Monocytic Differentiation Research Articles

NFκB and NLRP3/NLRC4 inflammasomes regulate differentiation, activation and functional properties of monocytes in response to distinct SARS-CoV-2 proteins

Increased recruitment of transitional and non-classical monocytes in the lung during SARS-CoV-2 infection is associated with COVID-19 severity. However, whether specific innate sensors mediate the activation or differentiation of monocytes in response to different SARS-CoV-2 proteins remain poorly characterized. Here, we show that SARS-CoV-2 Spike 1 but not nucleoprotein induce differentiation of monocytes into transitional or non-classical subsets from both peripheral blood and COVID-19 bronchoalveolar lavage samples in a NFκB-dependent manner, but this process does not require inflammasome activation. However, NLRP3 and NLRC4 differentially regulated CD86 expression in monocytes in response to Spike 1 and Nucleoprotein, respectively. Moreover, monocytes exposed to Spike 1 induce significantly higher proportions of Th1 and Th17 CD4 + T cells. In contrast, monocytes exposed to Nucleoprotein reduce the degranulation of CD8 + T cells from severe COVID-19 patients. Our study provides insights in the differential impact of innate sensors in regulating monocytes in response to different SARS-CoV-2 proteins, which might be useful to better understand COVID-19 immunopathology and identify therapeutic targets.

M1/M2 macrophages: origin, phenotype, methods of production, interaction with natural killer cells and trophoblast

This review presents current data on the origin of monocytes/macrophages, the conditions necessary for the differentiation of monocytes into M1 or M2 macrophages. Three subpopulations of peripheral blood monocytes are described: (I) classical – the main subpopulation (85-90%), effectively carrying out phagocytosis; (II) intermediate monocytes (5-10%) – participate in antigen processing and presentation, in angiogenesis, vascular endothelium restoration; (III) non-classical monocytes (10%) - "patrol" vascular network, remove cellular debris, participate in tissue remodeling. The review provides detailed characteristics for each subclass of macrophages: pro-inflammatory (M1) and anti-inflammatory (M2), which play different roles in the initiation and resolution of inflammation; their phenotype, the spectrum of secreted cytokines, the expression of transcription factors, and the functions performed are described. For the M2 population, the features of the subpopulation are described in detail: M2a, M2b, M2c, M2d. The review presents methods and approaches to obtaining polarized macrophages in vitro from both peripheral blood monocytes and cells of transplanted cultures based on signals received by macrophages in vivo; the phenotype, cytokine production and functional properties of artificially polarized macrophages depending on the conditions of their production are given. The review examines in detail the features of contact and distant interaction of macrophages of various subclasses with microenvironment cells on the example of natural killer cells and trophoblast cells, provides information on changes in the phenotype, transcriptional and secretory profile of interacting cells. The mechanisms of trophoblast control of macrophage differentiation into a unique M2 population of decidual macrophages controlling both the development and functioning of the trophoblast and its apoptosis are described. The review examines in detail the currently known variants of the interaction of macrophage subpopulations with natural killers. The influence of Mf on NK cells manifests itself in a change in the expression of transcription factors by the latter, which determine not only their differentiation, but also their functional activity. Macrophages are considered as cells that actively influence the functional state and differentiation of natural killers. The review examines the mechanisms of the relationship of all three types of cells: macrophages, trophoblast and natural killers in the area of uteroplacental contact. The study of the interactions of these cells will shed light not only on the features of intercellular relationships in the area of uteroplacental contact, but also on the relationship of tumor cells with NK cells and macrophages.

