Proliferation Of Peripheral Blood Mononuclear Cells Research Articles

Impact of platelet lysate on immunoregulatory characteristics of equine mesenchymal stromal cells.

Multipotent mesenchymal stromal cells (MSC) play an increasing role in the treatment of immune-mediated diseases and inflammatory processes. They regulate immune cells via cell-cell contacts and by secreting various anti-inflammatory molecules but are in turn influenced by many factors such as cytokines. For MSC culture, platelet lysate (PL), which contains a variety of cytokines, is a promising alternative to fetal bovine serum (FBS). We aimed to analyze if PL with its cytokines improves MSC immunoregulatory characteristics, with the perspective that PL could be useful for priming the MSC prior to therapeutic application. MSC, activated peripheral blood mononuclear cells (PBMC) and indirect co-cultures of both were cultivated in media supplemented with either PL, FBS, FBS+INF-γ or FBS+IL-10. After incubation, cytokine concentrations were measured in supernatants and control media. MSC were analyzed regarding their expression of immunoregulatory genes and PBMC regarding their proliferation and percentage of FoxP3+ cells. Cytokines, particularly IFN-γ and IL-10, remained at high levels in PL control medium without cells but decreased in cytokine-supplemented control FBS media without cells during incubation. PBMC released IFN-γ and IL-10 in various culture conditions. MSC alone only released IFN-γ and overall, cytokine levels in media were lowest when MSC were cultured alone. Stimulation of MSC either by PBMC or by PL resulted in an altered expression of immunoregulatory genes. In co-culture with PBMC, the MSC gene expression of COX2, TNFAIP6, IDO1, CXCR4 and MHC2 was upregulated and VCAM1 was downregulated. In the presence of PL, COX2, TNFAIP6, VCAM1, CXCR4 and HIF1A were upregulated. Functionally, while no consistent changes were found regarding the percentage of FoxP3+ cells, MSC decreased PBMC proliferation in all media, with the strongest effect in FBS media supplemented with IL-10 or IFN-γ. This study provides further evidence that PL supports MSC functionality, including their immunoregulatory mechanisms. The results justify to investigate functional effects of MSC cultured in PL-supplemented medium on different types of immune cells in more detail.

Activated and Naïve Allogenic Human Placental Mesenchymal Stromal Cells Exert an Immunomodulatory Effect on Hidradenitis Suppurativa Patient Peripheral Blood Mononuclear Cells.

This pilot study aimed to evaluate the immunomodulatory effect of placental mesenchymal stem/stromal cells (MSCs) on peripheral blood mononuclear cells (PBMCs) from patients with hidradenitis suppurativa (HS). Blood samples were collected from 3 healthy and 3 patients with HS. Isolated PBMCs were stained with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with phorbol 12-myristate 13-acetate (PMA)/Ionomycin solution. The PBMCs of patients with HS were co-cultured with naïve MSCs (n-MSCs), activated with tumor necrosis factor (TNF)-α (10 ng/mL) and interferon (IFN)-γ (10 ng/mL) MSCs (a-MSCs), or adalimumab (30 μg/mL). The division index (proliferation inhibition) of PBMCs was analyzed by flow cytometry using the Proliferation Modeling tool after 5 days of coculture. The relative inflammatory gene expression dynamics and cytokine secretion were quantified in triplicate using real-time polymerase chain reaction (PCR) and Luminex assays. PBMCs from the HS control group showed statistically significant increases in interleukin (IL)-6 and IFN-γ cytokine concentrations and IL-17A gene expression when compared with healthy subjects. Statistically significant reduction of the division index was found in the a-MSCs group (P = 0.04). Also, the Luminex assay revealed significantly reduced proinflammatory cytokine concentrations of IL-9 (P = 0.022) and IL-17A (P = 0.022) in the a-MSCs group with the same trend of numerical lowering in n-MSCs group when compared to HS control. The results of real-time PCR revealed a numerical increase in the expression of the IL-1β, IL-36α, and TNF-α genes in both the a-MSCs and n-MSCs groups compared with the HS control. In conclusion, our findings suggest that MSCs can effectively curb PBMCs proliferation and suppress the production of inflammatory cytokines. Moreover, the preactivation of MSCs with IFN-γ and TNF-α before use can enhance their therapeutic effectiveness. Nevertheless, a larger sample size is imperative to validate these results.

Combination of IFN-gamma with STING agonist and PD-1 immune checkpoint blockade: a potential immunotherapy for gastric cancer.

