion, ophiopogonin A can reduce the invasion and pathological damage of pathogens to lung tissue and exert an anti-inflammatory ability.

1496-P: Resatorvid, an Inhibitor of Toll-Like Receptor-4, Protects Allogenic Islets from Inflammatory and Immune Response

Purpose: Loss of islet mass by instant blood mediated inflammatory reaction (IBMIR) and immunological rejection are major hurdles currently facing the field of islet transplantation. Toll-like receptor 4 (TLR4) plays a central role in inflammation and shaping adaptive immune response. We investigated the protective effects of resatorvid (TAK-242) using in vitro models of islet transplantation. Methods: Soluble TAK-242 or TAK-242 conjugated to the surface of islets utilizing copper-free click chemistry with a cleavable chemical linker were used. Islets were mixed with allogenic blood for a period of 3 hours and plasma levels of miRNA-375 and miRNA-200c were analyzed by RT-qPCR. Allogenic peripheral blood mononuclear cells were cultured with islets. T cell and monocyte activation were monitored by flow cytometry. Cytokine and miRNA levels in culture supernatant were measured by Luminex assay and RT-qPCR respectively. Results: The presence of TAK-242 in soluble form as well as released by cleavable linker from islet surface significantly reduced the release of miRNA-375 (p&amp;lt;0.01) and miRNA-200c (p&amp;lt;0.01) indicating less islet damage. In allogenic co-culture of islets and PBMCs, TAK-242 inhibited the expression of CD69 in T cells as well as decreased (p&amp;lt;0.05) the expression of CD86 and CD80 in monocytes. The production of cytokines IL-1β (p&amp;lt;0.001), IL-6 (p=0.01), IL-8 (p&amp;lt;0.05), IP10 (p&amp;lt;0.05), MCP-1 (p&amp;lt;0.001) and TNFα (p=0.005) in culture supernatant was decreased in the presence of soluble TAK-242 as well as TAK-242 released from islet surfaces. Conclusions: Inhibition of TLR4 using small molecule, TAK-242, protected islets from innate damage due to IBMIR as well as adaptive responses of activation of T cells and monocytes. These results validate the future study of inhibition of TLR4 using in vivo transplant model(s) to further investigate the efficacy of TLR4 inhibition to improve islet transplant survival and function. Disclosure J.D.Mattke: None. B.Naziruddin: None. B.Kane: None.

Naringenin Impedes the Differentiation of Mouse Hematopoietic Stem Cells Derived from Bone Marrow into Mature Dendritic Cells, thereby Prolonging Allograft Survival.

The use of immature dendritic cells (imDCs) to induce donor-specific immunotolerance following in vivo stimulation is limited by their low rate of induction and their tendency to undergo maturation. We derived imDCs from bone marrow hematopoietic stem cells (HSCs-imDCs). We then tested the ability of naringenin (Nar) to impede the maturation of HSCs-imDCs for inducing transplantation immune tolerance. HSCs derived from bone marrow were collected and induced to differentiate into imDCs by treating with Nar (Nar-HSCs-imDCs). Flow cytometry was used to evaluate DC surface markers, apoptosis, and endocytic ability. The ability of DCs to influence the in vitro proliferation of T cells and of regulatory T cells (Tregs) was analyzed by mixed lymphocyte reaction assays. Enzyme-linked immunoassays were used to quantify cytokine levels in supernatants from co-cultured DCs and Tregs, as well as in the serum of experimental animals. The level of immunotolerance induced by Nar-HSCs-imDCs was evaluated by skin grafting in recipient Balb/c mice, while the Kaplan-Meier method was used to statistically evaluate graft survival. Compared with HSC-imDCs, Nar-HSCs-imDCs showed higher expression of cluster of differentiation 11c (CD11c), but lower expression levels of CD80, CD86, and major histocompatibility complex class II. Nar-HSCs-imDCs also showed stronger inhibition of T cells and higher Treg cell proliferation. Interleukin 2 (IL-2) and interferon gamma levels were downregulated in Nar-HSCs-imDCs, whereas IL-4, IL-10, and transforming growth factor beta levels were upregulated. The rate of apoptosis and endocytic capacity of Nar-HSCs-DCs increased significantly after treatment with lipopolysaccharide. HSCs-imDCs or Nar-HSCs-imDCs were injected into Balb/c mice via the tail vein 7 days before skin grafting. Significantly reduced donor-specific CD4+ T cells and induced proliferation of CD4+CD25+FoxP3+ Treg cells were observed in the spleen of mice from the Nar-HSCs-imDCs group, especially at a dose of 106 Nar-HSCs-imDCs. The latter group also showed significantly prolonged survival of skin grafts. Nar-HSCs-imDCs markedly improved the acceptance of organ allografts, offering a potentially new strategy for inducing immune tolerance in transplantation.