Role of CD4+ T-cells for regulating splenic myelopoiesis and monocyte differentiation after experimental myocardial infarction

Myocardial infarction (MI) induces the generation of proinflammatory Ly6Chigh monocytes in the spleen and the recruitment of these cells to the myocardium. CD4+ Foxp3+ CD25+ T-cells (Tregs) promote the healing process after myocardial infarction by engendering a pro-healing differentiation state in myocardial monocyte-derived macrophages. We aimed to study the effects of CD4+ T-cells on splenic myelopoiesis and monocyte differentiation. We instigated MI in mice and found that MI-induced splenic myelopoiesis is abrogated in CD4+ T-cell deficient animals. Conventional CD4+ T-cells promoted myelopoiesis in vitro by cell–cell-contact and paracrine mechanisms, including interferon-gamma (IFN-γ) signalling. Depletion of regulatory T-cells enhanced myelopoiesis in vivo, as evidenced by increases in progenitor cell numbers and proliferative activity in the spleen 5 days after MI. The frequency of CD4+ T-cells-producing factors that promote myelopoiesis increased within the spleen of Treg-depleted mice. Moreover, depletion of Tregs caused a proinflammatory bias in splenic Ly6Chigh monocytes, which showed predominantly upregulated expression of IFN-γ responsive genes after MI. Our results indicate that conventional CD4+ T-cells promote and Tregs attenuate splenic myelopoiesis and proinflammatory differentiation of monocytes.

Endothelial cell-derived extracellular vesicles expressing surface VCAM1 promote sepsis-related acute lung injury by targeting and reprogramming monocytes.

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a common life-threatening syndrome with no effective pharmacotherapy. Sepsis-related ARDS is the main type of ARDS and is more fatal than other types. Extracellular vesicles (EVs) are considered novel mediators in the development of inflammatory diseases. Our previous research suggested that endothelial cell-derived EVs (EC-EVs) play a crucial role in ALI/ARDS development, but the mechanism remains largely unknown. Here, we demonstrated that the number of circulating EC-EVs was increased in sepsis, exacerbating lung injury by targeting monocytes and reprogramming them towards proinflammatory macrophages. Bioinformatics analysis and further mechanistic studies revealed that vascular cell adhesion molecule 1 (VCAM1), overexpressed on EC-EVs during sepsis, activated the NF-κB pathway by interacting with integrin subunit alpha 4 (ITGA4) on the monocyte surface, rather than the tissue resident macrophage surface, thereby regulating monocyte differentiation. This effect could be attenuated by decreasing VCAM1 levels in EC-EVs or blocking ITGA4 on monocytes. Furthermore, the number of VCAM1+ EC-EVs was significantly increased in patients with sepsis-related ARDS. These findings not only shed light on a previously unidentified mechanism underling sepsis-related ALI/ARDS, but also provide potential novel targets and strategies for its precise treatment.

Mesenchymal Stem Cells from the Deciduous Tooth Pulp Lose their Ability to Suppress the Differentiation of Dendritic Cells during Long-Term Culturing.

A culture of cells expressing markers of mesenchymal stem cells (MSC) (CD73, CD90, CD44, CD29, and CD49b), but not hematopoietic cell markers, and capable of multilineage differentiation was isolated from the deciduous tooth pulp. Co-culturing with immature dendritic cells in the presence of LPS did not reveal an ability of the MSC to suppress the maturation of dendritic cells. On the contrary, co-culturing of MSC with monocytes in the presence of granulocyte-macrophage CSF and IL-4 led to complete suppression of monocyte differentiation into dendritic cells. However, long-term culturing of MSC from dental pulp showed that by the passage 11, they almost completely lose their suppressor ability. These results indicate that the immunological properties of MSC can change during culturing without changing their phenotypic markers. This should be taken into account when creating biomedical cell products.

Pathogenic GATA2 genetic variants utilize an obligate enhancer mechanism to distort a multilineage differentiation program

Mutations in genes encoding transcription factors inactivate or generate ectopic activities to instigate pathogenesis. By disrupting hematopoietic stem/progenitor cells, GATA2 germline variants create a bone marrow failure and leukemia predisposition, GATA2 deficiency syndrome, yet mechanisms underlying the complex phenotypic constellation are unresolved. We used a GATA2-deficient progenitor rescue system to analyze how genetic variation influences GATA2 functions. Pathogenic variants impaired, without abrogating, GATA2-dependent transcriptional regulation. Variants promoted eosinophil and repressed monocytic differentiation without regulating mast cell and erythroid differentiation. While GATA2 and T354M required the DNA-binding C-terminal zinc finger, T354M disproportionately required the N-terminal finger and N terminus. GATA2 and T354M activated a CCAAT/Enhancer Binding Protein-ε (C/EBPε) enhancer, creating a feedforward loop operating with the T-cell Acute Lymphocyte Leukemia-1 (TAL1) transcription factor. Elevating C/EBPε partially normalized hematopoietic defects of GATA2-deficient progenitors. Thus, pathogenic germline variation discriminatively spares or compromises transcription factor attributes, and retaining an obligate enhancer mechanism distorts a multilineage differentiation program.

