ß-glucans from Euglena gracilis or Saccharomyces cerevisiae effects on immunity and inflammatory parameters in dogs.

Considering the differences in molecular structure and function, the effects of ß-1,3-glucans from Euglena gracilis and ß-1,3/1,6-glucans from Saccharomyces cerevisiae on immune and inflammatory activities in dogs were compared. Four diets were compared: control without ß-glucans (CON), 0.15 mg/kg BW/day of ß-1,3/1,6-glucans (Β-Y15), 0.15 mg/kg BW/day of ß-1,3-glucans (Β-S15), and 0.30 mg/kg BW/day of ß-1,3-glucans (Β-S30). Thirty-two healthy dogs (eight per diet) were organized in a block design. All animals were fed CON for a 42-day washout period and then sorted into one of four diets for 42 days. Blood and faeces were collected at the beginning and end of the food intake period and analysed for serum and faecal cytokines, ex vivo production of hydrogen peroxide (H2O2) and nitric oxide (NO), phagocytic activity of neutrophils and monocytes, C-reactive protein (CRP), ex vivo production of IgG, and faecal concentrations of IgA and calprotectin. Data were evaluated using analysis of covariance and compared using Tukey's test (P<0.05). Dogs fed Β-Y15 showed higher serum IL-2 than dogs fed Β-S30 (P<0.05). A higher phagocytic index of monocytes was observed in dogs fed the B-S15 diet than in those fed the other diets, and a higher neutrophil phagocytic index was observed for B-S15 and B-Y15 than in dogs fed the CON diet (P<0.05). Monocytes from dogs fed B-S15 and B-S30 produced more NO and less H2O2 than those from the CON and B-Y15 groups (P<0.05). Despite in the reference value, CRP levels were higher in dogs fed B-S15 and B-S30 diets (P<0.05). ß-1,3/1,6-glucan showed cell-mediated activation of the immune system, with increased serum IL-2 and neutrophil phagocytic index, whereas ß-1,3-glucan acted on the immune system by increasing the ex vivo production of NO by monocytes, neutrophil phagocytic index, and serum CRP. Calprotectin and CRP levels did not support inflammation or other health issues related to ß-glucan intake. In conclusion, both ß-glucan sources modulated some immune and inflammatory parameters in dogs, however, different pathways have been suggested for the recognition and action of these molecules, reinforcing the necessity for further mechanistic studies, especially for E. gracilis ß-1,3-glucan.

Myeloperoxidase induces monocyte migration and activation after acute myocardial infarction.

Introduction: Myocardial infarction (MI) is a significant contributor to morbidity and mortality worldwide. Many individuals who survive the acute event continue to experience heart failure (HF), with inflammatory and healing processes post-MI playing a pivotal role. Polymorphonuclear neutrophils (PMN) and monocytes infiltrate the infarcted area, where PMN release high amounts of the heme enzyme myeloperoxidase (MPO). MPO has numerous inflammatory properties and MPO plasma levels are correlated with prognosis and severity of MI. While studies have focused on MPO inhibition and controlling PMN infiltration into the infarcted tissue, less is known on MPO's role in monocyte function. Methods and results: Here, we combined human data with mouse and cell studies to examine the role of MPO on monocyte activation and migration. We revealed a correlation between plasma MPO levels and monocyte activation in a patient study. Using a mouse model of MI, we demonstrated that MPO deficiency led to an increase in splenic monocytes and a decrease in cardiac monocytes compared to wildtype mice (WT). In vitro studies further showed that MPO induces monocyte migration, with upregulation of the chemokine receptor CCR2 and upregulation of inflammatory pathways identified as underlying mechanisms. Conclusion: Taken together, we identify MPO as a pro-inflammatory mediator of splenic monocyte recruitment and activation post-MI and provide mechanistic insight for novel therapeutic strategies after ischemic injury.

The amoeboid migration of monocytes in confining channels requires the local remodeling of the cortical actin cytoskeleton by cofilin-1.

