Differences In Monocytes Research Articles

Both biofilm cytotoxicity and monocytes' adhesion may be used as estimators of enterococcal virulence.

Our study aimed to identify markers of enterococci's virulence potential by evaluating the properties of strains of different sites of isolation. Enterococcal strains were isolated as commensals from faeces and as invasive strains from the urine and blood of patients from the University Clinical Centre, Gdańsk, Poland. Changes in monocytes' susceptibility to the cytotoxic activity of isolates of different origins and their adherence to biofilm were evaluated using a flow cytometer. The bacterial protein profile was estimated by matrix assisted laser desorption ionization-time of flight mass spectrometer. The cytotoxicity of biofilm and monocytes' adherence to it were the most accurate factors in predicting the prevalence of the strain in the specific niche. Additionally, a bacterial protein with mass-to-charge ratio (m/z) 5000 was found to be responsible for the increased bacterial cytotoxicity, while monocytes' decreased adherence to biofilm was linked with the presence of proteins either with m/z 3330 or 2435. The results illustrate that monocytes' reaction when exposed to the bacterial biofilm can be used as an estimator of pathogens' virulence potential. The observed differences in monocytes' response are explainable by the bacterial proteins' profile. Additionally, the results indicate that the features of both bacteria and monocytes impact the outcome of the infection.

Functional and Multi-Omics Effects of an Optimized CRISPR-Mediated FURIN Depletion in U937 Monocytes.

The pro-protein convertase FURIN (PCSK3) is implicated in a wide range of normal and pathological biological processes such as infectious diseases, cancer and cardiovascular diseases. Previously, we performed a systemic inhibition of FURIN in a mouse model of atherosclerosis and demonstrated significant plaque reduction and alterations in macrophage function. To understand the cellular mechanisms affected by FURIN inhibition in myeloid cells, we optimized a CRISPR-mediated gene deletion protocol for successfully deriving hemizygous (HZ) and nullizygous (NZ) FURIN knockout clones in U937 monocytic cells using lipotransfection-based procedures and a dual guide RNA delivery strategy. We observed differences in monocyte and macrophage functions involving phagocytosis, lipid accumulation, cell migration, inflammatory gene expression, cytokine release patterns, secreted proteomics (cytokines) and whole-genome transcriptomics between wild-type, HZ and NZ FURIN clones. These studies provide a mechanistic basis on the possible roles of myeloid cell FURIN in cardiovascular disorders.

INVESTIGATION OF EOSINOPHIL, LYMPHOCYTE AND MONOCYTE VALUES ACCORDING TO AGE AND GENDER IN INSECT BITES IN THE EMERGENCY DEPARTMENT

Objective: The purpose of this study was to look at the seasonal distribution, age and gender distribution, and eosinophil, lymphocyte, and monocyte values according to age and gender in cases of insect bites that were brought to the emergency room over the course of a year. 
 Materials and method: Retrospective analysis was performed on patients who were brought to the emergency room between 1.12.2021 and 1.12.2022 and had the ICD code W57 (Diagnosis Code - Bitten or stung by Nonvenomous Insects and Other Nonvenomous Arthropods). The following values were noted: age, gender, presenting season, CRP, Leukocyte, Platelet, Lymphocyte, Monocyte, Eosinophil, INR, PTZ, and Aptt levels.
 Findings: The study comprised a total of 694 patients—308 females and 386 males. The patients were 39.81 16.42 years old on average. Spring saw 9.4% of the patients, summer saw 67%, and fall saw 23.6%. According to the patients' gender, there were significant differences in the eosinophil (t:-3.535; p:0.0010.01) and monocyte (t:-4.909; p:0.0010.01) values. Regarding the season in which the patients were admitted, significant differences in lymphocyte (F:7.045; p:0.0010.01) and monocyte (F:3.208; p:0.0410.05) values were discovered. When the disparities in eosinophil, lymphocyte, and monocyte values were evaluated in relation to the patients' ages, significant differences in monocyte values were discovered (F:2.552; p:0.0270.05).
 Result: We commonly see insect bites in emergency rooms, which we can usually cure with straightforward remedies or occasionally without treatment, but in some unfortunate circumstances, we may have to deal with major issues and allergic responses (4). Almost little studies have been done on the seasonal distribution and evaluation of blood tests according to age and gender, despite the fact that there are many studies on this topic in the literature. We think that more study on this topic is necessary.

Dimethyl fumarate-related immune and transcriptional signature is associated with clinical response in multiple sclerosis-treated patients.