Single-cell RNA profiling reveals classification and characteristics of mononuclear phagocytes in colorectal cancer.

Colorectal cancer (CRC) is a major cause of cancer mortality and a serious health problem worldwide. Mononuclear phagocytes are the main immune cells in the tumor microenvironment of CRC with remarkable plasticity, and current studies show that macrophages are closely related to tumor progression, invasion and dissemination. To understand the immunological function of mononuclear phagocytes comprehensively and deeply, we use single-cell RNA sequencing and classify mononuclear phagocytes in CRC into 6 different subsets, and characterize the heterogeneity of each subset. We find that tissue inhibitor of metalloproteinases (TIMPs) involved in the differentiation of proinflammatory and anti-inflammatory mononuclear phagocytes. Trajectory of circulating monocytes differentiation into tumor-associated macrophages (TAMs) and the dynamic changes at levels of transcription factor (TF) regulons during differentiation were revealed. We also find that C5 subset, characterized by activation of lipid metabolism, is in the terminal state of differentiation, and that the abundance of C5 subset is negatively correlated with CRC patients' prognosis. Our findings advance the understanding of circulating monocytes' differentiation into macrophages, identify a new subset associated with CRC prognosis, and reveal a set of TF regulons regulating mononuclear phagocytes differentiation, which are expected to be potential therapeutic targets for reversing immunosuppressive tumor microenvironment.

Intestinal stroma guides monocyte differentiation to macrophages through GM-CSF

Stromal cells support epithelial cell and immune cell homeostasis and play an important role in inflammatory bowel disease (IBD) pathogenesis. Here, we quantify the stromal response to inflammation in pediatric IBD and reveal subset-specific inflammatory responses across colon segments and intestinal layers. Using data from a murine dynamic gut injury model and human ex vivo transcriptomic, protein and spatial analyses, we report that PDGFRA+CD142−/low fibroblasts and monocytes/macrophages co-localize in the intestine. In primary human fibroblast-monocyte co-cultures, intestinal PDGFRA+CD142−/low fibroblasts foster monocyte transition to CCR2+CD206+ macrophages through granulocyte-macrophage colony-stimulating factor (GM-CSF). Monocyte-derived CCR2+CD206+ cells from co-cultures have a phenotype similar to intestinal CCR2+CD206+ macrophages from newly diagnosed pediatric IBD patients, with high levels of PD-L1 and low levels of GM-CSF receptor. The study describes subset-specific changes in stromal responses to inflammation and suggests that the intestinal stroma guides intestinal macrophage differentiation.

VEGFR-3 signaling in macrophages: friend or foe in disease?

Lymphatic vessels have been increasingly appreciated in the context of immunology not only as passive conduits for immune and cancer cell transport but also as key in local tissue immunomodulation. Targeting lymphatic vessel growth and potential immune regulation often takes advantage of vascular endothelial growth factor receptor-3 (VEGFR-3) signaling to manipulate lymphatic biology. A receptor tyrosine kinase, VEGFR-3, is highly expressed on lymphatic endothelial cells, and its signaling is key in lymphatic growth, development, and survival and, as a result, often considered to be "lymphatic-specific" in adults. A subset of immune cells, notably of the monocyte-derived lineage, have been identified to express VEGFR-3 in tissues from the lung to the gut and in conditions as varied as cancer and chronic kidney disease. These VEGFR-3+ macrophages are highly chemotactic toward the VEGFR-3 ligands VEGF-C and VEGF-D. VEGFR-3 signaling has also been implicated in dictating the plasticity of these cells from pro-inflammatory to anti-inflammatory phenotypes. Conversely, expression may potentially be transient during monocyte differentiation with unknown effects. Macrophages play critically important and varied roles in the onset and resolution of inflammation, tissue remodeling, and vasculogenesis: targeting lymphatic vessel growth and immunomodulation by manipulating VEGFR-3 signaling may thus impact macrophage biology and their impact on disease pathogenesis. This mini review highlights the studies and pathologies in which VEGFR-3+ macrophages have been specifically identified, as well as the activity and polarization changes that macrophage VEGFR-3 signaling may elicit, and affords some conclusions as to the importance of macrophage VEGFR-3 signaling in disease.

