Increased Levels of CD107a and Intracellular Cytokines in IL-2 Stimulated PBMCs from Endometriosis Patients

It has been postulated that the immune system is impaired in individuals with endometriosis, with attention directed to natural killer (NK) cells. Specifically, it has been hypothesized that altered numbers of peripheral NK cells in blood are associated with the presence of endometriotic lesions. This study aimed to evaluate the level of the peripheral NK cell surface marker CD107a in endometriosis in the presence of IL-2 stimulation. Peripheral blood mononuclear cells (PBMCs) were obtained from 7 women with endometriosis and 7 women without endometriosis. The PBMCs were divided into two groups and either treated with recombinant IL-2 or left untreated. The cytotoxic activity of the PBMCs toward target cells (K562) was evaluated. Then, both groups were cocultured for 4 days. The expressions of CD107a, TNF-α, and IFN-γ were determined using flow cytometry analysis. There was no difference in the expression of CD107a prior to IL-2 stimulation in PBMCs from women with endometriosis compared to those from women without endometriosis. However, we observed upregulation of the expression of the surface marker CD107a after treatment in the endometriosis group. In addition, there was a significant difference in CD107a expression in the endometriosis group before versus after stimulation with IL-2 ( p < 0.01). We also found no difference in the production of TNF-α and IFN-γ before versus after treatment with IL-2 in either groups. The levels of CD107a were significantly enhanced in peripheral blood taken from women with endometriosis after treatment with IL-2.

CCL20/TNF/VEGFA Cytokine Secretory Phenotype of Tumor-Associated Macrophages Is a Negative Prognostic Factor in Cutaneous Melanoma

TAMs constitute a large fraction of infiltrating immune cells in melanoma tissues, but their significance for clinical outcomes remains unclear. We explored diverse TAM parameters in clinically relevant primary cutaneous melanoma samples, including density, location, size, and polarization marker expression; in addition, because cytokine production is a hallmark of macrophages function, we measured CCL20, TNF, and VEGFA intracellular cytokines by single-cell multiparametric confocal microscopy. The Kaplan–Meier method was used to analyze correlation with melanoma-specific disease-free survival and overall survival. No significant correlations with clinical parameters were observed for TAM density, morphology, or location. Significantly, higher contents of the intracellular cytokines CCL20, TNF, and VEGFA were quantified in TAMs infiltrating metastasizing compared to non-metastasizing skin primary melanomas (p < 0.001). To mechanistically explore cytokine up-regulation, we performed in vitro studies with melanoma-conditioned macrophages, using RNA-seq to explore involved pathways and specific inhibitors. We show that p53 and NF-κB coregulate CCL20, TNF, and VEGFA in melanoma-conditioned macrophages. These results delineate a clinically relevant pro-oncogenic cytokine profile of TAMs with prognostic significance in primary melanomas and point to the combined therapeutic targeting of NF-kB/p53 pathways to control the deviation of TAMs in melanoma.

Elevation of Neutrophil CEACAM1 Associated with Multiple Inflammatory Mediators was Related to Different Disease Progression in Ischemic Stroke Patients

Background Tissue damage, expecially blood-brain barrier (BBB) injury caused by inflammatory factor storm and stroke-associated infection (SAI) during cerebral stroke is an important factor affecting the prognosis of patients. We aim to analyze the level of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) together with the multiple inflammatory mediators at different stages of ischemic stroke (IS), as well as to investigate the role of CEACAM1 and the potential underlying mechanism in IS in the meanwhile. Methods The frequency of CEACAM1 positive neutrophils, neutrophil viability and levels of intracellular cytokines were detected by flow cytometry. The plasma CEACAM1, neutrophil gelatinase-associated lipocalin (NGAL), matrix metalloproteinases-9 (MMP-9), interleukin-10 (IL-10) and tumor necrosis factor (TNF) were measured by ELISA. Results Compared with the normal control, the percentage of CEACAM1 positive neutrophils in the peripheral blood in the IS patients showed significant elevation, and a significant increase was also noticed in the content of plasma CEACAM1 at the subacute stage. There was a positive correlation between CEACAM1 expression rate in the neutrophils and plasma CEACAM1 and IL-10 content in the subacute group. Compared with the acute group and healthy control group, there was an instinct elevation in the level of plasma MMP-9 in the subacute group. The plasma NGAL in the subacute and stable groups was significantly higher than that of the normal control. Conclusion Our data showed that there was rapid elevation of CEACAM1 in the neutrophils at the acute stage of cerebral IS. We speculated that CEACAM1 may serve as an inhibitory regulator involved in the progression of focal cerebral ischemia. Moreover, the elevation of serum NGAL at the acute and stable stages may involve in the BBB injury mediated by MMP-9.

