IL-1ra Production Research Articles

Sex-Specific Differences in Cytokine Production Capacity in Patients with Gout Compared to Controls

Gout, an inflammatory disease orchestrated by interleukin-1β activation and release, is more prevalent in men. The clinical profiles of patients with gout report differences by sex. This study aims to investigate sex-specific cytokine profiles in circulation and in stimulated peripheral blood mononuclear cells (PBMCs) of patients with gout and controls. Participants included in the gout group met the criteria of the American College of Rheumatology/European League Against Rheumatism (ACR/EULAR). The control group included individuals with varying levels of serum urate and absence of gout. PBMCs were stimulated in vitro for 24 h with various TLR ligands. Cytokines were determined in culture supernatants and plasma. Plasma IL-1Ra and high-sensitivity C-reactive protein (hsCRP) were higher in men with gout compared to men without gout whereas no significant differences in circulating cytokines were observed in women. PBMCs of patients with gout showed higher cytokine production of IL-1β, IL-1Ra, and TNF-α following 24 h stimulation, predominantly observed in women. We identified sex-specific cytokine production in gout in response to in vitro stimulation. While men with gout had higher levels of circulating cytokines, stimulated PBMCs of women with gout show an enhanced capacity for cytokine production. These data may suggest potentially different regulatory mechanisms of inflammation in men and women with gout.

Immunoregulatory properties of the dry extract of the herb <i>Cichorium intybus L.</i> in <i>in vitro</i> and <i>in vivo</i> systems

Aim: to evaluate the effect of a dry extract of the herb of Cichorium intybus L. on the production of cytokines by human peripheral mononuclear cells in the systems in vitro and on the mass (thymus, spleen) and number of cells (thymus, spleen, bone marrow) of immune organs of mice in a model experiment of immunodeficiency in the systems in vivo.
 Materials and methods. The study of the immunoregulatory properties of the dry extract of chicory herb was evaluated by the ability to change the production of cytokines on the model of mononuclear cells during co-cultivation under in vitro conditions and by the ability to restore the mass (thymus, spleen) and the number of cells (thymus, spleen, bone marrow) of the organs of the immune system of outbred mice under conditions of immunosuppression induced by methotrexate to the level of intact mice in vivo.
 Results. An analysis of the immunoregulatory properties of dry extract of chicory herb in the systems in vitro showed that a solution of chicory extract increased the concentration of the anti-inflammatory cytokine IL-10 and reduced the production of pro-inflammatory cytokines TNFα, IL-6, IFNγ and anti-inflammatory cytokine IL-1Ra. Evaluation of the effect of the dry extract of chicory herb in the systems in vivo found that the dry extract of the herb of Cichorium intybus L. had no effect on the mass (thymus, spleen) and number of cells (thymus, spleen, bone marrow) of the immune system organs in intact mice and restored these parameters of the immune system in animals treated with cytostatic MTX to the level of intact animals, and the effect of the extract was comparable to the effect of the reference drug “Immunal”.
 Conclusion. The dry extract of the herb of Cichorium intybus L. can be considered as a promising agent that normalizes the parameters of the immune system under conditions of MTX-induced immunosuppression, suppresses the synthesis of pro-inflammatory cytokines and stimulates the secretion of anti-inflammatory IL-10, and the activity of this extract is associated with high content of phenolic compounds. Given the above, further study of chicory herb and dry extract from it is expedient and aimed at creating new effective herbal preparations that regulate the functions of the body’s immune system.

PDZ Peptide of the ZO-1 Protein Significantly Increases UTP-Induced MUC8 Anti-Inflammatory Mucin Overproduction in Human Airway Epithelial Cells.

Mucus hyperproduction and hypersecretion are observed often in respiratory diseases. MUC8 is a glycoprotein synthesized by epithelial cells and generally expressed in the respiratory track. However, the physiological mechanism by which extracellular nucleotides induce MUC8 gene expression in human airway epithelial cells is unclear. Here, we show that UTP could induce MUC8 gene expression through P2Y2-PLCβ3-Ca2+ activation. Because the full-length cDNA sequence of MUC8 has not been identified, a specific siRNA-MUC8 was designed based on the partial cDNA sequence of MUC8. siRNA-MUC8 significantly increased TNF-α production and decreased IL-1Ra production, suggesting that MUC8 may downregulate UTP/P2Y2-induced airway inflammation. Interestingly, the PDZ peptide of ZO-1 protein strongly abolished UTP-induced TNF-α production and increased IL-1Ra production and MUC8 gene expression. In addition, the PDZ peptide dramatically increased the levels of UTP-induced ZO proteins and TEER (trans-epithelial electrical resistance). These results show that the anti-inflammatory mucin MUC8 may contribute to homeostasis, and the PDZ peptide can be a novel therapeutic candidate for UTP-induced airway inflammation.