Along with PaO2/FiO2 ratio and lymphopenia, low HLA-DR monocytes are the only additional parameter that independently predicts the clinical course of undifferentiated SARS-CoV-2 patients in emergency departments.

Because one-third of patients deteriorate after their admission to the emergency department, assessing the prognosis of COVID-19 patients is of great importance. However, to date, only lymphopenia and the partial pressure of oxygen/fraction of inspired oxygen (PaO2/FiO2) ratio have been reported as partly predictive of COVID-19-related further deterioration, and their association has not been evaluated. We asked whether other key biomarkers of SARS-CoV-2 immunologic defects-increase in circulating immature granulocytes, loss of monocyte HLA-DR (mHLA-DR) expression, and monocyte differentiation blockade-could also predict further COVID-19 deterioration. A series of 284 consecutive COVID-19 patients, with the sole inclusion criterion of being an adult, were prospectively enrolled at emergency department admission (day 0) of 2 different hospitals: 1 for the exploratory cohort (180 patients) and 1 for the confirmatory cohort (104 patients). Deterioration was assessed over the next 7 days. Neither increased immature granulocyte levels nor monocyte differentiation blockade predicted patient worsening. Among more than 30 clinical, biological, and radiological parameters, the value of decreased P/F ratio and lymphopenia for prediction of further COVID-19 deterioration was strongly confirmed, and the loss of mHLA-DR was the only additional independent marker. Combined together in a simple OxyLymphoMono score, the 3 variables perfectly predicted patients who did not worsen and correctly predicted worsening in 59% of cases. By highlighting lymphocyte and monocyte defects as preceding COVID-19 deterioration, these results point on early immunosuppression in COVID-19 deterioration. Combining P/F ratio, lymphopenia, and loss of mHLA-DR together in a simple and robust score could offer a pragmatic method for COVID-19 patient stratification.

Adipose-derived stem cell exosomes promote tumor characterization and immunosuppressive microenvironment in breast cancer

Adipose-derived stem cells (ASC) or autologous fat transplantation could be used to ameliorate breast cancer postoperative deformities. This study aims to explore the action of ASC and ASC-exosomes (ASC-exos) in breast cancer characterization and tumor microenvironment immunity, which provided a new method into the application of ASC-exos. ASC were extracted from human adipose tissue for the isolation and verification of ASC-exos. ASC-exos were co-cultured with CD4+T cells, CD14+ monocytes and MCF-7 cells, respectively. The tumor formation of nude mice was also constructed. Cell characterization was determined by CCK8, scratch assay, and Transwell. Hematoxylin–eosin (HE), immunohistochemistry (IHC) and immunofluorescence (IF) staining were used to observe the histopathology and protein expression. CD4+T cell and CD14+ monocytes differentiation was detected by flow cytometry. Western blot, qRT-PCR and RNAseq were used to detect the action of ASC-exos on gene and protein expression. CD4+T cells could take up ASC-exos. ASC-exos inhibited Th1 and Th17 differentiation and promoted Treg differentiation of CD4+T cells. ASC-exos inhibited M1 differentiation and promoted M2 differentiation of CD14+ monocytes. ASC-exos promoted the migration, proliferation, and invasion, while inhibited apoptosis of MCF-7 cells. ASC-exos promoted the tumor formation of breast cancer. The effect of ASC-exos on tumor microenvironment immunity was in accordance with the above in vitro results. TOX, CD4 and LYZ1 genes were upregulated, while Mettl7b and Serpinb2 genes were downregulated in ASC-exos group. Human T-cell leukemia virus 1 infection pathway was significantly enriched in ASC-exos. Thus, ASC-exos promoted breast cancer characterization and tumor microenvironment immunosuppression by regulating macrophage and T cell differentiation.