Dimethyl fumarate (DMF) is an immunomodulatory drug approved for the therapy of multiple sclerosis (MS). The identification of response biomarkers to DMF is a necessity in the clinical practice. With this aim, we studied the immunophenotypic and transcriptomic changes produced by DMF in peripheral blood mononuclear cells (PBMCs) and its association with clinical response. PBMCs were obtained from 22 RRMS patients at baseline and 12 months of DMF treatment. Lymphocyte and monocyte subsets, and gene expression were assessed by flow cytometry and next-generation RNA sequencing, respectively. Clinical response was evaluated using the composite measure "no evidence of disease activity" NEDA-3 or "evidence of disease activity" EDA-3 at 2 years, classifying patients into responders (n=15) or non-responders (n=7), respectively. In the whole cohort, DMF produced a decrease in effector (TEM) and central (TCM) memory T cells in both the CD4+ and CD8+ compartments, followed by an increase in CD4+ naïve T cells. Responder patients presented a greater decrease in TEM lymphocytes. In addition, responder patients showed an increase in NK cells and were resistant to the decrease in the intermediate monocytes shown by non-responders. Responder patients also presented differences in 3 subpopulations (NK bright, NK dim and CD8 TCM) at baseline and 4 subpopulations (intermediate monocytes, regulatory T cells, CD4 TCM and CD4 TEMRA) at 12 months. DMF induced a mild transcriptional effect, with only 328 differentially expressed genes (DEGs) after 12 months of treatment. The overall effect was a downregulation of pro-inflammatory genes, chemokines, and activators of the NF-kB pathway. At baseline, no DEGs were found between responders and non-responders. During DMF treatment a differential transcriptomic response was observed, with responders presenting a higher number of DEGs (902 genes) compared to non-responders (189 genes). Responder patients to DMF exhibit differences in monocyte and lymphocyte subpopulations and a distinguishable transcriptomic response compared to non-responders that should be further studied for the validation of biomarkers of treatment response to DMF.

Evaluation of leukocytes cells types counts in blood from patients with different severities of periodontal diseases

Background: Periodontal diseases are inflammatory disorders caused by the accumulation of oral biofilm and the host response to this accumulation which characterized by exaggerated leukocytes and neutrophils attraction to the sites of inflammation by chemoattractants which are a very important part of the pathogenesis of periodontal diseases. This study aimed to determine and compare the clinical periodontal parameters and the leukocyte cell types in the peripheral blood between patients with gingivitis and periodontitis with different severities compared to healthy controls. Materials and methods: This study included 150 male subjects aged between 35-50 years. They were divided into three groups: gingivitis group (n=30), periodontitis patients (n=90) which subdivided into Mild =30 patients, Moderate =30 patients, Severe =30 patients and a control group (n=30) with clinically healthy periodontium. Clinical periodontal parameters were recorded ((plaque index (PLI), gingival index (GI), bleeding on probing (BOP), probing pocket depth (PPD) and clinical attachment level (CAL)). Blood samples were collected then an automated blood analyzer evaluated leukocyte cell types. Results: Significant differences in The counts of neutrophils and lymphocytes exhibited significant differences among the study groups and subgroups. On contrary, differences in monocytes, eosinophils, and basophils counts were not significant. Additionally, severity of periosontitis was significantly correlated with the mean counts of the various leukocyte cell types; however, clinical periodontal characteristics did not show such correlation with these inflammatory cells. Conclusion: This study demonstrated that periodontal disease with different severities is associated with possible episodes of bacteremia that originate from periodontal lesions which mediate inflammatory conditions that in turn causing changes in the systemic markers especially leukocytes cells types.

Characteristic gene prognostic model of type 1 diabetes mellitus via machine learning strategy.

The present study was designed to detect possible biomarkers associated with Type 1 diabetes mellitus (T1DM) incidence in an effort to develop novel treatments for this condition. Three mRNA expression datasets of peripheral blood mononuclear cells (PBMCs) were obtained from the GEO database. Differentially expressed genes (DEGs) between T1DM patients and healthy controls were identified by Limma package in R, and using the DEGs to conduct GO and DO pathway enrichment. The LASSO-SVM were used to screen the hub genes. We performed immune correlation analysis of hub genes and established a T1DM prognosis model. CIBERSORT algorithm was used to identify the different immune cells in distribution between T1DM and normal samples. The correlation of the hub genes and immune cells was analyzed by Spearman. ROC curves were used to assess the diagnostic value of genes in T1DM. A total of 60 immune related DEGs were obtained from the T1DM and normal samples. Then, DEGs were further screened to obtain 3 hub genes, ANP32A-IT1, ESCO2 and NBPF1. CIBERSORT analysis revealed the percentage of immune cells in each sample, indicating that there was significant difference in monocytes, T cells CD8+, gamma delta T cells, naive CD4+ T cells and activated memory CD4+ T cells between T1DM and normal samples. The area under curve (AUC) of ESCO2, ANP32A-IT1 and NBPF1 were all greater than 0.8, indicating that these three genes have high diagnostic value for T1DM. Together, the findings of these bioinformatics analyses thus identified key hub genes associated with T1DM development.