Cytokine production profiles of immune cells of stressed beta2-adrenergic receptor knockout mouse model during Chlamydia muridarumgenital infection

Abstract Chlamydia genital infection by Chlamydia trachomatisis the most common bacterial sexually transmitted disease worldwide. The relationships between stress and chlamydia genital infection remain unknown. Beta2-adrenergic receptor(β2-AR), the major receptor of the stress hormone norepinephrine (NE), is known to impair the function of immune cells. Stress was induced by immersing mice in cold water for five minutes daily for 21 days before infection. This study determined profiles of immune cells of stressed β2-AR KO and stressed WT C57BL/6J after 48 h of Chlamydia muridarumgenital infection. Splenic T cells and differentiated bone marrow-derived macrophages and dendric cells (DCs) were purified by negative selection and stimulated by seeding T cells on anti-CD3/CD28 antibodies coated wells and by treating macrophages and DCs with LPS. After 48 h proliferation, cytokine levels in culture supernatants of the cells were determined using ELISA. Results show increased IFN-γ and IL-1β secretions but deceased IL-4 secretion by CD4+ T cells of β2-AR KO compared to WT (p&amp;lt;0.05). The secretion of IL-13, IL-5, and IL-10 but not IL-23 in CD4 T cells of stressed β2-AR KO mice was two-fold lower than that of stressed WT. TNF-α and IL-1β secretions in macrophages and DCs or IL-12 secretion in DCs was high in stressed β2-AR KO compared to WT (p&amp;lt;0.05). Overall, this study provides insights into the β2-AR signaling pathway’s role in suppressing the secretion of protective cytokines suggesting that inhibiting β2-AR signaling pathway may restore the function of immune cells during chlamydia genital infection. However, further study is warranted. This work was funded by NIH grant # 1R15AI124156-01 awarded to Bluefield State University

T-Helper 22 Cell Type Responses to Nickel in Contact Allergic Subjects Are Associated with T-Helper 1, T-Helper 2, and T-Helper 17 Cell Cytokine Profile Responses and Patch Test Reactivity

Introduction: Contact allergy to nickel (Ni) is a delayed-type hypersensitivity reaction mediated by Ni-reactive T cells producing the hallmark cytokines of several T-helper cell (Th) populations including IFN-γ (Th1), IL-4, IL-5 and IL-13 (Th2), and IL-17A (Th17). IL-22-expressing CD4+ cells, which could be either Th17 co-expressing IL-22 or Th22, expressing IL-22 in the absence of IL-17A, have also been found in Ni-provoked skin of allergic subjects. It has been unclear if Ni-reactive T cells consist of distinct Th cell type populations or if they secrete a mix of Th cell hallmark cytokines. The aim herein was to assess if cellular cytokine responses to Ni, in ex vivo-stimulated peripheral blood mononuclear cells (PBMCs) from Ni-allergic subjects, include not only Th1, Th2, and Th17 but also Th22 hallmark cytokines and to define if the cytokines are produced by distinct cell populations representing different Th profiles. Methods: PBMC from Ni-allergic subjects (n = 15) with different degrees of patch test reactivity and non-allergic controls (n = 5) were in vitro stimulated with Ni. Cytokine levels in PBMC supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA) (IFN-γ, IL-2, IL-3, IL-5, IL-6, IL-13, IL-17A, IL-22, and IL-31). FluoroSpot was used to assess if individual Ni-reactive cells produced single, or combinations of, cytokines representing different Th profiles. Cytokine combinations analyzed were IL-17A/IL-22/IFN-γ, IL-5/IL-17A/IFN-γ, IL-13/IL-22/IFN-γ, and IL-5/IL-13. Results: IL-22 as well as all other cytokines measured by ELISA were induced by Ni at higher levels in PBMC from allergic versus non-allergic subjects, with higher levels being associated with stronger patch test reactivity. The levels of most Ni-induced cytokines were positively correlated with each other; IL-2 displayed the highest correlation with other cytokines and IL-6 the lowest. FluoroSpot analysis showed that Th signature cytokines, IFN-γ (Th1), IL-5 and IL-13 (Th2), IL-17A (Th17), and IL-22 (Th22), were almost exclusively produced by distinct cell populations. Conclusion: Distinct Th cell populations, including Ni-reactive cells displaying Th1, Th2, Th17, and Th22 cytokine profiles, are all increased in PBMC from Ni-allergic subjects and positively associated with patch test reactivity. The relevance of these different Th profile populations for the up- or down-regulation of inflammatory reactions in the skin of Ni-allergic subjects remains to be clarified.

Blockade of neutrophil extracellular traps ameliorates toluene diisocyanate-induced steroid-resistant asthma.

