Distribution Of Monocyte Subsets Research Articles

A reactive monocyte subset characterized by low expression of CD91 is expanded during sterile and septic inflammation.

This study was undertaken to assess CD91 expression on monocytes and changes in monocyte subset distribution during acute tissue damage and bloodstream infection (BSI). We investigated blood specimens from healthy individuals, trauma and cardiac surgery patients as a model of tissue damage, and patients with BSI, by flow cytometry using a panel of antibodies comprising CD45, HLA-DR, CD14, CD16 and CD91 for the identification of monocyte subsets. While infrequent in healthy subjects, CD91low/neg monocyte levels were markedly high in BSI, trauma and after cardiac surgery. This monocyte subset expanded up to15-fold in both patient cohorts, whereas CD14+CD16+ inflammatory monocytes were multiplied by a factor of 5 only. CD14+CD91low monocytes displayed a significantly lower density of HLA-DR and markedly reduced expression of CD300e, compared to the other subsets. They also expressed high levels of myeloperoxidase and showed robust phagocytic and oxidative burst activity. Expansion of CD91low monocytes is a sensitive marker of acute inflammatory states of infectious and non-infectious etiology.

Contribution of plasma MicroRNA-21, MicroRNA-155 and circulating monocytes plasticity to childhood neuroblastoma development and induction treatment outcome

Neuroblastoma (NB) accounts for 15% of all pediatric cancer fatalities (NB). Biomarkers that facilitate early NB detection are needed because by the time of diagnosis, over half of NBs had spread. MicroRNA-21(miR-21) and miR-155 are involved in cancer biology due to their immune modulation functions. Altered monocyte subset distribution is thought to be involved in a number of solid tumors due to its immunological role. We aimed to investigate the expression levels of miR-21 and miR-155 and their association with circulating monocytes subsets in NB and to evaluate if they correlate to the disease pathogenesis and outcome. Patients and methodsThis case control study involved 79 children classified into 39 newly diagnosed NB children and 40 age and sex matched healthy children. Real-time PCR was used to assess the expression of plasma miR-21 and miR-155. The frequency of circulating monocytes subsets was assessed by flow cytometry. ResultsNB group showed significant up-regulation in expression of miR-21(20.9 folds) and miR-155 (1.8 folds) when compared to the control group (p < 0.001) and (p = 0.02) respectively. Also, frequency of circulating intermediate monocytes revealed significant up regulation in children with NB. In NB patients, there was a positive correlation between miR-21 and frequency of total and intermediate monocytes (r = 0.5 p < 0.001 and r = 0.7, p < 0.001, respectively). We found no discernible differences when we compared study markers between the high risk and intermediate risk groups. In addition, no significant difference was seen in study markers when patients were sub-grouped according to their induction treatment response. ROC curve analysis revealed that miR-21 up-regulation distinguished NB in childhood at an AUC of 0.94 (82% sensitivity and 100% specificity) while miR-155 up-regulation had less capacity to distinguish NB in childhood at an AUC of 0.65 (38% sensitivity and 93% specificity). ConclusionmiR-21 can be utilized as a sensitive biomarker for childhood NB development. In pediatric NB, miR-21 was linked to intermediate monocyte plasticity. Both, miR-21 and miR-155 had no impact on NB outcome.

Evaluation of Hematological Profiles and Monocyte Subpopulations in Water Buffalo Calves after Immunization with Two Different IBR Marker Vaccines and Subsequent Infection with Bubaline alphaherpesvirus-1.

Bubaline alphaherpesvirus-1 (BuAHV-1) and Bovine alphaherpesvirus-1 (BoAHV-1) are respiratory viruses that can cause an infection known as "Infectious Bovine Rhinotracheitis" (IBR) in both water buffalo and bovine species. As the main disease control strategy, vaccination can protect animals from clinical disease through the development of specific humoral and cell-mediated immune responses. In the present study, the time-related circulatory kinetics of hematological profile and bubaline monocyte subsets have been investigated in vaccinated buffalo calves after challenge infections with BuAHV-1. Thirteen buffalo calves were selected and grouped into the VAX-1 group, which received an IBR-live-attenuated gE-/tk-deleted marker vaccine; the VAX-2 group, which received an IBR-inactivated gE-deleted marker vaccine; the CNT group, which remained an unvaccinated control. Fifty-five days after the first vaccination, the animals were infected with 5 × 105.00 TCID50/mL of wild-type BuAHV-1 strain via the intranasal route. Whole blood samples were collected at 0, 2, 4, 7, 10, 15, 30, and 63 days post-challenge (PCDs) for the analysis of hematological profiles and the enumeration of monocyte subsets via flow cytometry. The analysis of leukocyte compositions revealed that neutrophils were the main leukocyte population, with a relative increase during the acute infection. On the other hand, a general decrease in the proportion of lymphocytes was observed early in the post-infection, both for the VAX-1 and VAX-2 groups, while in the CNT group, the decrease was observed later at +30 and +63 PCDs. An overall infection-induced increase in blood total monocytes was observed in all groups. The rise was especially marked in the animals vaccinated with an IBR-live-attenuated gE-/tK-deleted marker vaccine (VAX-1 group). A multicolor flow cytometry panel was used to identify the bubaline monocyte subpopulations (classical = cM; intermediate = intM; and non-classical = ncM) and to investigate their variations during BuAHV-1 infection. Our results showed an early increase in cMs followed by a second wave of intMs. This increase was observed mainly after stimulation with live-attenuated viruses in the VAX-1 group compared with the animals vaccinated with the inactivated vaccine or the non-vaccinated animal group. In summary, the present study characterized, for the first time, the hematological profile and distribution of blood monocyte subsets in vaccinated and non-vaccinated water buffalo in response to experimental infection with BuAHV-1. Although not experimentally proven, our results support the hypothesis of a linear developmental relationship between monocyte subsets.

