Percentages Of Non-classical Monocytes Research Articles

Effect of non-surgical periodontal therapy on CD14 + CD16+ monocyte counts in peripheral blood samples: a clinical interventional study

Monocytes and their macrophage progeny are thought to be involved in tissue and alveolar bone destruction in periodontal disease. It has been documented that the proportion of (CD14 + CD16+) non-classical monocytes in the blood are elevated in chronic periodontitis;A total of 20 chronic generalized periodontitis patients who were otherwise healthy, were recruited for this study. At baseline and 3 weeks after non-surgical periodontal treatment, peripheral blood was obtained to assess the levels of C-reactive protein (CRP) and the proportion of monocyte subsets. Monocyte subsets were assessed using flow cytometry;The mean percentage of CD14 + CD16+ non-classical monocytes in the peripheral blood sample at baseline was 13.95 + 2.09, that reduced to 8.94 + 1.23 3 weeks after non-surgical treatment. A distinct significant reduction in the percentage of non-classical monocytes and a concomitant increase in classical monocytes were observed following periodontal treatment compared to baseline. There was a significant reduction in the all the periodontal parameters and CRP levels 3 weeks post non-surgical periodontal treatment. A positive correlation between CRP and percentage of non-classical monocytes was also observed; Periodontal treatment potentially modulates the host response effectively.

Pre-vaccination immunotypes reveal weak and robust antibody responders to influenza vaccination.

Effective vaccine-induced immune responses are particularly essential in older adults who face an increased risk of immunosenescence. However, the complexity and variability of the human immune system make predicting vaccine responsiveness challenging. To address this knowledge gap, our study aimed to characterize immune profiles that are predictive of vaccine responsiveness using "immunotypes" as an innovative approach. We analyzed an extensive set of innate and adaptive immune cell subsets in the whole blood of 307 individuals (aged 25-92) pre- and post-influenza vaccination which we associated with day 28 hemagglutination inhibition (HI) antibody titers. Building on our previous work that stratified individuals into nine immunotypes based on immune cell subsets, we identified two pre-vaccination immunotypes associated with weak and one showing robust day 28 antibody response. Notably, the weak responders demonstrated HLA-DR+ T-cell signatures, while the robust responders displayed a high naïve-to-memory T-cell ratio and percentage of nonclassical monocytes. These specific signatures deepen our understanding of the relationship between the baseline of the immune system and its functional potential. This approach could enhance our ability to identify individuals at risk of immunosenescence. Our findings highlight the potential of pre-vaccination immunotypes as an innovative tool for informing personalized vaccination strategies and improving health outcomes, particularly for aging populations.

Impact of Psychological Distress on Immune Phenotype in CLL/SLL Patients Managed By Active Observation

Introduction: Asymptomatic patients with a new diagnosis of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) are usually managed by active observation; however, many patients report elevated levels of distress and worry during the ‘watchful waiting’ phase. Psychological distress may have clinical implications given that natural killer [NK] and T cells are not only sensitive to distress, but also critical in controlling disease progression. Thus, the purpose of this study was to examine associations of psychological functioning and quality of life (QOL) with biomarker expression and functional activity of immune cells from peripheral blood of CLL/SLL patients being managed with surveillance. Methods: We conducted a multisite, prospective study in patients with indolent B-cell neoplasms who were managed with surveillance. Participants completed measures of perceived stress (Perceived Stress Scale), anxiety and depressive symptoms (Hospital Anxiety and Depression Scale) and quality of life (FACT-G) at study entry and then every 6 months for up to 2 years or disease progression (whichever occurred first). Blood samples were collected at study entry and every 6 months for immune phenotyping using multiparametric flow cytometry (up to 13-color). Biomarkers of activation, viability, proliferation, exhaustion, and receptor expression were assessed on subsets of T, NK, and myeloid cells, and mean fluorescence intensity or percentage of cells staining were determined. Results: Between April 2016 and November 2020, 233 patients were enrolled at two academic institutions in Philadelphia, including 111 with a diagnosis of CLL/SLL. Of these, 76 patients also had viable samples for immune phenotyping. Participants were predominantly male (60.5%) with a mean age of 63.7 years (SD=9.1 years). Approximately 5% self-reported their race as Black or African American and 4% were of Hispanic ethnicity. At enrollment, the median time since diagnosis was 10.6 months (median follow-up time = 23.6 months). Multivariable regression analyses revealed that increased depressive symptoms were associated with a significant shift from lower percentages of non-classical monocytes (NCM) to higher percentages of classical monocytes (CM; see Figure 1). Similar significant population shifts from reduced NCM toward increased CM were also observed with increased levels of stress and anxiety. These results suggest that patients reporting increased stress, anxiety, and depressive symptoms have fewer of the “patrolling” NCM and increased “sensing” CM in peripheral blood. We also assessed expression of numerous innate immune receptors, including DNAM-1, which can stimulate responses by NK and T cells toward tumors. We observed lower expression of DNAM-1 on CD4 + helper T cells and CD8 + cytotoxic T cells in patients with increased depressive symptoms (p< 0.05). Similarly, patients reporting lower QOL also expressed lower levels of DNAM-1 on CD4 + and CD8 + T cells, as well as cytotoxic CD56 dim NK cells (p<0.05). As DNAM-1 is important for antitumor responses, our data suggest that patients with higher depressive symptoms have T and NK cells with reduced capacity to respond to CLL/SLL tumor cells. NKp30, another activating receptor on NK and CD8+ T cells promoting cytotoxicity, was also expressed at lower levels on CD56 dim NK cells and T cells in the peripheral blood of patients reporting depressive symptoms. Patients who reported higher levels of depressive symptoms had lower levels of expression of the activation marker CD69 on CD4+ and CD8+ T cells. Finally, longitudinal analyses revealed that higher levels of depressive symptoms predicted lower levels of PD-L1 on CD56 dim NK cells over time ( p = 0.04). Conclusion: In CLL/SLL patients, psychological functioning is associated with differential patterns of monocyte distribution. Greater depressive symptoms and lower quality of life predicted lower expression of innate immune receptors, such as DNAM-1 on CD4 + helper T cells and CD8 + cytotoxic T cells. Future directions will investigate whether these measures are associated with disease progression or the occurrence of second primary cancers, given the importance of DNAM-1 and NKp30 in immune surveillance for cancers and reactivation of latent viruses. These findings may have direct translational relevance and enable the early identification of patients at-risk for poor outcomes.