Association of White Blood Cell Subtypes and Derived Ratios with a Mortality Outcome in Adult Patients with Polytrauma.

Background. After trauma, the subtypes of white blood cells (WBCs) in circulation and the derived neutrophil-to-lymphocyte ratio (NLR), monocyte-to-lymphocyte ratio (MLR), and platelet-to-lymphocyte ratio (PLR) may undergo relative changes and reflect the patients’ immune-inflammatory status and outcome. This retrospective study was designed to investigate the relationship between these variables and the mortality outcomes in adult patients with polytrauma, which is defined as an abbreviated injury scale (AIS) score ≥ 3 in two or more different body regions. Methods. A comparison of the expression of subtypes of WBCs, NLR, MLR, and PLR upon arrival to the emergency department was performed in selected propensity score-matched patient cohorts created from 479 adult patients with polytrauma between 1 January 2015 and 31 December 2019. A multivariate logistic regression analysis was used to identify the independent risk factors for mortality. Results. There were no significant differences in monocyte, neutrophil, and platelet counts, as well as in MLR, NLR, and PLR, between deceased (n = 118) and surviving (n = 361) patients. In the propensity score-matched patient cohorts, which showed no significant differences in sex, age, comorbidities, and injury severity, deceased patients had significantly higher lymphocyte counts than survivors (2214 ± 1372 vs. 1807 ± 1162 [106/L], respectively, p = 0.036). In addition, the multivariate logistic regression analysis revealed that the lymphocyte count (OR, 1.0; 95% confidence interval [CI], 1.00–1.06; p = 0.043) was a significant independent risk factor for mortality in these patients. Conclusions. This study revealed that there was no significant difference in the counts of monocytes, neutrophils, and platelets, as well as in MLR, NLR, and PLR, between deceased and surviving patients with polytrauma. However, a significantly higher lymphocyte count may be associated with a worse mortality.

Machine Learning Reveals Ets2 as a Novel Target for Membranous Nephropathy Treatment and Its Role in Immune Infiltration.

BackgroundMembranous nephropathy (MN) is a common pathological phenotype for adult nephrotic syndrome (NS). The occurrence of MN is increasing across China, but diagnostic methods for MN still rely on kidney biopsy and PLA2R and THSD7A detection in plasma and kidney tissue, and there has been no new biomarker for MN discovered since 2014. Immune infiltration status in MN patients suffers from the dearth of associated studies. In the present study, we aimed to find new bio-markers for MN and evaluate the role of immune cells infiltration in MN pathology.MethodsWe downloaded MN expression profile from the Gene Expression Omnibus database and used R-project to screen differentially expressed genes (DEGs) and performed functional correlation analysis. Least absolute shrinkage and selection operator (LASSO) logistic regression and Radom Forest algorithms were used to screen and verify the bio-markers of MN. Finally, CIBERSORT was used to evaluate the infiltration of immune cells in MN tissues.ResultsA total of 463 DEGs were screened from the MN tissue in this study. ETS2 was identified as bio-marker for MN. The CIBERSORT results showed that there were statistical differences in monocytes, plasma cells, regulatory T cells, and memory B cells. In addition, ETS2 was positively related to monocytes, M1 phase macrophages, and neutrophils and negatively correlated to plasma cells, CD4+ T memory cells, M2 macrophages, CD8+ T cells, memory B cells, and resting mast cells.Conclusion(1) Machine learning algorithms reveals Ets2 as a novel target for membranous nephropathy patients. (2) Immune infiltration plays an important part in membranous nephropathy. (3) Ets2 expression is related to immune cells infiltration.

Monocyte/Lymphocyte Ratio and MCHC as Predictors of Collateral Carotid Artery Disease-Preliminary Report.

