Monocyte Subpopulations Research Articles

EFFECT OF LOW-LEVEL LASER ON CYTOKINE PRODUCTION BY MONOCYTES EXPOSED TO CISPLATIN AND STIMULATED WITH P. GINGIVALIS

Objective This study aimed to evaluate the effect of photobiomodulation (PBM) on monocytes exposed to cisplatin and P. gingivalis (Pg). Study Design Peripheral blood mononuclear cells from 10 healthy donors were exposed to cisplatin, irradiated with red laser (AlGaInP, 660nm, 0.028cm², 20mW, 0.71W/cm², 0.14J, 7s, 5J/cm²) and stimulated with Pg. Cell viability and cytokine expression by total monocytes and monocyte subpopulations were evaluated by flow cytometry. Results The treatments did not affect the viability of monocytes and TGF-β1 expression. In Pg-stimulated cells, cisplatin reduced the expression of IL-6, IL-8, and IL-10 in total and classical monocytes and increased IL-6 and TNF expression in the intermediate monocytes. In cisplatin exposed cells, PBM reduced the expression of IL-6 and IL-8 in total, classical, and intermediate monocytes, and IL-10 and TNF in total monocytes. In cisplatin and Pg-stimulated cells, PBM increased the expression of IL-6 and IL-8 in total and intermediate monocytes. Conclusion The red laser modulates the expression of cytokines by human monocytes treated with cisplatin, which may represent PBM mechanisms of action in the management of oral mucositis. Apoio: FAPEMIG APQ03601-16, FIP/PUC Minas 2020/24894-1S, and CAPES Code 001. This study aimed to evaluate the effect of photobiomodulation (PBM) on monocytes exposed to cisplatin and P. gingivalis (Pg). Peripheral blood mononuclear cells from 10 healthy donors were exposed to cisplatin, irradiated with red laser (AlGaInP, 660nm, 0.028cm², 20mW, 0.71W/cm², 0.14J, 7s, 5J/cm²) and stimulated with Pg. Cell viability and cytokine expression by total monocytes and monocyte subpopulations were evaluated by flow cytometry. The treatments did not affect the viability of monocytes and TGF-β1 expression. In Pg-stimulated cells, cisplatin reduced the expression of IL-6, IL-8, and IL-10 in total and classical monocytes and increased IL-6 and TNF expression in the intermediate monocytes. In cisplatin exposed cells, PBM reduced the expression of IL-6 and IL-8 in total, classical, and intermediate monocytes, and IL-10 and TNF in total monocytes. In cisplatin and Pg-stimulated cells, PBM increased the expression of IL-6 and IL-8 in total and intermediate monocytes. The red laser modulates the expression of cytokines by human monocytes treated with cisplatin, which may represent PBM mechanisms of action in the management of oral mucositis.

#3764 SINGLE-CELL RNA-SEQ REVEALS IMMUNE AND KIDNEY CELL TYPE–SPECIFIC MOLECULAR ASSOCIATIONS TO IGA NEPHROPATHY

Abstract Background and Aims IgA nephropathy is the most common primary glomerulonephritis worldwide. There is growing evidence of host immunity is closely related to the pathogenesis of IgAN, but the detailed mechanism in the early stages of IgAN remain largely unknown. In this study, we applied single-cell RNA-seq technology to investigate the transcriptomic changes in PBMCs and kidney tissues of early stage IgAN patients at the single-cell resolution. This study aims to establish a immune and kidney cell landscape of IgAN, and offers new insights for the diagnosis and treatment of IgAN. Method In strict accordance to the admission criteria, collect peripheral venous blood and kidney samples of healthy donors and newly diagnosed IgAN patients, prepare single cell solution, verify cell viability by flow cytometry, and perform single-cell RNA-seq using a BD Rhapsody platform. Meanwhile, the clinical data of healthy donors and IgAN patients were collected and analysed. Finally, the correlation and association of differential expressed genes (DEGs) and cell clusters-identified by single-cell RNA-seq with the clinical data of IgAN were investigated. Results First, we established immune cells and kidney cells landscape of IgAN. The differentially expressed genes (DEGs) between the control group and IgAN were mainly concentrated in the NK cell-mediated cytotoxicity and cell killing pathways. Interestingly, we found significant decreases in NK cell numbers and cytotoxicity, and NK cell numbers and marker genes were negatively correlated with clinical parameters, including urinary protein creatinine ratio (UPCR) and serum galactose-deficient IgA1 and IgA. In contrast, B cell DEGs were enriched in different viral infection pathways, and one specific B cell subgroup showed inhibition of NFκB signaling, which was positively correlated with clinical parameters. In addition, a subpopulation of monocytes expressing interferon-inducing genes was positively associated with clinical severity of IgAN. In case of the kidney tissue, compared with the control group, mesangial cells of IgAN patients up-regulated extracellular matrix, transcription factors, kidney development and genes related to the regulation of Wnt signaling pathway. The expression of structural protein ITGA8 on mesangial cells was significantly decreased, indicating the mesangial cells were damaged in early IgAN. Pseudo-time analysis indicated that extracellular matrix-related genes were involved in mesangial cell lesion. We also found that the patie'ts' glomerular endothelial cells expressed high level of angiogenesis and VEGF- related genes. Moreover, podocytes of IgAN patients expressed genes of cytoskeletal recombination and FGFR activation. Conclusion We successfully established the single-cell landscapes of peripheral blood immune cells and kidney cells in early IgAN patients, which revealed changes in the number and status of immune cells and kidney cells closely related to clinical manifestations and phenotypic transformation. Our study offers new insights for the diagnosis and treatment of IgAN.