Extracellular Cold Shock Protein YB-1 Induces Tolerance to GMDP and LPS in Mouse Macrophage Cell Line J774

Cold shock protein YB-1 is involved in the regulation of a huge number of fundamental biological processes. Previously, we showed that YB-1 is also involved in the process of recognition of muramylpeptide GMDP by the innate immune receptor NOD2 and is able upon preliminary administration to protect mice from death in a model of septic shock induced by Escherichia coli bacteria. We hypothesized that its protective effect may be associated with the development of a state of tolerance (“nonresponsiveness”). Changes in the cellular response were assessed by the level of mRNA expression of the target molecules by quantitative PCR analysis combined with reverse transcription. We tested the possibility of tolerance induction by the YB-1 protein in a model system on the J774 mouse macrophage cell line with the participation of E. coli bacterial cell wall components, immunostimulants GMDP (NOD2 receptor agonist) and LPS (TLR4 receptor agonist). Pretreatment of cells with YB-1 resulted in a significant decrease in the level of mRNA expression of pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 in response to further stimulation with GMDP and LPS, as well as significant changes in the expression of mRNA of RIP2 and MyD88 adapter molecules and components of transcriptional factor NF-κB. Our data show that YB-1 is able to induce tolerance to such as GMDP and LPS immunostimulants, apparently by increasing the production of the anti-inflammatory cytokine IL-1Ra and the SOCS1 inhibitor. A more precise characterization of the features of the YB-1-induced tolerogenic immune response requires further research.

Antigen-Induced IL-1RA Production Discriminates Active and Latent Tuberculosis Infection.

The IGRA (Interferon Gamma Release Assays) test is currently the standard specific test for Mycobacterium tuberculosis infection status. However, a positive test cannot distinguish between active tuberculosis disease (ATBD) and latent tuberculosis infection (LTBI). Developing a test with this characteristic is needed. We conducted longitudinal studies to identify a combination of antigen peptides and cytokines to discriminate between ATBD and LTBI. We studied 54 patients with ATBD disease and 51 with LTBI infection. Cell culture supernatant from cells stimulated with overlapping Mycobacterium tuberculosis novel peptides and 40 cytokines/chemokines were analyzed using the Luminex technology. To summarize longitudinal measurements of analyte levels, we calculated the area under the curve (AUC). Our results indicate that in vitro cell stimulation with a novel combination of peptides (Rv0849-12, Rv2031c-14, Rv2031c-5, and Rv2693-06) and IL-1RA detection in culture supernatants can discriminate between LTBI and ATBD.

Effect of exogenous IL-37 on immune cells from a patient carrying a potential IL37 loss-of-function variant: A case study.

Chronic inflammatory or autoimmune diseases are commonly treated with immunosuppressive medication such as NSAIDs, corticosteroids, or antibodies against specific cytokines (TNF, IL-1 IL-17, IL-23, etc.) or signalling cascades (e.g. JAK-STAT inhibitors). Using sequencing data to locate genetic mutations in relevant genes allows the identification of alternative targets in a patient-tailored therapy setting. Interleukin (IL)-37 is an anti-inflammatory cytokine with broad effects on innate and adaptive immune cell function. Dysfunctional IL-37 expression or signalling is linked to various autoinflammatory disorders. The administration of recombinant IL-37 to hyperinflammatory patients that are non-responsive to standard treatment bears the potential to alleviate symptoms. In this case study, the (hyper)responsiveness of immune cell subsets was investigated in a single patient with a seronegative autoimmune disorder who carries a heterozygous stop-gain variant in IL37 (IL37 Chr2(GRCh37):g.113670640G>A NM_014439.3:c.51G>A p.(Trp17*)). As the patient has been non-responsive to blockage of TNF or IL-1 by Etanercept or Anakinra, respectively, additional in-vitro experiments were set out to elucidate whether treatment with recombinant IL-37 could normalise observed immune cell functions. Characterisation of immune cell function showed no elevated overall production of acute-phase pro-inflammatory cytokines by patient PBMCs and neutrophils at baseline or upon stimulation. T-cell responses were elevated, as was the metabolic activity and IL-1Ra production of PBMCs at baseline. The identified stop-gain variant in IL37 does not result in the absence of the protein in circulation. In line with this, treatment with recombinant IL-37 did overall not dampen immune responses with the exception of the complete suppression of IL-17. The heterozygous stop-gain variant in IL37 (IL37 NM_014439.3:c.51G>A p.(Trp17*)) is not of functional relevance as we observed no clear pro-inflammatory phenotype in immune cells of a patient carrying this variant.

