Monocyte Production Research Articles

232 Immunomodulation with AM3 and antioxidants creates an adequate framework for skin repair and decreases the monocyte proinflammatory stage in smoker women

Tobacco smoke has been considered a crucial factor in promoting skin and systemic aging and is also associated with the development of a low-level, systemic, chronic inflammation known as “inflammaging” where monocytes play a pivotal role. In our study, 34 healthy smoker middle- aged women were 1:1 randomly assigned to AM3 plus antioxidants and placebo treatment for three months. Peripheral mononuclear blood cells count, and cutaneous biopsy were obtained before and after the treatment and flow cytometry and immunohystologycal studies, respectively, were performed. AM3 is a glucomannan of natural origin from Candida utilis cell wall. AM3 plus antioxidants treatment significantly reduced the monocyte production of the proinflammatory IL-1, TNFα and IL-6 cytokines and also increased the regulatory IL-10 in middle-aged smoker women. The active combination also stimulated a significative increase in reticular dermis depth as well as an increase in the expression of CD117 and CD31. CD117 (also known as c-kit) is a tyrosine kinase receptor that is found in mast cells and whose expression may correlate with the increase in reticular dermis depth due to the production of cytokines such as TGFβ. As seen in fibroblasts, TGFβ-1 and TGFβ-2 are potent enhancers of elastin gene expression. On the other hand, CD31 is a glycoprotein present on the surface of endothelial cells. The increase in the expression of CD31 is related with an enhancement in dermis vascularization. In conclusion, AM3 and antioxidants treatment reduces the systemic proinflammatory monocyte disturbance of heathy smoker middle- aged women and favors the generation of cutaneous repair mechanisms.

Different Concentrations of Lactobacillus acidophilus Cell Free Filtrate Have Differing Anti-Biofilm and Immunomodulatory Effects.

Probiotics such as various strains of Lactobacillaceae have been shown to have antimicrobial and immunomodulatory activity. In vitro studies have shown that Lactobacilli can decrease bacterial biofilm formation. Effects on immune cells have been unclear with most studies showing anti-inflammatory activity. The mechanism of effects has not been clearly elucidated. In these studies, we used different concentrations of live Lactobacillus acidophilus as well as cell free filtrate (CFF) derived from different concentrations of bacteria. Use of CFF is advantageous as a therapeutic because in vivo it can directly contact immune cells and its concentration is fixed. Both live cells and CFF inhibited Pseudomonas aeruginosa biofilm formation. Importantly, we show that high concentration CFF destroyed mature biofilm. This activity was not due to a lowered pH per se, as pH matched HCl did not remove mature biofilm. High concentration CFF totally inhibited P. aeruginosa growth and was bactericidal (>99.99%), but low concentration CFF was not bactericidal. To examine the immunomodulatory effects of L. acidophilus, we incubated THP-1 monocytes and derived macrophages with CFF and measured TNFα production. CFF did not significantly increase TNFα production in THP-1 monocytes. When cells were prestimulated with LPS, high concentration CFF increased TNFα production even further. In macrophages, high concentration CFF alone increased TNFα production but did not affect LPS prestimulated cells. In contrast, low concentration CFF decreased TNFα production in LPS prestimulated cells. To elucidate the possible mechanisms for these effects, we repeated the experiments using a NF-κB reporter THP-1 cell line. High concentration CFF increased NF-κB activity in monocytes and macrophages. In LPS prestimulated macrophages, only low concentration CFF reduced NF-κB activity. These results suggest that high concentration CFF alone induced NF-κB expression which could account partially for an increase in TNFα production. On the other hand, in macrophages, the lower non-bactericidal concentration of CFF reduced NF-κB expression and decreased TNFα production after LPS prestimulation. Taken together, the results provide evidence that different concentrations of L. acidophilus CFF possess varying bactericidal, anti-biofilm and immunomodulatory effects. This is important in vivo to evaluate the possible use of L. acidophilus CFF in different conditions.