P120 A novel population of classical monocytes is expanded in CD and defined by an upregulation of genes relating to mononuclear cell differentiation and response to microbes

Abstract Background In Crohn's disease (CD), differentiation of classical blood monocytes to intestinal macrophages in the mucosa generates inflammatory rather than regulatory cells. Microbial products and cytokines can systemically reprogramme monocytes via effects on myelopoiesis. We hypothesised that in CD transcriptional changes occur in monocytes prior to their recruitment to the intestine which predispose to pro-inflammatory signatures in the mucosa. Here, we used single cell RNAsequencing (scRNAseq) to explore monocyte heterogeneity and identify CD-associated changes in gene expression profiles. Methods PBMCs were isolated from newly diagnosed CD patients (n=10) and matched healthy controls (HC, n=10). scRNAseq was performed using the 10x Genomics Chromium 3’ assay at a target capture rate of 10,000 cells/sample and sequencing at ~50,000 reads/cell. A dataset was generated using Cell Ranger and following unsupervised clustering (using Seurat) the CD14+ and CD16+ monocyte populations were selected. Data were further analysed using Seurat, DeSEQ2, g:Profiler, GSEA and Cytoscape. Results In addition to non-classical and intermediate monocytes, five transcriptionally distinct clusters of classical monocytes were identified in both CD patients and controls. One cluster (‘cluster 3’) was significantly increased in abundance in CD; other clusters were present at similar frequency in health and CD. Cluster 3 was defined by gene ontology (GO) pathways related to response to bacteria, cell migration, lymphocyte proliferation and response to oxidative stress. In CD compared with health, the most upregulated genes included those encoding EGR (early growth response) proteins 1-3 that play a key role in the functional plasticity of monocytes, and the key inflammatory cytokine IL-1β, as well as cytokines and regulators of their signalling (e.g. CXCL2/3, SOCS3). Overall, in Cluster 3, GO pathways positively enriched in CD related to microbial response signalling, canonical WNT signalling and mononuclear cell differentiation. Conclusion Classical monocytes are heterogeneous and include a novel subset that is expanded in CD. In this population, the expression of genes and pathways related to functional plasticity, response to microbes and inflammatory activity are enhanced in CD. These data support a systemic reprogramming of monocytes in CD that predisposes to inflammatory activity upon recruitment to the intestine.

STIM2 is involved in the regulation of apoptosis and the cell cycle in normal and malignant monocytic cells.

Calcium is a ubiquitous messenger that regulates a wide range of cellular functions, but its involvement in the pathophysiology of acute myeloid leukemia (AML) is not widely investigated. Here, we identified, from an analysis of The Cancer Genome Atlas and genotype-tissue expression databases, stromal interaction molecule 2 (STIM2) as being highly expressed in AML with monocytic differentiation and negatively correlated with overall survival. This was confirmed on a validation cohort of 407 AML patients. We then investigated the role of STIM2 in cell proliferation, differentiation, and survival in two leukemic cell lines with monocytic potential and in normal hematopoietic stem cells. STIM2 expression increased at the RNA and protein levels upon monocyte differentiation. Phenotypically, STIM2 knockdown drastically inhibited cell proliferation and induced genomic stress with DNA double-strand breaks, as shown by increased levels of phosphorylate histone H2AXγ (p-H2AXγ), followed by activation of the cellular tumor antigen p53 pathway, decreased expression of cell cycle regulators such as cyclin-dependent kinase 1 (CDK1)-cyclin B1 and M-phase inducer phosphatase 3 (CDC25c), and a decreased apoptosis threshold with a low antiapoptotic/proapoptotic protein ratio. Our study reports STIM2 as a new actor regulating genomic stability and p53 response in terms of cell cycle and apoptosis of human normal and malignant monocytic cells.

CSF-1R in Cancer: More than a Myeloid Cell Receptor.

