Activation Of Peripheral Blood Mononuclear Cells Research Articles

Recombinant Human Proteoglycan‐4 Regulates Phagocytic Activation of Monocytes and Reduces IL‐1b Secretion by Urate Crystal Stimulated PBMCs

Introduction Gout is a chronic inflammatory disease due to monosodium urate (MSU) crystal deposits in articular joints. MSU phagocytosis by monocytes/macrophages results in interleukin-1 beta (IL-1β) secretion. Pharmacologic treatments of gout are inadequate and are associated with significant side effects. Interleukin-1 receptor antagonist (IL-1RA) is an endogenous inhibitor of IL-1b signaling and is efficacious in acute gout management where other treatments are contraindicated. Proteoglycan-4 (PRG4) is an anti-inflammatory glycoprotein that has shown efficacy in reducing MSU induced IL-1b release from macrophages. Our objective was to compare the activation of monocytes, of normal subjects and gout patients, by MSU crystals and examine the comparative efficacy of rhPRG4 and rhIL-1RA in regulating phagocytic activation of monocytes and IL-1b secretion. Methods Collection of peripheral blood mononuclear cells (PBMCs) and sera from patients with gout was approved by the IRB at RIH. PBMCs and sera of normal subjects were obtained from ATCC. IL-1b and IL-1RA levels were determined by ELISA. PBMCs were seeded (1 million cells/well) in 6 well plates and treated with Pam3CSK4 (1mg/mL; TLR2 agonist) ± rhIL-1RA or rhPRG4 for 24 h followed by MSU crystals (200mg/mL) for 6 h. Monocytes in PBMCs were identified by flow cytometry using anti-CD14-APC-Cy7 and anti-CD45-PE-A antibodies (BioLegend). Monocytes’ phagocytic activity was determined by co-incubation of PBMCs with FITC-labeled rabbit IgG-coated latex beads (Cayman Chemicals) for 2 h followed by determination of percent bead-positive monocytes. Statistical analyses included Student's t-test and one-way ANOVA followed by post-hoc Tukey's test. Results Sera from patients with gout had lower IL-1RA levels compared to sera from normal subjects (p<0.01; fig. 1A). The gating of monocytes and corresponding bead phagocytosis is shown in fig. 1B. Representative plots showing increased bead phagocytosis by gout PBMCs compared to normal PBMCs are shown in fig. 1C. Basal phagocytic activities of gout PBMCs were higher than corresponding activities of normal PBMCs (p<0.0001; fig. 1D). IL-1b secretion in MSU crystal stimulated gout PBMCs was higher than corresponding secretion in normal PBMCs (p<0.01; fig. 1E). rhIL-1A and rhPRG4 dose-dependently reduced IL-1b secretion by normal PBMCs (p<0.001; fig. 2A & 2B). rhPRG4 was efficacious in reducing phagocytic activities of monocytes from normal and goutsubjects (p<0.001; fig. 2C & 2E). rhPRG4 exhibited a non-significant trend towards reducing IL-1b secretion by gout PBMCs (p=0.13) and the efficacy of rhPRG4 was not different from IL-1RA (p>0.05; fig. 2D). Conclusion Gout PBMCs secreted more IL-1b associated with higher phagocytic activation of monocytes. Uniquely, rhPRG4 reduced phagocytic activation of monocytes, from normal and gout patients. rhIL-1RA and rhPRG4 showed comparable anti-inflammatory efficacy. rhPRG4 is a promising new systemic therapy in gout.

Paracoccidioidesbrasiliensis induces IL-32 and is controlled by IL-15/IL-32/vitamin D pathway in vitro

Paracoccidioidomycosis (PCM) is a systemic fungal disease caused by Paracoccidioides spp., whose clinical outcome depends on immune response. Interleukin 32 (IL-32) is a cytokine present in inflammatory and infectious diseases, including bacterial, virus and protozoan infections. Its role in fungal disease remains unclear. The axis IL-15, IL-32 and vitamin D leads to microbicidal capacity against intracellular pathogens. Thus, the aims of this study were to investigate the production of IL-32 during Paracoccidioides spp. infection and whether this cytokine and IL-15 can increase P. brasiliensis control in a vitamin D dependent manner. IL-32 was highly detected in oral lesions from patients with PCM. In addition, high production of this cytokine was intracellularly detected in peripheral blood mononuclear cells (PBMCs) from healthy donors after exposure to particulated P. brasiliensis antigens (PbAg). The IL-32γ isoform was predominantly expressed, but there was mRNA alternative splicing for IL-32α isoform. The induction of IL-32 was dependent on Dectin-1 receptor. Infection of PBMCs with P. brasiliensis yeasts did not significantly induce IL-32 production even after activation with exogenous IFN-γ or IL-15 treatments. Although IL-15 was a potent inducer of IL-32 production, treatment with this cytokine did not increase the fungal control unless vitamin D was present in high levels. In this case, both IL-15 and IL-32 increased fungicidal activity of PBMCs. Together, data showed that IL-32 is present in lesions of PCM, PbAg induces IL-32, and the axis of IL-15/IL-32/vitamin D can contribute to control fungal infection. The data suggest that exposure to molecules from P. brasiliensis, as β-glucans, is needed to induce IL-32 production since only heat-killed and sonicated P. brasiliensis yeasts were able to increase IL-32, which was blocked by anti-Dectin-1 antibodies. This is the first description about IL-15/IL-32/vitamin D pathway role in P. brasiliensis infection.