Background: Carotid artery disease accounts for 30% of ischemic strokes in the general population. Numerous biomarkers have been investigated for predicting either the progression or the severity of the disease. The aim of this retrospective study was to compare hematologic indices among patients referred for surgical interventions due to severe carotid disease. Methods: In total, 135 patients (87 (64.4%) men and 48 (35.6%) women) with a mean age of 70 ± 8 years who underwent surgical carotid intervention were enrolled into the study. Results: A Mann–Whitney test for independent samples revealed significant differences in monocyte to lymphocyte ratio (MLR) and mean corpuscular hemoglobin concentration (MCHC) between patients with one and two (collateral) carotid diseases. The cut-off value for MLR was 0.3 (AUC = 0.654, p = 0.048, 70.0% sensitivity and 74.6% specificity) and for MHCH was 21.6. (AUC = 0.730, p < 0.001, 70.0% sensitivity and 77.2% specificity). A multivariable model of logistic regression revealed two significant parameters for collateral carotid stenosis disease including MLR > 0.3 (OR 6.19 with 95% CI 2.02–19.01, p = 0.001) and MCHC > 21.6 (OR 7.76, 95% CI 2.54–23.72, p < 0.001). Conclusions: MLR above 0.3 and MCHC above 21.6 have predictive values for colleterial carotid stenosis and may be used as easily accessible indicators for atherosclerosis severity.

Functional and transcriptional differences in monocytes from children with obesity compared to children of healthy weight

Abstract Background and aim Cardiometabolic risk accrues across the entire life course and childhood is a key epoch for effective prevention. Obesity in childhood is the most prevalent modifiable risk factor for later cardiovascular disease (CVD). Inflammatory biomarkers and innate immune capacity are increased in adults with obesity, but childhood data are scarce. We aimed to investigate (i) innate immune cell activation in children with and without obesity; and (ii) whether weight loss impacts the innate immune inflammatory phenotype. Methods The innate immune phenotype of Peripheral Blood Mononuclear Cells (PBMCs) from 31 children with obesity (BMI z-score&amp;gt;2.5) and 22 children of healthy weight (−1.5≤BMIz≤1.5, sex, age and pubertal stage matched) was characterized by high dimensional flow cytometry, ex vivo stimulation assays with subsequent 27-plex cytokine measurements, and transcriptome analysis using RNA sequencing (Figure 1). Children with obesity participated to the Royal Children's Hospital Weight Management Service (median 5 years) and at follow-up, PBMCs were obtained again as well as anthropometric data and subclinical cardiovascular phenotypes. Results Flow cytometric analysis showed marked differences in cell composition between children with obesity and children of healthy weight. Specifically, children with obesity have significant changes in monocyte subsets and an increased expression of monocyte activation markers. Upon stimulation, monocytes of children with obesity show an increased cytokine production capacity. Finally, transcriptomic analysis shows significant differences between monocytes from obese children and healthy controls. Effects of weight loss on these immune parameters and correlations with preclinical CVD phenotypes are currently being analysed. Conclusions Monocytes from children with obesity have a pro-inflammatory phenotype compared to children of normal weight. Heightened inflammation may contribute to increased CVD risk later in life and may offer opportunities for early intervention. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): Dutch Scientific Organisation (NWO) - Rubicon grant to S.B. Dutch Heart Foundation - CVON IN CONTROL II to N.P.R. and D.B. Figure 1. Schematical overview of study

Functional and transcriptional differences in monocytes from children with obesity compared to children of healthy weight

Background and Aims: Cardiometabolic risk accrues across the entire life course and childhood is a key epoch for effective prevention. Obesity in childhood is the most prevalent modifiable risk factor for later cardiovascular disease (CVD). Inflammatory biomarkers and capacity are increased in adults with obesity, but childhood data are scarce. We aimed to investigate (i) innate immune cell activation in children with and without obesity ; and (ii) whether weight loss impacts the innate immune inflammatory phenotype.

MO132IMPACT OF MONOCYTE CHEMOATTRACTANTS ON IN-HOSPITAL MORTALITY IN RELATION TO KIDNEY FUNCTION IN PATIENTS WITH COVID-19