Colony-stimulating factor 1 receptor (CFS-1R) is a myeloid receptor with a crucial role in monocyte survival and differentiation. Its overexpression is associated with aggressive tumors characterized by an immunosuppressive microenvironment and poor prognosis. CSF-1R ligands, IL-34 and M-CSF, are produced by many cells in the tumor microenvironment (TME), suggesting a key role for the receptor in the crosstalk between tumor, immune and stromal cells in the TME. Recently, CSF-1R expression was reported in the cell membrane of the cancer cells of different solid tumors, capturing the interest of various research groups interested in investigating the role of this receptor in non-myeloid cells. This review summarizes the current data available on the expression and activity of CSF-1R in different tumor types. Notably, CSF-1R+ cancer cells have been shown to produce CSF-1R ligands, indicating that CSF-1R signaling is positively regulated in an autocrine manner in cancer cells. Recent research demonstrated that CSF-1R signaling enhances cell transformation by supporting tumor cell proliferation, invasion, stemness and drug resistance. In addition, this review covers recent therapeutic strategies, including monoclonal antibodies and small-molecule inhibitors, targeting the CSF-1R and designed to block the pro-oncogenic role of CSF-1R in cancer cells.

MULTILINEAGE DIFFERENTIATION OF C2C12 CELLS AND MONOCYTES IN VITRO WITHIN A 6-BROMOINDIRUBIN-3’-OXIME-INCORPORATED CHITOSAN-BASED SCAFFOLD FOR ENHANCED BONE REGENERATION

Critical size bone defects deriving from large bone loss are an unmet clinical challenge1. To account for disadvantages with clinical treatments, researchers focus on designing biological substitutes, which mimic endogenous healing through osteogenic differentiation promotion. Some studies have however suggested that this notion fails to consider the full complexity of native bone with respect to the interplay between osteoclast and osteoblasts, thus leading to the regeneration of less functional tissue2. The objective of this research is to assess the ability of our laboratory's previously developed 6-Bromoindirubin-3’-Oxime (BIO) incorporated guanosine diphosphate crosslinked chitosan scaffold in promoting multilineage differentiation of myoblastic C2C12 cells and monocytes into osteoblasts and osteoclasts1, 3, 4. BIO addition has been previously demonstrated to promote osteogenic differentiation in cell cultures5, but implementation of a co-culture model here is expected to encourage crosstalk thus further supporting differentiation, as well as the secretion of regulatory molecules and cytokines2.Biocompatibility testing of both cell types is performed using AlamarBlue for metabolic activity, and nucleic acid staining for distribution. Osteoblastic differentiation is assessed through quantification of ALP and osteopontin secretion, as well as osteocalcin and mineralization staining. Differentiation into osteoclasts is verified using SEM and TEM, qPCR, and TRAP staining.Cellular viability of C2C12 cells and monocytes was maintained when cultured separately in scaffolds with and without BIO for 21 days. Both scaffold variations showed a characteristic increase in ALP secretion from day 1 to 7, indicating early differentiation but BIO-incorporated sponges yielded higher values compared to controls. SEM and TEM imaging confirmed initial aggregation and fusion of monocytes on the scaffold's surface, but BIO addition appeared to result in smoother cell surfaces indicating a change in morphology. Late-stage differentiation assessment and co-culture work in the scaffold are ongoing, but initial results show promise in the material's ability to support multilineage differentiation.Acknowledgements: The authors would like to acknowledge the financial support of the Collaborative Health Research Program (CHRP) through CIHR and NSERC, as well as Canada Research Chair – Tier 1 in Regenerative Medicine and Nanomedicine, and the FRQ-S.

Cellular and microenvironmental cues that promote macrophage fusion and foreign body response.

During the foreign body response (FBR), macrophages fuse to form foreign body giant cells (FBGCs). Modulation of FBGC formation can prevent biomaterial degradation and loss of therapeutic efficacy. However, the microenvironmental cues that dictate FBGC formation are poorly understood with conflicting reports. Here, we identified molecular and cellular factors involved in driving FBGC formation in vitro. Macrophages demonstrated distinct fusion competencies dependent on monocyte differentiation. The transition from a proinflammatory to a reparative microenvironment, characterised by specific cytokine and growth factor programmes, accompanied FBGC formation. Toll-like receptor signalling licensed the formation of FBGCs containing more than 10 nuclei but was not essential for cell-cell fusion to occur. Moreover, the fibroblast-macrophage crosstalk influenced FBGC development, with the fibroblast secretome inducing macrophages to secrete more PDGF, which enhanced large FBGC formation. These findings advance our understanding as to how a specific and timely combination of cellular and microenvironmental factors is required for an effective FBR, with monocyte differentiation and fibroblasts being key players.