Short-term toll-like receptor 9 inhibition leads to left ventricular wall thinning after myocardial infarction.

Ischaemia-reperfusion injury (IRI) following myocardial infarction remains a challenging topic in acute cardiac care and consecutively arising heart failure represents a severe long-term consequence. The extent of neutrophil infiltration and neutrophil-mediated cellular damage are thought to be aggravating factors enhancing primary tissue injury. Toll-like receptor 9 was found to be involved in neutrophil activation as well as chemotaxis and may represent a target in modulating IRI, aspects we aimed to illuminate by pharmacological inhibition of the receptor. Forty-nine male adult Sprague-Dawley rats were used. IRI was induced by occlusion of the left coronary artery and subsequent snare removal after 30min. Oligonucleotide (ODN) 2088, a toll-like receptor 9 (TLR9) antagonist, control-ODN, or DNase, were administered at the time of reperfusion and over 24h via a mini-osmotic pump. The hearts were harvested 24h or 4weeks after left coronary artery occlusion and immunohistochemical staining was performed. Echocardiography was done after 1 and 4weeks to determine ventricular function. Inhibition of TLR9 by ODN 2088 led to left ventricular wall thinning (P=0.003) in association with drastically enhanced neutrophil infiltration (P=0.005) and increased markers of tissue damage. Additionally, an up-regulation of the chemotactic receptor CXCR2 (P=0.046) was found after TLR9 inhibition. No such effects were observed in control-ODN or DNase-treated animals. We did not observe changes in monocyte content or subset distribution, hinting towards neutrophils as the primary mediators of the exerted tissue injury. Our data indicate a TLR9-dependent, negative regulation of neutrophil infiltration. Blockage of TLR9 appears to prevent the down-regulation of CXCR2, followed by an uncontrolled migration of neutrophils towards the area of infarction and the exertion of disproportional tissue injury resulting in potential aneurysm formation. In comparison with previous studies conducted in TLR-/- mice, we deliberately chose a transient pharmacological inhibition of TLR9 to highlight effects occurring in the first 24h following IRI.

Monocyte subtype expression patterns in septic patients with diabetes are distinct from patterns observed in obese patients.

Sepsis causes a high rate of mortality and long-term morbidity, associated with an imbalance of innate immunity against infections and inflammation. Obesity and diabetes increase the risk for disease severity. Monocyte dysfunction plays a major role and justify further investigations. To investigate the distribution and inflammatory phenotypes in circulating monocyte subsets in patients manifesting with sepsis including septic shock with and without obesity and diabetes. A total of 235 blood samples were tested from critically ill adult patients registered at the intensive care unit (ICU). The cohorts were divided into non-diabetic groups with or without obesity and diabetic groups with or without obesity, suffering from sepsis or septic shock. We determined frequencies of total monocytes and of monocyte subsets in the circulation and density expression levels of functional markers, including CD14, CD16, HLA-DR, CD33, CD163, CD206, and arginase-1 by flow cytometric analysis. When progressing to septic shock in non-diabetic and diabetic patients, the percentages of total monocytes among the leukocyte population and of CD33+ and CD14+ monocytes among the monocyte population were consistently down-regulated compared to non-sepsis in non-diabetic and diabetic patients, respectively. Non-diabetic sepsis patients further presented with decreased CD33 and up-regulated CD163 expression density, which was absent in diabetic patients. We subsequently addressed obesity-related changes of monocytes in non-diabetic and diabetic septic patients. Obese septic patients with diabetes were unique in displaying increased monocytic CD16 and CD163 expression. However, obese septic patients without diabetes solely presented with lower amounts of non-classical monocytes. Body mass index (BMI) dependent changes were restricted to diabetic septic patients, with a significantly higher diminution of the classical monocyte subset and concomitantly increased CD16 expression densities. Distribution and phenotypes of monocyte subsets were differentially modulated in critically ill patients with and without metabolic disease when progressing to sepsis or septic shock. Only diabetic septic patients displayed decline of classical monocytes and increase of CD16 expression densities. Therefore, diabetes but not obesity appears to promote the inflammatory phenotype of circulating monocytes in critically ill patients.

Optimization of monocyte gating to quantify monocyte subsets for the diagnosis of chronic myelomonocytic leukemia.

