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NK cells in the peripheral blood of severe COVID-19 patients exhibit a unique profile characterized by activation and dysfunction. Previous studies have identified soluble factors, including type I IFN and TGF-β, that underlie this dysregulation. However, the role of cell-cell interactions in modulating NK cell function during COVID-19 remains unclear. To address this question, we combined cell-cell communication analysis on existing single-cell RNA sequencing data with in vitro primary cell coculture experiments to dissect the mechanisms underlying NK cell dysfunction in COVID-19. We found that NK cells are predicted to interact most strongly with monocytes and that this occurs via both soluble factors and direct interactions. To validate these findings, we performed in vitro cocultures in which NK cells from healthy human donors were incubated with monocytes from COVID-19+ or healthy donors. Coculture of healthy NK cells with monocytes from COVID-19 patients recapitulated aspects of the NK cell phenotype observed in severe COVID-19, including decreased expression of NKG2D, increased expression of activation markers, and increased proliferation. When these experiments were performed in a Transwell setting, we found that only CD56bright CD16- NK cells were activated in the presence of severe COVID-19 patient monocytes. O-link analysis of supernatants from Transwell cocultures revealed that cultures containing severe COVID-19 patient monocytes had significantly elevated levels of proinflammatory cytokines and chemokines, as well as TGF-β. Collectively, these results demonstrate that interactions between NK cells and monocytes in the peripheral blood of COVID-19 patients contribute to NK cell activation and dysfunction in severe COVID-19.

Long noncoding RNAs (lncRNAs) account for the largest portion of RNA from the transcriptome, yet most of their functions remain unknown. Here, we performed two independent high-throughput CRISPRi screens to understand the role of lncRNAs in monocyte function and differentiation. The first was a reporter-based screen to identify lncRNAs that regulate TLR4-NFkB signaling in human monocytes and the second screen identified lncRNAs involved in monocyte to macrophage differentiation. We successfully identified numerous noncoding and protein-coding genes that can positively or negatively regulate inflammation and differentiation. To understand the functional roles of lncRNAs in both processes, we chose to further study the lncRNA LOUP [lncRNA originating from upstream regulatory element of SPI1 (also known as PU.1)], as it emerged as a top hit in both screens. Not only does LOUP regulate its neighboring gene, the myeloid fate-determining factor SPI1, thereby affecting monocyte to macrophage differentiation, but knockdown of LOUP leads to a broad upregulation of NFkB-targeted genes at baseline and upon TLR4-NFkB activation. LOUP also harbors three small open reading frames capable of being translated and are responsible for LOUPs ability to negatively regulate TLR4/NFkB signaling. This work emphasizes the value of high-throughput screening to rapidly identify functional lncRNAs in the innate immune system.

BACKGROUNDPreclinical studies suggest that cholesterol accumulation leads to insulin resistance. We previously reported that alterations in a monocyte cholesterol metabolism transcriptional network (CMTN) - suggestive of cellular cholesterol accumulation - were cross-sectionally associated with obesity and type 2 diabetes (T2D). Here, we sought to determine whether the CMTN alterations independently predict incident prediabetes/T2D risk, and correlate with cellular cholesterol accumulation.METHODSMonocyte mRNA expression of 11 CMTN genes was quantified among 934 Multi-Ethnic Study of Atherosclerosis (MESA) participants free of prediabetes/T2D; cellular cholesterol was measured in a subset of 24 monocyte samples.RESULTSDuring a median 6-year follow-up, lower expression of 3 highly correlated LXR target genes - ABCG1 and ABCA1 (cholesterol efflux) and MYLIP (cholesterol uptake suppression) - and not other CMTN genes, was significantly associated with higher risk of incident prediabetes/T2D. Lower expression of the LXR target genes correlated with higher cellular cholesterol levels (e.g., 47% of variance in cellular total cholesterol explained by ABCG1 expression). Further, adding the LXR target genes to overweight/obesity and other known predictors significantly improved prediction of incident prediabetes/T2D.CONCLUSIONThese data suggest that the aberrant LXR/ABCG1-ABCA1-MYLIP pathway (LAAMP) is a major T2D risk factor and support a potential role for aberrant LAAMP and cellular cholesterol accumulation in diabetogenesis.FUNDINGThe MESA Epigenomics and Transcriptomics Studies were funded by NIH grants 1R01HL101250, 1RF1AG054474, R01HL126477, R01DK101921, and R01HL135009. This work was supported by funding from NIDDK R01DK103531 and NHLBI R01HL119962.

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.