Monocyte subsets associated with the efficacy of anti‑PD‑1 antibody monotherapy.

Immune checkpoint inhibitors (ICIs) are among the most notable advances in cancer immunotherapy; however, reliable biomarkers for the efficacy of ICIs are yet to be reported. Programmed death (PD)-ligand 1 (L1)-expressing CD14+ monocytes are associated with shorter overall survival (OS) time in patients with cancer treated with anti-PD-1 antibodies. The present study focused on the classification of monocytes into three subsets: Classical, intermediate and non-classical. A total of 44 patients with different types of cancer treated with anti-PD-1 monotherapy (pembrolizumab or nivolumab) were enrolled in the present study. The percentage of each monocyte subset was investigated, and the percentage of cells expressing PD-L1 or PD-1 within each of the three subsets was further analyzed. Higher pretreatment classical monocyte percentages were correlated with shorter OS (r=-0.32; P=0.032), whereas higher non-classical monocyte percentages were correlated with a favorable OS (r=0.39; P=0.0083). PD-L1-expressing classical monocytes accounted for a higher percentage of the total monocytes than non-classical monocytes with PD-L1 expression. In patients with non-small cell lung cancer (NSCLC), a higher percentage of PD-L1-expressing classical monocytes was correlated with shorter OS (r=-0.60; P=0.012), which is similar to the observation for the whole patient cohort. Comparatively, higher percentages of non-classical monocytes expressing PD-L1 were significantly associated with better OS, especially in patients with NSCLC (r=0.60; P=0.010). Moreover, a higher percentage of non-classical monocytes contributed to prolonged progression-free survival in patients with NSCLC (r=0.50; P=0.042), with similar results for PD-L1-expressing non-classical monocytes. The results suggested that the percentage of monocyte subsets in patients with cancer before anti-PD-1 monotherapy may predict the treatment efficacy and prognosis. Furthermore, more classical monocytes and fewer non-classical monocytes, especially those expressing PD-L1, are involved in shortening OS time, which may indicate the poor efficiency of anti-PD-1 treatment approaches.

Non-classical monocytes frequency and serum vitamin D3 levels are linked to diabetic foot ulcer associated with peripheral artery disease.

Peripheral artery disease (PAD) serves as a risk factor for diabetic foot ulcers (DFUs). PAD pathology involves atherosclerosis and impaired immunity. Non-classical monocytes are believed to have an anti-inflammatory role. 1,25-Dihydroxy vitamin D (vitamin D3 ) is claimed to have immune-modulating and lipid-regulating roles. Vitamin D receptor is expressed on monocytes. We aimed to investigate if circulating non-classical monocytes and vitamin D3 were implicated in DFUs associated with PAD. There were two groups of DFU patients: group 1 (n = 40) included patients with first-degree DFUs not associated with PAD, and group 2 (n = 50) included patients with DFU with PAD. The monocyte phenotypes were detected using flow cytometry. Vitamin D3 was assessed by enzyme-linked immunosorbent assay. DFU patients with PAD showed a significant reduction in the frequency of non-classical monocytes and vitamin D3 levels, when compared with DFU patients without PAD. The percentage of non-classical monocytes positively correlated with vitamin D3 level (r = 0.4, P < 0.01) and high-density lipoprotein (r = 0.5, P < 0.001), whereas it was negatively correlated with cholesterol (r = -0.5, P < 0.001). Vitamin D3 was negatively correlated with triglyceride/high-density lipoprotein (r = -0.4, P < 0.01). Regression analysis showed that a high vitamin D3 serum level was a protective factor against PAD occurrence. Non-classical monocytes frequency and vitamin D3 levels were significantly reduced in DFU patients with PAD. Non-classical monocytes frequency was associated with vitamin D3 in DFUs patients, and both parameters were linked to lipid profile. Vitamin D3 upregulation was a risk-reducing factor for PAD occurrence.