The presence of >94% classical monocytes (MO1, CD14++/CD16-) in peripheral blood (PB) has an excellent performance for the diagnosis of chronic myelomonocytic leukemia (CMML). However, the monocyte gating strategy is not well defined. The objective of the study was to compare monocyte gating strategies and propose an optimal one. This is a prospective, single center study assessing monocyte subsets in PB. First, we compared monocyte subsets using 13 monocyte gating strategies in 10 samples. Then we developed our own 10 color tube and tested it on 124 patients (normal white blood cell counts, reactive monocytosis, CMML and a spectrum of other myeloid malignancies). Both conventional and computational (FlowSOM) analyses were used. Comparing different monocyte gating strategies, small but significant differences in %MO1 and percentually large differences in %MO3 (nonclassical monocytes) were found, suggesting that the monocyte gating strategy can impact monocyte subset quantification. Then, we designed a 10-color tube for this purpose (CD45/CD33/CD14/CD16/CD64/CD86/CD300/CD2/CD66c/CD56) and applied it to 124 patients. This tube allowed proper monocyte gating even in highly abnormal PB. Computational analysis found a higher %MO1 and lower %MO3 compared to conventional analysis. However, differences between conventional and computational analysis in both MO1 and MO3 were globally consistent and only minimal differences were observed when comparing the ranking of patients according to %MO1 or %MO3 obtained with the conventional versus the computational approach. The choice of monocyte gating strategy appears relevant for the monocyte subset distribution test. Our 10-color proposal allowed satisfactory monocyte gating even in highly abnormal PB. Computational analysis seems promising to increase reproducibility in monocyte subset quantification.

Assessment of Dysgranulopoiesis By Optical Cytology Compared to Distribution of Monocyte Subsets By Flow Cytometry in Peripheral Blood for Predicting the Diagnosis of CMML

Introduction The diagnosis of chronic myelomonocytic leukemia (CMML) according to the WHO 2017 requires the presence of absolute (≥ 1x109/L) and relative (≥ 10%) monocytosis. Optical cytology represents the basis for its diagnosis and is essential to evaluate dysmyelopoiesis and to identify promonocytes and blasts. Although dysplasia is not a specific finding, its presence along with a persistent monocytosis is highly suggestive of CMML. In this sense, dysgranulopoiesis is an almost constant finding in CMML. The study of monocyte subsets distribution in peripheral blood (PB) by multiparametric flow cytometry (MFC) has proven to be a very useful tool, since the presence of >94% of classical phenotype monocytes (MO1) allows to predict the diagnosis of CMML with a sensitivity and specificity greater than 90% (Selimoglu-Buet et al. Blood 2015). The aim of this study was to evaluate the predictive capacity of morphological evaluation of PB smears for the diagnosis of CMML in a series of patients with absolute and relative monocytosis. Additionally, these results were compared with the analysis of monocyte subsets distribution in PB by MFC. Methods 162 patients with absolute and relative monocytosis were studied. 114 PB smears were available for morphological assessment and 160 MFC analysis of monocyte subsets distribution. The final diagnosis for these patients was: CMML (n=76), Phi negative chronic myeloproliferative neoplasia (MPN) (n=9) and reactive monocytosis (n=77). Genomic information (cytogenetics and NGS) was available in 66 CMML and all of MPN. After anonymizing PB smears, a cytological assessment was performed without additional clinical or analytical data (eg.: age, blood counts, biochemistry). This way we tried to compare the predictive capacity for the diagnosis of CMML of both techniques without the influence of additional data other that the known absolute and relative monocytosis. The cytological evaluation of PB smears consisted fundamentally of a qualitative evaluation of dysgranulopoiesis: nuclear hyposegmentation, chromatin hypercondensation and/or cytoplasmic hypo-degranulation. The sensitivity, specificity, Youden index, positive predictive value (PPV) and negative predictive value (NPV) of both techniques were analyzed. The Youden index allows to evaluate the balance between sensitivity and specificity ([sensitivity + specificity] -100). The capacity for diagnostic prediction of CMML for both techniques was assessed using the C-index. Finally, Cohen's Kappa index was used to evaluate the concordance between both techniques. Results Table 1 shows the results of sensitivity, specificity, Youden index, PPV, NPV and C-index of both techniques. The Kappa index showed a very good degree of agreement (K=0.715; p<0.001) between both techniques. Conclusions The presence of >94% MO1 had a better balance between sensitivity and specificity than the assessment of dysgranulopoiesis in PB. It also had a high sensitivity, demonstrating that it can be very useful screening tool. Despite this, the specificity and PPV of the assessment of dysgranulopoiesis were higher than those of the MFC, demonstrating that the identification of morphological dysplasia would allow the diagnosis of CMML to be predicted with a higher degree of certainty in the initial workup of absolute and relative monocytosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Two week interruption of statin therapy results in an exaggerated inflammatory phenotype in young myocardial infarction patients without standard modifiable risk factors