Within the bloodstream, monocytes must traverse the microvasculature to prevent leukostasis, which is the entrapment of monocytes within the confines of the microvasculature. Using the model cell line, THP-1, and VCAM-1 coated channels to simulate the microvasculature surface, we demonstrate that monocytes predominantly adopt an amoeboid phenotype, which is characterized by the formation of blebs. As opposed to cortical actin flow in leader blebs, cell movement is correlated with myosin contraction at the cell rear. It was previously documented that cofilin-1 promotes cortical actin turnover at leader bleb necks in melanoma cells. In monocytes, our data suggest that cofilin-1 promotes the local upregulation of myosin contractility through actin cytoskeleton remodeling. In support of this concept, cofilin-1 is found to localize to a single cell edge. Moreover, the widespread upregulation of myosin contractility was found to inhibit migration. Thus, monocytes within the microvasculature may avoid entrapment by adopting an amoeboid mode of migration.

Monocyte bioenergetics: An immunometabolic perspective in metabolic dysfunction-associated steatohepatitis.

Monocytes (Mos) are crucial in the evolution of metabolic dysfunction-associated steatotic liver disease (MASLD) to metabolic dysfunction-associated steatohepatitis (MASH), and immunometabolism studies have recently suggested targeting leukocyte bioenergetics in inflammatory diseases. Here, we reveal a peculiar bioenergetic phenotype in circulating Mos of patients with MASH, characterized by high levels of glycolysis and mitochondrial (mt) respiration. The enhancement of mt respiratory chain activity, especially complex II (succinate dehydrogenase [SDH]), is unbalanced toward the production of reactive oxygen species (ROS) and is sustained at the transcriptional level with the involvement of the AMPK-mTOR-PGC-1α axis. The modulation of mt activity with dimethyl malonate (DMM), an SDH inhibitor, restores the metabolic profile and almost abrogates cytokine production. Analysis of a public single-cell RNA sequencing (scRNA-seq) dataset confirms that in murine models of MASH, liver Mo-derived macrophages exhibit an upregulation of mt and glycolytic energy pathways. Accordingly, the DMM injection in MASH mice contrasts Mo infiltration and macrophagic enrichment, suggesting immunometabolism as a potential target in MASH.

Intermittent fasting alleviates IMQ-induced psoriasis-like dermatitis via reduced γδT17 and monocytes in mice.

Psoriasis is a chronic immune mediated inflammatory skin disease with systemic manifestations. It has been reported that caloric restriction could improve severity of psoriasis patients. However, the mechanism of intermittent fasting effects on psoriasis has not been investigated. Caloric restriction is known to reduce the number of circulating inflammatory monocytes in a CCL2-dependent manner. However, it is still unknown whether caloric restriction can improve psoriasis by regulating monocytes through CCL2. In this study, we used imiquimod (IMQ)-induced psoriasis-like mouse model to explore the effects and the mechanisms of intermittent fasting on psoriasis-like dermatitis. We found that intermittent fasting could significantly improve IMQ-induced psoriasis-like dermatitis, and reduce the number of γδT17 cells and IL-17 production in draining lymph nodes and psoriatic lesion via inhibiting proliferation and increasing death of γδT17 cells. Furthermore, intermittent fasting could significantly decrease monocytes in blood, and this was associated with decreased monocytes, macrophages and DC in psoriasis-like skin inflammation. Reduced monocytes in circulation and increased monocytes in BM of fasting IMQ-induced psoriasis-like mice is through reducing the production of CCL2 from BM to inhibit monocyte egress to the periphery. Our above data shads light on the mechanisms of intermittent fasting on psoriasis.

Comprehensive analysis of human monocyte subsets using full-spectrum flow cytometry and hierarchical marker clustering.