Suppression of the cGAS-STING pathway is an immune escape mechanism in cancer cells. The critical role of this pathway in gastric cancer (GC) is not fully understood. Herein, we evaluated the effect of the interferon-gamma (IFN-gamma), STING agonist, PD-1 immune checkpoint blockade, and their combination on the cGAS-STING pathway in GC. Expression of cGAS and STING in tumor tissue samples and adjacent normal tissue (ANT) biopsies of fifty new GC patients was evaluated by quantitative real-time PCR (qRT-PCR). Moreover, cGAS and STING expression levels were examined in Peripheral Blood Mononuclear Cells (PBMC) samples of forty GC patients and twenty-five healthy subjects. The apoptosis rate of cancer cells was analyzed by Annexin V-FITC/PI. Cell proliferation was measured by the BrdU assay. Also, IFN-β levels were evaluated in the supernatants of the treated groups. The cGAS expression was decreased in patients with distant metastasis. Co-cultures treated with IFN-gamma showed an elevated level of cGAS and STING expressions in PBMC and cancer cells. The rate of apoptosis increased in all the treatment groups. In addition, the rate of proliferation in PBMCs increased in different treated groups. The main role of PBMCs in cytotoxicity was determined by a comparative analysis of the viability of cells treated with all treatments, both with and without PBMCs. The production of IFN-β was elevated in all treated groups. The current study suggests that a combination therapy using IFN-gamma, STING agonist, and anti-PD-1 antibody can provide a promising approach to the treatment of GC.

Immunomodulatory Role of Rosmarinus officinalis L., Mentha x piperita L., and Lavandula angustifolia L. Essential Oils in Sheep Peripheral Blood Mononuclear Cells.

Recently, the uses of essential oils (EOs) as rumen modifiers, anti-inflammatory agents, and antioxidants were demonstrated in livestock. In the present study, the role of Mentha x piperita L. (MEO), Rosmarinus officinalis L. (REO), and Lavandula angustifolia L. (LEO) EOs in an in vitro sheep model of inflammation was investigated. With this aim, peripheral blood mononuclear cells (PBMCs) were treated with incremental concentrations (3, 5, 7, and 10%) of each EO to test their effects on cell viability and proliferation and on interleukin (IL)-6, IL-10, and IL-8 secretion. The PBMCs were stimulated by Concanavalin A (ConA) alone or in combination with lipopolysaccharide (LPS) mitogen. The positive and negative controls were represented by PBMCs in the presence or absence, respectively, of mitogens only. The cell viability and proliferation were determined by XTT and BrdU assays, while the cytokines were analyzed by ELISA. The EO treatments did not affect the viability; on the contrary, the PBMC proliferation increased in presence of all the EOs tested, according to the different percentages and mitogens used. The IL-10 secretion was higher in both the REO and the LEO tested at 3% than in the positive control; furthermore, the IL-8 level was influenced differently by the various EOs. The present data demonstrate that EOs may modulate the immune response activated by inflammation.

Cytotoxicity and anti-inflammatory properties of a GLP-1 receptor agonist in a model of human peripheral blood mononuclear cell culture

<p><strong>Introduction. </strong>GLP-1R agonists (GLP-1RAs) are a type of anti-hyperglycemic medications used for the management of type 2 diabetes mellitus (T2DM). In addition to glucose-lowering effects, GLP-1R agonists (GLP-1RAs) also provide further advantages by promoting weight loss and controlling blood pressure. Furthermore, GLP-1RAs have been reported for their therapeutic benefits in neurological, cardiovascular, endocrine, and metabolic diseases. Emerging evidence from many clinical and experimental studies, as well as in vitro researches, suggests that GLP-1RA agonists may reduce inflamma tion and modify the immunological response, but the underlying molecu lar mechanisms remain unclear. In this research, we examined the effect of GLP-1RA on the cytotoxicity, proliferative activity, and cytokine production of human peripheral blood mononuclear cells (PBMCs) in vitro.</p> <p><strong> Methods. </strong>The peripheral blood mononuclear cells (PBMCs) cultures from healthy volunteers were used. GLP-1RA (DMB), in different concentrations, was added to the cell cultures. A flow cytometry assay was used to assess cell viability, while MTT and CFSE dye dilution assays were used to quantify proliferative activity. Cytokine levels were measured using a sandwich en zyme-linked immunosorbent assay (ELISA).</p> <p><strong>Results.</strong> The cytotoxicity data demonstrated that only the highest DMB concentration (1000 nM) decreased the metabolic activity (viability) of PBMCs (p<0.05) in comparison to untreated cultures, while concentra tions of 200 nM (p<0.05) and 1000 nM (p<0.001) of GLP-1RA increased the proportion of late apoptotic and necrotic cells (p<0.001). The prolif eration of PBMCs was significantly decreased in the presence of GLP-1RA at a 100 nM concentration when compared to the control (p<0.05). The same concentration of GLP-1RA significantly reduced the production of IL-6, IL-1β, and TNF-α.</p> <p><strong>Conclusion.</strong> Our results suggested that GLP-1RA (DMB), at a non-toxic concentration of 100 nM, inhibited the proliferation and production of pro-inflammatory cytokines by human PBMCs in vitro. These findings open the perspective to study the immune response in detail in future experiments.</p>

Leucine-Rich Glioma-Inactivated 1 (LGI1) Protein Stimulates Proliferation and IL-10 Production in Peripheral Blood Mononuclear Cells of Patients with LGI1 Antibody-Mediated Autoimmune Encephalitis In Vitro.