Chelidonium majus crude extract induces activation of peripheral blood mononuclear cells and enhances their cytotoxic effect toward HeLa cells

ABSTRACT The aim of the study was to examine the immunomodulatory effect of crude Chelidonium majus L ethanolic extract on ex vivo harvested peripheral blood mononuclear cells (PBMNCs). PBMNCs were isolated by density gradient centrifugation. The PBMNC cytotoxicity assay was performed with HeLa tumor cells as target cells. MTT assay was used to estimate the proliferation effect of extract and cytotoxic efficiency of treated PBMNCs. Flow cytometric analysis was used for immunophenotyping. Treatment induced moderate proliferative response, perturbation in PBMNC ratios, and the emergence of some unconventional subpopulations. The percentage ratio of double positive CD4+ and CD8+ T lymphocytes and monocytes, ratio of T and B lymphocytes expressing CD14, and percentage of NK cells expressing CD57 increased after treatment, indicating activation of PBMNC subpopulations. Cytotoxic activity against HeLa cells was enhanced. Activation of PBMNCs and enhancement of their cytotoxic effect toward HeLa cells indicate the immunostimulatory effect of Ch. majus ethanolic extract.

Effect of suberoylanilide hydroxamic acid on peripheral blood mononuclear cell cytotoxicity towards tumor cells in canines.

Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase inhibitor (HDACi) that suppresses the growth of tumor cells in humans and canines. SAHA reportedly enhances the antitumor activity of human peripheral blood mononuclear cell (PBMC). However, it is unclear whether a similar effect is exerted in canines. The present study focused on the effect of SAHA on the cytotoxicity of IL-2 activated PBMC in three tumor cell lines (CTAC, CIPm, and MCM-N1). The mRNA expression of a ligand for the NKG2D receptor was upregulated in SAHA-treated cell lines. Moreover, the SAHA-treated cell lines, except MCM-N1 demonstrated a significantly higher PBMC cytotoxicity compared to the untreated cell lines. Therefore, the NKG2DL upregulation likely enhanced the interaction of NKG2D-NKG2DL, leading to enhanced cytotoxicity of PBMC. It was also revealed that activated PBMC treated with SAHA significantly attenuated their cytotoxicity toward all the cell lines. Although the NKG2D, NKp46, NKp44, and NKp30 receptors, involved in PBMC cytotoxicity, were presumed to be downregulated, there was no significant reduction in the mRNA expression of these receptors. This study revealed that SAHA not only sensitizes the canine tumor cells to cytotoxicity due to PBMC activation, but also suppresses the cytotoxicity of PBMC themselves. Therefore, our results highlight the necessity of avoiding this inhibitory action to enhance the antitumor effect of SAHA in canines.

Chronic Hyperglycemia Drives Functional Impairment of Lymphocytes in Diabetic INSC94Y Transgenic Pigs.

People with diabetes mellitus have an increased risk for infections, however, there is still a critical gap in precise knowledge about altered immune mechanisms in this disease. Since diabetic INSC94Y transgenic pigs exhibit elevated blood glucose and a stable diabetic phenotype soon after birth, they provide a favorable model to explore functional alterations of immune cells in an early stage of diabetes mellitus in vivo. Hence, we investigated peripheral blood mononuclear cells (PBMC) of these diabetic pigs compared to non-diabetic wild-type littermates. We found a 5-fold decreased proliferative response of T cells in INSC94Y tg pigs to polyclonal T cell mitogen phytohemagglutinin (PHA). Using label-free LC-MS/MS, a total of 3,487 proteins were quantified, and distinct changes in protein abundances in CD4+ T cells of early-stage diabetic pigs were detectable. Additionally, we found significant increases in mitochondrial oxygen consumption rate (OCR) and higher basal glycolytic activity in PBMC of diabetic INSC94Y tg pigs, indicating an altered metabolic immune cell phenotype. Thus, our study provides new insights into molecular mechanisms of dysregulated immune cells triggered by permanent hyperglycemia.

Human Adipose Tissue-Derived Mesenchymal Stromal Cells Inhibit CD4+ T Cell Proliferation and Induce Regulatory T Cells as Well as CD127 Expression on CD4+CD25+ T Cells.