Monocyte subsets and monocyte-related chemokines in Takayasu arteritis

The pathogenesis of Takayasu arteritis (TAK) is poorly understood and no previous studies have analyzed monocytes in TAK. This study evaluated monocyte subsets and monocyte-related chemokines in the peripheral blood of TAK patients and healthy controls (HC). Monocyte subsets were identified as classical (CD14+CD16−), intermediate (CD14+CD16dim), and non-classical (CD14dimCD16high) in the peripheral blood. The chemokines CCL (C–C chemokine ligand)2, CCL3, CCL4, CCL5, CCL7, CXCL (C-X-C motif ligand)10, and CX3CL (C-X3-C motif ligand)1 were measured in the sera. Thirty-two TAK patients and 30 HC were evaluated. Intermediate monocytes were higher in TAK than HC [25.0 cells ×106/L (16.7–52.0) vs. 17.2 cells ×106/L (9.2–25.3); p = 0.014]. Active disease was associated with monocytosis (p = 0.004), increased classical (p = 0.003), and intermediate (p < 0.001) subsets than HC. Prednisone reduced the percentage of non-classical monocytes (p = 0.011). TAK patients had lower CCL3 (p = 0.033) and CCL4 (p = 0.023) levels than HC, whereas CCL22 levels were higher in active TAK compared to the remission state (p = 0.008). Glucocorticoids were associated with lower CXCL10 levels (p = 0.012). In TAK, CCL4 correlated with total (Rho = 0.489; p = 0.005), classical and intermediate monocytes (Rho = 0.448; p = 0.010 and Rho = 0.412; p = 0.019). In conclusion, TAK is associated with altered counts of monocyte subsets in the peripheral blood compared to HC and CCL22 is the chemokine with the strongest association with active disease in TAK.

Inflammation parameters, monocyte subgroups and toll-like receptor expression before and after dialysis in patients with chronic kidney disease

Abstract Objectives The data of the monocyte subgroups and expressed toll like receptors (TLR) in the innate immune system response, which develop against chronic inflammation in patients with predialysis chronic kidney disease (CKD) and in patients who undergo dialysis treatment, are limited. We aimed to investigate the effect of the dialysis procedure on the current chronic inflammatory condition and which role of monocyte subgroups ratios, the expressions of TLR2/4 and serum Tumor necrosis factor alpha (TNF-α) levels involved in the innate immune response process. Methods We investigated monocyte subgroups, TLR2/TLR4 expressions and serum TNF-α levels in 30 predialysis CKD patients, 90 CKD patients undergoing dialysis and 30 healthy control subjects. Monocyte subgroup percentages and TLR2/TLR4 expressions were determined using the flow cytometry, serum TNF-α levels were investigated using the enzyme-linked immunosorbent assay (ELISA). Results In the dialysis patients, the percentages of classical (p=0.0001) and non-classical (p=0.078) monocytes were found to be higher when compared with the predialysis CKD patients. The percentages of TLR4 expression on non-classical monocytes was higher in dialysis and predialysis patients compared with the healthy controls (p&lt;0.0001, p=0.796). Serum TNF-α level was significantly higher in dialysis and predialysis patients compared with the healthy controls (p=0.013, p=0.022) and a positive correlation between the classical monocyte subgroup and TNF-α was observed (r=0.285, p=0.006). Conclusions Increased percentages of non-classical monocytes, TLR4 expressions and serum TNF-α levels observed in the predialysis CKD patients and dialysis patients might be related to inflammation.

The Role of Relapse Specific over-Expression of S100A8/A9 B Leukemic Cells in Promoting Chemoresistance