#5730 PERLECAN AS A POTENTIAL BIOMARKER OF CARDIOVASCULAR RISK: ROLE OF ENDOTHELIAL GLYCOCALYX IN CHRONIC KIDNEY DISEASE

Abstract Background and Aims Chronic kidney disease (CKD) is associated with cardiovascular disease (CVD). CVD is in turn related to endothelial dysfunction, endothelial dysfunction, and degradation of the endothelial glycocalyx (EG), releasing its components into the bloodstream. The EG consists of glycosaminoglycans and proteoglycans, such as Perlecan. The aim of the study is to analyse the levels of perlecan in plasma in different stages of CKD, and to relate them to age and inflammatory monocytes, as well as their adhesion capacity (CD54/ICAM-1). Method An observational cross-sectional study was carried out. 56 patients were included: advanced chronic kidney disease (ACKD) (n=13), haemodialysis (HD) (n=13), peritoneal dialysis (PD) (n=15) and transplant recipients (TX) (n=15). Thirteen healthy subjects (CT) were analysed. Plasma perlecan levels were quantified using ELISA and phenotyping of monocyte subpopulations (classical (CD14++CD16-), intermediate (CD14+CD16+) and non-classical (CD14+CD16++)), as well as CD54 (ICAM-1) expression by flow cytometry. Statistical analysis: ANOVA and Spearman correlation. Results Patients with CKD had higher plasma levels of perlecan than healthy participants (p-value = 0.044 vs ACKD, 0.028 vs HD and 0.002 vs PD) (Fig. 1). These levels were associated with higher percentage of classical monocytes (p=0,011) intermediate monocytes (p=0,009) and non-classical monocytes (p=0,003) expressing ICAM-1 but not with a higher expression of ICAM-1 per monocyte. Perlecan levels correlate negatively with age (p=0,03) (Table 1). Conclusion Patients with ACKD, HD and PD have elevated plasma perlecan levels compared to CT and TX. Elevated perlecan concentrations are associated with increased ICAM-1 expression on monocytes, which is important for its possible role in the development of atherosclerosis. Perlecan may be postulated as a molecule of interest to assess endothelial and cardiovascular damage in CKD patients.

Expression of arginase 1 and tyrosine kinase Mer by blood monocytes in the dynamics of physiological pregnancy

During pregnancy, the maternal immune system must maintain tolerance to paternal antigens, at the same time being able to eliminate pathogens, which is achieved by the weakening of adoptive immunity and the activation of innate immunity, in particular, monocytes. However, the question about the functional phenotype of monocytes, having not only pro-inflammatory, but also anti-inflammatory activity, remains open. In the given work, we have investigated the expression of M2-associated suppressive markers Arg1 and MerTK in monocyte subpopulations during uncomplicated pregnancy. Fifty-three pregnant women with uncomplicated gestation were recruited, including 14 pregnant in the 1st trimester, 20 – in the 2nd and 19 – in the third pregnancy trimester. The comparison group consisted of 15 fertile unpregnant women without aggravated somatic anamnesis, with a history of at least one childbirth. The findings showed that in the unpregnant group circulating Mo express Arg1 and MerTK, and the most relative number of Arg1+ and MerTK+ cells is concentrated in intermediate and nonclassic monocytes. During pregnancy the expression of researched molecules in monocytes reliably increases. An increase in MerTK expression is manifested by a simultaneous increase in the number of MerTK+ cells and the mean fluorescence intensity of this marker; it is observed in the 1st and 2nd trimesters and registered in all three monocyte subpopulations. At the same time, an increase in Arg1 expression is manifested either by an enhancement of Arg1+ cells, or an increase in receptor density; it is registered throughout pregnancy, including the 3rd trimester, and is maximally expressed in classic monocytes. There is a direct correlation between the number of Arg1+ and MerTK+ cells in intermediate Mo, which increases with the progression of pregnancy, and in the 3rd trimester is also detected in classical and non-classical Mo. In general, the revealed increase in the expression of Arg1 and MerTK by monocytes indicates an increase in the anti-inflammatory potential of monocytes during pregnancy, and the involvement of monocytes in the regulation of the inflammatory process at the system level. Moreover, the features of Arg1 and MerTK expression in various monocyte subpopulations during pregnancy suggest that monocytes expressing Arg1 and MerTK can mediate different mechanisms of immune adaptation during pregnancy.