Introduction: Exploring monocytes' roles within the tumor microenvironment is crucial for crafting targeted cancer treatments. Methods: This study unveils a novel methodology utilizing four 20-color flow cytometry panels for comprehensive peripheral immune system phenotyping, specifically targeting classical, intermediate, and non-classical monocyte subsets. Results: By applying advanced dimensionality reduction techniques like t-distributed stochastic neighbor embedding (tSNE) and FlowSom analysis, we performed an extensive profiling of monocytes, assessing 50 unique cell surface markers related to a wide range of immunological functions, including activation, differentiation, and immune checkpoint regulation. Discussion: This in-depth approach significantly refines the identification of monocyte subsets, directly supporting the development of personalized immunotherapies and enhancing diagnostic precision. Our pioneering panel for monocyte phenotyping marks a substantial leap in understanding monocyte biology, with profound implications for the accuracy of disease diagnostics and the success of checkpoint-inhibitor therapies. Key findings include revealing distinct marker expression patterns linked to tumor progression and providing new avenues for targeted therapeutic interventions.

Myeloid cell expression of CD200R is modulated in active TB disease and regulates Mycobacterium tuberculosis infection in a biomimetic model.

A robust immune response is required for resistance to pulmonary tuberculosis (TB), the primary disease caused by Mycobacterium tuberculosis (Mtb). However, pharmaceutical inhibition of T cell immune checkpoint molecules can result in the rapid development of active disease in latently infected individuals, indicating the importance of T cell immune regulation. In this study, we investigated the potential role of CD200R during Mtb infection, a key immune checkpoint for myeloid cells. Expression of CD200R was consistently downregulated on CD14+ monocytes in the blood of subjects with active TB compared to healthy controls, suggesting potential modulation of this important anti-inflammatory pathway. In homogenized TB-diseased lung tissue, CD200R expression was highly variable on monocytes and CD11b+HLA-DR+ macrophages but tended to be lowest in the most diseased lung tissue sections. This observation was confirmed by fluorescent microscopy, which showed the expression of CD200R on CD68+ macrophages surrounding TB lung granuloma and found expression levels tended to be lower in macrophages closest to the granuloma core and inversely correlated with lesion size. Antibody blockade of CD200R in a biomimetic 3D granuloma-like tissue culture system led to significantly increased Mtb growth. In addition, Mtb infection in this system reduced gene expression of CD200R. These findings indicate that regulation of myeloid cells via CD200R is likely to play an important part in the immune response to TB and may represent a potential target for novel therapeutic intervention.

Mycobacterium tuberculosis resides in lysosome-poor monocyte-derived lung cells during chronic infection.

Mycobacterium tuberculosis (Mtb) infects lung myeloid cells, but the specific Mtb-permissive cells and host mechanisms supporting Mtb persistence during chronic infection are incompletely characterized. We report that after the development of T cell responses, CD11clo monocyte-derived cells harbor more live Mtb than alveolar macrophages (AM), neutrophils, and CD11chi monocyte-derived cells. Transcriptomic and functional studies revealed that the lysosome pathway is underexpressed in this highly permissive subset, characterized by less lysosome content, acidification, and proteolytic activity than AM, along with less nuclear TFEB, a regulator of lysosome biogenesis. Mtb infection does not drive lysosome deficiency in CD11clo monocyte-derived cells but promotes recruitment of monocytes that develop into permissive lung cells, mediated by the Mtb ESX-1 secretion system. The c-Abl tyrosine kinase inhibitor nilotinib activates TFEB and enhances lysosome functions of macrophages in vitro and in vivo, improving control of Mtb infection. Our results suggest that Mtb exploits lysosome-poor lung cells for persistence and targeting lysosome biogenesis is a potential host-directed therapy for tuberculosis.

Sensor macrophages derived from human induced pluripotent stem cells to assess pyrogenic contaminations in parenteral drugs.