Background: We previously discovered a novel superenhancer (SE) adjacent to S100A8/9 at relapse in a majority of patient samples indicating a role in clonal evolution. In agreement with these findings, a metanalysis of gene expression studies demonstrated that S100A8 is the top overexpressed transcript at relapse in childhood ALL. S100A8 is a member of the S100 calcium-binding protein family and some studies suggest a role in mediating intrinsic drug resistance. However, secreted S100A8/9 proteins play a major role in inflammation and it is possible that their role in clonal evolution occurs by altering the tumor microenvironment (TME). Design/Method Isogenic B-ALL cell lines (697 and RS4;11) were created to overexpress an empty vector control (EV), S100A8 or S100A9 alone, or S100A8+S100A9 together. Protein expression and secretion were measured using an ELISA for human S100A8/9 (Abcam). Proliferation over a 7-day period was measured using the Beckman Coulter Vi-CELL counter. Chemoresistance was measured using Promega's CellTiter-Glo® assay following exposure to chemotherapeutic agents used in the treatment of ALL including mercaptopurine, prednisolone, methotrexate, vincristine, cytarabine, and etoposide for 72-120 hours. Colony forming potential was assessed after 14 days in MethoCult (STEMCELL Technologies). To determine that expression originated from leukemia cells one sample pair (SGZ) known to overexpress S100A8 at relapse using bulk RNA expression, was compared to 3 paired patient samples without overexpression using single cell RNAseq (scRNAseq). CD45+CD19+ (blasts) and CD45+CD19- (microenvironment) were sorted and then pooled to increase the fraction of cells from the microenvironment before scRNAseq using 10X Genomics. CellPhoneDB analysis was used to predict ligand-receptor interactions between different cell types. Results Western blot demonstrated stable expression of S100A8 and S100A9 proteins when co-expressed but protein was not detected when either was alone. Overexpression of S100A8+S100A9 was confirmed via ELISA using both cell lysate and condition media suggesting secretion. No differences in proliferation, colony formation, or chemoresistance were observed between cells overexpressing S100A8/9 compared to the empty vector control line, suggesting no cell intrinsic effect of S100A8/9 overexpression ssRNAseq analysis validated upregulation of S100A8/9 specifically in leukemic blasts (cluster 12, L2FC of 5.6 and 5.3 (p<.0001) for S100A8 and S100A9, respectively). In contrast, two myeloid monocytic-like populations showed minimal to no change in expression level from diagnosis to relapse (L2FC 1.13 and 0.18 for S100A8 and S100A9, respectively (p<.01). Pathway analysis of differentially regulated genes in SGZ (L2FC cutoff of 1 and p<.001, Enrichr, Panther 2016) revealed known relapse pathways, including Ras, p53, and p38 MAPK, but also those known to impact the TME including Inflammation mediated by chemokine and cytokine signaling, Toll receptor signaling, and TFG-beta signaling. Interestingly, we also observed an increase in, and higher percentage of non-classical monocytes within the non-leukemic cell population at relapse in SGZ (>20%) compared to minimal (<5%) in the other three patients. In SGZ, we found 455 and 573 significantly upregulated (p<.05, abs L2FC>.32) and 686 and 362 significantly downregulated genes (p <.05, absolute L2FC <.32) in classical monocytes and non-classical monocytes, respectively. In contrast, we found less than 20 differential genes within these clusters in any of the other patient pairs, suggesting a profound impact of S100A8/A9 expression on the transcriptome of these myeloid populations. Using CellPhone DB we studied the cell-cell communication mediated by ligand-receptor unique to SGZ by comparing the blast and the two monocytic populations. A number of unique interactions were seen at relapse, including CCL7-CCL3/CCR1, CCL3/IDE and CCL5/CCR5 known to induce the migration of leukocytes and myeloid cells. Other interactions involved in tumorigenesis and inflammation upregulated at relapse were seen: NRP2/VEGFA, LGALS9/SLC1A5. Conclusion Our results indicate that S100A8/A9 overexpression in B-ALL cells at relapse has no cell intrinsic impact but triggers remodeling of the TME by inducing an increase in myeloid subpopulations and unique cell to cell interactions.

Immunoparalysis in critically ill children.

Immunoparalysis is associated with poorer outcomes in the paediatric intensive care unit (PICU) setting. We aimed to determine the group of patients with higher chances of immunoparalysis and correlate this status with increased risks of nosocomial infection and adverse clinical parameters. We conducted an exploratory study with prospective data collection in a university-affiliated tertiary medical, surgical, and cardiac PICU. Fifteen patients with multiple organ dysfunction syndrome were included over a period of 6 months. Monocyte's human leucocyte antigen (HLA)-DR expression and tumour necrosis factor (TNF)-α and interleukin (IL)-6 production were measured by flow-cytometry at three time points (T1=1-2 days; T2=3-5 days; T3=6-8 days). Using the paediatric logistic organ dysfunction-2 score to assess initial disease severity, we established the optimal cut-off values of the evaluated parameters to identify the subset of patients with a higher probability of immunoparalysis. A comparative analysis was performed between them. Sixty per cent were males; the median age was 4.1 years. Considering the presence of two criteria in T1 (classical monocytes mean fluorescence intensity [MFI] for HLA-DR ≤ 1758.5, area under the curve (AUC)=0.775; and frequency of monocytes producing IL-6 ≤ 68.5%, AUC=0.905) or in T3 (classical monocytes MFI of HLA-DR ≤ 2587.5, AUC=0.675; and frequency of monocytes producing TNF-α ≤ 93.5%, AUC=0.833), a variable to define immunoparalysis was obtained (100% sensitivity, 81.5% specificity). Forty per cent of patients were assigned to the immunoparalysis group. In this: a higher frequency of nosocomial infection (p= 0.011), vasoactive inotropic score (p= 0.014) and length of hospital stay (p= 0.036) was observed. In the subgroup with the diagnosis of sepsis/septic shock (n= 5), patients showed higher percentages of non-classical monocytes (p= 0.004). No mortality was recorded. A reduction in classical monocytes HLA-DR expression with lower frequencies of monocytes producing TNF-α and IL-6 during the first week of critical illness, appears to be a good marker of immunoparalysis; these findings relate to an increased risk of nosocomial infection and deleterious outcomes. The increased frequency of non-classical monocytes in patients with sepsis/septic shock is suggestive of a better prognosis.