Abstract Background In patients with myocardial infarction (MI) it is recommended to start high intensity statin therapy, as early as possible, irrespective of lipid levels at presentation. In ST-elevated myocardial infarction (STEMI) patients that do not have standard modifiable risk factors (SMuRFs) there is lower use of statins at discharge compared to patients with at least 1 risk factor, and this is associated with a worse prognosis. Beside lipid lowering capacities, statins are known for their anti-inflammatory effects. Atherosclerosis is characterized as an inflammatory disease in which monocytes play a pivotal role. Circulating monocytes of atherosclerosis patients with SMuRFs have a pro-inflammatory phenotype. In the absence of other modifiable factors in young SMurfless patients, targeting inflammation seems crucial. The effects of (short term) cessation of statin treatment in young SMuRFless MI patients are not known. Purpose The aim of this study was to assess the short term effect of cessation of statin treatment on the inflammatory profile in young SMuRFless MI patients. Methods We included 19 SMuRFless patients (low-density Lipoprotein cholesterol (LDLc) &amp;lt;4.5 mmol/L), no hypertension, no diabetes mellitus, body mass index &amp;lt;30 kg/m2) who suffered a myocardial infarction between 1 and 4 years ago at an age &amp;lt;50 years, and were actively receiving statin therapy. Blood was drawn prior to- and after 14 days of statin cessation. At both time points we assessed circulating high sensitive-C-reactive protein (hs-CRP) levels and leukocyte counts. Using flow cytometry we determined monocyte (subset) phenotype. Monocyte function was studied by measuring cytokine production of peripheral blood mononuclear cells (PBMCs) after isolation and 24-hours incubation with Toll-like receptor (TLR) agonists. Results Short-term interruption of statin treatment resulted in a significant increase in circulating hs-CRP (0.6 vs 0.7 mg/L, p=0.03). Flow cytometry analysis revealed that short term cessation of statin treatment caused a shift in monocyte subset distribution (Figure 1). The CD14++CD16+ “intermediate” monocyte subset became significantly upregulated (p=0.026). Statin interruption also resulted in altered PBMC function after stimulation. No significant differences were seen in PBMCs stimulated with lipopolysaccharide (LPS). Pam3CysK4 (P3C) stimulated cells produces more IL-6 (p=0.004), IL-10 (p=0.010) and IL1Ra (p=0.047) after cessation of statin treatment (Figure 2). Conclusions In young SMuRFless MI patients, short-term interruption of statin treatment results in a heightened inflammatory profile, as demonstrated by elevated circulating CRP, a switch in monocyte phenotype and exaggerated monocyte cytokine production capacity. This difference was already noticeable 14 days after cessation of statin. This emphasizes the importance of statins therapy, irrespective of lipid levels or other SMuRFs. Funding Acknowledgement Type of funding sources: None.

Alterations of blood monocyte subset distribution and surface phenotype are linked to infection severity in COVID-19 inpatients.

Severe coronavirus disease 19 (COVID‐19) manifests with systemic immediate proinflammatory innate immune activation and altered iron turnover. Iron homeostasis, differentiation, and function of myeloid leukocytes are interconnected. Therefore, we characterized the cellularity, surface marker expression, and iron transporter phenotype of neutrophils and monocyte subsets in COVID‐19 patients within 72 h from hospital admission, and analyzed how these parameters relate to infection severity. Between March and November 2020, blood leukocyte samples from hospitalized COVID‐19 patients (n = 48) and healthy individuals (n = 7) were analyzed by flow cytometry enabling comparative analysis of 40 features. Inflammation‐driven neutrophil expansion, depletion of CD16+ nonclassical monocytes, and changes in surface expression of neutrophil and monocyte CD64 and CD86 were associated with COVID‐19 severity. By unsupervised self‐organizing map clustering, four patterns of innate myeloid response were identified and linked to varying levels of systemic inflammation, altered cellular iron trafficking and the severity of disease. These alterations of the myeloid leukocyte compartment during acute COVID‐19 may be hallmarks of inefficient viral control and immune hyperactivation and may help at risk prediction and treatment optimization.

Kinetic Changes of Peripheral Blood Monocyte Subsets and Expression of Co-Stimulatory Molecules during Acute Dengue Virus Infection.

Monocytes, one of the main target cells for dengue virus (DENV) infection, contribute to the resolution of viremia and to pathogenesis. We performed a longitudinal study by a detailed phenotypic comparison of classical (CD14++CD16−, non-classical (CD14+CD16++) and intermediate (CD14++CD16+) monocyte subsets in blood samples from dengue fever (DF) to the severe dengue hemorrhagic fever (DHF) and healthy individuals. Various costimulatory molecules of CD40, CD80, CD86 and inducible costimulatory ligand (ICOSL) expressed on these three monocyte subsets were also analyzed. DENV-infected patients showed an increase in the frequency of intermediate monocytes and a decrease in the classical monocytes when compared to healthy individuals. Although these differences did not correlate with disease severity, changes during the early phase of infection gradually returned to normal in the defervescence phase. Moreover, decreased frequency of classical monocytes was associated with a significant up-regulation of co-stimulatory molecules CD40, CD86 and ICOSL. Kinetics of these co-stimulatory molecule-expressing classical monocytes showed different patterns throughout the sampling times of acute DENV infection. Different distribution of monocyte subsets and their co-stimulatory molecules in the peripheral blood during acute infection might exacerbate immune responses like cytokine storms and ADE, and future studies on intracellular molecular pathways utilized by these monocyte linages are warranted.

Reconstitution of Monocyte Subsets and PD-L1 Expression but Not T Cell PD-1 Expression in Obstructive Sleep Apnea Patients upon PAP Therapy.