T-lymphocytes of the 2nd type of the immune response and their role in enhancing inflammation during the professional activities of firefighters

T lymphocytes of the 2nd type of immune response contribute to the development and exacerbation of inflammation, mainly allergic. Increased inflammation with prolonged exposure to adverse factors during the work of firefighters can lead to the development of various diseases. Evaluation of the immunity of firefighters is important for the appointment of adequate treatment and prevention of infectious and allergic diseases. This paper aimed to analyze the indicators of immunity in employees of the state fire service of EMERCOM of Russia, depending on the age and intensity of the professional workload. The surveyed were men (n = 79), mean age 31 years, work experience from 1 to 22 years, with different workload intensity. In peripheral blood, flow cytometry (Navios, FC 500, Beckman Coulter) was used to evaluate subpopulations of monocytes, the relative number of T lymphocytes of the 2nd type of immune response CD3+CD294+. The concentration of total immunoglobulin E (Immulite) was determined. From nasal secretions, the content of secretory immunoglobulin A (Vector Best) was evaluated (n = 30). Statistical processing of the results was performed using the Statistica 12.0 package (StatSoft). An increase in the number of CD3+CD294+ cells was observed in 16.5%. A direct correlation was found between the number of T lymphocytes 2 and the age of the examined persons (р < 0.05). In the group of firefighters with a more intense workload, an increase in the number of CD3+CD294+ cells were 5 times higher (р < 0.05). Among patients who had any disease of the respiratory tract, an increase in this population was observed statistically significantly more often – in 26% of cases versus 11.5%. A strong direct correlation was found between the number of T lymphocytes 2 and the duration of smoking experience (р < 0.05). A direct correlation was established between the number of T lymphocytes 2 and the concentration of total IgE (р < 0.05). A decrease in secretory IgA in the secret from the nasal passages was observed in 23% of firefighters, in 13% of the examined, the indicator went beyond the upper limit of the reference interval. Significantly more often revealed the deviation of this indicator from the reference values in firefighters with a high workload. An increase in the subpopulation of classical monocytes was established in the group of those examined with a high number of CD3+CD294+ cells (p < 0.05). Thus, with an increase in the professional workload of firefighters in unfavorable conditions of service, inhibition of protection to infection and aggravation of damage to the respiratory tract with an increase in the 2nd type of immune response are noted. Evaluation of the number of type 2 T lymphocytes in peripheral blood will reveal a predisposition to the T2 profile of immune inflammation, which will contribute to a personalized approach to patient management.

SUBPOPULATIONS OF LYMPHOCYTES AND MONOCYTES IN PATIENTS` VENOUS BLOOD WITH ATRIAL FIBRILLATION OR ATRIAL FLUTTER ASSOCIATED WITH HYPERTENSION

Objective: The most common reason of arrhythmia onset nowadays is hypertensive desease. The aim of the study is to compare the subpopulations of lymphocytes (LCTs) and monocytes (MCTs) in blood of patients (pts) with paroxysmal and persistent forms of AF or atrial flutter (AFL) associated with arterial hypertension. Design and method: The study involved 103 patients with atrial fibrillation and flutter that occurred on the backdround of hypertension. Depending on the form of arrhythmia, patients were divided into three main groups: group I – with paroxysmal form of atrial fibrillation, group II – with a persistent form of atrial fibrillation, group III – with a persistent form of atrial flutter. The control groups included patients with hypertension, but without these arrhythmias and healthy individuals who entered groups IV and V, respectively. The lymphocytes and monocytes subpopulation in peripheral blood was assessed by flow cytometry. Results: Analyzing the lymphocyte subpopulations in peripheral blood of patients with atrial fibrillation and flutter (groups I, II and III), it was found that the number of cells with cytotoxic activity (NK and NKT) in both absolute count and percentage values was significantly higher than in healthy individuals. A statistically significant decrease of T-regulatory cells number was found in patients with arrhythmias compared to control groups (p < 0.05). In patients with AF and AFL associated with hypertension, compared to patients with hypertension without these rhythm disturbances or healthy individuals, there is an increased number of classical and intermediate monocytes subpopulations. Conclusions: 1. In patients with atrial fibrillation or atrial flutter associated with hypertension, compared to patients with hypertension without these rhythm disturbances or healthy people, there is an increased number of classical and intermediate monocytes fraction and a decreased content of T-regulatory cells in peripheral blood, an increased number of cells with cytotoxic activity (NK and NKT) compared to healthy individuals. These changes can be one of the factors for the activation of systemic and local inflammatory processes that can lead to fibrosis in the myocardium and the associated electrical and structural remodeling typical for AF and AFL.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Background: A growing body of evidence has indicated impaired function or compositional changes of monocytes in inflammatory disorders, such as acute respiratory syndrome and covid-19. In myeloma tumor microenvironment, activation of type I interferon pathway and dysregulated expression of major histocompatibility complex type II genes are observed in classical monocytes, which result in loss of antigen presentation of monocytes. The proportions of BAFF+PD-L1+ monocytes in the bone marrow also correlate with survival of myeloma patients following chimeric antigen-receptor T cell therapy. Nevertheless, the mechanisms underlying PB and BM monocytes defects in myeloma remain poorly addressed, at least in part by the lack of large scale scRNA-seq studies. To resolve the heterogeneous bone marrow and peripheral blood monocyte subpopulations and their transcriptional factors between healthy donors and multiple myeloma patients. Methods: We conducted scRNA-seq on monocytes of 7 newly diagnosed myeloma patients and 12 healthy donors. Specifically, 3 and 5 bone marrow aspirates and 9 and 7 peripheral blood samples of 12 healthy controls and 7 newly diagnosed patients were obtained, respectively. Results: Heterogeneous bone marrow and peripheral blood monocyte subpopulations and their transcriptional factors were resolved. The function characteristics of monocyte were compared between MM patients and healthy donors, which indicated a stress signature induced by multiple myeloma. Furthermore, we identified two monocyte differentiation pathways, and discovered that bone marrow monocyte feature type I IFN-associated differentiation dysregulation in patients with MM as well as dysregulated patterns at transcriptome. Finally, we included 10 MM patients as a validation cohort, by tracking the stress signature over time in those patients undergoing first-line treatment, we founded that the stress signature was partially overcome by antitumor therapy. Conclusion: Our results provided further insight into transcriptional and differentiation changes occurring in the bone marrow and peripheral blood monocytes from patients with multiple myeloma and hint and mechanisms of immune evasion. Encore Abstract - previously submitted to EHA 2023 The research was funded by: This investigation was supported by the International Cooperation Projects of National Natural Science Foundation (grants 81920108006), CAMS Innovation Fund for Medical Sciences (CIFMS) (grants 2022-I2M-1-022), the National Natural Science Foundation (grants 81630007; grants 82270218, 81670202; grants 81900214), and the Atlas of Blood Cell Alliance. Keywords: genomics, epigenomics, and other -omics, microenvironment, multiple myeloma No conflicts of interests pertinent to the abstract.

