scholarly journals P068 The combination of JAK1 and TPL2 or IRAK4 inhibitors is more effective than single agents in reducing TLR-mediated cytokine responses in human monocyte populations

2021 ◽  
Vol 15 (Supplement_1) ◽  
pp. S173-S173
Author(s):  
E Hornsby ◽  
A N Yadon ◽  
A Clarke ◽  
E Grant ◽  
J O Lindsay ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Cytokine Production ◽  
Inflammatory Responses ◽  
Human Monocyte ◽  
Signalling Pathways ◽  
Tnf Alpha ◽  
Monocyte Subsets ◽  
Cytokine Responses ◽  
Erk Phosphorylation ◽  
The Impact

Abstract Background Dysregulated Toll-like receptor (TLR)-mediated responses in intestinal monocyte-derived cells contribute to pathology in inflammatory bowel disease (IBD). JAK inhibitors regulate the production of inflammatory mediators and are an emerging therapy in IBD. Recent data suggest that combination therapies may be more effective than single agents, and therapies targeting aspects of TLR signalling pathways are being developed. We assessed the impact of inhibitors of IRAK4, TPL2 and JAK1 (IRAK4i, TPL2i and JAK1i, respectively) either alone or in combination on TLR-mediated cytokine responses and associated signalling pathways in classical (CD14+CD16-), intermediate (CD14+CD16+) and non-classical (CD14lowCD16+) human blood monocyte subsets. Methods Peripheral Blood Mononuclear Cells (PBMCs) or CD14+ monocytes from healthy volunteers were pre-incubated with TPL2i, IRAK4i or JAK1i individually or with JAK1i in combination with TPL2i or IRAK4i for 1 hour at 37°C. Cells were then stimulated at 37°C with agonists of TLR4 (E.coli lipopolysaccharide; LPS) or TLR2 (Pam3CYSK4). Intra-cellular staining and flow cytometry were used to measure phosphorylation of signalling molecules (NF-kappaB p65, p38 MAPK and ERK) or cytokines (TNF-alpha and IL-1-beta) in the three monocyte subsets after stimulation for 15 minutes or 3 hours respectively. Results Pre-incubation of PBMCs or CD14+ monocytes with IRAK4i, TPL2i or JAK1i individually led to a significant dose-dependent reduction in TLR-stimulated cytokine production (the frequency of TNF-alpha+ cells and the per cell level of IL-1-beta) within the classical, intermediate and non-classical monocyte subsets. The combination of JAK1i with IRAK4i or TPL2i had an additive effect. TPL2i reduced LPS-stimulated ERK phosphorylation, but p65 and p38 phosphorylation in response to LPS was not affected by any of the inhibitors. Conclusion Small molecule inhibitors of TPL2, IRAK4 and JAK1 dampen TLR4- and TLR2- mediated inflammatory responses in human monocyte subsets, by affecting pathways independent of p65 or p38. This is due to a cell intrinsic effect on CD14+ monocytes. TPL2 inhibition may in part act to dampen cytokine production in monocytes through the reduction of ERK phosphorylation. Combining JAK1i with IRAK4i or TPL2i may be more effective in reducing inflammatory responses than single agents. Further work is required to elucidate the mechanisms by which IRAK4i and JAK1i dampen TLR-mediated inflammation in monocyte subsets.

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

2021 ◽  
Vol 12 ◽  
Author(s):  
Asimenia Angelidou ◽  
Joann Diray-Arce ◽  
Maria-Giulia Conti ◽  
Mihai G. Netea ◽  
Bastiaan A. Blok ◽  
...  
Keyword(s):  
Cytokine Production ◽  
Colorimetric Assay ◽  
Age Groups ◽  
Metabolic Activation ◽  
Human Monocyte ◽  
Autologous Serum ◽  
Live Attenuated Vaccine ◽  
Cytokine Responses ◽  
The Impact

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

scholarly journals Lymphokine overproduction in severe aplastic anemia is not related to blood transfusions

Blood ◽  
1989 ◽  
Vol 74 (8) ◽  
pp. 2713-2717 ◽  
Author(s):  
W Hinterberger ◽  
G Adolf ◽  
P Bettelheim ◽  
K Geissler ◽  
C Huber ◽  
...  
Keyword(s):  
Cyclosporin A ◽  
Aplastic Anemia ◽  
Mononuclear Cells ◽  
Cytokine Production ◽  
Severe Aplastic Anemia ◽  
Tnf Alpha ◽  
Blood Transfusions ◽  
Peripheral Blood Mononuclear ◽  
Ifn Gamma