Obstructive sleep apnea (OSA) is characterized by nocturnal breathing intermissions resulting in oxidative stress and eventually, a low-grade systemic inflammation. The study aimed to investigate the impact of positive airway pressure (PAP) therapy on the inflammatory milieu as measured by monocyte and T cell phenotypic alterations. Participants were assessed for their OSA severity before PAP therapy and about six months later, including patient-reported outcome and therapy usage by telemetry readout. The distributions of the CD14/CD16-characterized monocyte subsets as well as the CD4/CD8-characterized effector T cell subsets with regard to their PD-1 and PD-L1 expression were analyzed by flow cytometry from blood samples. Data of 25 patients revealed a significant reconstitution of the monocyte subset distribution and a decrease in PD-L1 expression on pan-monocytes and CD8+ T cells without an association to initial AHI and overweight. The PD-1 expression was still increased on T cell subsets, especially on CD4+ TH17/22 cells. We conclude that PAP therapy might have a rapid effect on the monocyte phenotype and overall PD-L1 expression levels. However, T cell immune alterations especially on TH17/22 cells persist longer, indicating an ongoing disturbance of the adaptive immune system.

Increased classical monocyte subsets in South Asians compared to White Caucasians at risk for coronary atherosclerosis

Abstract Background South Asians (SA) have an increased prevalence of coronary artery disease (CAD) and myocardial infarction compared with age- and sex-adjusted White Caucasians (WC). The mechanism for this increased risk is poorly understood. While classical CD14++CD16- monocytes act as independent predictors of cardiovascular disease, differences in the distribution of monocyte subsets between SA and WC have not been established. Purpose We aimed to determine if differences exist in monocyte subsets between SA and WC at risk for CAD. Methods Our cohort consisted of 119 consecutively enrolled patients (59 SA, 60 WC) at intermediate or higher risk for CAD by the INTERHEART score using self-reported history and physical exam. A single blood sample was collected prospectively for the purpose of monocyte analysis. Flow cytometry using dual colour fluorescence (CD14, CD16) within the monocyte gate was used to identify monocyte subsets (classical, intermediate and non-classical) by staff blinded to the individuals' characteristics. Variables were compared using Mann-Whitney U test and Chi-squared test, as appropriate. Eta coefficient was calculated to analyze the relationship between ethnicity and proportion of monocyte subsets. Eta squared values were calculated to assess the impact of ethnicity on monocyte subset proportions. Results The SA group consisted of 64% males with a mean age of 54 (± 9), while the WC group consisted of 55% males with a mean age of 59 (± 7). Both groups had similar body mass index, rates of hypertension, dyslipidemia and family history of premature CAD. Compared to WC, SA had higher prevalence of diabetes (36% vs. 13%, p=0.005) and hemoglobin A1C levels (6.0±1.1% vs. 5.6±0.6%, p&amp;lt;0.001). SA patients had a higher proportion (85.3±10.7% vs. 81.4±11.0%, p=0.009) and total level (449.0±180.4 vs. 388±127.4, p=0.010) of classical CD14++CD16- monocytes compared to WC. There was no difference between the two groups in the proportion of intermediate CD14++CD16+ and non-classical CD14+CD16++ monocytes. There was no association between diabetes and the proportion of monocyte subsets. Ethnicity had a moderate association with the proportion of classical CD14++CD16- monocytes (Eta coefficient = 0.525) with a large effect size (Eta squared = 27.5%). The association of ethnicity with intermediate CD14++CD16+ and non-classical CD14+CD16++ monocytes was either weak or negligible with minimal to no effect size. Conclusion In patients with substantive risk for CAD, SA had a significantly higher proportion and level of classical CD14++CD16- monocytes compared to WC. Our findings provide a novel insight into the potential mechanism of increased CAD susceptibility amongst SA compared to WC. Future studies are needed to determine whether these ethnic differences in the distribution of monocyte subsets can predict susceptibility to developing CAD and suffering atherothrombotic events. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Cardiology Academic Practice Plan grant at the University of British Columbia

A Novel Monocyte Subset as a Unique Signature of Atherosclerotic Plaque Rupture.

The evaluation of monocyte subset distribution among acute coronary syndrome (ACS) patients according to culprit coronary plaque morphology has never been explored. We evaluated whether there were significant differences in frequency of circulating monocyte subsets isolated from ACS patients according to optical coherence tomography (OCT) investigation of plaque erosion and rupture. We enrolled 74 patients with non-ST-elevation ACS (NSTE-ACS), 21 of them underwent OCT investigation of the culprit coronary plaque and local macrophage infiltration (MØI) assessment. As control, we enrolled 30 chronic coronary syndrome (CCS) patients. We assessed the frequency of monocyte subsets in the whole study population, in reliance on their CD14 and CD16 expression (classical, CM: CD14++CD16–; intermediates, IM: CD14++CD16+; non-classical, NCM: CD14+CD16++). Then, we tested the effect of lipopolysaccharide (LPS) (a CD14 ligand) on peripheral blood mononuclear cells (PBMCs) of NSTE-ACS patients, quantifying the inflammatory cytokine levels in cell-culture supernatants. Our data proved that monocyte subsets isolated from NSTE-ACS patients represent a peculiar biological signature of the pathophysiological mechanism lying beneath atherosclerotic plaque with a ruptured fibrous cap (RFC) as compared with plaque erosion. Moreover, the magnitude of LPS-mediated effects on IL-1β, IL-6, and IL-10 cytokine release in cell-culture supernatants appeared to be greater in NSTE-ACS patients with RFC. Finally, we described a fourth monocyte population never explored before in this clinical setting (pre-classical monocytes, PCM: CD14+CD16–) that was prevalent in NSTE-ACS patients as compared with CCS and, especially, in patients with RFC and culprit plaque with MØI.