Alteration of monocyte subsets and their functions in thalassemia patients

Infection is one of the leading causes of mortality in thalassemia patients. This study aimed to examine qualitative and quantitative changes in monocytes in thalassemia patients. Monocytes were isolated from peripheral blood mononuclear cells and separated into subpopulations by flow cytometry. Cytokine levels were measured using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and sandwich enzyme-linked immunosorbent assay (ELISA). The primary endpoint was monocyte-derived TNF-α expression. A total of 78 patients and 26 controls were included. The mean log (TNF-α fold-change) by qRT-PCR was significantly lower in all thalassemia groups, at 1.27 in controls, versus 0.97 (p = 0.0014) in non-transfusion-dependent thalassemia (NTDT), 0.96 (p = 0.0004) in non-splenectomized transfusion-dependent thalassemia (TDT-NS), and 0.87 (p < 0.0001) in splenectomized transfusion-dependent thalassemia (TDT-S). Similarly, the mean 2-h TNF-α level measured by sandwich ELISA assay was significantly lower in all thalassemia groups, at 98.16 pg/mL in controls, versus 56.45 pg/mL (p = 0.0093) in NTDT, 39.05 pg/mL (p = 0.0001) in TDT-NS and 32.37 pg/mL (p < 0.0001) in TDT-S. Likewise, TDT patients had a significantly decreased percentage of non-classical monocytes, by approximately half compared to controls. Our results show that thalassemia major patients have clearly impaired monocyte counts and function.

Modulation of Leukocyte Subsets Mobilization in Response to Exercise by Water Immersion Recovery.

Purpose: To investigate the effect of different water immersion temperatures on the kinetics of blood markers of skeletal muscle damage and the main leukocyte subpopulations. Methods: Eleven recreationally trained young men participated in four experimental sessions consisting of unilateral eccentric knee flexion and 90 min of treadmill running at 70% of peak oxygen uptake, followed by 15 min of water immersion recovery at 15, 28 or 38°C. In the control condition participants remained seated at room temperature. Four hours after exercise recovery, participants completed a performance test. Blood samples were obtained before and immediately after exercise, after immersion, immediately before and after the performance test and 24 h after exercise. The number of leukocyte populations and the percentage of lymphocyte and monocytes subsets, as well as the serum activity of creatine kinase and aspartate aminotransferase were determined. Results: Leukocytosis and increase in blood markers of skeletal muscle damage were observed after the exercise. Magnitude effect analysis indicated that post-exercise hot-water immersion likely reduced the exercise-induced lymphocytosis and monocytosis. Despite reduced monocyte count, recovery by 38°C immersion, as well as 28°C, likely increased the percentage of non-classical monocytes in the blood. The percentage of CD25+ cells in the CD4 T cell subpopulation was possibly lower after immersion in water at 28 and 15°C. No effect of recovery by water immersion was observed for serum levels of creatine kinase and aspartate aminotransferase. Conclusions: Recovery by hot-water immersion likely attenuated the leukocytosis and increased the mobilization of non-classical monocytes induced by a single session of exercise combining resistance and endurance exercises, despite no effect of water immersion on markers of skeletal muscle damage. The monocyte response mediated by hot water immersion may lead to the improvement of the inflammatory response evoked by exercise in the skeletal muscle.

Leukocyte inflammatory phenotype and function in migraine patients compared with matched non-migraine volunteers: a pilot study

BackgroundMigraine is a neurological condition characterized by chronic inflammation. However, not much is known about the potential role of peripheral blood immune cells in the pathophysiology of migraine.MethodsWe investigated the status of peripheral blood immune cells of 15 adults with frequent episodic or chronic migraine recruited chronologically from a randomized clinical trial (RCT) on Nutrition for Migraine (NCCIH 5R01AT007813-05) and 15 non-migraine, healthy volunteers (control) matched by age, gender, and Body Mass Index (BMI).Continuous variables were presented as means ± standard deviationas well as medians, and comparisons between patients and healthy volunteers were performed with non-parametric Wilcoxon signed rank tests. Statistical analysis was performed using Stata (StataCorp. 2019. Stata Statistical Software). Fluorescence-Activated Cell Sorting (FACS) data were processed using FlowJo software (Ashland, OR: Becton, Dickenson and Company; 2019).ResultsWe observed that migraineurs had a significantly lower percentage of non-classical monocytes (CD14+CD16++) in blood circulation, compared to the control group. In addition, Migraineurs also showed a significantly lower percentage of blood CD3+CD4+ helper T cells and CD4+CD25+ regulatory T cells, compared to controls. Differences in leukocyte surface markers between chronic migraine patients and their matched controls were more prominent than those between episodic migraine patients and their matched controls.ConclusionsOur results suggest that migraine is associated with dysregulated peripheral immune homeostasis and that inflammation and autoimmunity may play a role in its pathophysiology.