Resistin is a potential marker of the activity of skin diseases accompanied by urticarial rashes

Resistin is a cysteine-rich secretory protein. This protein expressed in various human organs and tissues, including white adipose tissue, lung, spleen, adrenal gland, pituitary gland, hypothalamus, intestinal epithelium, placenta, pancreas, stomach, skeletal muscle, skin and plasma. Resistin expression increases during the differentiation of monocytes into macrophages and induced by proinflammatory cytokines such as IL-1, IL-6, IL-12 and TNF-a. After translation, resistin is stored on the neutrophil membrane, as well as in azurophilic and specific granules. Resistin is released upon activation of neutrophils. Increased resistin levels lead to increased production of proinflammatory cytokines, creating a circle of chronic inflammation. Currently, the role of neutrophils in the pathogenesis of various chronic skin diseases is being widely studied, but the role of neutrophils in the pathogenesis of diseases accompanied by urticarial rashes remains poorly understood. Therefore it is of interest to study clinical and diagnostic significance of the level of resistin in peripheral blood in diseases accompanied by chronic urticarial rashes. Our aim was to determine resistin levels in chronic spontaneous urticaria and urticarial vasculitis and its correlation with the severity of these conditions. Materials and methods. A prospective observational study was conducted. The work included 30 patients with diagnoses of CSU, UV, HUV and HUVS, aged from 19 to 71 years, 42±15 [37;48], of which 25 were women and 5 men. Recruitment of patients was carried out at the Clinic of Skin and Venereal Diseases named after. V.A. Rakhmanov Sechenov University in the period from February 2022 to April 2023. Results. Resistin levels didn’t vary significantly depending on the diagnosis and were within normal values in most patients. However, when conducting a correlation analysis of the relationship between the level of serum resistin and the disease activity index, a moderately close direct relationship was established. In addition, when assessing the relationship between resistin levels and CRP levels, a weak direct relationship was established. No statistically significant relationships between CRP, ESR, leukocyte and neutrophil counts and diagnosis were found. Conclusion. Taking into account the results of our study, resistin can be considered as a biological marker of the activity of CSU, UV, HUV and HUVS. In addition, these data may be useful for a deeper and more detailed study of the pathogenesis of diseases, as well as for the development of innovative therapeutic methods.

Met-Flow analyses of the metabolic heterogeneity associated with different stages of cord blood-derived hematopoietic cell differentiation.

The differentiation of hematopoietic cells is significantly affected by cell metabolic activity. However, despite increasing interest in this field, there has been no comprehensive investigation of the metabolic functions of human hematopoietic cells during specific phases of differentiation. Thus, this study was conducted to develop a method for comparing hematopoietic cell lineage differentiation based on the metabolic functions of the cell. The metabolic activity of human umbilical cord-derived hematopoietic cells was examined during various phases of differentiation, specifically, hematopoietic stem cells (HSCs), hematopoietic progenitor cells, and differentiated blood cells. This approach was used to develop comprehensive metabolic maps corresponding to the different stages. HSCs were found to have robust fatty acid (FA) synthesis, FA oxidation, pentose phosphate pathway (PPP) activity, and glucose uptake, shown by their significantly higher expression of ACAC, CPT1A, G6PD, and GLUT1 as compared to differentiated pluripotent progenitor cells, common myeloid progenitors, megakaryocyte erythroid progenitors, lympho-myeloid primed progenitors, and granulocyte-macrophage progenitor cell populations. In monocytic differentiation, higher levels of ACAC, ASS1, ATP5A, CPT1A, G6PD, GLUT1, IDH2, PRDX2, and HK1 protein expression were evident in classical and intermediate monocytes relative to non-classical monocytes, consistent with high anabolic and catabolic levels. Compared with myelocytes and mature cells, the meta-myelocyte and pro-myelocyte populations of granulocytes show significantly elevated levels of ACAC, ASS1, ATP5A, CPT1A, G6PD, IDH2, PRDX2, and HK. In contrast to naïve and regulatory B cells, pro-B cells had higher levels of oxidative phosphorylation, while regulatory B cells showed greater PPP activity, glucose uptake, and tricarboxylic acid cycle activity. The analyses of T cells also indicated significantly higher ACAC, ASS1, ATP5A, CPT1A, G6PD, GLUT1, IDH2, PRDX2, and HK1 expression levels in CD4+ populations compared with CD8+ populations. The results provide comprehensive analytical methods and reference values for future systematic studies into the metabolic functions of various cord blood-derived hematopoietic cell populations in different pathological or physiological conditions. These findings could also contribute to research on the connection between cellular metabolism and cancer or aging.