Characterisation of cytotoxicity and immunomodulatory effects of glycolipid biosurfactants on human keratinocytes

Skin irritation and allergic reactions associated with the use of skincare products formulated with synthetically derived surfactants such as sodium lauryl ether sulphate (SLES) have encouraged the search for naturally derived and biocompatible alternatives. Glycolipid biosurfactants such as sophorolipids (SL) and rhamnolipids (RL) offer a potential alternative to SLES. However, most studies on the bioactive properties of microbial glycolipids were determined using their mixed congeners, resulting in significant inter-study variations. This study aims to compare the effects of highly purified SL (acidic and lactonic) and RL (mono-RL and di-RL) congeners and SLES on a spontaneously transformed human keratinocyte cell line (HaCaT cells) to assess glycolipids’ safety for potential skincare applications. Preparations of acidic SL congeners were 100% pure, lactonic SL were 100% pure, mono-RL were 96% pure, and di-RL were 97% pure. Cell viability using XTT assays, cell morphological analyses, and immunoassays revealed that microbial glycolipids have differing effects on HaCaT cells dependent on chemical structure. Compared with SLES, acidic SL and mono-RL have negligible effects on cell viability, cell morphology, and production of pro-inflammatory cytokines. Furthermore, at non-inhibitory concentrations, di-RL significantly attenuated IL-8 production and CXCL8 expression while increasing IL-1RA production and IL1RN expression in lipopolysaccharide-stimulated HaCaT cells. Although further studies would be required, these results demonstrate that as potential innocuous and bioactive compounds, microbial glycolipids could provide a substitute to synthetic surfactants in skincare formulations and perform immunopharmacological roles in topical skin infections such as psoriasis.Key points• Purified glycolipid congeners have differing effects on human keratinocytes.• Compared with SLES, acidic sophorolipids and mono-rhamnolipids have innocuous effects on keratinocytes.• Di-rhamnolipids and mono-rhamnolipids modulate cytokine production in lipopolysaccharide stimulated human keratinocytes.

Ex vivo cytokine production in psoriatic disease: Towards specific signatures in cutaneous psoriasis and peripheral psoriatic arthritis.

Psoriatic arthritis (PsA) and cutaneous psoriasis (PsO) are different phenotypes of psoriatic disease (PsD), whose underlying specific mechanisms remain incompletely understood. As cytokines are key elements to induce and tune up immune responses to drive inflammatory diseases, our objective was to assess whether clinical features, disease phenotype and PsA and PsO activity were associated with a particular ex vivo cytokine production profile. Forty-eight patients (37 PsA and 11 PsO) and 11 healthy subjects (HS) were studied. Cytokine production by peripheral blood mononuclear cells (PBMC) that were either unstimulated, or stimulated with LPS or anti-CD3/CD28 antibodies, were analysed by multiplex assay in the culture supernatants. Cytokine signature of PsD includes a high level of TNFα in supernatants of LPS-stimulated PBMC, higher levels of IL-6 and lower levels of IFN-γ and IL-17A after CD3-CD28 stimulation, as well as higher spontaneous IL-1RA and TNFα production compared to HS. High body mass index (BMI) was associated with lower levels of IL-1β, and metabolic syndrome with lower levels of IFN-γ after LPS stimulation. In PsD, dermatological activity was related with higher IL-17A level, while rheumatic activity was linked with lower levels of IFN-γ and TNFα. Comparing each PsD subtype to HS, IL-1β and IL-6 productions are higher when using LPS stimulation in PsO patients with higher levels of IL-1β and IL-1α in peripheral PsA patients after CD3/CD28 stimulation. LPS stimulation induced high levels of IL-17A in peripheral PsA compared to axial PsA. PsA patients with axial PsA share some features with PsO but shows a distinct cytokine pattern compared to peripheral PsA. PsO and the different PsA subtypes exhibit distinct ex vivo cytokine production profiles and common features of the so-called PsD. Analysis of IL-1 cytokine family and IL-6 seems to be of particular interest to distinguish PsO and peripheral PsA since it depends on monocytes in PsO and T-lymphocytes in peripheral PsA. Peripheral cytokine profiles are influenced by rheumatic and dermatological activity of the disease, and also by metabolic syndrome features. Our results highlight the crucial role of immune cell interactions with different patterns of interaction depending on clinical phenotype.