Immunomodulatory Activities of Carica papaya L. Leaf Juice in a Non-Lethal, Symptomatic Dengue Mouse Model

The role of Carica papaya L. leaf juice in immune dysregulation caused by dengue virus infection remains unclear. This study aimed to investigate the immunomodulatory activities of the freeze-dried C. papaya leaf juice (FCPLJ) on AG129 mice infected with a clinical DENV-2 (DMOF015) isolate. The infected AG129 mice were orally treated with 500 and 1000 mg/kg/day of FCPLJ, for three days. Platelet, leukocyte, lymphocyte and neutrophil counts were microscopically determined. The level of plasma proinflammatory cytokines was measured by multiplex immunoassay. The levels of intracellular cytokines and viral RNA were determined by RT-qPCR technique. The results showed that the FCPLJ treatment increased the total white blood cell and neutrophil counts in the infected mice. The FCPLJ treatment decreased the level of GM-CSF, GRO-alpha, IL-1 beta, IL-6, MCP-1 and MIP-1 beta in the plasma of the infected mice. The intracellular IL-6 and viral RNA levels in the liver of infected mice were decreased by the FCPLJ treatment. In conclusion, this study supports the potential immunomodulatory role of the FCPLJ in a non-lethal, symptomatic dengue mouse model. Further studies on the action mechanism of the C. papaya leaf juice and its possible use as adjunctive dengue immunotherapy are warranted.

Optimized Intracellular Staining Reveals Heterogeneous Cytokine Production Ability of Murine and Human Hematopoietic Stem and Progenitor Cells

Under stress conditions, hematopoietic stem and progenitor cells (HSPCs) can translate danger signals into a plethora of cytokine signals. These cytokines, or more precisely their combination, instruct HSPCs to modify the magnitude and composition of hematopoietic output in response to the threat, but investigations into the heterogeneous cytokine expression and regulatory mechanisms are hampered by the technical difficulty of measuring cytokine levels in HSPCs at the single-cell level. Here, we optimized a flow cytometry-based method for the simultaneous assessment of multiple intracellular cytokines in HSPCs. By selecting an optimal combination of cytokine restimulation reagents, protein transport inhibitors, and culture supplements, an optimized restimulation protocol for intracellular staining was developed. Using this method, we successfully examined expression levels of granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in murine and human HSPC subsets under steady-state or different stress conditions. Different cytokine expression patterns were observed, suggesting distinct regulatory modes of cytokine production dependent on the HSPC subset, cytokine, disease, organ, and species. Collectively, this technical advance may help to obtain a better understanding of the nature of HSPC heterogeneity on the basis of differential cytokine production.

Dysregulation of metabolic pathways in circulating natural killer cells isolated from inflammatory bowel disease patients

Background and aims Inflammatory Bowel Disease (IBD), comprising Crohn's disease (CD) and ulcerative colitis (UC) are chronic conditions characterized by severe dysregulation of innate and adaptive immunity resulting in the destruction of the intestinal mucosa. Natural Killer (NK) cells play a pivotal role in the dynamic interaction between the innate and adaptive immune response. There is an increasing appreciation for the key role immunometabolism plays in the regulation of NK cell function, yet little remains known about the metabolic profile, cytokine secretion and killing capacity of human NK cells during active IBD. Methods PBMC were isolated from peripheral blood of patients with moderate to severely active IBD and healthy controls. NK cells were stained with a combination of cell surface receptors, intracellular cytokines, proteins and analyzed by flow cytometry. For measurements of NK cell cytotoxicity, the calcein-AM release assay was performed. Metabolic profile was analyzed by extracellular flux analyzer. Results NK cells from IBD patients produce large quantities of pro-inflammatory cytokines, IL-17A and TNF-α ex vivo but have limited killing capability. Furthermore, patient NK cells have reduced mitochondrial mass and oxidative phosphorylation. mTORC1, an important cell and metabolic regulator, demonstrated limited activity in both freshly isolated cells and cytokine stimulated cells. Conclusions Our results demonstrate that circulating NK cells of IBD patients have an unbalanced metabolic profile, with faulty mitochondria and reduced capacity to kill. These aberrations in NK cell metabolism may contribute to defective killing and thus the secondary infections and increased risk of cancer observed in IBD patients.