Effect of pregnancy-specific β1-glycoprotein on myeloid-derived suppressor cell differentiation

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous cell population that primarily suppress T lymphocytes in healthy pregnancies and pathologies. MDSCs are one of the key regulators of immune responses. Finding ways to control them is important for the treatment of cancer, autoimmune diseases, miscarriage, and post-transplant complications. The mechanisms of immune suppression by MDSC are: expression of CD73, ADAM17, PD -L1, production of Arg 1, iNOS, IDO, IL -10 and TGF-b1.Pregnancy-specific b1-glycoprotein (PSG) has modulatory effects on dendritic cells and macrophages that mediate the shift of T cell phenotypes toward Th2 and Treg. We have previously shown that native PSG suppresses Th17 differentiation and cytokine production, stimulates the production of IDO by monocytes and the differentiation of Tregs.Considering the immunomodulatory properties of PSG and the key role of MDSCs in pathologies, the aim of our work was to investigate the effect of native and recombinant PSG on the differentiation of MDSCs in vitro.MDSCs were differentiated from CD11b+ peripheral blood cells. Cells were cultured for 7 days and received stepwise GM-CSF, IL-1b, and LPS. Native (n) (1; 10 and 100 mg/mL) and recombinant (r) (1 and 10 mg/mL) PSG were introduced into the cultures three days before the end of incubation. Flow cytometry was used to determine the percentage of MDSC among the cells in culture and the percentage of M-, PMN-, and e-MDSC among the total number of MDSCs.It was found that rPSG (1 mg/mL) increased the percentage of MDSCs in culture. Both nPSG (1 and 10 mg/mL) and rPSG (10 mg/mL) increased the proportion of M-MDSC, whereas rPSG (10 mg/mL) decreased the number of PMN-MDSC.Thus, the cytokine background in CD11b+ cell cultures favored the differentiation of predominantly M-MDSC, similar to the tumor microenvironment, whereas native and recombinant PSG enhanced this effect. Thus, nPSG and rPSG are able to modulate the differentiation of MDSCs by increasing their number, mainly due to the monocytic subpopulation. This fact opens perspectives for new research on targeted manipulation of MDSCs.

The dynamic changes of monocytes and cytokines during wound healing post-burn injury

BackgroundBurn injury is a sudden and traumatic injury that affects a large part of the population worldwide, who are placed at high risk of developing hypertrophic scars (HTS). HTS are a fibrotic scar resulting in painful contracted and raised scarring, affecting mobility in joints and work life, as well as cosmetically. The aim of this research was to enhance our understanding of the systematic response of monocytes and cytokines in wound healing after burn injury, in order to develop novel approaches to prevention and treatment of HTS. MethodsTwenty-seven burn patients and thirteen healthy individuals were recruited in this study. Burn patients were stratified by burn total body surface area (TBSA). Peripheral blood samples were taken post-burn injury. Serum and peripheral blood mononuclear cells (PBMCs) were separated from the blood samples. This research investigated cytokines IL-6, IL-8, IL1RA, IL-10, and chemokine pathways SDF-1/CXCR4, MCP-1/CCR2, RANTES/CCR5 during the wound healing process in burn patients with varying severity of injuries by using enzyme-linked immunosorbent assays. PBMCs were stained for monocytes and the chemokine receptors by flow cytometry. Statistical analysis was done by one-way ANOVA with a Tukey correction, and regression analysis was performed using Pearson’s Correlation analysis. ResultsThe CD14+ CD16- monocyte subpopulation is larger in patients who developed HTS at 4–7 days. The CD14+ CD16+ monocyte subpopulation is smaller in the first week of injury, where it is similar after 8 days. Burn injury increased CXCR4, CCR2, and CCR5 expressions in CD14+ CD16+ monocytes. Increases in MCP-1 at 0–3 days after burn injury was positively correlated with burn severity. IL-6, IL-8, RANTES, and MCP-1 significantly increased with increasing burn severity. ConclusionsMonocytes and their chemokine receptors, as well as systemic levels of cytokines in wound healing of burn patients and scar development will require ongoing assessment to enhance our understanding of the abnormal wound healing after burn injury.

Causal relationship between circulating immune cells and the risk of type 2 diabetes: a Mendelian randomization study.