Improved sensitivity of the skin pathergy test with polysaccharide pneumococcal vaccine antigens in the diagnosis of Behçet disease.

The skin pathergy test (SPT) is an important tool in the diagnosis of Behçet disease (BD), but its decreasing sensitivity over years has resulted in less frequent use in the clinical practice. This study aimed to improve the sensitivity of the SPT without compromising its specificity. BD patients, patients with other inflammatory diseases, recurrent aphthous stomatitis, and healthy controls comprised the study group. The SPT was conducted using 20G needle and 21G lancet pricks, or with additional application of 23-valent polysaccharide pneumococcal vaccine (PS-23), Alum, or ATP to the prick site. Development of erythema and induration at 24 h/48 h were evaluated by the same observer. Induration (≥2 mm) with erythema at 48 h was accepted as a positive reaction. Proinflammatory cytokine production following stimulation with lipopolysaccharide or PS-23 was investigated by whole-blood assay (WBA) in a subgroup. Stimulation of the forearm skin by PS-23 and a 20G needle prick showed the highest sensitivity and specificity in BD (64.3% and 100%, respectively), especially in patients with active disease (80.3% and 100%, respectively), compared with a sensitivity of 4.8% in all and 6.1% in active patients using a single 20G prick. A positive result was associated with active disease and no use of immunosuppressives. In WBA, increased IL-1β and IL-1Ra production in response to PS-23 was observed in the group with active BD, while the cytokine response to lipopolysaccharide was similar in all groups. The SPT conducted using a 20G needle prick and PS-23 antigens was shown to be a promising tool for the diagnosis of BD owing to its improved sensitivity compared with the standard approach.

334.8: Extracellular Vesicles as Mediators of Infectious Tolerance in Human Alloreactive Regulatory Cells

Introduction: We have developed a method for ex vivo expansion of donor antigen specific T regulatory cell enriched lines (ASTRLs) from stable human kidney transplant recipients (KTRs). Rat derived ASTRLs induced prolonged graft survival and donor specific tolerance in a rat kidney transplant model. Human ASTRLs are suppressive in their phenotype and function. We examined the various mechanisms of ASTRL mediated suppression including extracellular vesicle (EV)-mediated suppression. Method: ASTRLs were expanded ex vivo from PBMCs of stable KTRs by stimulation with donor allopeptides in presence of IL-2. ASTRLs were characterized for conventional Treg markers by flow cytometry. Functional characterization of ASTRLs were performed using standard suppression assays. Extracellular ATP hydrolysis was determined by malachite green assay. EVs were isolated from the ASTRL expansion media via differential ultracentrifugation and characterized by liquid chromatography-mass spectrometry. Results: The suppressive mechanism of ASTRLs was alloantigen-specific and dependent on cell-cell contact as shown in Figure 1 (top panel). ASTRLs upregulated the expression CD39 and CD73, ectonucleotidases associated with the adenosinergic pathway, and ASTRLs demonstrated increased extracellular ATP hydrolysis (eATP) that was inhibited by POM1, a CD39 specific inhibitor (Figure 1 left bottom panel) which also abrogated the suppressive effect of ASTRLs. In KTRs ASTRLs specific for one donor alloantigen were able to suppress an effector response against other donor alloantigens demonstrating bystander/ linked suppression and inhibit effector T cell proliferation in response to both direct and indirect allorecognition (Figure 1 bottom right panel). EVs isolated from ASTRLs enhanced the suppressive ability of ASTRLs and hydrolyze eATP independently, which was inhibited by POM1 (Fig. 2) and increase production of IL-4, IL-13, and IL-1RA by alloantigen stimulated T cells, indicating a biasing towards a regulatory response (Fig. 2). Global proteomics identified 1,709 unique proteins in the large EV fraction and 1,356 unique proteins in the small EV fraction. Pathway analysis revealed that both EV fractions under stimulation were enriched for proteins involved in T cell receptor (TCR) signaling and Treg differentiation (Fig. 2) with 145 differentially enriched proteins (DEPs) in the large and 162 DEPs in the small EVs (q<0.05). Pathway analysis of DEPs will be presented. Conclusion: ASTRLs expanded from stable KTRs suppress donor antigen specific effector responses predominantly via the adenosinergic pathway and demonstrate bystander/linked immunosuppression. ASTRL-EVs enhance the suppressive capacity of ASTRLs and exhibit an increase in CD39 function. These findings indicate that human ASTRLs may be useful as autologous regulatory cell therapy in transplant recipients and that ASTRL related EVs may be useful adjuncts to ASTRL cell therapy. Saxena Transplantation Fund.