Ensuring the safety of parenteral drugs before injection into patients is of utmost importance. New regulations around the globe and the need to refrain from using animals however, have highlighted the need for new cell sources to be used in next-generation bioassays to detect the entire spectrum of possible contaminating pyrogens. Given the current drawbacks of the Monocyte-Activation-Test (MAT) with respect to the use of primary peripheral blood mono-nuclear cells or the use of monocytic cell lines, we here demonstrate the manufacturing of sensor monocytes/macrophages from human induced pluripotent stem cells (iMonoMac), which are fully defined and superior to current cell products. Using a modern and scalable manufacturing platform, iMonoMac showed typical macrophage-like morphology and stained positive for several Toll like receptor (TLRs) such as TLR-2, TLR-5, TLR-4. Furthermore, iMonoMac derived from the same donor were sensitive to endotoxins, non-endotoxins, and process related pyrogens at a high dynamic range and across different cellular densities. Of note, iMonoMac showed increased sensitivity and reactivity to a broad range of pyrogens, demonstrated by the detection of interleukin-6 at low concentrations of LPS and MALP-2 which could not be reached using the current MAT cell sources. To further advance the system, iMonoMac or genetically engineered iMonoMac with NF-κB-luciferase reporter cassette could reveal a specific activation response while correlating to the classical detection method employing enzyme-linked immunosorbent assay to measure cytokine secretion. Thus, we present a valuable cellular tool to assess parenteral drugs safety, facilitating the future acceptance and design of regulatory-approved bioassays.

Monocytes prevent apoptosis of iPSCs and promote differentiation of kidney organoids.

BACKGROUND: Induced pluripotent stem cells (iPSCs)-derived kidney organoids are a promising model for studying disease mechanisms and renal development. Despite several protocols having been developed, further improvements are needed to overcome existing limitations and enable a wider application of this model. One of the approaches to improve the differentiation of renal organoids in vitro is to include in the system cell types important for kidney organogenesis in vivo, such as macrophages. Another approach could be to improve cell survival. Mesodermal lineage differentiation is the common initial step of the reported protocols. The glycogen synthase kinase-3 (GSK-3) activity inhibitor, CHIR99021 (CHIR), is applied to induce mesodermal differentiation. It has been reported that CHIR simultaneously induces iPSCs apoptosis that can compromise cell differentiation. We thought to interfere with CHIR-induced apoptosis of iPSCs using rapamycin. METHODS: Differentiation of kidney organoids from human iPSCs was performed. Cell survival and autophagy were analyzed using Cell counting kit 8 (CCK8) kit and Autophagy detection kit. Cells were treated with rapamycin or co-cultured with human monocytes isolated from peripheral blood or iPSCs-macrophages using a transwell co-culture system. Monocyte-derived extracellular vesicles (EVs) were isolated using polyethylene glycol precipitation. Expression of apoptotic markers cleaved Caspase 3, Poly [ADP-ribose] polymerase 1 (PARP-1) and markers of differentiation T-Box Transcription Factor 6 (TBX6), odd-skipped related 1 (OSR1), Nephrin, E-Cadherin, Paired box gene 2 (Pax2) and GATA Binding Protein 3 (Gata3) was assessed by RT-PCR and western blotting. Organoids were imaged by 3D-confocal microscopy. RESULTS: We observed that CHIR induced apoptosis of iPSCs during the initial stage of renal organoid differentiation. Underlying mechanisms implied the accumulation of reactive oxygen species and decreased autophagy. Activation of autophagy by rapamacin and by an indirect co-culture of differentiating iPSCs with iPSCs-macrophages and human peripheral blood monocytes prevented apoptosis induced by CHIR. Furthermore, monocytes (but not rapamycin) strongly promoted expression of renal differentiation markers and organoids development via released extracellular vesicles. CONCLUSION: Our data suggest that co-culturing of iPSCs with human monocytes strongly improves differentiation of kidney organoids. An underlying mechanism of monocytic action implies, but not limited to, an increased autophagy in CHIR-treated iPSCs. Our findings enhance the utility of kidney organoid models.