Though type 2 diabetes (T2D) has been known as a metabolic disease caused by multiple factors, the etiology remains insufficiently understood. Here, we aimed to figure out whether circulating immune cell profiles causally impact T2D liability. We applied one genome-wide association study (GWAS) summary statistics of blood traits in 563,085 participants from the Blood Cell Consortium and another GWAS of flow cytometric profile of lymphocyte subsets comprising 3,757 Sardinians to identify genetically predicted blood immune cells. We also obtained GWAS summary statistics in 898,130 individuals from the DIAGRAM Consortium to evaluate genetically predicted T2D. We primarily used inverse variance weighted (IVW) and weighted median methods to perform Mendelian randomization analyses and sensitivity analyses to evaluate heterogeneity and pleiotropy. For circulating blood leukocyte and its subpopulations, the increase of genetically predicted circulating monocyte count was causally correlated with a higher risk of T2D [odds ratio (OR) = 1.06, 95% confidence interval (CI) = 1.02-1.10, p = 0.0048]. For lymphocyte subsets, CD8+ T cell and CD4+ CD8dim T cell count were identified with causal effect on T2D susceptibility (CD8+ T cell: OR = 1.09, 95% CI = 1.03-1.17, p = 0.0053; CD4+ CD8dim T cell: OR = 1.04, 95% CI = 1.01-1.08, p = 0.0070). No pleiotropy was determined. These findings demonstrated that higher circulating monocyte and T-lymphocyte subpopulation predicted increased T2D susceptibility, which confirmed the immunity predisposition for T2D. Our results may have the potential to provide new therapeutic targets for the diagnosis and treatment of T2D.

Lymphocytes and monocytes subpopulation composition in peripheral blood in patients with atrial fibrillation and futter

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): NSC "Institute of cardiology, clinical and regenerative medicine named by acad. Strazhesko" NAMS of Ukraine The purpose of the trial is to compare the state of intracardiac and systemic hemodynamics in patients with paroxysmal and persistent forms of atrial fibrillation and flutter that appeared on the base of arterial hypertension, depending on the lymphocytes and monocytes subpopulation composition in peripheral blood. Materials and methods The study involved 103 patients with atrial fibrillation and flutter and were divided into three main groups: I – with paroxysmal form of atrial fibrillation, II – with a persistent form of atrial fibrillation, III – with a persistent form of atrial flutter. The subpopulation composition of lymphocytes and monocytes in peripheral blood was assessed by the method of flow cytometry. Hemodynamic parameters were evaluated using echocardiography, office blood pressure measurement, daily blood pressure monitoring, and transesophageal echocardiography. For control, the data of patients with hypertension but without the above-mentioned arrhythmias were used, and practically healthy people who entered groups IV and V, respectively. Results and discussion The number of cells with cytotoxic activity (NK and T-NK) in both absolute and percentage values was significantly higher in groups with arrhythmia than in practically healthy individuals. A mathematically significant decrease of T-regulatory cells number (p ≤0.05) in the second and third groups compared to the control groups was detected, same as increased number of classical and intermediate monocytes fractions. Comparing echocardiogram results indicators between groups I, II, III and I V, a mathematical significant difference (p≤0.05) between the LVID was revealed: LVIDd, LVEDV, LVESV, IVS, PW thickness; LA AP, RVmid diameter at end-diastole, E/A and LVMI. Compared to V the group difference was significant only between LVIDd, LA AP size, IVS thickness, and PW thikness. Office systolic blood pressure was significantly higher in patients with arrhythmias. According to daily blood pressure monitoring, the difference between the indicators of average and maximum diastolic pressure was significant. Conclusions In patients with atrial fibrillation and flutter as a result of hypertension, compared to patients without arrhythmias or healthy people, there is an increased content of pro-inflammatory monocytes subpopulation, T-cytotoxic cells and a decreased amount of T-regulatory cells. According to EchoCG, these patients had more severe structural changes in the myocardium: an increased LA AP and LV hypertrophy, a thickening of IVS and PW, and diastolic dysfunction. The recorded measurement indicators of SBP and DBP differed mathematically significantly, which indicates hemodynamic disorders caused by both rhythm disturbances and manifestations of arterial hypertension.

Monocyte pathology in human tuberculosis is due to plasma milieu changes and aberrant STAT signalling.

Monocyte-derived macrophages contribute centrally to immune protection in Mycobacterium tuberculosis infection and changes in monocyte phenotype characterize immunopathology in tuberculosis patients. Recent studies highlighted an important role of the plasma milieu in tuberculosis immunopathology. Here, we investigated monocyte pathology in patients with acute tuberculosis and determined tuberculosis plasma milieu effects on phenotype as well as cytokine signalling of reference monocytes. Patients with tuberculosis (n = 37) and asymptomatic contacts (controls n = 35) were recruited as part of a hospital-based study in the Ashanti region of Ghana. Multiplex flow cytometry phenotyping of monocyte immunopathology was performed and effects of individual blood plasma samples on reference monocytes prior to and during treatment were characterized. Concomitantly, cell signalling pathways were analysed to elucidate underlying mechanisms of plasma effects on monocytes. Multiplex flow cytometry visualization characterized changes in monocyte subpopulations and detected higher expression of CD40, CD64 and PD-L1 in monocytes from tuberculosis patients as compared to controls. Aberrant expression normalized during anti-mycobacterial treatment and also CD33 expression decreased markedly. Notably, higher CD33, CD40 and CD64 expression was induced in reference monocytes when cultured in the presence of plasma samples from tuberculosis patients as compared to controls. STAT signalling pathways were affected by the aberrant plasma milieu and higher levels of STAT3 and STAT5 phosphorylation was found in tuberculosis plasma-treated reference monocytes. Importantly, high pSTAT3 levels were associated with high CD33 expression and pSTAT5 correlated with CD40 as well as CD64 expression. These results suggested plasma milieu effects with potential implications on monocyte phenotype and function in acute tuberculosis.