MONOCYTE SUBSETS HETEROGENEITY AND CORONARY ATHEROSCLEROSIS IN SOUTH ASIANS COMPARED TO WHITE CAUCASIANS

BACKGROUND South Asians (SAs) have an increased prevalence of coronary artery disease (CAD) and myocardial infarction compared with age- and sex-adjusted White Caucasians (WCs). The mechanism for this increased risk is poorly understood. While classical CD14++CD16- monocytes act as independent predictors of cardiovascular disease, differences in the distribution of monocyte subsets between SA and WC have not been established. We aimed to determine if differences exist in monocyte subsets between SAs and WCs at risk for CAD. METHODS AND RESULTS 119 consecutive patients (59 SA, 60 WC) of at least intermediate CAD risk by the INTERHEART score were prospectively enrolled. Patients with history of malignancy, age > 70, GFR < 30 or on dialysis were excluded. A single blood sample was collected for monocyte analysis. Flow cytometry using dual colour fluorescence (CD14, CD16) within the monocyte gate was used to identify monocyte subsets (classical, intermediate, and non-classical) by staff blinded to the individuals’ characteristics. The SA group consisted of 64% males with a mean age of 54 (+/- 9), while the WC group consisted of 55% males with a mean age of 59 (+/- 7). Both groups had similar body mass index, rates of hypertension, dyslipidemia and family history of premature CAD. Compared to WCs, SAs had higher prevalence of diabetes (36% vs. 13%, p = 0.005) and hemoglobin A1C levels (6.0 +/- 1.1% vs. 5.6 +/- 0.6%, p < 0.001). SA patients had a higher proportion (85.3 +/- 10.7% vs. 81.4 +/- 11.0%, p = 0.009) and total level (449.0 +/- 180.4 vs. 388 +/- 127.4, p = 0.010) of classical CD14++CD16- monocytes compared to WCs. There was no difference between the two groups in the proportion of intermediate CD14++CD16+ and non-classical CD14+CD16++ monocytes (see Figure 1). There was no association between diabetes and the proportion of monocyte subsets. Ethnicity had a moderate association with the proportion of classical CD14++CD16- monocytes (Eta coefficient = 0.525) with a large effect size (Eta squared = 27.5%). The association of ethnicity with intermediate CD14++CD16+ and non-classical CD14+CD16++ monocytes was either weak or negligible with minimal to no effect size (see Table 1). CONCLUSION In patients with substantive risk for CAD, SAs had a significantly higher proportion and level of classical CD14++CD16- monocytes compared to WCs. Our findings provide a novel insight into the potential mechanism of increased CAD susceptibility amongst SAs compared to WCs. South Asians (SAs) have an increased prevalence of coronary artery disease (CAD) and myocardial infarction compared with age- and sex-adjusted White Caucasians (WCs). The mechanism for this increased risk is poorly understood. While classical CD14++CD16- monocytes act as independent predictors of cardiovascular disease, differences in the distribution of monocyte subsets between SA and WC have not been established. We aimed to determine if differences exist in monocyte subsets between SAs and WCs at risk for CAD. 119 consecutive patients (59 SA, 60 WC) of at least intermediate CAD risk by the INTERHEART score were prospectively enrolled. Patients with history of malignancy, age > 70, GFR < 30 or on dialysis were excluded. A single blood sample was collected for monocyte analysis. Flow cytometry using dual colour fluorescence (CD14, CD16) within the monocyte gate was used to identify monocyte subsets (classical, intermediate, and non-classical) by staff blinded to the individuals’ characteristics. The SA group consisted of 64% males with a mean age of 54 (+/- 9), while the WC group consisted of 55% males with a mean age of 59 (+/- 7). Both groups had similar body mass index, rates of hypertension, dyslipidemia and family history of premature CAD. Compared to WCs, SAs had higher prevalence of diabetes (36% vs. 13%, p = 0.005) and hemoglobin A1C levels (6.0 +/- 1.1% vs. 5.6 +/- 0.6%, p < 0.001). SA patients had a higher proportion (85.3 +/- 10.7% vs. 81.4 +/- 11.0%, p = 0.009) and total level (449.0 +/- 180.4 vs. 388 +/- 127.4, p = 0.010) of classical CD14++CD16- monocytes compared to WCs. There was no difference between the two groups in the proportion of intermediate CD14++CD16+ and non-classical CD14+CD16++ monocytes (see Figure 1). There was no association between diabetes and the proportion of monocyte subsets. Ethnicity had a moderate association with the proportion of classical CD14++CD16- monocytes (Eta coefficient = 0.525) with a large effect size (Eta squared = 27.5%). The association of ethnicity with intermediate CD14++CD16+ and non-classical CD14+CD16++ monocytes was either weak or negligible with minimal to no effect size (see Table 1). In patients with substantive risk for CAD, SAs had a significantly higher proportion and level of classical CD14++CD16- monocytes compared to WCs. Our findings provide a novel insight into the potential mechanism of increased CAD susceptibility amongst SAs compared to WCs.

Monocyte Dysfunction Detected by the Designed Ankyrin Repeat Protein F7 Predicts Mortality in Patients Receiving Veno-Arterial Extracorporeal Membrane Oxygenation.