Limbic encephalitis (LE) due to anti-leucine-rich glioma-inactivated 1 (LGI1) antibodies is an autoimmune disease characterized by distinct clinical features unique to LGI1 LE, such as faciobrachial dystonic seizures. However, it is unclear whether an additional disease-related LGI1 antigen-specific T cell response is involved in the pathogenesis of this disease. To address this question, we studied the effect of recombinant LGI1 on the proliferation and effector-specific cytokine production (IFN-γ, IL-5, IL-10, and IL-17) of peripheral blood mononuclear cells (PBMCs) from patients with LGI1 LE and healthy controls. We observed that recombinant LGI1 stimulated the proliferation of PBMCs from patients with LGI1 LE, but not from healthy controls. Cytokine measurement of cell culture supernatants from PBMCs incubated with recombinant LGI1 revealed a highly significant increase in IL-10 release in PBMCs from patients with LGI1 LE in comparison with healthy controls. These results suggest that LGI1-mediated stimulation of PBMCs from patients with LGI1 LE leads to the establishment of an IL-10-dominated immunosuppressive cytokine milieu, which may inhibit Th1 differentiation and support B cell proliferation, IgG production, and IgG subclass switching.

Anti-inflammatory effects of infliximab and methotrexate on peripheral blood and synovial fluid mononuclear cells: ex vivo study

Objective To investigate the effects of methotrexate (MTX) and the tumour necrosis factor inhibitor infliximab (IFX) on immune cells derived from peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) of inflammatory arthritis patients. Method Phytohaemagglutinin (PHA)-induced proliferation of healthy donors’ PBMCs and synovial intermediate monocytes (CD14+CD16+ cells) in SFMCs derived from psoriatic arthritis (PsA) and rheumatoid arthritis (RA) patients was determined by flow cytometry following co-culture with IFX and MTX. PHA-induced interferon-γ (IFN-γ) production in PBMCs was measured by enzyme-linked immunosorbent assay. The drugs’ effect on mRNA expression in SFMCs was determined by quantitative polymerase chain reaction. Results The combination of IFX 10 μg/mL + MTX 0.1 μg/mL had the strongest inhibitory effect on PBMC proliferation (91%), followed by MTX 0.1 μg/mL (86%) and IFX 10 μg/mL (49%). In PHA-stimulated PBMCs, IFN-γ production was reduced by IFX 10 μg/mL, MTX 0.1 μg/mL, and IFX 10 μg/mL + MTX 0.1 μg/mL by 68%, 90%, and 85%, respectively. In SFMCs, IFX 10 µg/mL significantly reduced CD14+CD16+ cells compared to medium (PsA 54%, p < 0.01; RA 46%, p < 0.05), while MTX had no effect on this population. IFX + MTX led to a similar suppression of CD14+CD16+ cells as achieved by IFX alone. The drugs had different impacts on SFMC gene expression. Conclusion Both IFX and MTX effectively inhibited PBMC proliferation and IFN-γ production, but only IFX reduced synovial monocytes and pro-inflammatory gene expression in SFMCs, suggesting a differential impact of IFX and MTX on critical inflammatory cell populations ex vivo.

Abstract C014: CXCR4 inhibition enhances anti-tumor immune response in an ex vivo autologous patient-derived organoids and PBMC model system of pancreatic ductal adenocarcinoma