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.

Living in endemic area for infectious diseases accelerates epigenetic age

Inflammaging is a low-grade inflammatory state generated by the aging process that can contribute to frailty and age-related diseases in the elderly. However, it can have distinct effects in the elderly living in endemic areas for infectious diseases. An increased inflammatory response may confer protection against infectious agents in these areas, although this advantage can cause accelerating epigenetic aging. In this study, we evaluated the inflammatory profile and the epigenetic age of infected and noninfected individuals from an endemic area in Brazil. The profile of cytokines, chemokines and growth factors analyzed in the sera of the two groups of individuals showed similarities, although infected individuals had a higher concentration of these mediators. A significant increase in IL-1ra, CXCL8, CCL2, CCL3 and CCL4 production was associated with leprosy infection. Notably, elderly individuals displayed distinct immune responses associated with their infection status when compared to adults suggesting an adaptive remodelling of their immune responses. Epigenetic analysis also showed that there was no difference in epigenetic age between the two groups of individuals. However, individuals from the endemic area had a significant accelerated aging when compared to individuals from São Paulo, a non-endemic area in Brazil. Moreover, the latter cohort was also epigenetically aged in relation to an Italian cohort. Our data shows that living in endemic areas for chronic infectious diseases results in remodelling of inflammaging and acceleration of epigenetic aging in individuals regardless of their infectious status. It also highlights that geographical, genetic and environmental factors influence aging and immunosenescence in their pace and profile.

The SARS-CoV-2 Nucleoprotein Induces Innate Memory in Human Monocytes.

The interaction of SARS-CoV-2 with the human immune system is at the basis of the positive or negative outcome of the infection. Monocytes and macrophages, which are major innate immune/inflammatory effector cells, are not directly infected by SARS-CoV-2, however they can react to the virus and mount a strong reaction. Whether this first interaction and reaction may bias innate reactivity to re-challenge, a phenomenon known as innate memory, is currently unexplored and may be part of the long-term sequelae of COVID-19. Here, we have tested the capacity of SARS-CoV-2 and some of its proteins to induce innate memory in human monocytes in vitro. Our preliminary results show that the Spike protein subunits S1 and S2 and the entire heat-inactivated virus have no substantial effect. Conversely, monocytes pre-exposed to the nucleocapsid N protein react to subsequent viral or bacterial challenges with an increased production of anti-inflammatory IL-1Ra, a response profile suggesting a milder response to new infections.

Lactic acid bacteria derived-bioactive molecules condition an in vitro cell model into a tolerogenic dendritic-like cell phenotype