Background: Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) is used for critically ill patients requiring hemodynamic support but has been shown to induce an inflammatory response syndrome potentially leading to severe complications and poor outcome. Monocytes are comprised of different subsets and play a central role in the innate immune system. The unique small binding proteins, Designed Ankyrin Repeat Protein “F7” and single chain variable fragment “MAN-1,” specifically detect the activated conformation of the leukocyte integrin Mac-1 enabling the highly sensitive detection of monocyte activation status. The aim of this study was to characterize monocyte function and heterogeneity and their association with outcome in VA-ECMO patients.Methods: VA-ECMO patients were recruited from the ICUs of the University Hospital in Freiburg, Germany. Blood was sampled on day 0 and day 3 after VA-ECMO placement, after VA-ECMO explantation and from healthy controls. Monocyte subset distribution, baseline activation and stimulability were analyzed by flow cytometry using the unique small binding proteins F7 and MAN-1 and the conventional activation markers CD163, CD86, CD69, and CX3CR1. Furthermore, expression of monocyte activation markers in survivors and non-survivors on day 0 was compared. Simple logistic regression was conducted to determine the association of monocyte activation markers with mortality.Results: Twenty two patients on VA-ECMO and 15 healthy controls were recruited. Eleven patients survived until discharge from the ICU. Compared to controls, baseline monocyte activation was significantly increased, whereas stimulability was decreased. The percentage of classical monocytes increased after explantation, while the percentage of intermediate monocytes decreased. Total, classical, and intermediate monocyte counts were significantly elevated compared to controls. On day 0, baseline binding of F7 was significantly lower in non-survivors than survivors. The area under the ROC curve associated with mortality on day 0 was 0.802 (p = 0.02).Conclusions: Distribution of monocyte subsets changes during VA-ECMO and absolute classical and intermediate monocyte counts are significantly elevated compared to controls. Monocytes from VA-ECMO patients showed signs of dysfunction. Monocyte dysfunction, as determined by the unique tool F7, could be valuable for predicting mortality in patients receiving VA-ECMO and may be used as a novel biomarker guiding early clinical decision making in the future.

Monocyte subset distribution and surface expression of HLA-DR and CD14 in patients after cardiopulmonary resuscitation

Systemic inflammation is a major feature of the post-cardiac arrest syndrome. The three monocyte subpopulations are thought to play an important role in this inflammatory state because they are endowed with numerous pattern recognition receptors, such as CD14, that have been associated with ischemia–reperfusion injury. By contrast, an exaggerated antiinflammatory response has also been described following cardiac arrest, which may be mediated by downregulation of antigen presentation receptor HLA-DR. We report the composition of monocyte subpopulations and the expression of CD14 and HLA-DR following cardiac arrest. Blood specimens were collected from 32 patients at three timepoints in the first 48 h after cardiac arrest. Monocyte subset composition was determined by flow cytometry based on the expression of CD14, CD16, and HLA-DR. Monocyte subset composition and the expression of CD14 and HLA-DR were correlated with patient outcomes. The results were compared to 19 patients with coronary artery disease. Cardiac arrest patients showed a significant decline in the percentage of nonclassical monocytes. Monocyte CD14 expression was upregulated after 24 h and correlated with the time to return of spontaneous circulation. Downregulation of HLA-DR expression was observed mainly among classical monocytes and significantly correlated with the dose of norepinephrine used to treat shock. Downregulation of HLA-DR among nonclassical and intermediate monocytes was significantly associated with disease severity. Our data demonstrate the disturbance of monocyte subset composition with a significant decline in nonclassical monocytes at an early stage following cardiac arrest. Our findings suggest the simultaneous presence of hyperinflammation, as evidenced by upregulation of CD14, and monocyte deactivation, characterized by downregulation of HLA-DR. The extent of monocyte deactivation was significantly correlated with disease severity.

Immune Phenotyping of Patients With Acute Vogt-Koyanagi-Harada Syndrome Before and After Glucocorticoids Therapy.

Previous studies have established that disturbed lymphocytes are involved in the pathogenesis of Vogt-Koyanagi-Harada (VKH) syndrome. Accordingly, glucocorticoids (GCs), with their well-recognized immune-suppressive function, have been widely used for treatment of VKH patients with acute relapses. However, the systemic response of diverse immune cells to GC therapy in VKH is poorly characterized. To address this issue, we analyzed immune cell subpopulations and their phenotype, as well as cytokine profiles in peripheral blood from VKH patients (n=25) and health controls (HCs, n=21) by flow cytometry and luminex technique, respectively. For 16 patients underwent GC therapy (methylprednisolone, MP), the aforementioned measurements as well as the transcriptome data from patients before and after one-week’s GC therapy were also compared to interrogate the systemic immune response to GC therapy. Lymphocyte composition in the blood was different in VKH patients and HCs. VKH patients had significantly higher numbers of T cells with more activated, polarized and differentiated phenotype, more unswitched memory B cells and monocytes, as compared to HCs. MP treatment resulted in decreased frequencies of T cells and NK cells, inhibited NK cell activation and T cell differentiation, and more profoundly, a marked shift in the distribution of monocyte subsets. Collectively, our findings suggest that advanced activation and differentiation, as well as dysregulated numbers of peripheral lymphocytes are the major immunological features of VKH, and GC therapy with MP not only inhibits T cell activation directly, but also affects monocyte subsets, which might combinatorically result in the inhibition of the pathogenic immune response.