Abstract Introduction Immune checkpoint blockade either alone or in combination with chemotherapy has been ineffective in pancreatic ductal adenocarcinoma (PDAC) likely due to underlying immunosuppressive pathways. The CXC chemokine receptor type 4 (CXCR4)/chemokine (CXC motif) ligand 12 (CXCL12) axis is one key immunosuppressive pathway. CXCL12 (released by cancer-associated fibroblasts (CAF)) binds to CXCR4 on CD8+ T cells (CTL) and decreases its infiltration into the tumor. Encouraging preclinical studies from murine PDAC models and human PDAC tumor sections have demonstrated infiltration of T cells with tumors after co-inhibition of CXCR4 and PD1/PDL1, however with limited tumor regressions in mouse model. We established an innovative ex vivo autologous patient-derived immune/organoid (PDIO) co-culture system that allows studying the interactions between distinct immune cells and tumor cells. Methods Patient-derived tumor organoids (PDTOs) generated from tumors resected from patients diagnosed with pancreas cancer were grown to confluence in Matrigel domes. We conducted ex vivo migration assays with autologous peripheral blood mononuclear cells (PBMCs) which were placed in the surrounding liquid phase of the matrigels. We separately treated PBMCs and PDTOs with AMD3100, an inhibitor of CXCR4, and tested the role of CXCR4 inhibition on PBMCs and PDTOs in T cell exclusion and immune cell activation. Results Pre-treatment of PBMCs with AMD3100 increased CD8+ T cell mobilization towards autologous PDTOs in matrigel dome migration assays by up to 11-fold. After co-culture in the liquid interphase of PDTOs matrigel domes, AMD3100 pre-treated PBMCs exhibit a more robust proliferative response compared to untreated PBMCs and a significant increase in the number of reactive T cells migrated inside the PDTO matrigel dome and expressing activation markers (p=0.02). Nearly 50% of T cells in proximity of tumor cells produce the cytotoxic factor Granzyme B (p=0.002), with AMD3100 treatment of PBMC increasing it by up to 3-fold compared to untreated PBMC. CXCR4 inhibition of PDTOs resulted in modulation of immune checkpoint ligands involved in the tumor immune response and tumor cell immunogenicity. Combination treatment of PBMCs and PDTOs with AMD3100 and anti-PD1 antibody ultimately resulted in increased tumor cell death with up to 45% of PDTOs exhibiting apoptosis markers after coculture with AMD3100-treated PBMC, implicating a role of this combination in the clinic. Conclusions We demonstrate that inhibition of CXCR4, not only increases migration of autologous T cells towards PDTOs, but also results in proliferation of PBMCs and activation of T cells. Furthermore, CXCR4 inhibition of PDTOs increases antigen presentation and tumor cell death. Inhibition of CXCR4 is an attractive therapeutic target as it enhances anti-tumor immune response via dual targeting of immune and neoplastic compartments. We are testing this combination in a multicenter investigator-initiated clinical trial in treatment-naïve metastatic PDAC patients. Citation Format: Ilenia Pellicciotta. CXCR4 inhibition enhances anti-tumor immune response in an ex vivo autologous patient-derived organoids and PBMC model system of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C014.

Weight loss and vitamin D improve hyporesponsiveness to corticosteroids in obese asthma.

Obesity negatively impacts on asthma response to inhaled corticosteroids by unknown mechanisms. Objective: To demonstrate that the poor response to inhaled corticosteroids in obese asthma is associated with an impaired anti-inflammatory activity of corticosteroids and vitamin D deficiency, both improved by weight loss. 23 obese asthmatics (OA) [18 females; median age (interquartile range) 56 (51-59) years], 14 non-obese asthmatics (NOA) [11 females; 53 (43-60) years], 15 obese (O) [13 females; 47 (45-60) years], and 19 healthy controls (HC) [14 females; 43 (34-56) years] were enrolled. 10 OA and 11 O patients were evaluated at baseline (V1) and six months after (V2) bariatric surgery. Corticosteroid response was measured by dexamethasone inhibition of peripheral blood mononuclear cell (PBMC) proliferation. Lung function, serum levels of leptin, adiponectin, and vitamin D were measured at V1 and V2. We found a reduced response to dexamethasone in PBMCs of O and OA patients with respect to NOA and HC subjects, that inversely correlated with the diponectin/leptin ratio and vitamin D levels. Bariatric surgery improved corticosteroid responses in O and OA patients and normalized adiponectin/leptin ratio and vitamin D levels. Exposure of PBMCs to vitamin D potentiated the antiproliferative effects of corticosteroids. Dexamethasone and vitamin D induced similar MKP-1 expression in O and OA patients. We found a reduced response to dexamethasone in PBMCs of O and OA patients with respect to NOA and HC subjects, that inversely correlated with the adiponectin/leptin ratio and vitamin D levels. Bariatric surgery improved corticosteroid responses in O and OA patients and normalized adiponectin/leptin ratio and vitamin D levels. Exposure of PBMCs to vitamin D potentiated the antiproliferative effects of corticosteroids. Dexamethasone and vitamin D induced similar MKP-1 expression in O and OA patients.

Development of a novel in vitro cell model for evaluation of nanofiber mats immunogenicity

Immunological safety of nanofibers remains poorly reported within the scientific literature and lacks specific in vitro testing models distinct from those used to test nanoparticles. To address the challenges of currently used conventional setups being described in the literature, we developed a novel in vitro model for nanofiber mats immunogenicity testing, which enables standardization of tested surface area, excludes nanofiber mat edges, and ensures stable contacts of cells with nanofibers during the experiment. The effect of nanofibers was assessed on peripheral blood mononuclear cells (PBMCs) by measuring their metabolic activity using MTS cell proliferation assay, where key performance parameters, i.e. cell number, phytohemagglutinin-L (PHA-L) concentration, incubation time and cell lysis were optimized. Repeatability of results obtained with non-activated and PHA-L-activated PBMCs in contact with differently thick polycaprolactone nanofiber mats was compared using both models. Our model provided more reproducible results with lower variability, exhibiting its higher reliability and accuracy than the conventional one. Furthermore, results showed the presence of thicker mats resulted in reduced metabolic activity and PBMC proliferation without any observed cytotoxicity, providing additional insights into their non-immunogenic characteristics. The developed model enables more accurate biological assessment that can support new guidelines for in vitro nanofiber testing and formulation.