Increased Abundances of CD16+ Non-Classical Monocytes Accompany with Elevated Monocytic PD-L1 and CD4+ T Cell Disturbances in Oropharyngeal Cancer.

Background: Patients with human papilloma virus (HPV)-related oropharyngeal cancer have a better prognosis than nonvirally associated patients, most likely because of better immune responses. Increased infiltration of T lymphocytes into the oropharyngeal tumor tissue has been observed, but the dynamics of circulating lymphocytes and monocytes are not fully understood. The aim of this study was to understand the population dynamics of circulating monocyte subsets in oropharyngeal cancer (OPC) patients with regard to the clinicopathological parameters and accompanying immunological consequences in view of the CD4/CD8 T cell subset composition, and the expression of checkpoint pathway proteins programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1). Materials and Methods: The abundance of circulating monocyte subsets and peripheral blood CD4/CD8 T cells of oropharyngeal cancer patients and their PD-L1 and PD-1 expression levels were analyzed by flow cytometry. Results: The studied oropharyngeal cancer patients revealed heterogeneous individual redistributions of CD14++CD16− (classical), CD14++CD16+ (intermediate), and CD14dim+CD16+ (nonclassical) monocyte subsets compared with healthy donors. These differences in monocyte subset alterations were independent in patients with TNM or HPV status but entailed further immunological consequences. Increased percentages of nonclassical monocytes significantly correlated with increased levels of monocytic PD-L1 expression. We observed significantly decreased levels of CD4+ effector T cells, which were accompanied by increased CD4+ effector memory T cells in OPC patients compared with healthy donors, each having a stronger effect in patients with decreased levels of classical monocytes. Conclusion: We conclude that oropharyngeal cancer, as a malignancy from a lymphoid-tissue-rich anatomical region, has a strong systemic impact on the differentiation and regulation of circulating innate and adaptive immune cells. Further comprehensive investigations are required for the possible future usability of the described immunological alterations as bioliquid parameters for prognosis or therapy response prediction.

Reduced Percentage of CD14dimCD16+SLAN+ Monocytes Producing TNF and IL-12 as an Immunological Sign of CLL Progression.

Monocytes are one of the least studied immune cells with a potentially important role in the pathogenesis of chronic lymphocytic leukemia (CLL). Nevertheless, data regarding the role of subpopulations of monocytes in the CLL microenvironment are still limited. For the very first time, this study presents an assessment of monocyte subsets divided according to SLAN and CD16 expression in CLL patients. The study involved 70 freshly diagnosed CLL patients and 35 healthy donors. Using flow cytometry, monocyte subpopulations were assessed among PBMCs. CD14+ monocytes can be divided into: “classical” (CD14+CD16−SLAN−), “intermediate” (CD14+CD16+SLAN−) and “non-classical” (CD14dimCD16+SLAN+). In our study, we noted an increased percentage of non-classical monocytes with intracellular expression of TNF and IL-12. On the other hand, among the intermediate monocytes, a significantly higher percentage of cells synthesizing anti-inflammatory IL-10 was detected. The percentage of CD14dimCD16+SLAN+ monocytes producing TNF and IL-12 decreased with the stage of CLL and inversely correlated with the expression of the prognostic factors ZAP-70 and CD38. Moreover, the percentage of CD14dimCD16+SLAN+ monocytes producing TNF and IL-12 was lower in CLL patients requiring treatment. This may indicate the beneficial effect of non-classical monocytes on the anti-tumor response.

Fenofibrate Increases the Population of Non-Classical Monocytes in Asymptomatic Chagas Disease Patients and Modulates Inflammatory Cytokines in PBMC.