Abstract The production of interferons (IFNs), IFN-gamma, tumor necrosis factors (TNFs) and TNF-alpha (TNF-alpha) by peripheral blood mononuclear cells (PBMNCs) of untransfused and transfused, but otherwise untreated patients with severe aplastic anemia (SAA) was determined using bioassays and immunoassays. In untransfused and pretransfused SAA patients, spontaneous and lectin-induced production of these cytokines by PBMNCs was strongly enhanced. Cytokine production in untransfused SAA patients did not differ from that in pretransfused patients. Similar relative frequencies of activated (HLA-DR+) lymphocyte subpopulations present in the PBMNCs demonstrated cytokine overproduction per cells. Cytokine production was studied in three SAA patients before and after blood cell transfusions. Spontaneous and lectin-induced production of these cytokines was abnormally high and unaffected by blood transfusions. In another patient exhibiting abnormal cytokine production, the hematopoietic response to cyclosporin- A in vivo was accompanied by normalization of cytokine production in vitro. We conclude that overproduction of IFN-gamma and TNF-alpha by lectin-stimulated PBMNCs is an intrinsic abnormality of SAA unrelated to blood transfusions. Normalization of production of IFN-gamma and TNF- alpha accompanying a clinical response to cyclosporin-A may cautiously be taken as further evidence suggesting a pathogenetic role of cytokine overproduction in SAA.

Impact of Alloantigens and Storage-associated Factors on Stimulated Cytokine Response in an In Vitro  Model of Blood Transfusion

2002 ◽  
Vol 97 (5) ◽  
pp. 1102-1109 ◽  
Author(s):  
Andreas E. Biedler ◽  
Sven O. Schneider ◽  
Ullrich Seyfert ◽  
Hauke Rensing ◽  
Sasha Grenner ◽  
...  
Keyword(s):  
Whole Blood ◽  
Mononuclear Cells ◽  
Autologous Blood ◽  
Immunosuppressive Effect ◽  
Tnf Alpha ◽  
Major Surgery ◽  
Enzyme Linked Immunosorbent Assay ◽  
Fresh Blood ◽  
The Impact ◽  
And Storage

Background Transfusion of blood may contribute to immunosuppression in major surgery. The authors assessed the impact of alloantigens and storage on function of peripheral blood mononuclear cells cultured in their physiologic environment. Methods Blood units (whole blood, packed erythrocytes) were prepared with or without prestorage leukodepletion and stored for 24-26 days. Blood samples were coincubated with allogeneic fresh blood, autologous, or allogeneic stored blood. Endotoxin-stimulated release of tumor necrosis factor-alpha (TNF-alpha) and interleukin 10 (IL-10) was measured after 24 h of culture by enzyme-linked immunosorbent assay. Results Coincubation with equal amounts of allogeneic fresh blood showed almost no influence on TNF-alpha (-12%, not significant) and IL-10 (+11%, not significant) release. Stored allogeneic whole blood resulted in a significant TNF-alpha depression (-61%) and IL-10 induction (+221%). These effects were diminished but not prevented by prestorage leukodepletion (TNF-alpha -42%, IL-10 +110%) and required the presence of soluble factors (TNF-alpha suppression) and cellular components (IL-10 induction). TNF-alpha decrease and IL-10 increase were in the same order of magnitude (-40%, +134% with, -65%, +314% without leukodepletion) after coincubation with autologous blood. In contrast, allogeneic erythrocytes had only little effects (TNF-alpha -6%, IL-10 +36%) even at this high transfusion equivalent. Conclusion These data suggest that banked whole blood has an immunosuppressive effect that is largely attributable to storage-dependent factors. These factors are partially removed by prestorage leukodepletion, while the contribution of alloantigens is of minor significance. Immunosuppressive effects are least apparent with leukodepleted erythrocytes, suggesting that the presence of plasma during storage is required for the immunosuppressive effect to develop.

scholarly journals Nutritional Route Affects ERK Phosphorylation and Cytokine Production in Hepatic Mononuclear Cells