Abstract Retinoic acid (RA) is a vitamin A metabolite; CD103+ dendritic cells (DCs) acquire a tolerogenic phenotype through an RA-dependent mechanism, driving the differentiation of gut-homing regulatory T cells in the gut mucosa. Growing evidence shows bioactive molecules derived from lactic acid bacteria exhibit immunomodulatory activity by interacting with antigen presenting cells (APCs), stimulating their differentiation and altering their phenotype to further shape adaptive immunity. We utilized the monocytic THP-1 cell line as an in vitro model to examine the impact of the Lacticaseibacillus rhamnosus secretome (LrS) and interactions with RA on APC differentiation into a tolerogenic dendritic-like cell (DLC) phenotype. THP-1 monocytes were treated with Phorbol 12-myristate 13-acetate to drive differentiation into macrophages, then conditioned with the LrS and/or RA for 24, 48 or 72 hours. Expression of key DC markers CD11b, CD11c, DC-SIGN, CD83, CD80 and CD103 increased, while expression of proinflammatory marker CD64, co-stimulatory marker CD86 and MHC class II molecule HLA-DR was reduced. Cytokine profiling revealed LrS + RA co-treatment increased the production of immunoregulatory cytokines IL-10 and IL-1RA. Expression of NFKB1 and NFKB2, key genes involved in the activation of the NF-κB canonical and non-canonical pathways was induced by 48 hours of LrS conditioning, accompanied by increased expression of CD40 and IDO1, indicating an ability to drive DLC maturation and induce co-stimulatory capacity. Deciphering the mechanisms behind these types of microbiota-host interactions gives insight into how microbial metabolites can influence immune maturation and maintain immune homeostasis. Supported by Natural Sciences and Engineering Research Council of Canada Discovery Grant RGPIN-2017-05237

Study of the anti-inflammatory and immunotropic activity of the secretome from multipotent mesenchymal stromal cells induced by erythropoietin, valproic acid or dexamethasone &lt;i&gt;in vitro&lt;/i&gt;

The aim of this study was to evaluate the effect of treatment with valproic acid, erythropoietin, and dexamethasone on the anti-inflammatory and immunosuppressive activity of the secretome of adipose-derived multipotent mesenchymal stromal cells (MMSCs) in an in vitro experiment.Materials and methods. MMSCs were isolated from the fat of 6 healthy donors. The cells were grown in the culture up to passage 4. Then they were treated with valproic acid, erythropoietin or dexamethasone for 3 hours, washed from preparations, and incubated in a serum-free medium for 48 hours. Some of the cells were not treated with preparations. Supernatants from the cell cultures were concentrated by ultrafiltration, and protein standardization was performed using a nanophotometer. Then the supernatants were sterilized and added to mononuclear cells from peripheral blood of 8 healthy donors. The mononuclear cells were isolated by Ficoll density gradient centrifugation according to the standard protocol. Concentrations of TNFα, IL-2, IL-4, IL-6, IL-10, and IFNγ cytokines in 24-hour cultures and IL-9, IL-10, IL-17A, and IL-21 cytokines in 48-hour cultures were determined using multiplex analysis.Results. The production of IL-2, IL-6, TNFα, and IL-10 was reduced by the secretome of MMSCs treated with valproic acid. The production of IL-2, IL-6, and TNFα decreased during incubation of the mononuclear cells with the secretome of MMSCs treated with erythropoietin. The secretome of dexamethasone-treated MMSCs suppressed the production of IFNγ, IL-1β, IL-1ra, IL-2, IL-6, IL-9, IL-10, and IL-17A. No statistically significant differences were revealed in the production of IL-4, IL-5, IL-9, and IL-21.Conclusion. Among the studied inducers, dexamethasone enhanced the anti-inflammatory and immunosuppressive activity of MMSCs the most, which was manifested through the effect of their supernatants on peripheral blood mononuclear cells.

Development of a 3D psoriatic skin model optimized for infiltration of IL-17A producing T cells: Focus on the crosstalk between T cells and psoriatic keratinocytes