Chronic Chagas disease cardiomyopathy (CCC) is the most important clinical manifestation of infection with Trypanosma cruzi (T. cruzi) due to its frequency and effects on morbidity and mortality. Peripheral blood mononuclear cells (PBMC) infiltrate the tissue and differentiate into inflammatory macrophages. Advances in pathophysiology show that myeloid cell subpopulations contribute to cardiac homeostasis, emerging as possible therapeutic targets. We previously demonstrated that fenofibrate, PPARα agonist, controls inflammation, prevents fibrosis and improves cardiac function in a murine infection model. In this work we investigated the spontaneous release of inflammatory cytokines and chemokines, changes in the frequencies of monocyte subsets, and fenofibrate effects on PBMC of seropositive patients with different clinical stages of Chagas disease. The results show that PBMC from Chagas disease patients display higher levels of IL-12, TGF-β, IL-6, MCP1, and CCR2 than cells from uninfected individuals (HI), irrespectively of the clinical stage, asymptomatic (Asy) or with Chagas heart disease (CHD). Fenofibrate reduces the levels of pro-inflammatory mediators and CCR2 in both Asy and CHD patients. We found that CHD patients display a significantly higher percentage of classical monocytes in comparison with Asy patients and HI. Besides, Asy patients have a significantly higher percentage of non-classical monocytes than CHD patients or HI. However, no difference in the intermediate monocyte subpopulation was found between groups. Moreover, monocytes from Asy or CHD patients exhibit different responses upon stimulation in vitro with T. cruzi lysates and fenofibrate treatment. Stimulation with T. cruzi significantly increases the percentage of classical monocytes in the Asy group whereas the percentage of intermediate monocytes decreases. Besides, there are no changes in their frequencies in CHD or HI. Notably, stimulation with T. cruzi did not modify the frequency of the non-classical monocytes subpopulation in any of the groups studied. Moreover, fenofibrate treatment of T. cruzi-stimulated cells, increased the frequency of the non-classical subpopulation in Asy patients. Interestingly, fenofibrate restores CCR2 levels but does not modify HLA-DR expression in any groups. In conclusion, our results emphasize a potential role for fenofibrate as a modulator of monocyte subpopulations towards an anti-inflammatory and healing profile in different stages of chronic Chagas disease.

Non-Classical Monocyte Abundance Is an Independent Adverse Risk Factor for Relapse in Pediatric B-ALL

BackgroundAcute Lymphoblastic Leukemia (ALL) is the most common pediatric cancer and while curable in the majority of cases, 15%-20% of children will relapse with only 50% surviving long-term. Treatment failures arise from the outgrowth of pre-existing or acquired sub-clones that are genetically or epigenetically primed to resist treatment. In addition, the bone marrow microenvironment is known to provide a protective niche. We performed the first mapping of the human B-cell ALL (B-ALL) immune bone marrow (BM) microenvironment at single cell resolution at diagnosis, remission and relapse (Witkowski, 2020). We uncovered a striking rewiring of the myeloid compartment during B-ALL progression with significant over-representation of a leukemia-associated monocyte subpopulation expressing high levels of the Macrophage Colony Stimulating Factor Receptor (M-CSFR/CSF1R). Using both peripheral blood (PB) complete blood count analysis and RNA-seq data, we demonstrated that high monocyte abundance at B-ALL diagnosis is predictive of inferior pediatric and adult overall survival in two large independent clinical cohorts. To determine the association of non-classical monocyte abundance in BM and PB with risk of relapse, we examined a cohort of clinical samples from children enrolled on Children's Oncology Group (COG) protocols.MethodsUsing an unmatched case-control design, we established a preliminary cohort of PB and BM samples collected at diagnosis from 24 B-ALL patients with eventual relapse and 24 patients in long-term remission. Four remission samples from an NYU Langone cohort were used to validate the expansion of this population in the presence of B-ALL. We applied a customized flow cytometry based assay to identify CD115-expressing human monocyte subsets: classical (CD45 +CD56 -CD14 +CD16 -), non-classical (CD45 +CD56 -CD14 -CD16 +), as well as B-cells (CD19, CD22, CD10) and T/NK cells (CD3, CD56). We then performed univariate and multivariable analysis of outcome (relapse versus long-term remission) compared to monocyte subset abundance, adjusting for potential confounding factors (age, gender, CNS status, NCI risk, genetic subtype, WBC at diagnosis, and end of induction minimal residual disease).ResultsWe observed a significantly higher percentage of non-classical monocytes in the diagnostic BM from the COG cohort when compared to remission samples (COG diagnostic B-ALL BM non-classical percentage mean 52.19% vs NYU B-ALL remission BM non-classical percentage mean 1.775%, P = 0.0001). We also observed a strong correlation between the percentage of non-classical monocytes in the PB when compared to their matched BM specimens (r = 0.6, P = 0.0001). Multivariable analysis revealed a significant association between PB non-classical monocyte percentage at diagnosis and patient outcome (remission cohort non-classical monocyte percentage [mean, range]: 52.4%, 33.3-68.1%, n = 24, relapse cohort non-classical monocyte percentage: 65.9%, 56.4-84.7%, n = 24, P = 0.021). Similarly, a strong trend was observed in the BM, although it did not reach statistical significance. Flow cytometric analysis confirmed CD115 (M-CSFR/CSF1R) expression in this non-classical monocyte population, thereby validating a novel target for intervention.ConclusionsThese findings validate the presence of a unique monocyte subpopulation associated with childhood B-ALL and suggests that assessing this population in PB at diagnosis may be of prognostic significance. The availability of small molecule inhibitors and monoclonal antibodies targeting CSF1R-expressing monocytes may offer a novel approach to treating B-ALL. [Display omitted] DisclosuresRaetz: Pfizer: Research Funding; Celgene: Other: DSMB member. Teachey: Sobi: Consultancy; NeoImmune Tech: Research Funding; Janssen: Consultancy; BEAM Therapeutics: Consultancy, Research Funding. Aifantis: AstraZeneca: Research Funding; Foresite (FL2020-010) LLC: Consultancy.