Abstract 11: Single Cell Eccite-seq Analysis Reveal An Important Role Of Monocyte-specific Nlrp3 Inflammasome For Salt-sensitivity Of Hypertension In Humans

Salt sensitivity of blood pressure is an independent predictor of death due to cardiovascular events. Diagnosis of salt-sensitivity is not feasible in the clinic, making it difficult to investigate therapeutic strategies. We hypothesized that NLRP3-inflammasome and IL-1β production in monocytes plays a role in salt-sensitive hypertension. We phenotyped salt-sensitivity of blood pressure using an acute inpatient Weinberger protocol of an isocaloric high salt diet and rapid intravenous salt-loading, followed by low salt diet and furosemide-induced salt-depletion. Ambulatory blood pressure was continuously monitored and averaged for the days of salt-loading and salt-depletion. Blood samples were obtained at baseline, salt-loading, and after salt-depletion. Median age was 54 years (44-55), 3 of the 5 subjects were female, screening systolic blood pressure was 140 mmHg (134-148), diastolic blood pressure was 88 mmHg (84-99), and BMI was 35 kg/m 2 (30-39). Using cell hashing and ECCITE-seq analysis, we profiled transcriptomes in multiple immune cell types using antibody-derived tags (ADTs). UMAP clustering of different cell types were identified by ADTs including monocyte markers CD14 and CD16. Interestingly, UMAP visualization of CD14+ and CD16+ clusters indicated a greater decrease in CD14+ clusters after salt-depletion in the salt-resistant subjects than the salt-sensitive; however the salt-sensitive subjects had a greater decrease in CD16+ clusters than the salt-resistant group after both salt-loading and salt-depletion. These data were confirmed using flow cytometry. Unlike in salt-resistant participants, we found that within monocyte clusters, salt-sensitivity was associated with down regulation of the inflammasome components NLRP3 (0.386 ± 1.18 vs. 0.197 ± 0.778) and IL-1β (0.858 ± 2.32 vs. 0.159 ± 0.925) following salt-depletion. Using flow cytometry, we found Δ% isoLG+ CD14+/CD16+ monocytes correlated with salt-sensitivity of blood pressure (r=0.88, 95% CI, p=0.05). These results suggest that the inflammasome and monocyte activation are dynamically regulated by dietary salt in vivo and can serve as a potential diagnostic biomarker for salt-sensitivity of blood pressure.

Monocyte dysfunction in decompensated cirrhosis is mediated by the prostaglandin E2-EP4 pathway

Background & AimsInfection is a major problem in advanced liver disease secondary to monocyte dysfunction. Elevated prostaglandin (PG)E2 is a mediator of monocyte dysfunction in cirrhosis; thus, we examined PGE2 signalling in outpatients with ascites and in patients hospitalised with acute decompensation to identify potential therapeutic targets aimed at improving monocyte dysfunction.MethodsUsing samples from 11 outpatients with ascites and 28 patients hospitalised with decompensated cirrhosis, we assayed plasma levels of PGE2 and lipopolysaccharide (LPS); performed quantitative real-time PCR on monocytes; and examined peripheral blood monocyte function. We performed western blotting and immunohistochemistry for PG biosynthetic machinery expression in liver tissue. Finally, we investigated the effect of PGE2 antagonists in whole blood using polychromatic flow cytometry and cytokine production.ResultsWe show that hepatic production of PGE2 via the cyclo-oxygenase 1–microsomal PGE synthase 1 pathway, and circulating monocytes contributes to increased plasma PGE2 in decompensated cirrhosis. Transjugular intrahepatic sampling did not reveal whether hepatic or monocytic production was larger. Blood monocyte numbers increased, whereas individual monocyte function decreased as patients progressed from outpatients with ascites to patients hospitalised with acute decompensation, as assessed by Human Leukocyte Antigen (HLA)–DR isotype expression and tumour necrosis factor alpha and IL6 production. PGE2 mediated this dysfunction via its EP4 receptor.ConclusionsPGE2 mediates monocyte dysfunction in decompensated cirrhosis via its EP4 receptor and dysfunction was worse in hospitalised patients compared with outpatients with ascites. Our study identifies a potential drug target and therapeutic opportunity in these outpatients with ascites to reverse this process to prevent infection and hospital admission.Lay summaryPatients with decompensated cirrhosis (jaundice, fluid build-up, confusion, and vomiting blood) have high infection rates that lead to high mortality rates. A white blood cell subset, monocytes, function poorly in these patients, which is a key factor underlying their sensitivity to infection. We show that monocyte dysfunction in decompensated cirrhosis is mediated by a lipid hormone in the blood, prostaglandin E2, which is present at elevated levels, via its EP4 pathway. This dysfunction worsens when patients are hospitalised with complications of cirrhosis compared with those in the outpatients setting, which supports the EP4 pathway as a potential therapeutic target for patients to prevent infection and hospitalisation.