Background and AimsPatients with chronic kidney disease (CKD) are at higher risk of severe complications and mortality due to Covid-19 than patients with other known risk factors. The association between CKD and mortality persist in analyses adjusted for covariates known to associate with worse Covid-19 outcomes, suggesting that CKD confers a risk beyond that associated to comorbid conditions. The mechanisms underlying the increased susceptibility to severe Covid-19 in CKD remains unclear but morphologic and functional differences in monocytes have been associated with prolonged hospitalization in other cohorts of patients. The monocyte is capable to contribute to the pathophysiology through different mechanisms. There is however insufficient information on factors that orchestrate different aspects of monocyte function and how these factors relate to outcomes. Increased knowledge into which features of monocyte function that contribute to risks in CKD patients with Covid-19 is important to guide treatment strategies. The aim of the present study was to examine the concentrations of monocyte chemoattractant markers MCP-1 (Monocyte Chemoattractant Protein-1; CCL2) and MIP-1α (Macrophage Inflammatory Protein 1-α; CCL3) in patients with Covid-19 and normal or impaired kidney function and to compare that to CKD patients matched for sex and eGFR, and sex matched healthy subjects. We analyzed the impact of these monocyte chemoattractant markers on in-hospital and 30 days mortality by logistic and multiple regression analyses. We related this to established risk factors for morbidity and mortality in Covid-19 patients, e.g. CRP and IL-6.MethodWe prospectively included 110 patients with Covid‐19 (mean age 59 yr., mean eGFR 75 ml/min/1.73m2) admitted to Danderyd University Hospital, Stockholm, Sweden, during the first pandemic wave in April to May 2020 and 33 sex and eGFR-matched patients (mean age 51 yr., eGFR 52 ml/min/1.73m2) with CKD and 35 sex matched healthy subjects (mean age 47 yr., eGFR 101 ml/min/1.73m2).We used Luminex assays to analyze MCP-1, MIP-1α and IL-6 and routine laboratory tests to determine white blood cell count (WBC) and CRP.ResultsPatients with Covid-19 had significantly lower concentrations of MIP-1α (p<0.001), and higher IL-6 (p<0.001) and CRP (p<0.001) than patients with CKD and healthy subjects (Kruskal-Wallis), there were no differences in MCP-1 between groups. We found significant negative correlations between MCP-1 (p<0.05), MIP-1α (p<0.05) and IL-6 (p<0.05) with eGFR in patients with Covid-19 (Spearman´s rank correlation). Logistic regression analysis (Odds ratio, OR, 95% Confidence Intervals (CI)) and Cox proportional hazard models (Hazard ratio, HR), both adjusted for age, showed significant associations between in-hospital mortality and WBC, CRP, IL-6, MCP-1 and MIP-1α (Table, Figure). Similar findings were observed also for 30 days mortality.MO132 Table.Logistic regression analysis, odds ratio (OR) and Cox proportional hazard ratio (HR) both adjusted for age with 95% CI of in-hospital mortality in patients with Covid-19Logistic regression analysisCox proportional hazard analysisp-valueOR95% CIp-valueHR95% CILowerUpperLowerUpperWhite BC =0.001 1.4381.1671.771 <0.001 1.2511.1281.387CRP =0.01 1.0081.0021.015 0.004 1.0081.0031.013IL-6 =0.05 1.0071.0001.013 0.001 1.0011.0011.002MCP-1 <0.05 1.0011.0001.002 0.001 1.0001.0001.001MIP-1α =0.006 1.0061.0021.011 0.001 1.0051.0021.008ConclusionWe demonstrate that factors related to monocyte recruitment and activation, MCP-1 and MIP-1α, are associated with in-hospital and 30-days mortality in CKD patients with Covid-19. In this patient group general inflammatory markers as IL-6 and CRP are also associated with risk of mortality. These data contribute to an increased understanding of the impact of monocyte activation in Covid-19 and may be of value when treatment strategies are evaluated.

Activation profiles of monocyte-macrophages and HDL function in healthy women in relation to menstrual cycle and in polycystic ovary syndrome patients.

Hormonal status and menopause affect human macrophage function and cardiometabolic risk. In polycystic ovary syndrome (PCOS) patients the cardiometabolic risk increases through mechanisms that are largely unknown. We tested the hypotheses that macrophage activation is influenced by menstrual cycle and that ovarian dysfunction in PCOS patients is associated with altered macrophage inflammatory responses and cholesterol efflux capacity of serum HDL. Blood samples were obtained in the follicular and luteal phases from cycling women (n = 10) and on a single visit from PCOS patients with ovarian dysfunction (n = 11). Monocyte-derived macrophage activation and monocyte subsets were characterized ex vivo using flow cytometry. The capacity of HDL to promote cell cholesterol efflux through the main efflux pathways, namely aqueous diffusion, ATP-binding cassette A1 and G1, was also evaluated. Hormone and metabolic profiles differed as expected in relation to menstrual cycle and ovulatory dysfunction. Overall, macrophage responses to activating stimuli in PCOS patients were blunted compared with cycling women. Macrophages in the follicular phase were endowed with enhanced responsiveness to LPS/interferon-γ compared with the luteal phase and PCOS. These changes were not related to baseline differences in monocytes. HDL cholesterol efflux capacity through multiple pathways was significantly impaired in PCOS patients compared to healthy women, at least in part independent from lower HDL-cholesterol levels. Regular menstrual cycles entailed fluctuations in macrophage activation. Such dynamic pattern was attenuated in PCOS. Along with impaired HDL function, this may contribute to the increased cardiometabolic risk associated with PCOS.