Recombinant Human Thrombopoietin Reduces the Risk of Acute Graft-Versus-Host-Disease and Its Mechanisms

Objective: Acute graft-versus-host-disease (aGVHD) is an important complication that is observed after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We aimed to evaluate the effects of recombinant human thrombopoietin (TPO; rhTPO) on aGVHD using clinical data, mice models, and peripheral blood mononuclear cells (PBMCs). Methods: From 2016 to 2018, 162 cases of allo-HSCT in our hospital were recruited in this study. The correlation between TPO use and aGVHD was analyzed. After a GVHD model was constructed in Balb-C mice, HE staining was performed to compare the pathophysiological changes of the liver, intestine, lung, kidney, and other organs of the mice. The levels of lymphocyte subsets and cytokines in the mice spleens in the different groups were detected. PBMCs were isolated from the peripheral blood of healthy persons and patients with allo-HSCT via Ficoll gradient centrifugation and treated with different concentrations of rhTPO. The proliferation of PBMCs was detected via the CCK8 method, and the differentiation of PBMCs into lymphocytes was analyzed via flow cytometry. Results: 1) The occurrence of aGVHD was observed in 10 patients (47.6%) in the control group and 27 (23.1%) in the treatment group. The difference between the two groups was statistically significant (χ2=5.465, P=0.019). 2) The results of the aGVHD mice proved that rhTPO decreased the damage to the mouse liver, intestine, lung, and kidney tissues. In addition, splenic cell subpopulations CD4+cells, CD8+cells, T cells, and NK cells increased in the GVHD group but decreased due to rhTPO (P&amp;lt;0.05). After rhTPO treament, T cell subpopulations Th2 and Th17 cells decreased in the GVHD mice (P&amp;lt;0.05). Cytokines CD8+IL-17A+ and CD8+IL-4+IFNγ showed no difference between the four groups (P&amp;gt;0.05). Compared to GVHD mice, CD8+IL-4+ decreased in TPO-treated GVHD mice, and the effect of TPO at a high dose was more significant (P&amp;lt;0.05). 3) The results of the PMBCs suggested that a low dose of rhTPO could promote the proliferation of PMBCs, while a high dose could inhibit the proliferation. rhTPO promoted the differentiation of PBMCs into the CD3-PC5 subset. Conclusions: Altogether, this study indicates that rhTPO reduces the risk of aGVHD and is a protective factor against the occurrence of aGVHD.

Hypoxia and inflammatory factor preconditioning enhances the immunosuppressive properties of human umbilical cord mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have great potential for the treatment of various immune diseases due to their unique immunomodulatory properties. However, MSCs exposed to the harsh inflammatory environment of damaged tissue after intravenous transplantation cannot exert their biological effects, and therefore, their therapeutic efficacy is reduced. In this challenging context, an in vitro preconditioning method is necessary for the development of MSC-based therapies with increased immunomodulatory capacity and transplantation efficacy. To determine whether hypoxia and inflammatory factor preconditioning increases the immunosuppressive properties of MSCs without affecting their biological characteristics. Umbilical cord MSCs (UC-MSCs) were pretreated with hypoxia (2% O2) exposure and inflammatory factors (interleukin-1β, tumor necrosis factor-α, interferon-γ) for 24 h. Flow cytometry, polymerase chain reaction, enzyme-linked immunosorbent assay and other experimental methods were used to evaluate the biological characteristics of pretreated UC-MSCs and to determine whether pretreatment affected the immunosuppressive ability of UC-MSCs in coculture with immune cells. Pretreatment with hypoxia and inflammatory factors caused UC-MSCs to be elongated but did not affect their viability, proliferation or size. In addition, pretreatment significantly decreased the expression of coagulation-related tissue factors but did not affect the expression of other surface markers. Similarly, mitochondrial function and integrity were retained. Although pretreatment promoted UC-MSC apoptosis and senescence, it increased the expression of genes and proteins related to immune regulation. Pretreatment increased peripheral blood mononuclear cell and natural killer (NK) cell proliferation rates and inhibited NK cell-induced toxicity to varying degrees. In summary, hypoxia and inflammatory factor preconditioning led to higher immunosuppressive effects of MSCs without damaging their biological characteristics.

Human Amniotic Fluid Stem Cells Exert Immunosuppressive Effects on T Lymphocytes in Allergic Rhinitis.