The Effect of Orally Administered Multi-Strain Probiotic Formulation (Lactobacillus, Bifidobacterium) on the Phagocytic Activity and Oxidative Metabolism of Peripheral Blood Granulocytes and Monocytes in Lambs.

Probiotic feed additives have attracted considerable research interest in recent years because the effectiveness of probiotics can differ across microbial strains and the supplemented macroorganisms. The present study was conducted on 16 lambs divided equally into two groups (C-control and E-experimental). The examined lambs were aged 11 days at the beginning of the experiment and 40 days at the end of the experiment. The diet of group E lambs was supplemented with a multi-strain probiotic formulation (Lactobacillus plantarum AMT14, Lactobacillus plantarum AMT4, Lactobacillus rhamnosus AMT15, and Bifidobacterium animalis AMT30), whereas group C lambs did not receive the probiotic additive. At the beginning of the experiment (day 0) and on experimental days 15 and 30, blood was sampled from the jugular vein to determine and compare: phagocytic activity (Phagotest) and oxidative metabolism (Phagoburst) of peripheral blood granulocytes and monocytes by flow cytometry. An analysis of the phagocytic activity of granulocytes and monocytes revealed significantly higher levels of phagocytic activity (expressed as the percentage of phagocytic cells and mean fluorescence intensity) in lambs that were administered the multi-strain probiotic formulation compared with lambs in the control group. The probiotic feed additive also exerted a positive effect on the oxidative metabolism of both granulocytes and monocytes (expressed as the percentage of oxidative metabolism and mean fluorescence intensity) after stimulation with Escherichia coli bacteria and with PMA (4-phorbol-12-ß-myristate-13-acetate). These findings suggest that the tested probiotic formulation may have a positive effect on the immune status of lambs.

Monocytic Differentiation of Human Acute Myeloid Leukemia Cells: A Proteomic and Phosphoproteomic Comparison of FAB-M4/M5 Patients with and without Nucleophosmin 1 Mutations.

Even though morphological signs of differentiation have a minimal impact on survival after intensive cytotoxic therapy for acute myeloid leukemia (AML), monocytic AML cell differentiation (i.e., classified as French/American/British (FAB) subtypes M4/M5) is associated with a different responsiveness both to Bcl-2 inhibition (decreased responsiveness) and possibly also bromodomain inhibition (increased responsiveness). FAB-M4/M5 patients are heterogeneous with regard to genetic abnormalities, even though monocytic differentiation is common for patients with Nucleophosmin 1 (NPM1) insertions/mutations; to further study the heterogeneity of FAB-M4/M5 patients we did a proteomic and phosphoproteomic comparison of FAB-M4/M5 patients with (n = 13) and without (n = 12) NPM1 mutations. The proteomic profile of NPM1-mutated FAB-M4/M5 patients was characterized by increased levels of proteins involved in the regulation of endocytosis/vesicle trafficking/organellar communication. In contrast, AML cells without NPM1 mutations were characterized by increased levels of several proteins involved in the regulation of cytoplasmic translation, including a large number of ribosomal proteins. The phosphoproteomic differences between the two groups were less extensive but reflected similar differences. To conclude, even though FAB classification/monocytic differentiation are associated with differences in responsiveness to new targeted therapies (e.g., Bcl-2 inhibition), our results shows that FAB-M4/M5 patients are heterogeneous with regard to important biological characteristics of the leukemic cells.

Nano(bio)Materials Do Not Affect Macrophage Phenotype-A Study Conducted by the REFINE Project.