Bacitracin and Rutin Regulate Tissue Factor Production in Inflammatory Monocytes and Acute Myeloid Leukemia Blasts

Simple SummaryAberrant tissue factor (TF) expression by transformed myeloblasts and inflammatory monocytes contributes to coagulation activation in acute myeloid leukemia (AML). TF procoagulant activity (PCA) is regulated by protein disulfide isomerase (PDI), an oxidoreductase with chaperone activity, but its specific role in AML-associated TF biology is unclear. Here, we provide novel mechanistic insights into this interrelation. We show that bacitracin and rutin, two pan-inhibitors of the PDI family, prevent lipopolysaccharide (LPS)-induced monocyte TF production under inflammatory conditions and constitutive TF expression by THP1 cells and AML blasts, thus exerting promising anticoagulant activity. Downregulation of the TF protein was mainly restricted to its non-coagulant, cryptic pool and was at least partially regulated on the mRNA level in LPS-stimulated monocytes. Collectively, our study indicates a complex role of thiol isomerases in the regulation of myeloid TF PCA, with the most abundant PDI being a promising therapeutic target in the management of AML-associated coagulopathies.Aberrant expression of tissue factor (TF) by transformed myeloblasts and inflammatory monocytes drives coagulation activation in acute myeloid leukemia (AML). Although regulation of TF procoagulant activity (PCA) involves thiol-disulfide exchange reactions, the specific role of protein disulfide isomerase (PDI) and other thiol isomerases in AML-associated TF biology is unclear. THP1 cells and peripheral blood mononuclear cells (PBMCs) from healthy controls or AML patients were analyzed for thiol isomerase-dependent TF production under various experimental conditions. Total cellular and membrane TF antigen, TF PCA and TF mRNA were analyzed by ELISA, flow cytometry, clotting or Xa generation assay and qPCR, respectively. PBMCs and THP1 cells showed significant insulin reductase activity, which was inhibited by bacitracin or rutin. Co-incubation with these thiol isomerase inhibitors prevented LPS-induced TF production by CD14-positive monocytes and constitutive TF expression by THP1 cells and AML blasts. Downregulation of the TF antigen was mainly restricted to the cryptic pool of TF, efficiently preventing phosphatidylserine-dependent TF activation by daunorubicin, and at least partially regulated on the mRNA level in LPS-stimulated monocytes. Our study thus delineates a complex role of thiol isomerases in the regulation of myeloid TF PCA, with PDI being a promising therapeutic target in the management of AML-associated coagulopathies.

Impact of cholesterol on proinflammatory monocyte production by the bone marrow.

AimPreclinical work indicates that low-density lipoprotein cholesterol (LDL-C) not only drives atherosclerosis by directing the innate immune response at plaque level but also augments proinflammatory monocyte production in the bone marrow (BM) compartment. In this study, we aim to unravel the impact of LDL-C on monocyte production in the BM compartment in human subjects.Methods and resultsA multivariable linear regression analysis in 12 304 individuals of the EPIC-Norfolk prospective population study showed that LDL-C is associated with monocyte percentage (β = 0.131 [95% CI: 0.036–0.225]; P = 0.007), at the expense of granulocytes (β = −0.876 [95% CI: −1.046 to −0.705]; P < 0.001). Next, we investigated whether altered haematopoiesis could explain this monocytic skewing by characterizing CD34+ BM haematopoietic stem and progenitor cells (HSPCs) of patients with familial hypercholesterolaemia (FH) and healthy normocholesterolaemic controls. The HSPC transcriptomic profile of untreated FH patients showed increased gene expression in pathways involved in HSPC migration and, in agreement with our epidemiological findings, myelomonocytic skewing. Twelve weeks of cholesterol-lowering treatment reverted the myelomonocytic skewing, but transcriptomic enrichment of monocyte-associated inflammatory and migratory pathways persisted in HSPCs post-treatment. Lastly, we link hypercholesterolaemia to perturbed lipid homeostasis in HSPCs, characterized by lipid droplet formation and transcriptomic changes compatible with increased intracellular cholesterol availability.ConclusionsCollectively, these data highlight that LDL-C impacts haematopoiesis, promoting both the number and the proinflammatory activation of circulating monocytes. Furthermore, this study reveals a potential contributory role of HSPC transcriptomic reprogramming to residual inflammatory risk in FH patients despite cholesterol-lowering therapy.

Herbal melanin induces interleukin-1β secretion and production by human THP-1 monocytes via Toll-like receptor 2 and p38 MAPK activation.