The study aims to investigate the immunomodulatory effect of Amniotic fluid stem (AFS) cells to Th2-skewed allergic rhinitis (AR) on T-lymphocyte proliferation, viability, activation and cytokine production. AFS cells can suppress peripheral blood mononuclear cells (PBMCs) proliferation and display immunomodulatory properties, but AFS cells' immunoregulation on AR has not been defined. Human AFS cells were derived from magnetic cell sorting and co-cultured with PBMCs from AR patients stimulated by phytohemagglutinin (PHA). The AFS cells-associated suppressive proliferation was analyzed using CellTrace™ Violet assay; the T lymphocytes proliferation, viability, activation and the Foxp3+ Treg cells were determined by flow cytometry; cytokine levels were measured using an enzyme- linked immunosorbent assay. We determined that AFS cells significantly inhibited PHA-induced CD3+ T lymphocyte proliferation at the ratio higher than 1:50 (AFS cells: PBMCs) (P<0.05); AFS cells obviously increased the T lymphocytes viability (P<0.01), inhibited the apoptosis of T lymphocytes (P<0.001), compared to PBMCs alone; AFS cells suppressed CD3+CD25+ T lymphocytes activated by PHA (P<0.05); AFS cells significantly promote Treg cells expansion in house dust mite (HDM)-stimulated PBMCs from AR patients (P<0.05). Compared with HDM-stimulated PBMCs, AFS cell co-culture predominantly decreased IL-4 level (P<0.05), but increased IFN-γ and IL-10 levels (P<0.01). AFS cells modulate the T-cells' immune imbalance towards Th2 suppression in AR, which can be used as a new cell banking for allergic airway diseases.

Impact of Chlorogenic Acid on Peripheral Blood Mononuclear Cell Proliferation, Oxidative Stress, and Inflammatory Responses in Racehorses during Exercise.

Green coffee extract is currently of great interest to researchers due to its high concentration of chlorogenic acid (CGA) and its potential health benefits. CGA constitutes 6 to 10% of the dry weight of the extract and, due to its anti-inflammatory properties, is a promising natural supplement and agent with therapeutic applications. The purpose of our study was to discover the effects of CGA on peripheral blood mononuclear cell proliferation, and the production of pro- and anti-inflammatory cytokines as well as reactive oxidative species (ROS) in horses during exercise. According to the findings, CGA can affect the proliferation of T helper cells. In addition, at a dose of 50 g/mL, CGA increased the activation of CD4+FoxP3+ and CD8+FoxP3+ regulatory cells. Physical activity decreases ROS production in CD5+ monocytes, but this effect depends on the concentration of CGA, and the effect of exercise on oxidative stress was lower in CD14+ than in CD5+ cells. Regardless of CGA content, CGA significantly increased the release of the anti-inflammatory cytokine IL-10. Moreover, the production of IL-17 was greater in cells treated with 50 g/mL of CGA from beginners compared to the control and advanced groups of horses. Our findings suggest that CGA may have immune-enhancing properties. This opens new avenues of research into the mechanisms of action of CGA and possible applications in prevention and health promotion in sport animals.

A Bidens pilosa L. Non-Polar Extract Modulates the Polarization of Human Macrophages and Dendritic Cells into an Anti-Inflammatory Phenotype.

Different communities around the world traditionally use Bidens pilosa L. for medicinal purposes, mainly for its anti-inflammatory, antinociceptive, and antioxidant properties; it is used as an ingredient in teas or herbal medicines for the treatment of pain, inflammation, and immunological disorders. Several studies have been conducted that prove the immunomodulatory properties of this plant; however, it is not known whether the immunomodulatory properties of B. pilosa are mediated by its ability to modulate antigen-presenting cells (APCs) such as macrophages (MØs) and dendritic cells (DCs) (through polarization or the maturation state, respectively). Different polar and non-polar extracts and fractions were prepared from the aerial part of B. pilosa. Their cytotoxic and immunomodulatory effects were first tested on human peripheral blood mononuclear cells (PBMCs) and phytohemagglutinin (PHA)-stimulated PBMCs, respectively, via an MTT assay. Then, the non-cytotoxic plant extracts and fractions that showed the highest immunomodulatory activity were selected to evaluate their effects on human MØ polarization and DC maturation (cell surface phenotype and cytokine secretion) through multiparametric flow cytometry. Finally, the chemical compounds of the B. pilosa extract that showed the most significant immunomodulatory effects on human APCs were identified using gas chromatography coupled with mass spectrometry. The petroleum ether extract and the ethyl acetate and hydroalcoholic fractions obtained from B. pilosa showed low cytotoxicity and modulated the PHA-stimulated proliferation of PBMCs. Furthermore, the B. pilosa petroleum ether extract induced M2 polarization or a hybrid M1/M2 phenotype in MØs and a semi-mature status in DCs, regardless of exposure to a maturation stimulus. The immunomodulatory activity of the non-polar (petroleum ether) extract of B. pilosa on human PBMC proliferation, M2 polarization of MØs, and semi-mature status in DCs might be attributed to the low-medium polarity components in the extract, such as phytosterol terpenes and fatty acid esters.