Mesenchymal stromal cells (MSC) exert their immunomodulatory potential on several cell types of the immune system, affecting and influencing the immune response. MSC efficiently inhibit T cell proliferation, reduce the secretion of pro-inflammatory cytokines, limit the differentiation of pro-inflammatory Th subtypes and promote the induction of regulatory T cells (Treg). In this study, we analyzed the immunomodulatory potential of human adipose tissue-derived MSC (ASC), on CD4+ T cells, addressing potential cell-contact dependency in relation to T cell receptor stimulation of whole human peripheral blood mononuclear cells (PBMC). ASC were cultured with not stimulated or anti-CD3/CD28-stimulated PBMC in direct and transwell cocultures; PBMC alone were used as controls. After 7 days, cocultures were harvested and we analyzed: (1) the inhibitory potential of ASC on CD4+ cell proliferation and (2) phenotypic changes in CD4+ cells in respect of Treg marker (CD25, CD127 and FoxP3) expression. We confirmed the inhibitory potential of ASC on CD4+ cell proliferation, which occurs upon PBMC stimulation and is mediated by indoleamine 2,3-dioxygenase. Importantly, ASC reduce both pro- and anti-inflammatory cytokine secretion, without indications on specific Th differentiation. We found that stimulation induces CD25 expression on CD4+ cells and that, despite inhibiting overall CD4+ cell proliferation, ASC can specifically induce the proliferation of CD4+CD25+ cells. We observed that ASC induce Treg (CD4+CD25+CD127−FoxP3+) only in not stimulated cocultures and that ASC increase the ratio of CD4+CD25+CD127+FoxP3− cells at the expense of CD4+CD25+CD127−FoxP3− cells. Our study provides new insights on the interplay between ASC and CD4+ T cells, proposing that ASC-dependent induction of Treg depends on PBMC activation which affects the balance between the different subpopulations of CD4+CD25+ cells expressing CD127 and/or FoxP3.

Systemic Response of Antioxidants, Heat Shock Proteins, and Inflammatory Biomarkers to Short-Lasting Exercise Training in Healthy Male Subjects.

Regular physical activity can enhance immune function and effectively prevents the spread of the cytokine response, thus reducing systemic low-grade inflammation and improving various immune markers. Moreover, regular exercise maintains redox homeostasis in skeletal muscle and other tissues, including immune cells, but the interconnection between the anti-inflammatory effects of exercise with the redox status of immune cells is still poorly understood. With the aim to verify the overall beneficial effect of regular training on the immune system, we have examined the acute and short-term effect of a 5-day exercise program on the modulation of protein and lipid oxidation, antioxidants (i.e., superoxide dismutase-1 (SOD1) and superoxide dismutase-2 (SOD2), glutathione peroxide 1 (GPx1), thioredoxin reductase-1 (TrxR1), and catalase (CAT)), and heat shock protein expression (i.e., heat shock protein-70 (HSP70) and heat shock protein-27 (HSP27)), at both mRNA and protein levels, as well as the activation of the nuclear factor kappa light chain enhancer of activated B cells (NFκB) in peripheral blood mononuclear cells (PBMCs). Moreover, plasmatic markers of oxidative stress, inflammation, and stress response (i.e., protein carbonyl content, interleukin-6 (IL6), interleukin-8 (IL8), interleukin-10 (IL10), interleukin-17E (IL17E), interleukin-17F (IL17F), interleukin-21 (IL21), interleukin-22 (IL22), and interleukin-23 (IL23)) were analyzed in active untrained young adult subjects. Even in the absence of an increased amount of protein or lipid oxidation, we confirmed a PBMC upregulation of SOD1 (1.26 ± 0.07 fold change, p < 0.05), HSP70 (1.59 ± 0.28 fold change, p < 0.05), and HSP27 gene expression (1.49 ± 0.09 fold change, p < 0.05) after 3 hours from the first bout of exercise, followed by an increase in proteins' amount at 24 hours (SOD1, 1.80 ± 0.34 fold change; HSP70, 3.40 ± 0.58 fold change; and HSP27, 1.81 ± 0.20 fold change, p < 0.05) and return to basal levels after the 5 days of aerobic training. Indeed, the posttraining basal levels of oxidized molecules in plasma and PBMCs were statistically lower than the pretraining levels (carbonyl content, 0.50 ± 0.05 fold change, p < 0.01), paralleled by a lower expression of SOD2, Gpx1, and TrxR1, at mRNA (SOD2, 0.63 ± 0.06; GPx1, 0.69 ± 0.07; and TrxR1, 0.69 ± 0.12 fold change, p < 0.05) and protein (TrxR1, 0.49 ± 0.11 fold change, p < 0.05) levels. These results verified the existence of an early phase of redox adaptation to physical exercise already achievable after 5 days of moderate, regular aerobic training. More interestingly, this phenomenon was paralleled by the degree of NFκB activation in PBMCs and the decrease of plasmatic proinflammatory cytokines IL8, IL21, and IL22 in the posttraining period, suggesting an interconnected, short-term efficacy of aerobic exercise towards systemic oxidative stress and inflammation.