Macrophages are well known for their involvement in the biocompatibility, as well as biodistribution, of nano(bio)materials. Although there are a number of rodent cell lines, they may not fully recapitulate primary cell responses, particularly those of human cells. Isolation of tissue-resident macrophages from humans is difficult and may result in insufficient cells with which to determine the possible interaction with nano(bio)materials. Isolation of primary human monocytes and differentiation to monocyte-derived macrophages may provide a useful tool with which to further study these interactions. To that end, we developed a standard operating procedure for this differentiation, as part of the Regulatory Science Framework for Nano(bio)material-based Medical Products and Devices (REFINE) project, and used it to measure the secretion of bioactive molecules from M1 and M2 differentiated monocytes in response to model nano(bio)materials, following an initial assessment of pyrogenic contamination, which may confound potential observations. The SOP was deployed in two partner institutions with broadly similar results. The work presented here shows the utility of this assay but highlights the relevance of donor variability in responses to nano(bio)materials. Whilst donor variability can provide some logistical challenges to the application of such assays, this variability is much closer to the heterogeneous cells that are present in vivo, compared to homogeneous non-human cell lines.

Krüppel-like Factor-4-Mediated Macrophage Polarization and Phenotypic Transitions Drive Intestinal Fibrosis in THP-1 Monocyte Models In Vitro.

Background and Objectives: Despite the fact that biologic drugs have transformed inflammatory bowel disease (IBD) treatment, addressing fibrosis-related strictures remains a research gap. This study explored the roles of cytokines, macrophages, and Krüppel-like factors (KLFs), specifically KLF4, in intestinal fibrosis, as well as the interplay of KLF4 with various gut components. Materials and Methods: This study examined macrophage subtypes, their KLF4 expression, and the effects of KLF4 knockdown on macrophage polarization and cytokine expression using THP-1 monocyte models. Co-culture experiments with stromal myofibroblasts and a conditioned medium from macrophage subtype cultures were conducted to study the role of these cells in intestinal fibrosis. Human-induced pluripotent stem cell-derived small intestinal organoids were used to confirm inflammatory and fibrotic responses in the human small intestinal epithelium. Results: Each macrophage subtype exhibited distinct phenotypes and KLF4 expression. Knockdown of KLF4 induced inflammatory cytokine expression in M0, M2a, and M2c cells. M2b exerted anti-fibrotic effects via interleukin (IL)-10. M0 and M2b cells showed a high migratory capacity toward activated stromal myofibroblasts. M0 cells interacting with activated stromal myofibroblasts transformed into inflammatory macrophages, thereby increasing pro-inflammatory cytokine expression. The expression of IL-36α, linked to fibrosis, was upregulated. Conclusions: This study elucidated the role of KLF4 in macrophage polarization and the intricate interactions between macrophages, stromal myofibroblasts, and cytokines in experimental in vitro models of intestinal fibrosis. The obtained results may suggest the mechanism of fibrosis formation in clinical IBD.

Dynamic changes in RNA m6A and 5 hmC influence gene expression programs during macrophage differentiation and polarisation.

RNA modifications are essential for the establishment of cellular identity. Although increasing evidence indicates that RNA modifications regulate the innate immune response, their role in monocyte-to-macrophage differentiation and polarisation is unclear. While m6A has been widely studied, other RNA modifications, including 5 hmC, remain poorly characterised. We profiled m6A and 5 hmC epitranscriptomes, transcriptomes, translatomes and proteomes of monocytes and macrophages at rest and pro- and anti-inflammatory states. Transcriptome-wide mapping of m6A and 5 hmC reveals enrichment of m6A and/or 5 hmC on specific categories of transcripts essential for macrophage differentiation. Our analyses indicate that m6A and 5 hmC modifications are present in transcripts with critical functions in pro- and anti-inflammatory macrophages. Notably, we also discover the co-occurrence of m6A and 5 hmC on alternatively-spliced isoforms and/or opposing ends of the untranslated regions (UTR) of mRNAs with key roles in macrophage biology. In specific examples, RNA 5 hmC controls the decay of transcripts independently of m6A. This study provides (i) a comprehensive dataset to interrogate the role of RNA modifications in a plastic system (ii) a resource for exploring different layers of gene expression regulation in the context of human monocyte-to-macrophage differentiation and polarisation, (iii) new insights into RNA modifications as central regulators of effector cells in innate immunity.