Herbal melanin (HM), extracted from Nigella sativa, is known for its immunogenic properties through the modulation of cytokine production via Toll-like receptor (TLR)4. TLRs play a crucial role in the host defense through the regulation of innate and adaptive immune responses. However, the potential effect of HM on the production of interleukin-1β (IL-1β), the main immunoregulatory cytokine secreted by activated monocytes, has not been reported. The present study aimed to investigate the effects of HM on IL-1β secretion and production, detected by enzyme-linked immunosorbent assay, western blotting and mRNA expression monitored by reverse transcription-PCR, in human monocytes and a monocytic cell line, THP-1. Signaling pathways involved in the HM-induced IL-1β production was investigated in the THP-1 cells. It was shown that HM upregulated the IL-1β mRNA in the THP-1 cells and induced the secretion of IL-1β in the monocytes and THP-1 cells, in a dose-dependent manner, compared to the untreated cells. HM increased the protein expression of IL-1β, TLR2, the main receptor for IL-1β production, and activated p38 mitogen-activated protein kinase (MAPK), a key mediator for stress-induced IL-1β gene expression. The blockade of the p38 MAPK pathway, with the pharmacological inhibitor SB202190, and TLR2 receptor with a neutralization antibody, resulted in the decrease of HM-induced IL-1β production in THP-1 cells. The TLR4 receptor blockade also decreased HM-induced IL-1β production, but to a lesser extent than TLR2 blockade. In conclusion, the present study demonstrated that HM stimulates IL-1β production in monocytes and THP-1 cells, in a TLR2/p38 MAPK pathway-dependent manner, suggesting promising immunoregulatory potentials of HM against inflammatory-associated diseases.

TNF\u03b1 increases tyrosine hydroxylase expression in human monocytes

Most, if not all, peripheral immune cells in humans and animals express tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine synthesis. Since TH is typically studied in the context of brain catecholamine signaling, little is known about changes in TH production and function in peripheral immune cells. This knowledge gap is due, in part, to the lack of an adequately sensitive assay to measure TH in immune cells expressing lower TH levels compared to other TH expressing cells. Here, we report the development of a highly sensitive and reproducible Bio-ELISA to quantify picogram levels of TH in multiple model systems. We have applied this assay to monocytes isolated from blood of persons with Parkinson’s disease (PD) and to age-matched, healthy controls. Our study unexpectedly revealed that PD patients’ monocytes express significantly higher levels of TH protein in peripheral monocytes relative to healthy controls. Tumor necrosis factor (TNFα), a pro-inflammatory cytokine, has also been shown to be increased in the brains and peripheral circulation in human PD, as well as in animal models of PD. Therefore, we investigated a possible connection between higher levels of TH protein and the known increase in circulating TNFα in PD. Monocytes isolated from healthy donors were treated with TNFα or with TNFα in the presence of an inhibitor. Tissue plasminogen activator (TPA) was used as a positive control. We observed that TNFα stimulation increased both the number of TH+ monocytes and the quantity of TH per monocyte, without increasing the total numbers of monocytes. These results revealed that TNFα could potentially modify monocytic TH production and serve a regulatory role in peripheral immune function. The development and application of a highly sensitive assay to quantify TH in both human and animal cells will provide a novel tool for further investigating possible PD immune regulatory pathways between brain and periphery.

Toll‐like receptor 10 controls TLR2‐induced cytokine production in monocytes from patients with Parkinson's disease

Peripheral inflammation, particularly mediated by monocytes, can cause neuroinflammation in Parkinson's disease (PD). We investigated the mechanism of TLR2-induced cytokine impairment in peripheral monocytes from PD patients and the association between the presence of CD14+ TLR10+ monocytes and PD severity. Peripheral blood mononuclear cells from PD patients and healthy individuals were evaluated for TLR expression on monocyte subsets (CD14 and CD16 expression) using flow cytometry. Moreover, cytokines were evaluated using flow cytometry after stimulation with Pam3 Cys (TLR2/TLR1 agonist) in the absence or presence of neutralizing antibodies to TLR10. The severity of PD was assessed using the unified PD rating scale (UPDRS) and motor activity, anxiety (BAI), depression (BDI), and fatigue (PD Fatigue Scale-16) scales. The frequency of CD14+ TLR10+ monocytes and expression intensity of TLR2 and TLR10 were higher in patients with PD than healthy individuals. The frequency of intermediate monocytes (CD14++ CD16+ ) was not significantly increased in patients with PD, but was the main monocyte subset expressing TLR10. The TLR2/TLR1-impaired cytokine production (IL-6, TNFα, IL-8, and IL-10) in PD patients was reversed by neutralizing TLR10. The high frequency of total CD14+ TLR10+ monocytes was associated with a reduction in the severity of PD according to the evaluation of motor and nonmotor symptoms. Peripheral monocytes from patients with PD showed phenotypic and functional alterations. The expression of TLR10 on monocytes can protect against PD by controlling TLR2-induced cytokine production. Furthermore, data suggested that a low frequency of CD14+ TLR10+ monocytes indicates the severity of PD. The results identified new opportunities for the development of novel PD neuroprotective therapies.