Transcriptome analysis of Mycoplasma bovis stimulated bovine peripheral blood mononuclear cells

Mycoplasma bovis is a pathogenic bacterium in bovines that causes huge global economic losses. Numerous factors play important roles in M. bovis pathogenesis; however, the host immune response involved in M. bovis infection has not been fully elucidated. We aimed to determine the characteristics of the host immune response to Mycoplasma infection. We evaluated the responsiveness of bovine peripheral blood mononuclear cells (PBMCs) stimulated with M. bovis via microarray analysis. The transcriptional abundance of innate immune-related genes IL-36A, IL-27, IFN-γ, and IL-17 in PBMCs increased after M. bovis exposure. Upon M. bovis infection, there was increased expression of the lymphocyte activated genes basic leucine zipper transcription factor (BATF) and signaling lymphocytic activation molecule family members 1 and 7 (SLAMF 1 and SLAMF 7) in PBMCs compared with that in unstimulated cells. The study revealed that the transcriptional abundance of innate immunity genes in PBMCs increased during M. bovis infection. This induced the activation of PBMCs, giving rise to an immune response, which is followed by the development of the inflammatory response. The results from this study could be used as the basis for the development of novel vaccine candidates against M. bovis.

Regulation of toll-like receptors expression in muscle cells by exercise-induced stress.

ObjectiveThis study investigates the expression patterns of toll-like receptors (TLRs) and intracellular mediators in horse muscle cells after exercise, and the relationship between TLRS expression in stressed horse muscle cells and immune cell migration toward them.MethodsThe expression patterns of the TLRs (TLR2, TLR4, and TLR8) and downstream signaling pathway-related genes (myeloid differentiation primary response 88 [MYD88]; activating transcription factor 3 [ATF3]) are examined in horse tissues, and horse peripheral blood mononuclear cells (PBMCs), polymorphonuclear cells (PMNs) and muscles in response to exercise, using the quantitative reverse transcription-polymerase chain reaction (qPCR). Expressions of chemokine receptor genes, i.e., C-X-C motif chemokine receptor 2 (CXCR2) and C-C motif chemokine receptor 5 (CCR5), are studied in PBMCs and PMNs. A horse muscle cell line is developed by transfecting SV-T antigen into fetal muscle cells, followed by examination of muscle-specific genes. Horse muscle cells are treated with stressors, i.e., cortisol, hydrogen peroxide (H2O2), and heat, to mimic stress conditions in vitro, and the expression of TLR4 and TLR8 are examined in stressed muscle cells, in addition to migration activity of PBMCs toward stressed muscle cells.ResultsThe qPCR revealed that TLR4 message was expressed in cerebrum, cerebellum, thymus, lung, liver, kidney, and muscle, whereas TLR8 expressed in thymus, lung, and kidney, while TLR2 expressed in thymus, lung, and kidney. Expressions of TLRs, i.e., TLR4 and TLR8, and mediators, i.e., MYD88 and ATF3, were upregulated in muscle, PBMCs and PMNs in response to exercise. Expressions of CXCR2 and CCR5 were also upregulated in PBMCs and PMNs after exercise. In the muscle cell line, TLR4 and TLR8 expressions were upregulated when cells were treated with stressors such as cortisol, H2O2, and heat. Migration of PBMCs toward stressed muscle cells was increased by exercise and oxidative stresses, and combinations of these. Treatment with methylsulfonylmethane (MSM), an antioxidant on stressed muscle cells, reduced migration of PBMCs toward stressed muscle cells.ConclusionIn this study, we have successfully cultured horse skeletal muscle cells, isolated horse PBMCs, and established an in vitro system for studying stress-related gene expressions and function. Expression of TLR4, TLR8, CXCR2, and CCR5 in horse muscle cells was higher in response to stressors such as cortisol, H2O2, and heat, or combinations of these. In addition, migration of PBMCs toward muscle cells was increased when muscle cells were under stress, but inhibition of reactive oxygen species by MSM modulated migratory activity of PBMCs to stressed muscle cells. Further study is necessary to investigate the biological function(s) of the TLR gene family in horse muscle cells.

From the Literature

From the Literature

Sirt1 Activity in PBMCs as a Biomarker of Different Heart Failure Phenotypes.

Heart Failure (HF) is a syndrome, which implies the existence of different phenotypes. The new categorization includes patients with preserved ejection fraction (HFpEF), mid-range EF (HFmrEF), and reduced EF (HFrEF) but the molecular mechanisms involved in these HF phenotypes have not yet been exhaustively investigated. Sirt1 plays a crucial role in biological processes strongly related to HF. This study aimed to evaluate whether Sirt1 activity was correlated with EF and other parameters in HFpEF, HFmrEF, and HFrEF. Seventy patients, HFpEF (n = 23), HFmrEF (n = 23) and HFrEF (n = 24), were enrolled at the Cardiology Unit of the University Hospital of Salerno. Sirt1 activity was measured in peripheral blood mononuclear cells (PBMCs). Angiotensin-Converting Enzyme 2 (ACE2) activity, Tumor Necrosis Factor-alpha (TNF-α) and Brain Natriuretic Peptide (BNP) levels were quantified in plasma. HFpEF showed lower Sirt1 and ACE2 activities than both HFmrEF and HFrEF (p < 0.0001), without difference compared to No HF controls. In HFmrEF and HFrEF a very strong correlation was found between Sirt1 activity and EF (r2 = 0.899 and r2 = 0.909, respectively), and between ACE2 activity and Sirt1 (r2 = 0.801 and r2 = 0.802, respectively). HFrEF showed the highest TNF-α levels without reaching statistical significance. Significant differences in BNP were found among the groups, with the highest levels in the HFrEF. Determining Sirt1 activity in PBMCs is useful to distinguish the HF patients’ phenotypes from each other, especially HFmrEF/HFrEF from HFpEF.