Psoriasis is a chronic inflammatory skin disease involving several cell types, including T cells, via the IL-23/IL-17 axis. IL-17A acts on the surrounding epithelial cells thus resulting in an inflammatory feedback loop. The development of immunocompetent models that correctly recapitulate the complex phenotype of psoriasis remains challenging, which also includes both the T cell isolation and activation methods. The purpose of this work was to develop an advanced in vitro 3D psoriatic skin model that enables the study of the impact of T cells on psoriatic epithelial cells. To reach that aim, healthy and psoriatic fibroblasts and keratinocytes were used to reproduce this tissue-engineered skin model in which activated T cells, isolated beforehand from human whole blood, have been incorporated. Our study showed that isolation of T cells with the EasySep procedure, followed by activation with PMA/ionomycin, mimicked the psoriatic characteristics in an optimal manner with the production of inflammatory cytokines important in the pathogenesis of psoriasis, as well as increased expression of Ki67, S100A7, elafin and involucrin. This psoriatic model enriched in activated T cells displayed enhanced production of IL-17A, IFN-ƴ, CCL2, CXCL10, IL-1ra, IL-6 and CXCL8 compared with the healthy model and whose increased secretion was maintained over time. In addition, anti-IL17A treatment restored some psoriatic features, including epidermal thickness and basal keratinocytes proliferation, as well as a downregulation of S100A7, elafin and involucrin expression. Altogether, our study demonstrated that this model reflects a proper psoriatic inflammatory environment and is effective for the investigation of epidermal and T cell interaction over time. Statement of significanceThe aim of this study was to provide an innovative 3D immunocompetent human psoriatic skin model. To our knowledge, this is the first immunocompetent model that uses skin cells from psoriatic patients to study the impact of IL-17A on pathological cells. Through the use of this model, we demonstrated that the T-cell enriched psoriatic model differs from T-cell enriched healthy model, highlighting efficient crosstalk between pathologic epithelial cells and T cells. This advanced preclinical model further mimics the original psoriatic skin and will prove relevant in predicting clinical outcomes, thereby decreasing inaccurate predictions of compound effects.

Human Newborn Monocytes Demonstrate Distinct BCG-Induced Primary and Trained Innate Cytokine Production and Metabolic Activation In Vitro.

BackgroundNewborns exhibit distinct immune responses and are at high risk of infection. Neonatal immunization with BCG, the live attenuated vaccine against tuberculosis (TB), is associated with broad protection against a range of unrelated pathogens, possibly reflecting vaccine-induced training of innate immune cells (“innate memory”). However, little is known regarding the impact of age on BCG-induced innate responses.ObjectiveEstablish an age-specific human monocyte in vitro training platform to characterize and compare BCG-induced primary and memory cytokine responses and immunometabolic shifts.Design/MethodsHuman neonatal and adult CD33-selected monocytes were stimulated for 24h with RPMI (control) or BCG (Danish strain) in 10% autologous serum, washed and cultured for 5 additional days, prior to re-stimulation with the TLR4 agonist LPS for another 24h. Supernatants were collected at Day 1 (D1) to measure primary innate responses and at Day 7 (D7) to assess memory innate responses by ELISA and multiplex cytokine and chemokine assays. Lactate, a signature metabolite increased during trained immunity, was measured by colorimetric assay.ResultsCytokine production by human monocytes differed significantly by age at D1 (primary, BCG 1:750 and 1:100 vol/vol, p<0.0001) and D7 (innate memory response, BCG 1:100 vol/vol, p<0.05). Compared to RPMI control, newborn monocytes demonstrated greater TNF (1:100, 1:10 vol/vol, p<0.01) and IL-12p40 (1:100 vol/vol, p<0.05) production than adult monocytes (1:100, p<0.05). At D7, while BCG-trained adult monocytes, as previously reported, demonstrated enhanced LPS-induced TNF production, BCG-trained newborn monocytes demonstrated tolerization, as evidenced by significantly diminished subsequent LPS-induced TNF (RPMI vs. BCG 1:10, p <0.01), IL-10 and CCL5 production (p<0.05). With the exception of IL-1RA production by newborn monocytes, BCG-induced monocyte production of D1 cytokines/chemokines was inversely correlated with D7 LPS-induced TNF in both age groups (p<0.0001). Compared to BCG-trained adult monocytes, newborn monocytes demonstrated markedly impaired BCG-induced production of lactate, a metabolite implicated in immune training in adults.ConclusionsBCG-induced human monocyte primary- and memory-innate cytokine responses were age-dependent and accompanied by distinct immunometabolic shifts that impact both glycolysis and training. Our results suggest that immune ontogeny may shape innate responses to live attenuated vaccines, suggesting age-specific approaches to leverage innate training for broad protection against infection.

Recombinant Production and One-Step Purification of IL-1Ra in Escherichia coli and Evaluation of its IL-1 Antagonizing Efficacy.