Chemokine receptor hetero-oligomers regulate monocyte chemotaxis.

It is known that stress influences immune cell function. The underlying molecular mechanisms are unclear. We recently reported that many chemokine receptors (CRs) heteromerize with α1-adrenoceptors (α1-ARs) through which CRs are regulated. Here, we show that arginine vasopressin receptor 1A (AVPR1A) heteromerizes with all human CRs, except chemokine (C-X-C motif) receptor (CXCR)1, in recombinant systems and that such heteromers are detectable in THP-1 cells and human monocytes. We demonstrate that ligand-free AVPR1A differentially regulates the efficacy of CR partners to mediate chemotaxis and that AVPR1A ligands disrupt AVPR1A:CR heteromers, which enhances chemokine (C-C motif) receptor (CCR)1-mediated chemotaxis and inhibits CCR2-, CCR8-, and CXCR4-mediated chemotaxis. Using bioluminescence resonance energy transfer to monitor G protein activation and CRISPR/Cas9 gene-edited THP-1 cells lacking AVPR1A or α1B-AR, we show that CRs that share the propensity to heteromerize with α1B/D-ARs and AVPR1A exist and function within interdependent hetero-oligomeric complexes through which the efficacy of CRs to mediate chemotaxis is controlled. Our findings suggest that hetero-oligomers composed of CRs, α1B/D-ARs, and AVPR1A may enable stress hormones to regulate immune cell trafficking.

Independent and joint associations of monocyte to high-density lipoprotein-cholesterol ratio and body mass index with cardiorenal syndrome: insights from NHANES 2003-2020.

BACKGROUND: With the development of pathophysiology, cardiorenal syndrome (CRS), a complex and severe disease, has received increasing attention. Monocyte to high-density lipoprotein-cholesterol ratio (MHR) and body mass index (BMI) are independent risk factors for cardiovascular diseases, but their association with CRS remains unexplored. This study aims to explore the independent and joint effects of MHR and BMI on CRS. METHODS: We included 42,178 NHANES participants. The determination of CRS referred to the simultaneous presence of cardiovascular disease (identified through self-report) and chronic kidney disease (eGFR < 60 mL/min per 1.73 m²). We employed multivariate weighted logistic regression to evaluate the odds ratio (OR) and 95% confidence interval (CI) for the independent and joint associations of MHR and BMI with CRS. We also conducted restricted cubic spines to explore nonlinear associations. RESULTS: The prevalence of CRS was 3.45% among all participants. An increase in both MHR and BMI is associated with a higher risk of CRS (MHR: OR = 1.799, 95% CI = 1.520-2.129, P < 0.001, P-trend < 0.001; BMI: OR = 1.037, 95% CI = 1.023-1.051, P < 0.001). Individuals who simultaneously fall into the highest quartile of MHR and have a BMI of 30 or more face the highest risk of CRS compared to those in the lowest MHR quartile with a BMI of less than 25 (OR = 3.45, 95% CI = 2.40-4.98, P < 0.001). However, there is no interactive association between MHR and BMI with CRS. CONCLUSIONS: Higher MHR and BMI are associated with higher odds of CRS. MHR and BMI can serve as tools for early prevention and intervention of CRS, respectively.

Senotherapeutic Peptide 14 Suppresses Th1 and M1 Human T Cell and Monocyte Subsets In Vitro.