2007 ◽  
Vol 245 (4) ◽  
pp. 642-650 ◽  
Author(s):  
Tomoyuki Moriya ◽  
Kazuhiko Fukatsu ◽  
Yoshinori Maeshima ◽  
Fumie Ikezawa ◽  
Chikara Ueno ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Cytokine Production ◽  
Erk Phosphorylation

scholarly journals Erythropoietin Selectively Attenuates Cytokine Production and Inflammation in Cerebral Ischemia by Targeting Neuronal Apoptosis

2003 ◽  
Vol 198 (6) ◽  
pp. 971-975 ◽  
Author(s):  
Pia Villa ◽  
Paolo Bigini ◽  
Tiziana Mennini ◽  
Davide Agnello ◽  
Teresa Laragione ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Cytokine Production ◽  
Inflammatory Responses ◽  
Human Peripheral Blood ◽  
Inflammatory Cells ◽  
Artery Occlusion ◽  
Experimental Stroke ◽  
Peripheral Blood Mononuclear ◽  
Monocyte Chemoattractant Protein 1 ◽  
Cerebral Artery Occlusion

Ischemic brain injury resulting from stroke arises from primary neuronal losses and by inflammatory responses. Previous studies suggest that erythropoietin (EPO) attenuates both processes. Although EPO is clearly antiapoptotic for neurons after experimental stroke, it is unknown whether EPO also directly modulates EPO receptor (EPO-R)–expressing glia, microglia, and other inflammatory cells. In these experiments, we show that recombinant human EPO (rhEPO; 5,000 U/kg body weight, i.p.) markedly reduces astrocyte activation and the recruitment of leukocytes and microglia into an infarction produced by middle cerebral artery occlusion in rats. In addition, ischemia-induced production of the proinflammatory cytokines tumor necrosis factor, interleukin 6, and monocyte chemoattractant protein 1 concentration is reduced by >50% after rhEPO administration. Similar results were also observed in mixed neuronal-glial cocultures exposed to the neuronal-selective toxin trimethyl tin. In contrast, rhEPO did not inhibit cytokine production by astrocyte cultures exposed to neuronal homogenates or modulate the response of human peripheral blood mononuclear cells, rat glial cells, or the brain to lipopolysaccharide. These findings suggest that rhEPO attenuates ischemia-induced inflammation by reducing neuronal death rather than by direct effects upon EPO-R–expressing inflammatory cells.

Early Erythroid Development Is Enhanced with Hypoxia and Terminal Maturation with Normoxia in a 3D Ex Vivo Physiologic Eythropoiesis Model

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2453-2453
Author(s):  
Susana Brito dos Santos ◽  
Mark C. Allenby ◽  
Athanasios Mantalaris ◽  
Nicki Panoskaltsis
Keyword(s):  
Mononuclear Cells ◽  
Cytokine Production ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Erythroid Progenitors ◽  
Variable Intensity ◽  
Erythroid Progenitor ◽  
Tnf Α ◽  
The Impact