A Hydrogen Sulfide Donor NSAID Influences Inflammatory Cell Responses Following Exertional Heat Stroke

One of the consequences of exposure to exertional heat stroke (EHS) is marked damage to the GI tract. GI damage is believed to promote EHS‐induced activation of the immune system and to be a major contributor to the morbidity and mortality of EHS. We hypothesized that the use of non‐steroidal anti‐inflammatory drugs (NSAIDS), so commonly used in exercising humans, would amplify this damage, make subjects more susceptible to EHS and result in greater GI damage and inflammation. We also hypothesized that the use of next generation NSAIDs containing a hydrogen sulfide (H2S) donor, shown to protect the GI tract, would suppress the damaging effects of unmodified NSAIDs. In this study we focus on the impact of both forms of NSAIDs on immune cell populations in the blood following EHS. To test this, we utilized a preclinical mouse model of EHS in mice, previously developed in our laboratory.METHODSC57BL6/J male mice were implanted with temperature telemetry devices. The mice were exercise trained for 3 weeks prior to undergoing a forced running wheel EHS protocol at 37.5°C. The EHS endpoint was loss of consciousness, occurring at core temperatures of ~42.2°C. During 48 hours prior to the EHS protocol, the animals were given ad libitum access to a chow embedded with analgesic doses Ibuprofen (IBU), Diclofenac (DC), or Diclofenac + H2S (HS) (i.e. ATB‐337, Antibe Corp) or placebo (PL). Three hours post‐EHS, the mice were sacrificed and blood was collected for hematological analyses.RESULTSTotal white blood cell count was lower in the DC and IBU groups compared to the control group (p=0.0079, p=0.0011 respectively). There was no difference detected between the control and the HS group. Lower monocyte (p=0.0017, p=0.0032) and neutrophil counts (p=0.0076, p= 0.0002) were observed in the DC and IBU groups compared to controls. No differences in monocyte or neutrophil counts were observed between the control and HS groups. EHS decreased mean corpuscular volume (MCV), presumably due to hemoconcentration during heat exposure. Treatment with IBU and DC attenuated this decrease in MCV compared to both PL and HS donor.DISCUSSIONThe data are consistent with the hypothesis that, following EHS, there is a greater migration of circulating monocytes and neutrophils from the blood into damaged organs such as the GI tract. With the NSAID‐H2S donor, this effect was attenuated. Previous studies have shown that IBU and DC directly interact with RBC membranes, altering their morphology such that the RBCs resemble stomatocytes. The H2S donor appears to prevent this change. Overall, the H2S donor blocks the hematological associated‐effects of traditional NSAIDs on the responses to EHS. These data suggest that the H2S donors may prevent some negative impacts of heat related injury created by use of traditional NSAIDs during EHS. Author's Views not official US Army or DoD Policy.Support or Funding InformationSupported by the DoD W81XWH‐15‐2‐0038This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differences in monocyte: lymphocyte ratio and Tuberculosis disease progression in genetically distinct populations of macaques

Monocyte:lymphocyte ratio (M:L) has been identified as a risk factor in development of TB disease in children and those undergoing treatment for HIV in co-infected individuals. Retrospective analysis was performed using M:L data collected from TB modelling studies performed in Rhesus macaques of Indian genotype (RM), cynomolgus macaque of Chinese genotype (CCM) and cynomolgus macaque of Mauritian genotype (MCM), which found that the more susceptible populations (RM and MCM) had higher M:L ratios than the least susceptible population (CCM). Following Mycobacterium tuberculosis exposure, significant increases in M:L ratio were observed in susceptible RM and MCM within 12 weeks of TB infection, whereas M:L in CCM remained stable, suggesting that changes in M:L ratio may also act as a biomarker of TB disease progression. The frequency of PPD-specific interferon gamma (IFNγ) secreting cells (SFU) were compared, with the more susceptible macaque populations showing an association between M:L and IFNγ SFU frequency. Investigation of the genes associated with monocyte-derived antigen presenting cells revealed differences between RM and CCM, highlighting differences in their monocyte populations, as well as overall M:L ratio. Differences in M:L ratio between macaque populations could be used to explore immunological mechanisms in susceptible populations that would complement human population studies.