P0161TO EVALUATE THE TH PROFILE (TH1/TH2/TH17 ) AMONG GESTATIONAL DIABETES AND NORMAL PREGNANCY

Background and Aims Gestational diabetes mellitus (GDM) is a common pregnancy complication, characterized by insulin resistance and low-grade systemic inflammation with a pro-inflammatory immune system response. Our objective was to study the peripheral Th1, Th2, Th17 and Treg response in GDM compared to normal pregnancy. Method Th1, Th2, Th17 and Treg subsets was determined by flow cytometry based on staining for specific intracellular cytokines levels and their CRP levels were also assessed.. The health status of all offspring was also assessed 6 months post-delivery. Results Α total of 32 adult pregnant women were enrolled into a GDM (n=18) and Control (n=14) group. At the third trimester of pregnancy, the GDM group had a higher proportion of Th2, Th17 and Treg cells compared to control. Contrary to the control group, the GDM group exhibited no significant change in the Th1/Th2/Th17/Treg profile postpartum. A higher circulating CRP were noted in the GDM group compared to controls. At the 6-month post-delivery assessment, 23.6% of the offspring from the GDM group were found to have developed atopic dermatitis or food allergy compared to none from the control group. Conclusion Compared to an uncomplicated pregnancy, GDM exhibits a significantly different peripheral T-cell profile at the third pregnancy trimester characterized by higher proportion of Th2, Th17 and Treg cells which persist six months post-delivery, while the increased high sensitivity CRP (hsCRP) levels stressed the low-grade inflammatory profile of this disease

SAT0004 INCREASED M1 INFLAMMATORY PHENOTYPE OF CIRCULATING MONOCYTES IS ASSOCIATED WITH HISTORY OF CARDIOVASCULAR EVENTS IN RA PATIENTS

Background:Cardiovascular (CV) Disease is the main cause of death in Rheumatoid Arthritis (RA). Current tools like Framingham or European SCORE underestimate CV risk in RA patients. Efforts to improve the assessment including RA biomarkers (disease activity) have been only partially successful. There is a need for better biomarkers to identify AR patients at high risk for CV disease. Monocytes have an important role in plaque development. Monocytes differentiates into 2 main phenotypes M1 and M2 (1). In RA and in post-MI patients M1 monocytes are expanded (2). mTORC influences monocyte phenotypein vitroand has been associated with development of atheromatous plaque (3).Objectives:To evaluate the phenotype of circulating monocyte in RA patient with or without previous CV events (RA-CV(-)RA-CV(+)), and its possible association with mTORC activity.Methods:9 RA-CV(+)patients aged between 18 and 65 yo with RA (EULAR/ACR 2010 criteria), were paired with RA-CV(-)patients. 6 healthy individuals (HI) were also studied. Pairing criteria were classic CV risk factors (AHA 2018), sex, age, years since RA diagnosis, comorbidities, number of DMARDs previously used and use of bDMARDS. M1 and M2 circulating Monocytes were evaluated in PBMC obtained from patients and controls by flow cytometry analysis. Intracellular inflammatory cytokines (IL1, Il6) and phosphorylated S6R (P-S6R) as a measure of mTORC activation was also evaluated. M1 was defined as CD14+HLA-DR+CCR2+ and M2 CD14+CD163+CCR2-. DAS28-RCP, DAS28-ESR and Lipid profile was also measured. The differences among groups was analysed using Mann–Whitney U nonparametric. The relationship between variables with Spearman rank correlation test.Results:There were no differences in demographic, RA characteristic and CV risk factors between RA-CV (+) and RA-CV (-) patients. Male/Female 4/5, age 62±3 and 63±2 respectively. HI were younger than RA patients (32.5±7). CV events were 8 patients with MI and one Stroke. DAS28-RCP was 2.96±0.23 and 2.88±0.43 respectively. One patient in each group had failed to more than 2 sDMARDs and one in each group was receiving bDMARD. M1 circulating monocytes were expanded in RA as compared to HI. This difference was at RA-CV (+) expense. RA monocytes had higher Intracellular levels of IL-1b and IL-6 as compared to HI. M1 from RA-CV (+) had higher intracellular levels of IL-1b and IL-6 than RA-CV (-). M1 monocytes have higher levels of inflammatory cytokines than M2. P-S6R protein, (mTORC activation), was higher in RA patients than HI. The highest levels of P-S6R was observed in M1 monocytes from RA-CV(+) population.FIGURE 1.Circulating monocytes phenotype, intracellular cytokines and phosphorylated S6R in HI and RA-CV (+), RA-CV (-) and the combined RA patients. A) *=0,02; B) *=0,016; C) ****=0,0001, **=0,002; D) ****=0,0001, ***=0,0008, **=0,001, *=0,01; E) ****0,0001, **=0,002; F) ****=0,0001, **=0,001, **=0,003.Conclusion:RA-CV+ patients, have a significantly higher number of pro-inflammatory circulating monocytes, using a multiparametric classification method. These monocytes also express higher levels of inflammatory cytokines and higher activation of mTORC, which also participate in the development of atheromatous plaque, suggesting that these monocytes could be a key element in the non-clarified-yet, excess of CV risk of RA patients.References:[1] Fukui S, et al. M1 and M2 Monocytes in Rheumatoid Arthritis: A Contribution of Imbalance of M1/M2 Monocytes to Osteoclastogenesis. Front Immunol. 2017;8:1958.2. Zhuang J, et al. Comparison of circulating dendritic cell and monocyte subsets at different stages of atherosclerosis: insights from optical coherence tomography. BMC Cardiovasc Disord. 2017 Oct 18;17(1):270.Disclosure of Interests:None declared