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

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

Bacterial species-specific modulatory effects on phenotype and function of camel blood leukocytes

BackgroundRecent studies have reported pathogen-species-specific modulating effects on the innate immune system. Escherichia coli, Staphylococcus aureus, and Streptococcus agalactiae are important pathogenic bacteria responsible for different infectious diseases in several animal species. In the present study, a whole blood culture with S. aureus, E. coli, or S. agalactiae and flow cytometry were used to investigate, whether stimulation with different bacterial species induces different immunomodulation patterns in camel leukocytes. The expression of different cell surface myeloid markers and cell adhesion molecules on monocytes and neutrophils was investigated. In addition, the capacity of monocytes and neutrophils to produce reactive oxygen species (ROS) was analyzed.ResultsStimulation with either of the bacterial species resulted in the expansion of the camel CD14highMHCIIhigh monocyte subset with a reduced fraction of CD14highMHCIIlow monocytes. For the CD14lowMHCIIhigh monocytes, however, only stimulation with S. aureus or S. agalactiae increased their fractions in blood. Although all bacterial species elicited the upregulation of cell surface MHC class II molecules on granulocytes, the increase was, however, highest on cells stimulated with S. aureus. The expression levels of the two adhesion molecules, CD11a and CD18, on neutrophils and monocytes were differently affected by bacterial stimulation. Functionally, E. coli failed to stimulate ROS production in monocytes, while induced a strong ROS production response in granulocytes. S. agalactiae elicited a week ROS production in granulocytes when compared to the other two pathogens.ConclusionsThe different responsiveness of monocytes and granulocytes toward different bacterial species indicates different host-pathogen interaction mechanisms for the two cell populations. In addition, the phenotypic and functional differences between cells stimulated with E. coli, S. aureus, or S. agalactiae suggests pathogen-species-specific modulating effects of the bacterial pathogens on the camel innate myeloid cells.

The role of IL-32 in Bacillus Calmette-Guérin (BCG)-induced trained immunity in infections caused by different Leishmania spp.

BackgroundCells of the innate immune system undergo long-term functional reprogramming in response to Bacillus Calmette-Guérin (BCG) exposure via a process called trained immunity, conferring nonspecific protection to unrelated infections. Here, we investigate whether BCG-induced trained immunity is able to protect against infections caused by different Leishmania spp., protozoa that cause cutaneous and mucosal or visceral lesions. MethodsWe used training models of human monocytes with BCG and subsequent infection by L. braziliensis, L. amazonensis and L. infantum, and the vaccination of wild-type and transgenic mice for IL-32γ before in vivo challenge with parasites. ResultsWe demonstrated that monocytes trained with BCG presented enhanced ability to kill L. braziliensis, L. amazonensis and L. infantum through increased production of reactive oxygen species. Interleukin (IL)-32 appears to play an essential role in the development of trained immunity. Indeed, BCG exposure induced IL-32 production in human primary monocytes, both mRNA and protein. We have used a human IL-32γ transgenic mouse model (IL-32γTg) to study the effect of BCG vaccination in different Leishmania infection models. BCG vaccination decreased lesion size and parasite load in infections caused by L. braziliensis and reduced the spread of L. amazonensis to other organs in both infected wild-type (WT) and IL-32γTg mice. In addition, BCG reduced the parasite load in the spleen, liver and bone marrow of both WT and IL-32γTg mice infected with L. infantum. BCG vaccination increased inflammatory infiltrate in infected tissues caused by different Leishmania spp. In all infections, the presence of IL-32γ was not mandatory, but it increased the protective and inflammatory effects of BCG-induced training. ConclusionsBCG's ability to train innate immune cells, providing protection against leishmaniasis, as well as the participation of IL-32γ in this process, pave the way for new treatment strategies for this neglected infectious disease.

Assessing the Functional Heterogeneity of Monocytes in Human Septic Shock: a Proof-of-Concept Microfluidic Assay of TNFα Secretion.