Abstract Reproduction of dynamic physiologic erythropoiesis in vitro requires a three-dimensional (3D) architecture, erythroblast-macrophage interactions and cytokines such as erythropoietin (EPO). The role of oxygen concentration gradients in this process is unclear. We have created a 3D bone marrow (BM) biomimicry using collagen-coated polyurethane scaffolds (5mm3) to expand cord blood mononuclear cells (CBMNCs) in a cytokine-free environment for 28 days (D). Addition of EPO to this system induces mature erythropoiesis. We hypothesised that physiologic concentrations of cytokines - stem cell factor (SCF) / EPO - and a hypoxia (H)/normoxia (N) schedule to mimic BM oxygen gradients would enhance erythropoiesis. CBMNCs were seeded (4x106 cells/scaffold) in 3D serum-free cultures supplemented with 10ng/mL SCF (D0-D28), and 100mU/mL EPO (D7-D28), with medium exchange every 3D. Three conditions were compared: N (20%), H (5%) and 2-step oxygenation HN (H D0-D7 and N thereafter). Erythroid maturation was monitored weekly by flow cytometry (CD45/CD71/CD235a) both in situ (i.e., in scaffolds) and in supernatant (S/N) cells. D0-7 H was more efficient in early induction of CD235a in the absence of exogenous EPO (H 13% vs N 8% CD45loCD71+CD235alo cells, p<0.05). This maturation profile was also observed in D10 S/N cells, in which CD45loCD71+CD235a+ cells were proportionately more in H (30%) and HN (27%) than in N (16%, p<0.05). By D14, N and HN stimulated the appearance of CD45-CD71+CD235a+ cells, whereas H maintained the CD45loCD71+CD235a-/lo phenotype. By D21, a CD45-CD71+CD235a+ mature population was clearly distinguished in all conditions, most notably in N (16%) and HN (21%) vs H (9%). At D28, more mature CD45-CD71loCD235a+ cells were observed in normoxia conditions, N 3% and HN 4%, vs H 0.3%. A renewed population of erythroid progenitors was also evident at this time (H 62%, N 51% and HN 46% CD45loCD71lo/+CD235a- cells). In order to assess the impact of H and N on erythroid gene transcription, we evaluated erythroid signatures by qRT-PCR. GATA-1 expression was detected from D7, highest for H at D14 (p<0.05), and decreased thereafter. GATA-2 expression was up-regulated only at D28, in particular in N (p<0.05), and correlated with emerging erythroid progenitors identified at this stage. At D14, EPOR expression was maximal, especially in HN (p<0.05), simultaneous with high pSTAT5 levels, suggesting activation of EPOR signalling. Also at D14, H upregulated γ-globin (p<0.05). By Western Blot, only H and HN still produced γ-globin whereas β-globin expression was clearly detected in all conditions by D28. In situ production of cytokines was evaluated by cytometric bead array in the exhausted media. IL-6, G-CSF, GM-CSF, IL-1, TNF-α and IL-17 were detected at higher concentrations during the first 7 days, declining to undetectable thereafter. IL-21 was not detected at any point. IL-3 was detected from D13, with highest expression in H (p<0.05, D22). VEGF was also expressed after D7, highest in H (p<0.05, D16 & D19), concurrent with HIF-1α up-regulation observed at D7 and D14. TNF-α was produced with variable intensity from D4. These data suggested that D7-D14 was a crucial period for culture dynamics, in particular for H and HN, with up-regulation of erythroid transcription factors, EPOR signalling, and endogenous cytokine production. BFU-E and CFU-E also dominated the first 14 days of culture. Scanning electron microscopy at D17 and D25 revealed niche-like structures in situ, which expressed STRO-1, osteopontin and vimentin at D19 by confocal immunofluorescent microscopy, indicative of an endogenous stromal cell microenvironment. CD68+ cells were also detected at D19 in proximity to CD71+ cells suggesting formation of erythroblastic islands. In this 3D ex vivo biomimicry using near-physiologic cytokine and oxygen conditions, H induced initial erythroid commitment and established an early erythroid progenitor population. N was required at later maturational stages and enhanced the γ-globin to β-globin switch. We identified D7-D14 as a crucial timeframe in this system wherein endogenous cytokine production as well as up-regulation of GATA-1, EPOR and HIF-1α was observed. We propose that a combined HN schedule in this 3D BM biomimicy may enable a more robust and physiologic culture platform to study normal and abnormal erythroid differentiation. Disclosures No relevant conflicts of interest to declare.

scholarly journals The fate and lifespan of human monocyte subsets in steady state and systemic inflammation

2017 ◽  
Vol 214 (7) ◽  
pp. 1913-1923 ◽  
Author(s):  
Amit A. Patel ◽  
Yan Zhang ◽  
James N. Fullerton ◽  
Lies Boelen ◽  
Anthony Rongvaux ◽  
...  
Keyword(s):  
Steady State ◽  
Inflammatory Responses ◽  
Dynamic Equilibrium ◽  
Human Monocyte ◽  
Monocyte Subsets ◽  
Deuterium Labeling ◽  
Developmental Relationship ◽  
Endotoxemia Model ◽  
Classical Monocytes

In humans, the monocyte pool comprises three subsets (classical, intermediate, and nonclassical) that circulate in dynamic equilibrium. The kinetics underlying their generation, differentiation, and disappearance are critical to understanding both steady-state homeostasis and inflammatory responses. Here, using human in vivo deuterium labeling, we demonstrate that classical monocytes emerge first from marrow, after a postmitotic interval of 1.6 d, and circulate for a day. Subsequent labeling of intermediate and nonclassical monocytes is consistent with a model of sequential transition. Intermediate and nonclassical monocytes have longer circulating lifespans (∼4 and ∼7 d, respectively). In a human experimental endotoxemia model, a transient but profound monocytopenia was observed; restoration of circulating monocytes was achieved by the early release of classical monocytes from bone marrow. The sequence of repopulation recapitulated the order of maturation in healthy homeostasis. This developmental relationship between monocyte subsets was verified by fate mapping grafted human classical monocytes into humanized mice, which were able to differentiate sequentially into intermediate and nonclassical cells.