Artificial Microvesicles Isolated from Mesenchymal Stem Cells with IL2 Overexpression Activate CD8+ T-Killers to Kill Triple Negative Breast Cancer Cells

Almost all human cells release extracellular vesicles (EVs) participating in intercellular communication. EVs are rounded structures surrounded by the cytoplasmic membrane, which embody cytoplasmic contents of the parent cells. Since the orientation of surface receptors persists during EV formation, it is EVs that are of interest as a therapeutic tool for cell-free therapy that has targeted delivery. The ideal cell type for the EV production are mesenchymal stem cells (MSCs) as they exhibit a homing behaviour to tumor niches. In this study, human MSCs were isolated from adipose tissue (hADSCs) and genetically modified to overexpress interleukin-2 (IL2) (hADSCs-IL2) or red fluorescent protein (hADSCs-RFP). Extracellular vesicle release from native and genetically modified hADSCs was induced by cytochalasin B treatment. Size and immunophenotype of isolated Cytochalasin B induced microvesicles (CIMVs) were analysed using flow cytometry. To evaluate immunomodulating properties of microvesicles from native (native CIMVs) or genetically modified (CIMVs-IL2) hADSCs, peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood and co-cultured with CIMVs (50 μg/ml) for 72 hours. Activation of PBMC populations was determined by flow cytometry using staining with antibodies specific for surface markers of various human immune cell populations. To analyze the antitumor activity of PBMCs after interaction with CIMVs-IL2, activated PBMCs were transferred to triple negative breast cancer cells MDA-MB-231 which were pre-seeded on 16-well xCelligence plate (3 × 103 cells per well) or 6-well plate (5 × 104 cells per well) in 1:3 ratio (tumor cells:PBMCs). Proliferative activity of tumor cells was evaluated using an RTCA xCelligence instrument (ACEA Biosciences, USA) for 72 hours. Tumor cell viability was assessed 24 hours after culturing with PBMCs using the APC Annexin V Apoptosis Detection Kit with PI (#640932, Biolegend, USA) on FACS Aria III flow cytometer (BD, USA). Isolated CIMVs were mostly 50-200 nm in diameter and positive for CD44, CD90 and CD105 MSC surface markers, but CD29 and CD73 levels were significantly decreased. The number of activated T-killers (CD3+CD8+CD4-CD38+HLA-DR+) was significantly increased after incubation with CIMVs-IL2 (205.6 ± 4.5%) compare to native PBMCs (100.0 ± 9%) and PBMCs incubated with native CIMVs (116.7 ± 4.5%) (Fig. 1A). The number Th1 cells was decreased in the sample of PBMCs incubated with CIMVs-IL2 (92.3 ± 1.6%) compared with native PBMCs (100.0 ± 1%) (Fig. 1B), while the number of Th2 cells (109.0 ± 0.1%) was increased compared to native PBMCs (100.0 ± 1%) (Fig. 1C), number of Tregs was unchanged (Fig. 1D). After the addition of PBMCs, activated with CIMVs-IL2, the proliferation of MDA-MB-231 cells has been significantly decreases. Twenty four hours after the addition of CIMVs-IL2-activated PBMCs the proliferation of MDA-MB-231 was decreased 4-fold (to 25.0 ± 5.6%) compared to native MDA-MB-231 (100.0 ± 2.9%) and MDA-MB-231 incubated with native PBMCs (110.8 ± 8.7%) and PBMCs after native CIMVs (103.7 ± 2.4%) (Fig. 1E,G). This decrease in proliferation is likely due to the fact that part of the tumor cells underwent apoptosis and then died which is confirmed by the MDA-MB-231 cell viability after 24 hours of cultivation with PBMCs activated with CIMVs-IL2 (Fig. 1F). The number of healthy MDA-MB-231 cells after 24 hours of cultivation with PBMCs after CIMVs-IL2 was reduced by 35% (46.1 ± 3.7%) compared to native MDA-MB-231 (78.2 ± 2.1%) and MDA-MB-231 incubated with native PBMCs (70.1 ± 2.8%) and PBMCs after native CIMVs (62.1 ± 3.2%). Viable MDA-MB-231 cells in the sample of PBMCs after CIMVs-IL2 continue to proliferate, however their proliferative activity remains reduced (41.2 ± 4.7%) after 72 hours of cultivation compared to native MDA-MB-231 (100.0 ± 21.0%) and MDA-MB-231 incubated with native PBMCs (109.7 ± 2.5%) and PBMCs after native CIMVs (82.5 ± 20.5%) (Fig. 1H). The use of CIMVs-IL2 can be effective in the treatment of triple negative breast cancer, since CIMVs-IL2 are able to activate and stimulate the proliferation of T-killers, which in turn are able to induce apoptosis in breast cancer cells. However, further studies of CIMVs efficiency in animal tumor models are required. This study was supported by the Russian Science Foundation grant 18-74-10044 and the Russian Government Program of Competitive Growth of KFU. Figure Disclosures No relevant conflicts of interest to declare.