3521 Distinct single cell gene expression in peripheral blood monocytes correlates with treatment response groups to TNF-alpha inhibition in rheumatoid arthritis

OBJECTIVES/SPECIFIC AIMS: The cellular mechanisms that underlie the IFNβ/α ratio that predicts response are not known. Effects of IFN on single immune cells may be masked in whole blood or mixed cell populations. By studying the effect of IFNβ/α activity ratio on individual monocytes, we can determine the functional impact of the IFN ratio and suggest the cellular mechanisms that underlie response/non-response to TNFi therapy in RA. METHODS/STUDY POPULATION: We used single cell analysis to investigate whether monocyte gene expression differs significantly between RA patients according to their pre-TNFi serum IFN-β/α ratio. Single classical (CL) and non-classical (NC) blood-derived monocytes were isolated from 15 seropositive RA subjects prior to biologic therapy. Subjects were grouped by pre-TNFi serum IFN-β/α ratio into two groups, those with a high IFN-β/α ratio (≥1.3, n = 6) and those with a low IFN-β/α ratio (&lt;1.3, n = 9). 87 target genes were analyzed. Genes that varied significantly between the groups by categorical analyses were tested in multivariate logistic regression models. RESULTS/ANTICIPATED RESULTS: Every participant was seropositive for rheumatoid factor and antibodies to cyclic citrullinated peptide. Among the participants in the groups, there were no significant differences in age or DAS scores (P&gt;0.05). The treatments were comparable and none were being treated with biologic therapy. There were striking differences in monocyte gene expression between patients with pre-treatment blood IFNβ/α activity &lt;1.3 and ≥1.3. Expression of (1) key type I IFN pathway genes (JAK1, STAT2, IFIT2, IFIH1, PRDM1); (2) IL12; (3) CD36; and (4) CTLA4 were the strongest differentiators between groups (p&lt;0.0001 for each, corrected for multiple comparisons). DISCUSSION/SIGNIFICANCE OF IMPACT: In this study we were able to measure gene expression in single monocytes from seropositive RA patients prior to biologic treatment. Within-cell co-expression patterns demonstrate biological differences in monocytes of RA patients with an IFNβ/α ≥1.3, the ratio of type I IFNs which predicts non-response to TNFi. The data suggest that there may be differential IFN production and pathway activation in patients who do not respond to TNFi. The increased expression of CD36 in monocytes from RA patients with high IFN β/α activity may be a reflection of increased “foam cells” in the inflamed tissue of patients who do not respond to TNFi. Enrichment of CTLA4 in those with high serum IFNβ/α suggests that CTLA4-Ig may be less likely to be an effective alternative for someone who is not likely to respond to TNFi. Current work includes determining whether the peripheral blood findings reflect altered cellular composition, type I IFN production and signaling in the synovium. Significance: This work will help to develop a more individualized approach to therapy in RA and determine an immunological basis of response/non-response to TNFi.

IRF5 Is a Key Regulator of Macrophage Response to Lipopolysaccharide in Newborns.

Infections are a leading cause of mortality and morbidity in newborns. The high susceptibility of newborns to infection has been associated with a limited capacity to mount protective immune responses. Monocytes and macrophages are involved in the initiation, amplification, and termination of immune responses. Depending on cues received from their environment, monocytes differentiate into M1 or M2 macrophages with proinflammatory or anti-inflammatory and tissue repair properties, respectively. The purpose of this study was to characterize differences in monocyte to macrophage differentiation and polarization between newborns and adults. Monocytes from umbilical cord blood of healthy term newborns and from peripheral blood of adult healthy subjects were exposed to GM-CSF or M-CSF to induce M1 or M2 macrophages. Newborn monocytes differentiated into M1 and M2 macrophages with similar morphology and expression of differentiation/polarization markers as adult monocytes, with the exception of CD163 that was expressed at sevenfold higher levels in newborn compared to adult M1 macrophages. Upon TLR4 stimulation, newborn M1 macrophages produced threefold to sixfold lower levels of TNF than adult macrophages, while production of IL-1-β, IL-6, IL-8, IL-10, and IL-23 was at similar levels as in adults. Nuclear levels of IRF5, a transcription factor involved in M1 polarization, were markedly reduced in newborns, whereas the NF-κB and MAP kinase pathways were not altered. In line with a functional role for IRF5, adenoviral-mediated IRF5 overexpression in newborn M1 macrophages restored lipopolysaccharide-induced TNF production. Altogether, these data highlight a distinct immune response of newborn macrophages and identify IRF5 as a key regulator of macrophage TNF response in newborns.