ObjectiveThe development of advanced single-cell technologies to decipher inter-cellular heterogeneity has enabled the dynamic assessment of individual cells behavior over time, overcoming the limitation of traditional assays. Here, we evaluated the feasibility of an advanced microfluidic assay combined to fluorescence microscopy to address the behavior of circulating monocytes from septic shock patients.MethodsSeven septic shock patients and ten healthy volunteers were enrolled in the study. Using the proposed microfluidic assay we investigated the production over time of LPS-elicited TNFα by single monocytes encapsulated within droplets. Cellular endocytic activity was assessed by internalization of magnetic nanoparticles. Besides, we assessed HLA-DR membrane expression and LPS-induced TNFα production in monocytes through classical flow cytometry assays.ResultsConsistent with the flow cytometry results, the total number of TNFα molecules secreted by encapsulated single monocytes was significantly decreased in septic shock patients compared to healthy donors. TNFα production was dampened as soon as 30 and 60 minutes after LPS stimulation in monocytes from septic patients. Furthermore, the microfluidic assay revealed heterogeneous individual behavior of monocytes from septic shock patients. Of note, monocytes from both healthy donors and patients exhibited similar phagocytic activities over time.ConclusionThe microfluidic assay highlights the functional heterogeneity of monocytes, and provides in-depth resolution in assessing the hallmark monocyte deactivation encountered in post-septic immunosuppression.

Underlying Inflammation Impacts Immune Modulation Following Exposure to Platelet Concentrates and Packed Red Blood Cells

IntroductionTransfusion of platelet concentrates (PC) and packed red blood cells (PRBC) are lifesaving treatments for patients suffering from thrombocytopenia, haematological disorders, severe trauma and cancer. Platelets play a central role in mediating coagulation and wound healing, inflammation and innate immune responses. Red blood cells are crucial for delivery of oxygen to surrounding tissues and translocation of carbon dioxide in the lungs. In Australia, PC and PRBC are routinely stored for up to 5- or 42- days respectively prior to transfusion. Storage related changes of PC and PRBC may contribute to adverse patient outcomes and include increased release of biological mediators (sCD40L, IL-8), partial platelet activation, loss of 2,3-diphosphoglycerate and ATP, change in oxygen dissociation and altered morphology. Despite the therapeutic benefits of PC and PRBC transfusion, clinical studies suggest transfusion-associated modulation of the recipient immune response contributes to adverse outcomes including infection and mortality. Underlying inflammation is an important factor contributing to the development of transfusion-related acute lung injury (TRALI), a pro-inflammatory response, whereas in vitro transfusion models largely report leucocyte suppression following exposure to PC and PRBC. The relationship between the level of underlying inflammation and the impact on cell function post-transfusion remains unclear. We investigated the relationship between inflammation and modulation of dendritic cell (DC) and monocyte phenotype following exposure to PC and PRBC.MethodsAn in-vitro transfusion model was used to investigate changes in DC and monocyte phenotype following exposure to PC-supernatants (PC-SN) or PRBC-supernatants (PRBC-SN) in co-culture with a range of lipopolysaccharide concentrations (LPS; 0 µg/mL (no LPS), 0.25 µg/mL, 0.5 µg/mL, 0.75 µg/mL, 1 µg/mL LPS) to model different levels of underlying inflammation. Based on results of recent randomised controlled trials, we used fresh PC- and PRBC- SN. Freshly collected whole blood was cultured with RPMI media and day 2 (D2) PC-SN or D2 PRBC-SN at doses representing a 1 (10%), or 2-3 (25%) unit transfusion. Golgi-plug (containing Brefeldin-a) was used to inhibit protein release to facilitate intracellular staining of cytokines (IL-6, IL-8, IL-10, IL-12, IL-1α, TNF-α, MIP-1α, MIP-1β, MCP-1, IP-10) from DC and monocytes using via flow cytometry. Changes in monocyte and DC cytokine and chemokine production at different concentrations of LPS were assessed by a repeated measures one-way ANOVA with Tukey's post-test (P<0.05).ResultsCulture with LPS alone resulted in a concentration dependent decrease in the production of most monocyte and DC cytokines. Modelling a low dose (1 unit) transfusion with D2 PC-or PRBC- SN resulted in a reduction in monocyte and DC cytokine production in the presence of 0.25 µg/mL and 0.5 µg/mL LPS. This effect was less evident when higher concentrations of LPS (0.75 µg/mL, 1 µg/mL) were used. When modelling a high dose (2-3 unit) transfusion, exposure to D2 PC-or PRBC- SN resulted in a predominant suppression of monocyte and DC responses at all LPS concentrations tested.ConclusionOur data provides evidence that the level of underlying inflammation differentially affects DC and monocyte responses following exposure to PC-SN or PRBC-SN. Dose in combination with level of underlying inflammation significantly impacts monocyte and DC responses which may contribute and increase the risk of transfusion-associated poor patient outcomes including infection. DisclosuresNo relevant conflicts of interest to declare.