The regulatory role of peripheral blood mononuclear cell function by A20

Peripheral blood mononuclear cells (PBMC) consist of lymphocytes (T cells, B cells, natural killer cells), monocytes and dendritic cells and play important roles in initiating and regulating immunity against pathogens or immunotolerance to allergens. Activation of PBMCs is induced upon exposure to multiple stimuli by the binding with toll-like receptors (TLRs), recognition elements of the innate immune system. A20 is a negative regulator of nuclear factor (NF)-κB-dependent immune reaction in response to TLR ligands. A20-deficient mice display severe inflammation, tissue damage in multiple organs, cachexia and premature mortality. Single nucleotide polymorphisms (SNPs) at A20 gene region in humans reduce the binding capacity of A20 to NF‐κB subunits, resulting in reduced expression and function of A20 and leading to the pathogenesis of autoimmune and cancers. Although the inhibitory role of A20 on immune cells including B, T and DC functions has been previously reported, the effect of A20 on PBMC function is not mentioned yet. The present study, therefore, explored whether A20 expression is involved in immunophenotypic changes, the release of cytokine production, cell migration, and apoptosis. To this end, immonophenotypic profile and cell apoptosis were examined by flow cytometry, secretion of inflammatory cytokines by ELISA and cell migration by a transwell migration assay. As a result, percentages of CD3+CD25+, CD19+CD25+, and CD11b+CD40+ expressing cells, the release of TNF-α and IL-1β and cell migration were enhanced in A20-silenced PBMCs. However, cell apoptosis was independent of the presence of A20 in PBMCs. In conclusion, these results attained in this study suggested that A20 expression might modulate the immune response in autoimmune disease and cancers.

Ku70 and Ku80 participate in LPS-induced pro-inflammatory cytokines production in human macrophages and monocytes.

In human macrophages and monocytes, lipopolysaccharide (LPS) induces nuclear factor kappa B (NFκB) activation and pro-inflammatory cytokines production. We tested the possible involvement of Ku70 and Ku80 in the process. In THP-1 macrophages and primary human peripheral blood mononuclear cells (PBMCs), shRNA-induced double knockdown of Ku70 and Ku80 potently inhibited LPS-induced production of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6). Additionally, we developed CRISPR/Cas-9 gene-editing methods to knockout both Ku70 and Ku80 in THP-1 cells and PBMCs. Double knockout (DKO) largely inhibited LPS-induced pro-inflammatory cytokines production. Conversely, in THP-1 cells exogenous overexpression of both Ku70 and Ku80 enhanced the pro-inflammatory cytokines production by LPS. Ku70 and Ku80 co-immunoprecipitated with p65-p52 NFκB complex in the nuclei of LPS-treated THP-1 cells. Significantly, LPS-induced NFκB activation was inhibited by Ku70 plus Ku80 double knockdown or DKO. It was however enhanced with Ku70 and Ku80 overexpression. Together, Ku70 and Ku80 promote LPS-induced NFκB activation and pro-inflammatory response in THP-1 cells and human PBMCs.

Inhibitors of Tumor Necrosis Factor Synthesis as a New Approach for theTreatment of Rheumatoid Arthritis

Objective: The treatment of rheumatoid arthritis (RA) is based on the inhibition of TNF. Here we evaluated whether drugs that might inhibit TNF, such as pentoxifylline (PTX), rupatadine (RUP), rolipram (ROL) and thalidomide (THA), could be an alternative for RA treatment. Methods: In wistar male rats the changes in paw thickness, plasma TNF and by the activity of basic aminopeptidase (APB) in soluble fraction of synovial tissue and peripheral blood mononuclear cells (PBMC) evaluated after daily injection for 30 days were taken as anti-inflammatory outputs, while hepatotoxicity was assessed by measuring plasma alanine transaminase (ALT) and aspartate transaminase (AST) activity. The content of IL1-?, IL-6 in serum and synovial fluid and the histology of the injured tissue were determined only for ROL, THA and ROL+THA. Prednisolone was used as a standard drug. Results: Collagen treatment induced paw thickness, histological changes in the tibiotarsal joint, increase in synovial fluid of both cytokines and synovial tissue of APB activity. Furthermore, the APB activity in PBMC was reduced and ALT and AST activity were enhanced. The most effective drug schedule in reducing arthritis induced changes described above, as well as recovering from control levels TNF, IL1-β, APB in synovial tissue and AST activities were THA and the association of ROL and THA. However, only THA alone reduced the levels of ALT. Conclusion: The synthesis of TNF in RA models can be blocked by drugs acting at different targets. We show that THA and THA+ROL emerges as simple and effective therapeutic alternatives for RA.

Neutrophil extracellular trap from Kawasaki disease alter the biologic responses of PBMC.