Toluene diisocyanate (TDI)-induced asthma is characterized by mixed inflammation dominated by neutrophils, and is refractory to steroid treatment. Neutrophil extracellular traps (NETs) play an important role in severe asthma, but their role in TDI-induced asthma models is unclear. This study focused on the role and mechanism of NETs in steroid-resistant TDI-induced asthma. Induced sputum was collected from 85 asthmatic patients and 25 healthy controls to detect eDNA. A murine TDI-induced asthma model was prepared, and asthmatic mice were given dexamethasone or DNase I. In vitro, the human bronchial epithelial cell line HBE was stimulated with NETs or TDI-human serum albumin (TDI-HSA). Asthma patients had higher sputum eDNA compared to healthy subjects. In asthma patients, eDNA was positively correlated with sputum neutrophils, and negatively correlated with FEV1%predicted. Airway inflammation, airway reactivity, Th2 cytokine levels in lymph supernatant, and levels of NETs were significantly increased in the TDI-induced asthmatic mice. These increases were suppressed by DNase I, but not by dexamethasone. Inhibition of NETs improved interleukin (IL)-8 and MKP1 mRNA expression, and reduced phosphorylation of GR-S226 induced by TDI. Inhibition of NETs improved airway epithelial barrier disruption, as well as p38 and ERK signaling pathways in TDI-induced asthmatic mice. In vitro, NETs promoted the expression of IL-8 mRNA in HBE cells, and reduced the expression of MKP1. IL-8 elevation induced by NETs was suppressed by a p38 inhibitor or ERK inhibitor, but not by dexamethasone. Pretreatment with RAGE inhibitor reduced NETs induced p38/ERK phosphorylation and IL-8 levels in HBE cells. Our data suggest that targeting NETs might effectively improved TDI-induced airway inflammation and airway epithelial barrier function. This may potentially be a treatment for patients with steroid-resistance asthma.

Optimal LC-MS metabolomic profiling reveals emergent changes to monocyte metabolism in response to lipopolysaccharide.

Immunometabolism examines the links between immune cell function and metabolism. Dysregulation of immune cell metabolism is now an established feature of innate immune cell activation. Advances in liquid chromatography mass spectrometry (LC-MS) technologies have allowed discovery of unique insights into cellular metabolomics. Here we have studied and compared different sample preparation techniques and data normalisation methods described in the literature when applied to metabolomic profiling of human monocytes. Primary monocytes stimulated with lipopolysaccharide (LPS) for four hours was used as a study model. Monocytes (n=24) were freshly isolated from whole blood and stimulated for four hours with lipopolysaccharide (LPS). A methanol-based extraction protocol was developed and metabolomic profiling carried out using a Hydrophilic Interaction Liquid Chromatography (HILIC) LC-MS method. Data analysis pipelines used both targeted and untargeted approaches, and over 40 different data normalisation techniques to account for technical and biological variation were examined. Cytokine levels in supernatants were measured by ELISA. This method provided broad coverage of the monocyte metabolome. The most efficient and consistent normalisation method was measurement of residual protein in the metabolite fraction, which was further validated and optimised using a commercial kit. Alterations to the monocyte metabolome in response to LPS can be detected as early as four hours post stimulation. Broad and profound changes in monocyte metabolism were seen, in line with increased cytokine production. Elevated levels of amino acids and Krebs cycle metabolites were noted and decreases in aspartate and β-alanine are also reported for the first time. In the untargeted analysis, 154 metabolite entities were significantly altered compared to unstimulated cells. Pathway analysis revealed the most prominent changes occurred to (phospho-) inositol metabolism, glycolysis, and the pentose phosphate pathway. These data report the emergent changes to monocyte metabolism in response to LPS, in line with reports from later time points. A number of these metabolites are reported to alter inflammatory gene expression, which may facilitate the increases in cytokine production. Further validation is needed to confirm the link between metabolic activation and upregulation of inflammatory responses.

Evaluation of the relationship between mesenchymal stem cells and immune system in vitro conditions.