Imatinib and M351-0056 enhance the function of VISTA and ameliorate the development of SLE via IFN-I and noncanonical NF-κB pathway.

V-domain immunoglobulin suppressor of T-cell activation (VISTA), an important negative checkpoint protein, participates in immunoregulation. Systemic lupus erythematosus (SLE) is an autoimmune disease in which patients exhibit high levels of autoantibodies and multi-organ tissue injury, primarily involving the kidney and skin. In wild-type (WT) mice and Vsir-/- mice with pristane-induced lupus-like disease, we found that VISTA deficiency exacerbated the lupus-like disease in mice, possibly through aberrant activation of type I interferon (IFN-I) signaling, CD4+ T cell, and noncanonical nuclear factor-κB (NF-κB) pathway. Surface plasmon resonance results showed that imatinib, an FDA-approved tyrosine kinase inhibitor, may have a high affinity for human VISTA-ECD with a KD value of 0.2009 μM. The biological activities of imatinib and VISTA agonist M351-0056 were studied in monocytes and T cells and in lupus-like disease murine model of chronic graft-versus-host disease (cGVHD) and lupus-prone MRL/lpr mice. VISTA small-molecule agonist reduced the cytokine production of peripheral blood mononuclear cells (PBMCs) and Jurkat cells and inhibited PBMCs proliferation. Moreover, they attenuated the levels of autoantibodies, renal injury, inflammatory cytokines, chemokines, and immune cell expansion in the cGVHD mouse model and MRL/lpr mice. Our findings also demonstrated that VISTA small-molecule agonist ameliorated the development of SLE through improving aberrantly activated IFN-I signaling and noncanonical NF-κB pathway. In conclusion, VISTA has a protective effect on the development and progression of SLE. VISTA agonist M351-0056 and imatinib have been firstly demonstrated to attenuate SLE, suggesting interventions to enhance VISTA function may be effective in treating SLE. VISTA deficiency exacerbates pristane-induced lupus-like disease in mice by promoting activation of the IFN-I and noncanonical NF-κB pathway. Imatinib was screened as a small-molecule VISTA agonist by molecular docking, SPR, and cellular level experiments. VISTA agonists (M351-0056 and imatinib) alleviated lupus-like disease progression in the cGVHD mouse model and MRL/lpr mice by inhibiting activation of IFN-I and noncanonical NF-κB pathway.

Effect of Cannabidiol on Human Peripheral Blood Mononuclear Cells and CD4+ T Cells.

Cannabidiol (CBD), the main non-psychoactive component of Cannabis sativa L., is widely used in therapy for the treatment of different diseases and as an adjuvant drug. Our aim was to assess the effects of CBD on proinflammatory cytokine production and cell proliferation in human peripheral blood mononuclear cells (PBMCs) and on CD4+ T lymphocyte differentiation, and, furthermore, to test CBD's ability to affect the functional properties of regulatory T cells (Treg). Experiments were performed on isolated PBMCs and purified CD4+ T lymphocytes obtained from the buffy coats of healthy subjects. Cytokines produced by CD4+ T cells were evaluated by flow cytometry and intracellular cytokine staining techniques. PBMC cytokine production was measured by an ELISA assay. Real-time PCR was used to assess the mRNA expression of cytokines and the key transcription factors (TFs) of CD4+ T cells. Finally, the proliferation of PBMC and CD4+ T effector cells (Teff), alone and in the presence of Treg, was assessed by flow cytometry. Results showed that CBD affects both the frequency of IL-4-producing CD4+ and of IFN-γ/IL-17-producing cells and dramatically decreases the mRNA levels of all TFs. Stimuli-induced cytokine mRNA expression was decreased while protein production was unaffected. CBD was unable to affect the ability of Treg to prevent Teff cell proliferation while it slightly increased PBMC proliferation. In conclusion, CBD may inhibit the expression of proinflammatory cytokines; however, the effect of CBD on cell proliferation suggests that this cannabinoid exerts a complex activity on human PBMCs and CD4+ T cells which deserves further investigation.

Graphene oxide accelerates TGFβ-mediated epithelial-mesenchymal transition and stimulates pro-inflammatory immune response in amniotic epithelial cells.