scholarly journals Modulation of Innate Cytokine Responses by Products of Helicobacter pylori

2000 ◽  
Vol 68 (11) ◽  
pp. 6265-6272 ◽  
Author(s):  
Frank Meyer ◽  
Keith T. Wilson ◽  
Stephen P. James
Keyword(s):  
Immune Response ◽  
Helicobacter Pylori ◽  
Lymphocyte Proliferation ◽  
Mononuclear Cells ◽  
Cytokine Production ◽  
Peripheral Blood Mononuclear ◽  
Cytokine Responses ◽  
Ifn Γ ◽  
H Pylori ◽  
Th1 Polarization

ABSTRACT The gastric inflammatory and immune response in Helicobacter pylori infection may be due to the effect of different H. pylori products on innate immune mechanisms. The aim of this study was to determine whether bacterial components could modulate cytokine production in vitro and thus contribute to Th1 polarization of the gastric immune response observed in vivo. The effect of H. pylori recombinant urease, bacterial lysate, intact bacteria, and bacterial DNA on proliferation and cytokine production by peripheral blood mononuclear cells (PBMCs) from H. pylori-negative donors was examined as a model for innate cytokine responses. Each of the different H. pylori preparations induced gamma interferon (IFN-γ) and interleukin-12p40 (IL-12p40), but not IL-2 or IL-5, production, and all but H. pylori DNA stimulated release of IL-10. Addition of anti-IL-12 antibody to cultures partially inhibited IFN-γ production. In addition, each bacterial product inhibited mitogen-stimulated IL-2 production by PBMCs and Jurkat T cells. The inhibitory effect of bacterial products on IL-2 production correlated with inhibition of mitogen-stimulated lymphocyte proliferation, although urease inhibited IL-2 production without inhibiting proliferation, suggesting that inhibition of IL-2 production alone is not sufficient to inhibit lymphocyte proliferation. The results of these studies demonstrate that Th1 polarization of the gastric immune response may be due in part to the direct effects of multiple different H. pylori components that enhance IFN-γ and IL-12 production while inhibiting both IL-2 production and cell proliferation that may be necessary for Th2 responses.

Differential effects of platelets and platelet inhibition by ticagrelor on TLR2- and TLR4-mediated inflammatory responses

2015 ◽  
Vol 113 (05) ◽  
pp. 1035-1045 ◽  
Author(s):  
Andre J. van der Ven ◽  
Niels Riksen ◽  
Gerard Rongen ◽  
Sabine Tacke ◽  
T. N. A. van den Berg ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Inflammatory Responses ◽  
Platelet Inhibition ◽  
P2y12 Receptor ◽  
Oral Dosage ◽  
Induced Response ◽  
Double Blind ◽  
Peripheral Blood Mononuclear ◽  
Cytokine Responses ◽  
Anti Inflammatory

SummaryPlatelets and platelet-monocyte interaction play an important role in inflammation. Both pro- and anti-inflammatory effects of platelet inhibition have been reported in animal models. This study aimed to investigate the effect of platelets and platelet inhibition by the new P2Y12 receptor antagonist ticagrelor on monocyte function, as assessed by cytokine responses to Toll-like Receptor (TLR) ligands. In a set of in vitro experiments, peripheral blood mononuclear cells (PBMC) incubated with the TLR2 ligand Pam3CSK4 produced less cytokines in the presence of platelets, whereas platelets increased the production of cytokines when PBMC were exposed to TLR4 ligand lipopolysaccharide (LPS). These effects of platelets were dependent on direct platelet-leukocyte aggregation and for the Pam3CSK4-induced response, on phagocytosis of platelets by monocytes. In a double blind, placebo-controlled crossover trial in healthy volunteers, a single oral dosage of 180 mg ticagrelor reduced platelet-monocyte complex (PMC) formation. This was associated with an increase in pro-inflammatory cytokines in blood exposed to Pam3CSK4, but a decrease in these cytokines in blood exposed to LPS. These findings show that platelets differentially modulate TLR2- and TLR4-mediated cytokine responses of PBMC. Through inhibition of platelet-leukocyte interaction, P2Y12 receptor antagonists may either exert a pro- or anti-inflammatory effect during infections depending on the TLR primarily involved.

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