Importance of circulating monocyte subpopulations and Fcγ receptors in immune thrombocytopenia.

Monocytes have been linked to the pathogenesis of immune thrombocytopenia (ITP) because of their role in autoantibody-mediated platelet phagocytosis. However, monocytes constitute unique populations with major differences in expression for surface Fcγ receptors (FcγRs). Thus, we evaluated monocytes in whole blood samples from patients with newly diagnosed and chronic ITP. Monocyte subpopulations were identified phenotypically by flow cytometry and defined according to the surface expression of CD14 (lipopolysaccharide receptor) and of CD16 (low-affinity Fcγ receptor III) into classical (CLM), intermediate (INTM) and nonclassical (non-CLM) monocytes. We also examined the expression of FcγRI/CD64 and FcγRIII/CD16 by monocyte subpopulations. Newly diagnosed patients showed a decrease in non-CLM, expressed as a relative percentage of total monocytes compared with controls and chronic ITP patients. Both non-CLM and INTM of newly diagnosed patients closely correlated with platelet count. These monocyte subpopulations showed significantly enhanced CD64 expression in newly diagnosed patients. On the contrary, patients with chronic ITP presented higher non-CLM in percentage than controls and concomitant lower CLM and total monocytes, in percentage and number. The expression of CD64 was increased by all monocyte subpopulations, CLM, INTM, and non-CLM in chronic patients. In conclusion, differences in monocyte subpopulations, together with enhanced expression of FcγRI/CD64 are evident in patients with ITP.

Innate immune side of STAT1 GOF

STAT1 gain-of-function (GOF) mutations underlie an inborn error of immunity hallmarked by chronic mucocutaneous candidiasis (CMC). Beyond the fungal susceptibility, attributed to Th17 failure, over half of the reported patients suffer from autoimmune manifestations, which remain mechanistically unexplained. Surprisingly, little is known about the biological consequences of augmented STAT1 signaling in innate immune cells. Considering the importance of innate immune mechanisms in antifungal defense and autoimmune complications, we examined the phenotype and function of monocytes and dendritic cells in a cohort of patients with STAT1 GOF.We demonstrated that human ex vivo STAT1 GOF monocytes are characterized by proinflammatory phenotype and strong inflammatory skew of their secretory cytokine profile. The distribution of monocytes subpopulations’ is altered and they exhibit various functional abnormalities, which might represent a contributing mechanism in the pathogenesis of STAT1 GOF-associated autoimmunity, e.g., a selectively enhanced responsiveness to TLR7/8 stimulation, but not to other TLR ligands. Importantly, some of these features extend to STAT1 GOF myeloid dendritic cells, suggesting that the alterations observed in monocytes are, in fact, intrinsic due to STAT1 mutation, and not mere secondary consequence of the chronic inflammatory milieu.To follow up on the ex vivo data, we employed an in vitro model of monocyte-derived DC (moDC) and tolerogenic DC (tDC). Functional and signaling studies, co-culture experiments and RNA sequencing demonstrated that STAT1 GOF DCs were profoundly altered in their phenotype and functions, characterized by loss of tolerogenic functions, proinflammatory skew and decreased capacity to induce Th17. Cytokine signaling, autophagy and metabolic processes were identified as the most prominently altered cellular processes.In conclusion, we present first-ever data implying a direct involvement of monocytes and DCs in the STAT1 GOF-associated immune pathology, possibly contributing to both autoimmune manifestations and the failure of antifungal defense. Our work thus highlights a yet understudied innate immune background in STAT1 GOF which should be dissected in details by future research.

A single-cell view on host immune transcriptional response to in vivo BCG-induced trained immunity

Bacillus Calmette-Guérin (BCG) vaccination is a prototype model for the study of trained immunity (TI) in humans, and results in a more effective response of innate immune cells upon stimulation with heterologous stimuli. Here, we investigate the heterogeneity of TI induction by single-cell RNA sequencing of immune cells collected from 156 samples. We observe that both monocytes and CD8+ Tcells show heterologous transcriptional responses to lipopolysaccharide, with an active crosstalk between these two cell types. Furthermore, the interferon-γ pathway is crucial in BCG-induced TI, and it is upregulated in functional high responders. Data-driven analyses and functional experiments reveal STAT1 to be one of the important transcription factors for TI shared in all identified monocyte subpopulations. Finally, we report the role of type I interferon-related and neutrophil-related TI transcriptional programs in patients with sepsis. These findings provide comprehensive insights into the importance of monocyte heterogeneity during TI in humans.