Anakinra (Kineret®), an IL-1 receptor antagonist, is the first FDA-approved biologic drug for antagonizing IL-1 in patients with Rheumatoid arthritis. The less expensive production of this drug might help reduce the final therapeutic costs. To evaluate the possibility of producing biologically active recombinant IL-1Ra by a single-step purification procedure mediated by a self-cleavable intein. Soluble expression of the rIL-1Ra was performed in E. coli BL21 (DE3) infusion to intein1 of pTWIN-1 vector and its cleavage induction using an elution buffer (pH 6.8) at room temperature. Evaluation of the antagonizing efficacy of this protein in various concentrations was performed on A375 and HEK293 cells treated by a constant concentration of IL-1β (2 ng/mL). IPTG induction of E. coli BL21 (DE3) transformed with the recombinant pTWIN-1, revealed a band approximately in 45 kDa, which is related to the intein1-rIL-1Ra fusion protein in the SDS-PAGE. Moreover, protein purification was confirmed by observing a band in 18 kDa. Finally, the percentage of inhibition effects of rIL-1Ra and Kineret® against IL-1β was not statistically significant in IL-1-responsive A375 cells. The inhibition percentage was calculated as 86% in cells treated with 15µg/mL of rIL-1Ra, which was 96% for the inhibitory effects of the standard drug. In this study, biologically active soluble rIL1-Ra was successfully produced with high purity through a one-step procedure. This method can reduce the cost and time of production for this protein and might be applicable other biological products.

Eucalyptus Citriodora Extract Regulates Cutaneous Homeostasis Including Immune Dysregulation and Skin Barrier Dysfunction Via the Modulation of Peroxisome Proliferator-Activated Receptor-β/δ (PPAR-β/δ) Pathway

Perturbation of cutaneous homeostasis including immune dysregulation and skin barrier dysfunction evokes skin disorders. In this study, we examined the effect of Eucalyptus citriodora (Euc-c) extract on cytokine production, cell proliferation and cell differentiation in HaCaT cells to elucidate its influence on cutaneous homeostasis. Euc-c suppressed significantly LPS-induced IL-6 and TNF-a-induced IL-8 production from HaCaT cells. Conversely IL-1ra production was significantly enhanced by Euc-c. The expressions of IVL, CERS3 and CERS4, keratinocyte differentiation markers, were upregulated to 3.1, 2.8 and 2.7-fold respectively by Euc-c treatment, compared to the control, while the proliferation was downregulated. The lipid contents in Euc-c-treated cells tended to increase, compared with non-treated cells. To explore the underlying mechanism of these effect, we next performed siRNA experiments against PPAR-b/d. Euc-c enhanced PPAR-b/d mRNA expression to 3.25-fold, while PPAR-b/d mRNA expression in transfected cells was suppressed. The expressions of IVL, CERS3 and CERS4 in transfected cells were suppressed to 1.48, 0.82 and 0.72-fold respectively, concomitant with suppression of PPAR-b/d mRNA expression. These results indicated that Euc-c exerts anti-inflammatory effects and regulates keratinocyte differentiation via the modulation of PPAR-b/d pathway. Therefore, the application of Euc-c is expected to exert beneficial effect on skin disorders evoked by perturbation of skin homeostasis.

Multi-Species Probiotic Modulates Cytokine Production and the Interplay between Immune and Colon Cancer Cells

The current study aimed to investigate the effect of a multi-species probiotic (MSP) on cytokine production by human peripheral blood mononuclear cells (PBMCs) and their immune dialogue with HT-29 colon cancer cells. PBMCs were incubated with MSP and their effect on cell proliferation and TNFα, IL-1β, IL-2, IL-6, IFNγ, IL-10, and IL-1ra production was evaluated. The impact of MSP on the cytokine production by PBMC stimulated by HT-29 cells was detected. Not-stimulated PBMC incubated with MSP showed increased production of TNFα, IL-1β, IL-6, and IL-10, but no change in IL-6, IFNγ, and IL-1ra. The stimulatory effect of MSP on lipopolysaccharide (LPS)-promoted PBMC was less pronounced for TNFα, IL-1β, and IFNγ, and the IL-6 production was decreased; phorbol 12-myristate 13- acetate (PMA)-induced IL-2 and IFNγ secretion was inhibited. The addition of MSP to co-cultures of PBMC and HT-29 cancer cells caused a remarkable increase in TNFα and IL-1β secretion, with no change in remaining cytokines. The multi-species probiotics modulated cytokine production by PBMC and affected the cross-talk between PBMC and HT-29 cancer cells. We conclude that probiotics may serve as supplements to the therapeutic strategies applied for the treatment of chronic inflammatory and malignant diseases, especially colorectal cancers.