Kawasaki disease (KD), also known as mucocutaneous lymph node syndrome, is an acute systemic vasculitis syndrome that mainly occurs in infants under 5 years of age. In the current manuscript, we were aiming to analyze the role of neutrophil extracellular traps (NETs) in the pathogenesis of KD, especially their interplay with peripheral blood mononuclear cells (PBMCs). Neutrophils were exposed to 20 nM phorbol myristate acetate (PMA), we found that neutrophils of KD patients were more likely to form NETs compared with healthy controls (HCs). Furthermore, PBMCs were cultured with NETs for 24 h, and we observed that NETs significantly increased the cell viability, suppressed cell apoptosis, and enhanced the pro-inflammatory cytokines production and NF-κB activation in PBMCs from KD patients. In addition, with the stimulation of NETs, the expression of vascular endothelial growth factor A (VEGF-A) and hypoxia-inducible factor-1α (HIF-1α) were increased, which were related with the pathological mechanism of KD. At last, we examined the activation of phosphoinositide 3 kinase (PI3K)/Akt signaling, and we found NETs treatment obviously enhanced the activation of PI3K and Akt. In conclusion, these findings suggested that the formation of NETs may alter the biologic responses of PBMC and affect the vascular injury in KD.

The Effects of Hydroalcoholic Extract from Alhagi on Matrix Metalloproteinase- 9 Production in Peripheral Blood Mononuclear Cells from Patients with Rheumatoid Arthritis

Background: The up-regulation of matrix metalloproteinase 9 (MMP-9) along with the imbalanced ratio of MMP-9 to tissue inhibitor of metalloproteinase 1 (TIMP-1) is important in the pathogenesis of Rheumatoid Arthritis (RA). Here, we investigated whether hydroalcoholic extract from the root of Alhagi camelorum Fisch can affect the levels of MMP-9 and TIMP-1 in peripheral blood mononuclear cells (PBMCs) of RA patients. Objective: In the current study, we suggest that Alhagi may have an inhibitory effect on MMP-9 production, which is mainly responsible for joint destruction in RA. In addition, we would like to stress that our findings, along with others, can provide the basis for future studies, which might help in determining the role of chemical ingredients of Alhagi as therapeutic targets for RA treatment. Methods: PBMCs were isolated from 12 RA patients and 12 healthy subjects and treated with two concentrations of Alhagi extract (100 and 500 μg/ml) for 24 h. MMP-9 gene expression and protein production, TIMP-1 levels and nitric oxide (NO) production were evaluated using standard methods. Results: Alhagi (500 μg/ml) caused a significant reduction in the expression and activity of MMP-9 in PBMCs from healthy (p=0.003 for both of them) and patient (p= 0.05 and p=0.02 respectively) subjects. Moreover, Alhagi (100 μg/ml) decreased MMP-9 production in the healthy subjects’ group (p=0.02). Conclusion: The present study reveals the inhibitory effects of Alhagi on the production of MMP-9 as the main responsible cause of joint destruction in RA.

Abstract 4551: Preclinical characterization of novel anti-CTLA-4 prodrug antibodies with an enhanced therapeutic index

Abstract Background: Blockade of the CTLA-4 pathway with ipilimumab (IPI) as monotherapy or in combination with nivolumab (anti–PD-1) is an effective treatment for a variety of cancers. To enhance the therapeutic index of CTLA-4–directed therapy, a proprietary Probody® therapeutics (PB-Tx) technology platform was used to generate anti–CTLA-4 monoclonal antibodies (mAbs) with a masking peptide to attenuate CTLA-4 binding in the periphery. Increased protease activity in the tumor microenvironment is hypothesized to preferentially cleave the masking peptide. Here, we describe the preclinical characterization of 2 novel anti–CTLA-4 PB mAbs: anti–CTLA-4 PB (BMS-986249) is a peptide-masked version of IPI, and anti–CTLA-4 nonfucosylated (NF) PB (BMS-986288) is a peptide-masked version of anti–CTLA-4 NF, which has enhanced antibody-dependent cellular cytotoxicity (ADCC) and regulatory T-cell (Treg) depletion compared with IPI. Methods: Antibody binding to CD16 was studied by surface plasmon resonance. IL-2 release from staphylococcal enterotoxin B (SEB)–stimulated normal human peripheral blood mononuclear cells (PBMCs) and CD4+ T cells was measured; ADCC function was evaluated by IL-2–activated, NK-cell–induced lysis of CD4+ T cells or activated CD4+Foxp3+ Tregs. Tumor growth was measured in transgenic (human CTLA-4 knock-in) mice implanted with MC38 tumors. Peripheral pharmacodynamic (PD) markers (ICOS, Ki-67) of anti–CTLA-4 activity were assessed using Tregs and effector T cells from the spleens of treated mice. PD was evaluated in cynomolgus (CYNO) macaques after administration of customized adenovirus-5 vector vaccines. Results: With the masking peptide intact, both anti–CTLA-4 PB and anti–CTLA-4 NF PB showed decreased activity vs IPI and anti–CTLA-4 NF, respectively, in non–protease containing in vitro assays. Anti–CTLA-4 PB exhibited a 40-fold reduction in CTLA-4 binding affinity vs IPI and decreased activity in SEB-activated PBMCs. Anti–CTLA-4 NF PB showed decreased ADCC activity vs anti–CTLA-4 NF in vitro. When tested in a MC38 tumor model, anti–CTLA-4 PB and anti–CTLA-4 NF PB showed equivalent antitumor activity to IPI and anti–CTLA-4 NF, respectively. Both anti–CTLA-4 PB and anti–CTLA-4 NF PB showed equivalent intratumoral PD activity and reduced peripheral PD activity relative to their parental mAbs. Similarly, anti–CTLA-4 PB and anti–CTLA-4 NF PB resulted in reduced inflammation and peripheral PD responses relative to their parental mAbs in CYNO macaques. Conclusion: These data demonstrate the potential of the PB-Tx technology platform to improve the therapeutic indices of anti–CTLA-4 PB and anti–CTLA-4 NF PB relative to their parental mAbs. The safety and antitumor activity of anti–CTLA-4 PB (NCT03369223) and anti–CTLA-4 NF PB (NCT03994601) are being investigated in patients with advanced solid cancers in ongoing phase 1 studies. Citation Format: John Engelhardt, Rahima Akter, John Loffredo, Natalie Bezman, Paula So, Kimberly Tipton, Bryan Irving, James West, Wendy Freebern, Todd Bunch, Karen Price. Preclinical characterization of novel anti-CTLA-4 prodrug antibodies with an enhanced therapeutic index [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4551.