Triple immune modulator therapy for aberrant hyperinflammatory responses in severe COVID-19

A dysregulated hyperinflammatory response is a key pathogenesis of severe COVID-19, but optimal immune modulator treatment has not been established. To evaluate the clinical effectiveness of double (glucocorticoids and tocilizumab) and triple (plus baricitinib) immune modulator therapy for severe COVID-19, a retrospective cohort study was conducted. For the immunologic investigation, a single-cell RNA sequencing analysis was performed in serially collected PBMCs and neutrophil specimens. Triple immune modulator therapy was a significant factor in a multivariable analysis for 30-day recovery. In the scRNA-seq analysis, type I and II IFN response-related pathways were suppressed by GC, and the IL-6-associated signature was additionally downregulated by TOC. Adding BAR to GC and TOC distinctly downregulated the ISGF3 cluster. Adding BAR also regulated the pathologically activated monocyte and neutrophil subpopulation induced by aberrant IFN signals. Triple immune modulator therapy in severe COVID-19 improved 30-day recovery through additional regulation of the aberrant hyperinflammatory immune response.

Qualification of a multidonor mixed lymphocyte reaction assay for the functional characterization of immunomodulatory extracellular vesicles

Background aimsExtracellular vesicles (EVs), including exosomes and microvesicles, are released by almost all cells and found in all body fluids. Unknown proportions of EVs transmit specific information from their cells of origin to specific target cells and are key mediators in intercellular communication processes. Depending on their origin, EVs can modulate immune responses, either acting as pro- or anti-inflammatory. With the aim to analyze the immunomodulating activities of EV preparations, especially those from mesenchymal stromal cells (MSCs) in vitro, a multi-donor mixed lymphocyte reaction (mdMLR) assay was established and stressed for its reproducibility. MethodsTo this end, human peripheral blood-derived mononuclear cells (PBMCs) of 12 different healthy donors were pooled warranting mutual allogeneic cross-reactivity, even following an optimized freezing and thawing procedure. After thawing, mixed PBMCs were cultured for 5 days in the absence or presence of EVs to be tested. Reflecting allogeneic reactions, in the absence of EVs, pooled PBMCs form characteristic satellite colonies whose appearance can be modulated by EVs. More quantifiable, the strength of the allogenic reaction is reflected by the content of activated CD4 and CD8 T cells being recognized by means of their CD25 and CD54 expression. ResultsOf note, connected to the use of primary cells, independent multi-donor PBMC pools differed in their capability to activate their cultured T cells. Thus, throughout the study, only pooled PBMC batches were used whose activated T-cell contents exceeded 25% of the total T-cell population at culture day 5 and whose contents were reproducibly reduced in the presence of immunomodulatory active MSC-EVs. T-cell activation–suppressing effects of the MSC-EV preparations tested were in all cases accompanied by the impact on monocytes. In the presence of immunomodulatory active MSC-EVs, more monocytes were harvested from mdMLR cultures than in their absence. Furthermore, in the absence of immunomodulatory EVs, most monocytes appeared as non-classical (CD14+CD16+) monocytes, whereas immunomodulatory active MSC-EVs promoted the appearance of classical (CD14++CD16–) and intermediate (CD14++CD16+) monocyte subpopulations. ConclusionsOverall, the obtained results qualify the mdMLR assay as a robust experimental tool for the evaluation of immunomodulatory potentials of given MSC-EV samples. However, further assay development is required to develop and qualify an authority-acceptable potency assay for clinically applicable MSC-EV products.

Analysis of monocyte subpopulations with cardiovascular, burn and other pathologies (2010 classification)

A classification of monocyte subpopulations developed in 2010 with coverage of the immunobiological properties of cells, their functional activity and participation in various pathological processes (inflammatory, cardiovascular diseases, strokes, myocardial infarctions, aortic aneurysms, surgical modification of heart valves, diabetes, burns, etc.). The diagnostic and prognostic aspects of the analysis of monocytic subpopulations are considered. The unique data obtained by the staff of the Gamaleya Institute of epidemiology and microbiology. They consist in the fact that various physical forms of unmodified native type 1 collagen are powder, i.e. crushed bundles of collagen fibers, a hydrogel or a solution of extracellular matrix peptides, as well as a suspension of collagen fibers obtained from a powder, when applied to the surface of acute and chronic and diabetic wounds, bedsores, trophic ulcers, etc., can provide a pronounced anti-inflammatory, reparative, remodulating and regenerative effect on condition of wounds, providing their accelerated healing due to the local accumulation of “regenerative” subpopulations of Mon3 monocytes, which can be most directly used in burn tissue lesions. In this case, the analysis of monocytic subpopulations is of paramount importance. Moreover, possible potentiating effects of additional use in burns under the control of the analysis of monocyte subpopulations of powerful modern pluripotent immunomodulators – polyoxidonium, galavit and their possible combination with local use of collagen preparations are discussed. Finally, we obtained preliminary data indicating the development in burned patients of a deficiency in the absolute and relative content of the most important “patrolling” non-classical subpopulation of CD14+CD16++ monocytes compared with healthy primary (non-professional) donors, which can be a very important finding in the diagnosis and prognosis and substantiation of new methods of treatment of burns.