The application of biomaterials on immune regenerative strategies to deal with unsolved pathologies is getting attention in the field of tissue engineering. In this context, graphene oxide (GO) has been proposed as an immune-mimetic material largely used for developing stem cell-based regenerative therapies, since it has shown to influence stem cell behavior and modulate their immune response. Similarly, amniotic epithelial stem cells (AECs) are getting an increasing clinical interest as source of stem cells due to their great plasticity and immunomodulatory paracrine activities, even though GO bio-mimetic effects still remain unknown. To this aim, GO-functionalized glass coverslips have been used for AECs culture. The results demonstrated how GO-coating is able to induce and accelerate the Epithelial-Mesenchymal Transition (EMT), in a process mediated by the intracellular activation of TGFβ1-SMAD2/3 signaling pathway. The trans-differentiation towards mesenchymal phenotype provides AECs of migratory ability and substantially changes the pattern of cytokines secretion upon inflammatory stimulus. Indeed, GO-exposed AECs enhance their pro-inflammatory interleukins production thus inducing a more efficient activation of macrophages and, at the same time, by slightly reducing their inhibitory action on peripheral blood mononuclear cells proliferation. Therefore, the adhesion of AECs on GO-functionalized surfaces might contribute to the generation of a tailored microenvironment useful to face both the phases of the inflammation, thereby fostering the regenerative process.

Short-term assays for mesenchymal stromal cell immunosuppression of T-lymphocytes.

Trauma patients are susceptible to coagulopathy and dysfunctional immune responses. Mesenchymal stromal cells (MSCs) are at the forefront of the cellular therapy revolution with profound immunomodulatory, regenerative, and therapeutic potential. Routine assays to assess immunomodulation activity examine MSC effects on proliferation of peripheral blood mononuclear cells (PBMCs) and take 3-7 days. Assays that could be done in a shorter period of time would be beneficial to allow more rapid comparison of different MSC donors. The studies presented here focused on assays for MSC suppression of mitogen-stimulated PBMC activation in time frames of 24 h or less. Three potential assays were examined-assays of apoptosis focusing on caspase activation, assays of phosphatidyl serine externalization (PS+) on PBMCs, and measurement of tumor necrosis factor alpha (TNFα) levels using rapid ELISA methods. All assays used the same initial experimental conditions: cryopreserved PBMCs from 8 to 10 pooled donors, co-culture with and without MSCs in 96-well plates, and PBMC stimulation with mitogen for 2-72 h. Suppression of caspase activity in activated PBMCs by incubation with MSCs was not robust and was only significant at times after 24 h. Monitoring PS+ of live CD3+ or live CD4+/CD3+ mitogen-activated PBMCs was dose dependent, reproducible, robust, and evident at the earliest time point taken, 2 h, although no increase in the percentage of PS+ cells was seen with time. The ability of MSC in co-culture to suppress PBMC PS+ externalization compared favorably to two concomitant assays for MSC co-culture suppression of PBMC proliferation, at 72 h by ATP assay, or at 96 h by fluorescently labeled protein signal dilution. TNFα release by mitogen-activated PBMCs was dose dependent, reproducible, robust, and evident at the earliest time point taken, with accumulating signal over time. However, suppression levels with MSC co-culture was reliably seen only after 24 h. Takeaways from these studies are as follows: (1) while early measures of PBMC activation is evident at 2-6 h, immunosuppression was only reliably detected at 24 h; (2) PS externalization at 24 h is a surrogate assay for MSC immunomodulation; and (3) rapid ELISA assay detection of TNFα release by PBMCs is a robust and sensitive assay for MSC immunomodulation at 24 h.

Eribulin promotes proliferation of CD8+ T cells and potentiates T cell-mediated anti-tumor activity against triple-negative breast cancer cells.

Chemotherapeutic agents exert immunomodulatory effects on triple-negative breast cancer (TNBC) cells and immune cells. Eribulin favorably affects the immunological status of patients with breast cancer. However, the effects of eribulin on the immune cells remain unexplored. The aim of this study was to investigate the effects of eribulin on immune cells. Peripheral blood mononuclear cells (PBMCs) from healthy donors and mouse splenocytes were stimulated with anti-CD3 and anti-CD28 antibodies. The effects of eribulin and paclitaxel on cell proliferation and differentiation status were analyzed using flow cytometry. RNA sequencing was performed to assess alterations in gene expression in CD8+ T cells following eribulin and paclitaxel treatment. Using TNBC cell lines (MDA-MB-231, Hs578T, and MDA-MB-157), the anti-tumor activity of CD3/CD28-stimulated T cells combined with eribulin or paclitaxel was evaluated. Eribulin did not affect CD3/CD28-stimulated PBMCs proliferation. However, eribulin significantly decreased the CD4/CD8 ratio in T cells, indicating that eribulin facilitates CD8+ T cell proliferation. Furthermore, eribulin significantly increased the frequency of less differentiated CD45RA+, CCR7+, and TCF1+ subsets of CD8+ T cells. RNA sequencing revealed that eribulin enhanced the expression of gene sets related to cell proliferation and immune responses. Moreover, eribulin augmented the anti-tumor effects of CD3/CD28-stimulated T cells against TNBC cells. These results were not observed in experiments using paclitaxel. Eribulin promoted CD8+ T cell proliferation, repressed effector T cell differentiation, and harnessed T cell-mediated anti-tumor effects. These mechanisms may be one of the cues that eribulin can improve the immunological status of tumor-bearing hosts.