1141-P: Short-Chain Fatty Acid Acetate Increases TNF-a-Induced MCP-1 Production in Monocytes via the ACSL-1/MAPK/NF-?B Axis: Significance for Inflammation and Insulin Resistance

Short-chain fatty acids (SCFAs) are formed as by-products of dietary fiber metabolism by gut bacteria and have multiple immunomodulatory functions in different cells involved in the inflammatory and immune responses. In general, the anti-inflammatory role of SCFAs is well documented. Here, we investigated whether SCFAs could modulate the TNF-α-mediated induction of chemokine MCP-1 (also known as CCL2) in monocytes/macrophages; and if so, which mechanisms were involved. Monocytic cells were exposed to SCFAs including acetate, propionate, and butyrate, with or without TNF-α, and the release of MCP-1 was measured. We found that none of the SCFAs suppressed TNF-α-induced MCP-1 release in monocytes. However, challenge with acetate in combination with TNF-α resulted in substantially greater MCP-1 release from monocytic cells than challenge with TNF-α alone, indicating synergistic effects. Interestingly, such effects of acetate were not seen in macrophages. Synergistic upregulation of MCP-1 was mediated through the activation of acyl-CoA synthetase 1 (ACSL1). Acetate/TNFα induced phosphorylation of p38, ERK1/2, and NF-κB was observed during the secretion of MCP-1. Acetate/TNF-α mediated synergistic expression of MCP-1 was significantly reduced by the inhibition of p38 MAPK, ERK1/2 and NF-κB signaling pathways. Furthermore, inhibition of the ACSL1 activity also attenuates the TNF-α-mediated phosphorylation of p38 MAPK, ERK1/2, and NF-κB. ACSL1 inhibition led to a lower induction of NF-κB/AP-1 activity in response to co-stimulation with acetate and TNF-α. In conclusion, these data support the proinflammatory, rather than anti-inflammatory, effects of acetate on TNF-α-mediated MCP-1 expression in monocytes involving the ACSL1/MAPK/NF-kB axis. These findings point to the differential effects of SCFA acetate in the settings of obesity and metabolic inflammation/insulin resistance. Disclosure R. Ahmad: None. A. Abu alroub: None. F. Alrashed: None. A. Al-sayyar: None. N. Akhter: None. S. P. Kochumon: None. F. Almulla: None. S. T. Sindhu: None.

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

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.

Impact of aging on monocyte subset production by bone marrow progenitors

Abstract Monocytes can patrol the circulation or be recruited into tissues to become macrophages or monocyte-derived dendritic cells (moDCs). We previously showed that monocytes arise via two independent pathways in mouse bone marrow: Granulocyte-Monocyte Progenitors (GMPs) produce neutrophils and monocytes, and Monocyte-DC Progenitors (MDPs) yield monocytes and conventional and plasmacytoid DCs (cDCs and pDCs). Notably, both pathways produce monocytes that can differentiate into macrophages, but moDCs arise exclusively via the MDP pathway. We also showed that the microbial stimuli LPS and CpG promote monocyte production by GMPs and MDPs, respectively. We are now investigating how aging impacts the balance of monocyte production by GMPs and MDPs. scRNAseq analysis revealed elevated Cd74 and H2 gene expression by steady-state classical monocytes from old mice compared to young mice, with a larger proportion of monocytes expressing these genes. Consistent with this, we observed proportionally more monocytes expressing CD74 and MHCII in the bone marrow and circulation of old mice by flow cytometry, and a higher yield of CD11c+ MHCII+ moDCs in GM-CSF cultures of MDPs and monocytes from old bone marrow. These findings are indicative of elevated moDC production during aging, perhaps reflecting the more inflammatory microenvironment. In ongoing studies, we are investigating whether imbalanced monocyte production by the GMP and MDP pathways underlies increased moDC production during aging.