Abstract 2292: Novel method for the detection and evaluation of disease biomarkers on white blood cells from liquid biopsies

Abstract Purpose: Blood based liquid biopsies of circulating tumor cells (CTCs) are a non-invasive tool with proven effectiveness in the prognosis, treatment selection, and stratification of cancer patients. In this study we demonstrate a novel application of the RareCyte platform, which was developed to find and analyze rare CTCs from blood samples, in evaluating white blood cell (WBC) sub-populations for disease biomarkers. Experimental Procedures: Using AccuCyte®, an unbiased density-based method for collecting nucleated cells from whole blood, we transferred the nucleated cells to slides and stained for CD14, CD66b, and CK/EpCAM to identify monocytes (CD14+/CD66b-), granulocytes (CD14-/CD66b+), and CTCs (CK/EpCAM+), as well as a drug-target biomarker to evaluate its expression across all cells. These slides were then imaged with CyteFinder®, an automated multiparameter immunofluorescent (IF) microscopy system that applies machine learning algorithms for cell identification. Each slide contains millions of cells, so we developed novel scanning and analysis algorithms to quantify WBC sub-populations in addition to rare CTCs. Results: In addition to detection and enumeration of CTCs, the RareCyte system was able to detect individual white blood cells and quantify monocyte and granulocyte subpopulations. Our results correlated very well with standard CBC (Complete Blood Count) readings from multiple donors. Additionally, the monocyte and granulocyte populations were evaluated for expression of a drug-target biomarker. While expression was observed on both granulocytes and monocytes, monocytes had a significantly higher expression of the drug-target biomarker, a critical finding for future implementation of this drug. Conclusions: Our results show that through modifications to the RareCyte platform we can identify rare and non-rare cell populations simultaneously and evaluate expression of biomarkers and drug-targets across multiple cells of interest. This new application combines the benefits of high throughput data analysis of flow cytometry with the specificity and sensitivity of rare cell detection. Additionally, high-resolution multiplexed immunofluorescent imaging provides cell morphology and biomarker localization information. Our ability to add multiple biomarkers in conjunction with CTC and other rare cell detection provides multiple avenues for future research. One possible application is characterizing white blood cells that cluster with CTCs, which are known to portend a worse prognosis for patients. Our modified workflow could be implemented to precisely characterize these WBCs that contribute to the metastatic process. Utilization of the RareCyte platform for both rare and non-rare cell analysis presents an exciting opportunity for a deeper evaluation of patients and their prognosis through liquid biopsies. Citation Format: Erin Bayer, Jon Ladd, Joshua Nordberg, Arturo B. Ramirez, Jessica A. Baker, Thomas Daly. Novel method for the detection and evaluation of disease biomarkers on white blood cells from liquid biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2292.

Abstract 2270: Combination of CXCR4 antagonist and anti-PD1 therapy results in significant mobilization and increased infiltration of myeloid cells into the metastatic liver microenvironment of PDAC

Abstract Pancreatic adenocarcinoma (PDAC) is extremely lethal and resistant to checkpoint immunotherapy, characterized by an immunosuppressive tumor microenvironment consisting of stromal and myeloid cells. Prior studies have demonstrated the utility of targeting the chemokine signaling axis CXCR4-CXCL12 (SDF-1), a highlighted feature in PDACs, to overcome the CXCL12-driven immobilization of T cells and thus facilitate their antitumor role within the tumor. Based on these findings, we have conducted a phase 2 trial evaluating the effects of plerixafor, a CXCR4 antagonist, and cemiplimab, a PD1 inhibitor antibody, in patients with metastatic PDAC who have progressed after one line of systemic chemotherapy (NCT04177810). To determine the immunological responses to therapy, blood samples and tissue biopsies were obtained at baseline and during treatment. Hematological assessment confirmed the activity of CXCR4 antagonist in mobilizing hematopoietic precursors (CD34+), immature myeloid cells, and lymphoid cells, as well as monocytic and granulocytic cell populations. Suspension mass cytometry analysis of peripheral blood mononuclear cells revealed that mobilized monocytic subpopulations had high expressions of chemokine receptors CCR2, CCR5, and CXCR2. Histopathologic evaluation of the serial tissue biopsies from the liver metastatic PDAC revealed increased levels of inflammation upon treatment. To further characterize the cellular constituents of the observed inflammation, multiplexed immunohistochemistry by imaging mass cytometry was performed, demonstrating strong trends toward increased infiltration of not only effector T cells but also macrophages and granulocytic cells into the tumor microenvironment. Taken together, these findings suggest that mobilization of myeloid cells by CXCR4 antagonism results in the recruitment of additional myeloid cells from circulation and that alternative chemokine signaling pathways are sufficient for doing so. This implicates a potential mode of resistance against CXCR4-targeted therapies. Furthermore, these observations reinforce the value of ongoing research efforts in the field to subvert the recruitment or immunosuppressive function of myeloid cells, which would be particularly relevant in the setting of CXCR4 antagonism. Citation Format: Sarah M. Shin, Alexei Hernandez, Erin Coyne, Zhehao Zhang, Sarah Mitchell, Jennifer Durham, Xuan Yuan, Hongqui Yang, Elana J. Fertig, Elizabeth M. Jaffee, Katherine M. Bever, Dung T. Le, Won Jin Ho. Combination of CXCR4 antagonist and anti-PD1 therapy results in significant mobilization and increased infiltration of myeloid cells into the metastatic liver microenvironment of PDAC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2270.