Leukocytes from Patients with Drug-Sensitive and Multidrug-Resistant Tuberculosis Exhibit Distinctive Profiles of Chemokine Receptor Expression and Migration Capacity.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains as a leading infectious cause of death worldwide. The increasing number of multidrug-resistant TB (MDR-TB) cases contributes to the poor control of the TB epidemic. Currently, little is known about the immunological requirements of protective responses against MDR-TB. This is of major relevance to identify immune markers for treatment monitoring and targets for adjuvant immunotherapies. Here, we hypothesized that MDR-TB patients display unique immunophenotypical features and immune cell migration dynamics compared to drug-sensitive TB (DS-TB). Hence, we prospectively conducted an extensive characterization of the immune profile of MDR-TB patients at different time points before and after pharmacological therapy. For this purpose, we focused on the leukocyte expression of chemokine receptors, distribution of different monocyte and lymphocyte subsets, plasma levels of chemotactic factors, and in vitro migration capacity of immune cells. Our comparative cohort consisted of DS-TB patients and healthy volunteer donors (HD). Our results demonstrate some unique features of leukocyte migration dynamics during MDR-TB. These include increased and prolonged circulation of CD3+ monocytes, CCR4+ monocytes, EM CD4+ T cells, EM/CM CD8+ T cells, and CXCR1+CXCR3+ T cells that is sustained even after the administration of anti-TB drugs. We also observed shared characteristics of both MDR-TB and DS-TB that include CCR2+ monocyte depletion in the blood; high plasma levels of MPC-1, CCL-7, and IP-10; and increased responsiveness of leukocytes to chemotactic signals in vitro. Our study contributes to a better understanding of the MDR-TB pathobiology and uncovers immunological readouts of treatment efficacy.

Helminth species specific expansion and increased TNF-alpha production of non-classical monocytes during active tuberculosis.

Both Mycobacterium tuberculosis infection and helminths may affect innate immune mechanisms such as differential effects on monocytes towards the non-classical and intermediate subsets that favor bacterial persistence. Our aim, was to investigate helminth species specific effects on the frequency and functional activity of monocyte subsets in patients with active tuberculosis and healthy subjects. HIV-negative patients with active pulmonary tuberculosis (PTB) and community controls (CCs) in Gondar, Ethiopia were screened for helminth infection by stool microscopy. Flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) and ex vivo stimulation with purified protein derivative (PPD) and helminth antigens were used to characterize the distribution of monocyte subsets and their function. A total of 74 PTB patients and 57 CCs with and without helminth infection were included. Non-classical monocytes were increased in PTB patients with Ascaris and hookworm infection but not in Schistosoma-infected patients. Ascaris had the strongest effect in increasing the frequency of non-classical monocytes in both PTB patients and CCs, whereas PTB without helminth infection did not affect the frequency of monocyte subsets. There was a helminth specific increase in the frequency of TNF-α producing non-classical monocytes in hookworm infected PTB patients, both with and without PPD-stimulation. Low-to-intermediate TB disease severity associated with increased frequency of non-classical monocytes only for helminth-positive PTB patients, and the frequency of TNF-α producing monocytes were significantly higher in intermediate and non-classical monocytes of helminth positive PTB patients with an intermediate disease score. Helminth infection affected the frequency of monocyte subsets and function both in TB patients and controls which was helminth species dependent in TB patients. The clinical role of this potential immunomodulatory effect needs further study and may affect the response and protection to tuberculosis in areas where helminth infections are endemic.

Systemic Inflammation and the Breakdown of Intestinal Homeostasis Are Key Events in Chronic Spinal Cord Injury Patients.

Our aim was to investigate the subset distribution and function of circulating monocytes, proinflammatory cytokine levels, gut barrier damage, and bacterial translocation in chronic spinal cord injury (SCI) patients. Thus, 56 SCI patients and 28 healthy donors were studied. The levels of circulating CD14+highCD16−, CD14+highCD16+, and CD14+lowCD16+ monocytes, membrane TLR2, TLR4, and TLR9, phagocytic activity, ROS generation, and intracytoplasmic TNF-α, IL-1, IL-6, and IL-10 after lipopolysaccharide (LPS) stimulation were analyzed by polychromatic flow cytometry. Serum TNF-α, IL-1, IL-6 and IL-10 levels were measured by Luminex and LPS-binding protein (LBP), intestinal fatty acid-binding protein (I-FABP) and zonulin by ELISA. SCI patients had normal monocyte counts and subset distribution. CD14+highCD16− and CD14+highCD16+ monocytes exhibited decreased TLR4, normal TLR2 and increased TLR9 expression. CD14+highCD16− monocytes had increased LPS-induced TNF-α but normal IL-1, IL-6, and IL-10 production. Monocytes exhibited defective phagocytosis but normal ROS production. Patients had enhanced serum TNF-α and IL-6 levels, normal IL-1 and IL-10 levels, and increased circulating LBP, I-FABP, and zonulin levels. Chronic SCI patients displayed impaired circulating monocyte function. These patients exhibited a systemic proinflammatory state characterized by enhanced serum TNF-α and IL-6 levels. These patients also had increased bacterial translocation and gut barrier damage.