Inflammation contributes to the onset and exacerbation of numerous age-related diseases, often manifesting as a chronic condition during aging. Given that cellular senescence fosters local and systemic inflammation, senotherapeutic interventions could potentially aid in managing or even reducing inflammation. Here, we investigated the immunomodulatory effects of the senotherapeutic Peptide 14 (Pep 14) in human peripheral blood mononuclear cells (PBMCs), monocytes, and macrophages. We found that, despite failing to significantly influence T cell activation and proliferation, the peptide promoted a Th2/Treg gene expression and cytokine signature in PBMCs, characterized by increased expression of the transcription factors GATA3 and FOXP3, as well as the cytokines IL-4 and IL-10. These observations were partially confirmed through ELISA, in which we observed increased IL-10 release by resting and PHA-stimulated PBMCs. In monocytes from the U-937 cell line, Pep 14 induced apoptosis in lipopolysaccharide (LPS)-stimulated cells and upregulated IL-10 expression. Furthermore, Pep 14 prevented LPS-induced activation and promoted an M2-like polarization in U-937-derived macrophages, evidenced by decreased expression of M1 markers and increased expression of M2 markers. We also showed that the conditioned media from Pep 14-treated macrophages enhanced fibroblast migration, indicative of a functional M2 phenotype. Taken together, our findings suggest that Pep 14 modulates immune cell function towards an anti-inflammatory and regenerative phenotype, highlighting its potential as a therapeutic intervention to alleviate immunosenescence-associated dysregulation.

Discrepant Phenotyping of Monocytes Based on CX3CR1 and CCR2 Using Fluorescent Reporters and Antibodies.

Monocytes, as well as downstream macrophages and dendritic cells, are essential players in the immune system, fulfilling key roles in homeostasis as well as in inflammatory conditions. Conventionally, driven by studies on reporter models, mouse monocytes are categorized into a classical and a non-classical subset based on their inversely correlated surface expression of Ly6C/CCR2 and CX3CR1. Here, we aimed to challenge this concept by antibody staining and reporter mouse models. Therefore, we took advantage of Cx3cr1GFP and Ccr2RFP reporter mice, in which the respective gene was replaced by a fluorescent reporter protein gene. We analyzed the expression of CX3CR1 and CCR2 by flow cytometry using several validated fluorochrome-coupled antibodies and compared them with the reporter gene signal in these reporter mouse strains. Although we were able to validate the specificity of the fluorochrome-coupled flow cytometry antibodies, mouse Ly6Chigh classical and Ly6Clow non-classical monocytes showed no differences in CX3CR1 expression levels in the peripheral blood and spleen when stained with these antibodies. On the contrary, in Cx3cr1GFP reporter mice, we were able to reproduce the inverse correlation of the CX3CR1 reporter gene signal and Ly6C surface expression. Furthermore, differential CCR2 surface expression correlating with the expression of Ly6C was observed by antibody staining, but not in Ccr2RFP reporter mice. In conclusion, our data suggest that phenotyping strategies for mouse monocyte subsets should be carefully selected. In accordance with the literature, the suitability of CX3CR1 antibody staining is limited, whereas for CCR2, caution should be applied when using reporter mice.

Enhanced CD95 and interleukin 18 signalling accompany T cell receptor Vß21.3+ activation in multi-inflammatory syndrome in children.

Multisystem inflammatory syndrome in children is a post-infectious presentation SARS-CoV-2 associated with expansion of the T cell receptor Vß21.3+ T-cell subgroup. Here we apply muti-single cell omics to compare the inflammatory process in children with acute respiratory COVID-19 and those presenting with non SARS-CoV-2 infections in children. Here we show that in Multi-Inflammatory Syndrome in Children (MIS-C), the natural killer cell and monocyte population demonstrate heightened CD95 (Fas) and Interleuking 18 receptor expression. Additionally, TCR Vß21.3+ CD4+ T-cells exhibit skewed differentiation towards T helper 1, 17 and regulatory T cells, with increased expression of the co-stimulation receptors ICOS, CD28 and interleukin 18 receptor. We observe no functional evidence for NLRP3 inflammasome pathway overactivation, though MIS-C monocytes show elevated active caspase 8. This, coupled with raised IL18 mRNA expression in CD16- NK cells on single cell RNA sequencing analysis, suggests interleukin 18 and CD95 signalling may trigger activation of TCR Vß21.3+ T-cells in MIS-C, driven by increased IL-18 production from activated monocytes and CD16- Natural Killer cells.