Role and origin of neutrophil-like regulatory monocytes and comparison with neutrophils

My colleagues and I previously found a subset of neutrophil-like Ly6Chi monocytes, named "regulatory monocytes", that expand in the bone marrow during the late phase of inflammation. Regulatory monocytes migrate to injured tissue where they promote tissue repair. Unlike classical Ly6Chi monocytes, regulatory monocytes arise from GMP through proNeu1, which was previously thought to be committed to becoming neutrophils. G-CSF not only stimulates neutrophil differentiation but also drives the expansion of regulatory monocytes in the absence of inflammatory stimuli. The human parallel to mouse regulatory monocytes was found in the peripheral blood CD14hiCD16lo monocyte fraction. These monocytes can be distinguished from classical CD14hiCD16lo monocytes by neutrophil marker CXCR1 expression. Like mouse regulatory monocytes, human CXCR1+ monocytes arise from neutrophil progenitors in response to G-CSF. CXCR1+CD14hiCD16lo monocytes suppressed the proliferation of syngeneic T cells in vitro, which suggests an immunosuppressive phenotype. Overall, these findings indicate that the process of differentiation of regulatory monocytes from progenitors of neutrophil lineage is maintained across humans and mice, and may aid in resolution of excess inflammation.

The Modulation of in Vitro Differentiation of Monocyte-derived Macrophage by Trypanosoma evansi Antigens in the Dromedary Camel

Studies on the camel immune response to Trypanosoma (T.) evansi, the causative agent of Surra, are very limited. In the present study, flow cytometry was employed to investigate the modulatory effects of different T. evansi antigens on the in vitro differentiation of camel blood monocytes into macrophages. For this, in vitro, separated camel monocytes were differentiated into monocyte-derived macrophages (MDM) in the presence or absence (control) of formalin-fixed (inactivated) T. evansi whole parasite (T. evansi group) or the purified Ro Tat 1.2 antigen (Ro Tat 1.2 group). The analysis of the antimicrobial functions of MDM (phagocytosis and reactive oxygen species (ROS) production) revealed reduced phagocytosis activity of camel MDM generated in the presence of T. evansi antigens. In addition, a lack of ROS-response was observed in camel MDM generated in the presence of T. evansi antigens after stimulation with PMA. These results indicated a compromising effect of T. evansi on the innate defense mechanisms in camels. Phenotypic analysis revealed the upregulation of major histocompatibility complex (MHC) class II molecules together with the lower abundance of the scavenger receptor for haptoglobin–hemoglobin complexes (CD163) on MDM generated in the presence of whole T. evansi parasites, indicating a polarizing effect of T. evansi on the differentiation of camel monocytes into an M1 phenotype. However, the reduced antimicrobial functions of these cells argue against their pro-inflammatory nature. Although both MDM generated in the presence of whole T. evansi antigens or their purified Ro Tat 1.2 proteins indicated similar expression levels of CD14 and MHCII molecules, the different abundance of the cell surface molecules CD172a, CD163, CD45, and CD44 indicated different phenotypes of the two MDMs. The results of the present study revealed compromising effects of T. evansi antigens on camel macrophages differentiated in vitro from blood monocytes. Whether these effects contribute to the in vivo pathogenesis of T. evansi in camels remains to be determined in future studies.