Trait positive affect buffers the association between experimental sleep disruption and inflammation

BackgroundSleep disturbances and insufficient sleep are highly prevalent. Both clinical sleep disorders and multiple forms of experimental sleep loss predict heightened inflammation. As such, it is necessary to investigate potential protective factors. Given that trait positive affect (PA) is associated with reduced inflammation, and buffers the proinflammatory effects of stress, it is possible that high trait positive affect might protect individuals from an inflammatory response to sleep disruption. The present study tested this hypothesis in an experimental sleep disruption paradigm with assessment of cellular inflammation. MethodsData were drawn from good sleeping adults (n = 79) who participated in a randomized, within-subjects crossover experiment comparing the effects of two nights of sleep disruption versus two nights of uninterrupted sleep. Stimulated monocytic production of intracellular proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6) were assayed using flow cytometric methods and indexed as the percentage of monocytes expressing TNF, IL-6, or co-expressing both. Hypotheses were evaluated using linear mixed effects models. ResultsControlling for negative affect, body mass index, age, and sex, PA significantly moderated the associations between sleep condition and stimulated monocyte production of IL-6 (b = −1.03, t = −2.02, p = .048) and its co-expression with TNF (b = −0.93, t = −2.00, p = .049), such that inflammatory responses were blunted among those high in PA with increases principally among those low in PA. The effect on TNF was similar in terms of effect size, but marginally significant. ConclusionsActivation of cellular inflammation in response to sleep disruption is buffered by PA independent of negative affect. Interventions that promote PA might protect persons from the inflammatory activation following sleep loss, with the potential to mitigate the adverse health consequences of sleep disturbance.

Exploring neural mechanisms of the health benefits of gratitude in women: A randomized controlled trial

BackgroundGratitude has received growing interest as an emotion that can bring greater happiness and health. However, little is known about the effects of gratitude on objective measures of physical health or the neural mechanisms that underlie these effects. Given strong links between gratitude and giving behavior, and giving and health, it is possible that gratitude may benefit health through the same mechanisms as giving to others. Thus, this study investigated whether gratitude activates a neural ‘caregiving system’ (e.g., ventral striatum (VS), septal area (SA)), which can downregulate threat responding (e.g., amygdala) and possibly cellular inflammatory responses linked to health. MethodsA parallel group randomized controlled trial examined the effect of a six-week online gratitude (n = 31) vs. control (n = 30) writing intervention on neural activity and inflammatory outcomes. Pre- and post-intervention, healthy female participants (ages 35–50) reported on support-giving behavior and provided blood samples to assess circulating plasma levels and stimulated monocytic production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)). Post-intervention, participants completed a gratitude task and a threat reactivity task in an fMRI scanner. ResultsThere were no significant group differences (gratitude vs. control intervention) in neural responses (VS, SA, or amygdala) to the gratitude or threat tasks. However, across the entire sample, those who showed larger pre- to- post-intervention increases in self-reported support-giving showed larger reductions in amygdala reactivity following the gratitude task (vs. control task). Additionally, those who showed larger reductions in amygdala reactivity following the gratitude task showed larger pre-to-post reductions in the stimulated production of TNF-α and IL-6. Importantly, gratitude-related reductions in amygdala reactivity statistically mediated the relationship between increases in support-giving and decreases in stimulated TNF-α production. ConclusionThe observed relationships suggest that gratitude may benefit health (reducing inflammatory responses) through the threat-reducing effects of support-giving.

Glutathione Metabolism Contributes to the Induction of Trained Immunity.

The innate immune system displays heterologous memory characteristics, which are characterized by stronger responses to a secondary challenge. This phenomenon termed trained immunity relies on epigenetic and metabolic rewiring of innate immune cells. As reactive oxygen species (ROS) production has been associated with the trained immunity phenotype, we hypothesized that the increased ROS levels and the main intracellular redox molecule glutathione play a role in the induction of trained immunity. Here we show that pharmacological inhibition of ROS in an in vitro model of trained immunity did not influence cell responsiveness; the modulation of glutathione levels reduced pro-inflammatory cytokine production in human monocytes. Single nucleotide polymorphisms (SNPs) in genes involved in glutathione metabolism were found to be associated with changes in pro-inflammatory cytokine production capacity upon trained immunity. Also, plasma glutathione concentrations were positively associated with ex vivo IL-1β production, a biomarker of trained immunity, produced by monocytes of BCG-vaccinated individuals. In conclusion, glutathione metabolism is involved in the induction of trained immunity, and future studies are warranted to explore its functional consequences in human diseases.