Mesenchymal stem cells (MSCs), are a novel therapeutic option as the most common cell source, play an important role in the immunomodulation. In this study, it was aimed to determine the effect of MSCs on cytokines secreted by the immune system cells. Intracellular cytokine levels (Interleukin-4 (IL-4), Interferon-γ (IFN-γ), and Interleukin-17 (IL-17)) detected by flow cytometry before and after co-culture between peripheral blood mononuclear cells (PBMCs) and MCSs. At the same time, supernatant cytokine levels were measured using the ELISA. In our study, MSCs were isolated from cord blood (CB) and Wharton's Jelly (WJ), and their surface markers (CD44 (100%), CD73 (99.6%), CD90 (100%), CD105 (88%)) shown by flow cytometry method. Both CB-MSCs and WJ-MSCs were used in co-culture MSC/PBMC ratios of 1/5 and 1/10, incubation times of 24h and 72h. In the present study, when we compared co-cultures of CB-MSC or WJ-MSC with PBMCs, intracellular levels of cytokines IFN-γ, IL-17 (pro-inflamatory) and IL-4 (anti-inflamatory) were increased, and supernatant levels were decreased significantly (p < 0.05). The level of transforming growth factor beta (TGF-β) (anti-inflamatory) was significantly decreased for both CB-MSC and WJ-MSC in supernatant (p < 0.05). It was investigated pro-inflammatory and anti-inflammatory effects of CB-MSCs and WJ-MSCs on PBMCs with the obtained results. According to the results, MSCs demonstrated different immunologic effects after the incubation time and ratios. For further studies, it should be known between interaction of MSCs and immune system.

Cytokine production by blood cells and tumor samples and its coupling to microRNA expression in breast cancer patients

Breast tumors show a complex structure and are highly heterogeneous. The study of cytokines, which exert great influence on tumor cells, and microRNAs, which, along with their influence on the proliferation and migration of neoplastic cells, may affect the work of cytokines, will contribute to a deeper understanding of pathological processes occurring in breast cancer. The aim of our work was to analyze the relationship of cytokine production with expression of miR-181a and miR-25in patients with invasive breast carcinoma of a non-specific type (IBC NST) with various molecular subtypes.Patients with IBC NST were divided into five subgroups according to the molecular genetics subtype of the tumor classified by immunohistochemical analysis of estrogen receptor (ER), progesterone (PR), epidermal growth factor 2 (HER2) and proliferation marker Ki-67. Using enzyme immunoassay, the concentration of 14 cytokines was determined in the supernatants of immunocompetent blood cells and tumors: IL-2, IL-6, IL-8, IL-10, IL-17, IL-18, IL-1β, IL-1ra, TNFα, IFNγ, G-CSF, GM-CSF, VEGF and MCP-1. The expression of miR-181a and miR-25 microRNAs isolated from the patients’ blood serum was evaluated using digital droplet polymerase chain reaction (ddPCR).In the luminal A subtype, cytokine concentrations and expression of miR-181a and miR-25 are significantly lower compared to other subtypes. Patients with the luminal B HER2-negative subtype were characterized by significantly increased expression of both studied microRNAs, especially when compared with the luminal A subtype. At the same time, patients with a triple negative molecular subtype, on the contrary, were characterized by high concentrations of cytokines in the supernatants of tumor samples and blood cells compared to other subtypes. In the general group of patients with IBC NST, direct correlations were found between the expression of both studied microRNAs and the concentration of vascular endothelial growth factor (VEGF) in the supernatant of tumor samples, which may presume mutual interactions existing between miR-181a and miR-25, and the process of angiogenesis in the tumor.The levels of cytokines in blood supernatants and tumors in invasive breast carcinoma may vary, depending on distinct molecular subtypes of the tumor. Moreover, they also have direct links with the levels of miR-181a and miR-25 in blood serum. Particularly noteworthy were the results of measuring the cytokines and microRNAs concentrations in luminal A, luminal B HER2-negative and triple negative molecular subtypes.

Isoalantolactone protects against ethanol-induced gastric ulcer via alleviating inflammation through regulation of PI3K-Akt signaling pathway and Th17 cell differentiation

Gastric ulcer (GU) is one of the most prevalent digestive system diseases in humans, and it has been linked to inflammation. Previous studies have demonstrated the anti-inflammatory potential of isoalantolactone (IAL), a sesquiterpene lactone isolated from Radix Inulae. However, the pharmacological effects of IAL on GU and its mechanism of action are still unclear. Hence, the present study is aimed to investigate the anti-inflammatory potential of IAL on GU. Firstly, we assessed the effect of IAL on ethanol-induced injury of human gastric epithelial cells and the levels of inflammatory cytokines in cell culture supernatants. Then, the anti-inflammatory effects of IAL were confirmed in vivo using zebrafish inflammation models. Furthermore, the mechanism of IAL against GU was preliminarily discussed through network pharmacology and molecular docking studies. Quantitative real-time PCR assays were also used to confirm the mechanism of IAL action. ALB, EGFR, SRC, HSP90AA1, and CASP3 were found for the first time as the key targets of the IAL anti-GU. PI3K-Akt signaling pathway and Th17 cell differentiation were identified to play a crucial role in the anti-GU effects of IAL. In conclusion, we found that IAL has anti-inflammatory effects both in vitro and in vivo, and showed potential protective effects against ethanol-induced GU.