Proximal and distal stress in myeloid cells in women with breast cancer

Diagnosis with breast cancer is a major life stressor. Stress is associated with changes to immune cell populations, including alterations in myeloid lineage cells. These changes may have implications for cancer outcomes; indeed, the ratio of myeloid to lymphoid lineage cells has been prospectively associated with cancer-related mortality, and specific monocyte subsets may contribute to cancer recurrence. In this study, we examined how proximal (cancer-related stress) and distal (childhood maltreatment) stressors may have implications for myeloid lineage cells (granulocytes, monocytes, and monocyte subtypes) in women recently diagnosed with early-stage breast cancer. We used flow cytometry to quantify cell populations in 110 women following cancer diagnosis and before the onset of any adjuvant chemo- or radiation therapy. In unadjusted bivariate analyses, childhood maltreatment was associated with having fewer granulocytes, F(1,108) = 5.35, p = 0.023, and a lower ratio of granulocytes to lymphocytes, F(1,108) = 4.82, p = 0.030, but no differences in monocytes, ps > 0.24. Higher cancer-related stress was associated with a smaller percentage of classical monocytes (CD14+/CD16−) in the leukocyte population, r = −0.23, p = 0.016. Psychosocial factors may have important implications for myelopoietic processes in the aftermath of cancer diagnosis, and may constitute risk factors for long-term health outcomes in women with breast cancer.

Inosine Triphosphate Pyrophosphohydrolase Expression: Decreased in Leukocytes of HIV-Infected Patients Using Combination Antiretroviral Therapy.

In HIV-infected patients, the enzyme Inosine triphosphate pyrophosphohydrolase (ITPase), involved in purine nucleotide homeostasis, was found to be decreased in erythrocytes. Since purine analogues are pivotal in the HIV treatment, a better understanding of ITPase expression in CD4 lymphocytes may lead to better understanding of nucleotide metabolism and (adverse) effects. Cross-sectional, cohort, observational study. HIV-infected and control patients above 18 years were included. All DNA samples were genotyped for the 2 functional ITPA SNPs; c.94C>A (rs1127354) and g.IVS+21A>C (rs7270101). ITPase expression was determined by flow cytometry in all leukocyte subsets. Fifty-nine HIV-infected patients and 50 controls were included. Leukocyte subtype distribution showed no difference in monocytes and granulocytes, but lymphocytes were higher in HIV-infected patients (P < 0.001). ITPase expression was highest in activated monocytes and lowest in lymphocytes. In HIV-infected patients, the percentage of ITPase positive cells was less in all leukocyte and lymphocyte subsets compared with controls (P < 0.01). In HIV-infected patients, 97.4% of CD4 lymphocytes were ITPase positive versus 99.9% in controls (P = 0.002) and 85.9% versus 99.6% of CD8 lymphocytes (P < 0.0001), respectively. Stratification according to genotype revealed no significant differences in ITPase expression in leukocytes in HIV-infected and control patients. HIV-infection seems to be interfering with the nucleotide metabolism in leukocytes, including CD4 lymphocytes, by decreasing ITPase expression, independently of ITPA genotype. Given that active metabolites of purine-analogue reverse transcriptase inhibitors are potential substrates for ITPase, these results warrant further research towards effectiveness and adverse events of purine analogues and ITPase activity.

IFNL3/4 genotype is associated with altered immune cell populations in peripheral blood in chronic hepatitis C infection.

Single-nucleotide polymorphisms near the interferon lambda 3 (IFNL3) gene predict outcomes to infection and anti-viral treatment in hepatitis C virus (HCV) infection. To identify IFNL3 genotype effects on peripheral blood, we collected phenotype data on 400 patients with genotype 1 chronic hepatitis C (CHC). The IFNL3 responder genotype predicted significantly lower white blood cells (WBCs), as well as lower absolute numbers of monocytes, neutrophils and lymphocytes for both rs8099917 and rs12979860. We sought to define the WBC subsets driving this association using flow cytometry of 67 untreated CHC individuals. Genotype-associated differences were seen in the ratio of CD4CD45RO+ to CD4CD45RO− CD8CD45RO+ to CD8CD45RO−, NK CD56 dim to bright and monocyte numbers and percentages. Whole blood expression levels of IFNL3, IFNLR1 (interferon lambda receptor 1), IFNLR1-mem (a membrane-associated receptor), IFNLR1-sol (a truncated soluble receptor), MxA and T- and NK (natural killer) cell transcription factors TBX21, GATA3, RORC, FOXP3 and EOMES in two subjects were also determined. CHC patients demonstrated endogenous IFN activation with higher levels of MxA, IFNLR1, IFNLR1-mem and IFNLR1-sol, and IFNL3 genotype-associated differences in transcription factors. Taken together, these data provide evidence of an IFNL3 genotype association with differences in monocyte, T- and NK cell levels in the peripheral blood of patients with CHC. This could underpin genotype associations with spontaneous and treatment-induced HCV clearance and hepatic necroinflammation.