SARS-CoV-2 nucleocapsid suppresses host pyroptosis by blocking Gasdermin D cleavage.

SARS‐CoV‐2 is an emerging coronavirus that causes dysfunctions in multiple human cells and tissues. Studies have looked at the entry of SARS‐CoV‐2 into host cells mediated by the viral spike protein and human receptor ACE2. However, less is known about the cellular immune responses triggered by SARS‐CoV‐2 viral proteins. Here, we show that the nucleocapsid of SARS‐CoV‐2 inhibits host pyroptosis by blocking Gasdermin D (GSDMD) cleavage. SARS‐CoV‐2‐infected monocytes show enhanced cellular interleukin‐1β (IL‐1β) expression, but reduced IL‐1β secretion. While SARS‐CoV‐2 infection promotes activation of the NLRP3 inflammasome and caspase‐1, GSDMD cleavage and pyroptosis are inhibited in infected human monocytes. SARS‐CoV‐2 nucleocapsid protein associates with GSDMD in cells and inhibits GSDMD cleavage in vitro and in vivo. The nucleocapsid binds the GSDMD linker region and hinders GSDMD processing by caspase‐1. These insights into how SARS‐CoV‐2 antagonizes cellular inflammatory responses may open new avenues for treating COVID‐19 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.

B regulatory cells and monocyte subpopulations in patients with chronic graft-vs-host disease

AimTo assess the correlations of B regulatory cells (Bregs) and monocyte subsets in peripheral blood with the National Institutes of Health (NIH)-consensus-defined clinical manifestations of chronic graft-vs-host disease (cGvHD), in an attempt to establish their role as cellular biomarkers.MethodsThis multidisciplinary prospective study enrolled adult cGVHD patients treated in the University Hospital Center Zagreb and University of Zagreb School of Medicine. Immunophenotypic subpopulations of CD24highCD38high Bregs (CD27-, CD27+, and total) and monocyte (classical, intermediate, and non-classical) counts were correlated with demographic, transplant, and cGVHD-related data. Bivariate correlation analysis was performed to evaluate the correlations between Bregs and monocytes subsets and cGVHD organ involvement, as well as cGVHD severity and immunosuppression intensity.ResultsTwenty-two adult patients (54.5% female) with cGVHD were enrolled. The median (range) age was 44.5 years (24-65). All patients were transplanted for hematologic malignancies and 40.9% had severe NIH cGVHD global score. The median time from cGVHD diagnosis to the analysis was 16.6 months (0-176). The organ most frequently affected with cGVHD were the eyes (68.2%), skin (45.5%), lungs (45.5%), and liver (40.9%). Lower total and CD27-Bregs counts were correlated with worse cGVHD severity, higher immunosuppression intensity, and lung cGVHD, in terms of cell count, but also with skin cGVHD, in terms of percentages. Patients with liver and joint/fascia cGVHD had a lower percentage of non-classical monocytes and patients with more severe global NIH score had a higher classical monocytes count.ConclusionDifferent organs affected by cGVHD are differently associated with different subpopulations of Bregs and monocytes.

Patterns of dendritic cell and monocyte subsets are associated with disease severity and mortality in liver cirrhosis patients

Liver cirrhosis is often complicated by an immunological imbalance known as cirrhosis-associated immune dysfunction. This study aimed to investigate disturbances in circulating monocytes and dendritic cells in patients with acute decompensation (AD) of cirrhosis. The sample included 39 adult cirrhotic patients hospitalized for AD, 29 patients with stable cirrhosis (SC), and 30 healthy controls (CTR). Flow cytometry was used to analyze monocyte and dendritic cell subsets in whole blood and quantify cytokines in plasma samples. Cirrhotic groups showed higher frequencies of intermediate monocytes (iMo) than CTR. AD patients had lower percentages of nonclassical monocytes than CTR and SC. Cirrhotic patients had a profound reduction in absolute and relative dendritic cell numbers compared with CTR and showed higher plasmacytoid/classical dendritic cell ratios. Increased plasma levels of IL-6, IL-10, and IL-17A, elevated percentages of CD62L+ monocytes, and reduced HLA-DR expression on classical monocytes (cMo) were also observed in cirrhotic patients. Patients with more advanced liver disease showed increased cMo and reduced tissue macrophages (TiMas) frequencies. It was found that cMo percentages greater than 90.0% within the monocyte compartment and iMo and TiMas percentages lower than 5.7% and 8.6%, respectively, were associated with increased 90-day mortality. Monocytes and dendritic cells are deeply altered in cirrhotic patients, and subset profiles differ between stable and advanced liver disease. High cMo and low TiMas frequencies may be useful biomarkers of disease severity and mortality in liver cirrhosis.