Aging affects responsiveness of peripheral blood mononuclear cells to immunosuppression of periodontal ligament stem cells.

ObjectivesThe effect of age on the response of peripheral blood mononuclear cells (PBMCs) to immunosuppression induced by human periodontal ligament stem cells (hPDLSCs) is unclear. The identity of the cytokines most effective in inducing the PBMC immune response remains unknown. This study investigated the effects of age on immunophenotype, proliferation, activation, and cytokine secretion capacities of PBMCs following co-culture with hPDLSCs.MethodsPBMCs were collected from younger (16–19 years) and older (45–55 years) donors, then co-cultured with confirmed hPDLSCs for various lengths of time. T lymphocyte proliferation and cell surface marker expression were analyzed by flow cytometry. Cytokine expression levels were measured by quantitative polymerase chain reaction assays and enzyme-linked immunosorbent assays.ResultsCD28 expression by T lymphocytes decreased with age, indicating reduced proliferation; CD95 expression increased with age, indicating enhanced apoptosis. Moreover, hPDLSCs inhibited T lymphocyte proliferation in both age groups; this inhibition was stronger in cells from older donors than in cells from younger donors. Age reduced the secretion of interleukin-2 and interferon-γ, whereas it increased the secretion of tumor necrosis factor-β by PBMCs cultured with hPDLSCs.ConclusionsAging may have a robust effect on the response of PBMCs towards hPDLSC-induced immunosuppression.

Moderate-intensity functional training improves mitochondrial capability and redox state in peripheral blood mononuclear cells of metabolic syndrome women

Evidence exists that both oxidative stress and mitochondrial dysfunction play a pivotal role in the metabolic syndrome (MetS), and also, likely then extent for peripheral blood mononuclear cells (PBMCs). Despite knowledge of the beneficial effects of physical exercises, studies about the modality of functional training (FT) on remodeling in mitochondrial metabolism and redox states, especially at PBMCs from patients with MetS, are rare and still controversial. Thus, this study intends to examine how FT can influence PBMCs metabolism, mitochondrial capability, and redox state in women with MetS. Untrained women over 45-years-old were classified as having three or more pre-defined components of MetS. The participants (n = 10) completed three training sessions, and each session lasted about 60-min sessions per week for 12-weeks. We performed baseline and posttest measures in PBMCs, which were obtained before training (baseline) and 72 h after the end of the training protocol to avoid acute exercise effects. Adherence was defined as completing 85% of exercise sessions. FT produced an increase of 96.6% in lactate dehydrogenase activity (p = 0.002) of PBMCs, while citrate synthase activity (p = 0.49) remained unaltered. The training protocol also demonstrated an increase of 39.7% DCF levels (p = 0.0039) exercise-induced formation, as well as antioxidant system adaptation (CAT activity) (78.5%; p = 0.005). Electron transfer capability from CI (112.7%; p = 0.002), CII (89.1%; p = 0.046) and CIII (26.8%; p = 0.046) increased after training, however, no changes in CIV (p = 0.72) were observed. No significant changes in BMI or body weight were found, but the training protocol resulted in a significant increase in VO2max of 18% at posttest (p = 0.0035). We provided novel evidence that a 12-week FT program modifies mitochondrial capability and redox state in PBMCs, it seems to be some extent women’s aerobic fitness improvement. Therefore, our data suggest that PBMCs could be used to detect and assess changes in cellular profiles from unhealthy patients, as well as biomarkers source for physical exercise control and quality.