BackgroundAdvancing age is a major risk factor for respiratory viral infections. The infections are often prolonged and difficult to resolve resulting hospitalizations and mortality. The recent COVID-19 pandemic has highlighted this as elderly subjects have emerged as vulnerable populations that display increased susceptibility and severity to SARS-CoV-2. There is an urgent need to identify the probable mechanisms underlying this to protect against future outbreaks of such nature. Innate immunity is the first line of defense against viruses and its decline impacts downstream immune responses. This is because dendritic cells (DCs) and macrophages are key cellular elements of the innate immune system that can sense and respond to viruses by producing inflammatory mediators and priming CD4 and CD8 T-cell responses.ResultsWe investigated the changes in innate immune responses to SARS-CoV-2 as a function of age. Our results using human PBMCs from aged, middle-aged, and young subjects indicate that the activation of DCs and monocytes in response to SARS-CoV-2 is compromised with age. The impairment is most apparent in pDCs where both aged and middle-aged display reduced responses. The secretion of IL-29 that confers protection against respiratory viruses is also decreased in both aged and middle-aged subjects. In contrast, inflammatory mediators associated with severe COVID-19 including CXCL-8, TREM-1 are increased with age. This is also apparent in the gene expression data where pathways related host defense display an age dependent decrease with a concomitant increase in inflammatory pathways. Not only are the inflammatory pathways and mediators increased after stimulation with SARS-CoV-2 but also at homeostasis. In keeping with reduced DC activation, the induction of cytotoxic CD8 T cells is also impaired in aged subjects. However, the CD8 T cells from aged subjects display increased baseline activation in accordance with the enhanced baseline inflammation.ConclusionsOur results demonstrate a decline in protective anti-viral immune responses and increase in damaging inflammatory responses with age indicating that dysregulated innate immune responses play a significant role in the increased susceptibility of aged subjects to COVID-19. Furthermore, the dysregulation in immune responses develops early on as middle-aged demonstrate several of these changes.

BACKGROUND: Oxidized phospholipids play a key role in the atherogenic potential of lipoprotein(a) (Lp[a]); however, Lp(a) is a complex particle that warrants research into additional proinflammatory mediators. We hypothesized that additional Lp(a)-associated lipids contribute to the atherogenicity of Lp(a). METHODS: Untargeted lipidomics was performed on plasma and isolated lipoprotein fractions. The atherogenicity of the observed Lp(a)-associated lipids was tested ex vivo in primary human monocytes by RNA sequencing, ELISA, Western blot, and transendothelial migratory assays. Using immunofluorescence staining and single-cell RNA sequencing, the phenotype of macrophages was investigated in human atherosclerotic lesions. RESULTS: Compared with healthy individuals with low/normal Lp(a) levels (median, 7 mg/dL [18 nmol/L]; n=13), individuals with elevated Lp(a) levels (median, 87 mg/dL [218 nmol/L]; n=12) demonstrated an increase in lipid species, particularly diacylglycerols (DGs) and lysophosphatidic acid (LPA). DG and the LPA precursor lysophosphatidylcholine were enriched in the Lp(a) fraction. Ex vivo stimulation with DG(40:6) demonstrated a significant upregulation in proinflammatory pathways related to leukocyte migration, chemotaxis, NF-κB (nuclear factor kappa B) signaling, and cytokine production. Functional assessment showed a dose-dependent increase in the secretion of IL (interleukin)-6, IL-8, and IL-1β after DG(40:6) and DG(38:4) stimulation, which was, in part, mediated via the NLRP3 (NOD [nucleotide-binding oligomerization domain]-like receptor family pyrin domain containing 3) inflammasome. Conversely, LPA-stimulated monocytes did not exhibit an inflammatory phenotype. Furthermore, activation of monocytes by DGs and LPA increased their transendothelial migratory capacity. Human atherosclerotic plaques from patients with high Lp(a) levels demonstrated colocalization of Lp(a) with M1 macrophages, and an enrichment of CD68+IL-18+TLR4+ (toll-like receptor) TREM2+ (triggering receptor expressed on myeloid cells) resident macrophages and CD68+CASP1+ (caspase) IL-1B+SELL+ (selectin L) inflammatory macrophages compared with patients with low Lp(a). Finally, potent Lp(a)-lowering treatment (pelacarsen) resulted in a reduction in specific circulating DG lipid subspecies in patients with cardiovascular disease with elevated Lp(a) levels (median, 82 mg/dL [205 nmol/L]). CONCLUSIONS: Lp(a)-associated DGs and LPA have a potential role in Lp(a)-induced monocyte inflammation by increasing cytokine secretion and monocyte transendothelial migration. This DG-induced inflammation is, in part, NLRP3 inflammasome dependent.

Childhood maltreatment has been repeatedly linked to a higher incidence of health conditions with an underlying proinflammatory component, such as asthma, chronic obstructive pulmonary disease, stroke, and cardiovascular disease. Childhood maltreatment has also been linked to elevated systemic inflammation prior to the onset of disease. However, childhood maltreatment is highly comorbid with other risk factors which have also been linked to inflammation, namely major depression. The present analysis addresses this issue by assessing the association of maltreatment with genome-wide transcriptional profiling of immune cells collected from four orthogonal groups of adolescents (aged 13-17): maltreated and not maltreated in childhood, with and without major depressive disorder. Maltreatment and psychiatric history were determined using semi-structured clinical interviews and cross-validated using self-report questionnaires. Dried whole blood spots were collected from each participant (n = 133) and assayed to determine the extent to which maltreatment in childhood was associated with a higher prevalence of transcriptional activity among differentially expressed genes, specific immune cell subtypes, and up- or down-regulation of genes involved in immune function after accounting for current major depression. Maltreatment was associated with increased interferon regulatory factor (IRF) transcriptional activity (p = 0.03), as well as nuclear factor erythroid-2 related factor 1 (NRF1; p = 0.002) and MAF (p = 0.01) among up-regulated genes, and increased activity of nuclear factor kappa beta (NF-κB) among down-regulated genes (p = 0.01). Non-classical CD16+ monocytes were implicated in both the up- and down-regulated genes among maltreated adolescents. These data provide convergent evidence supporting the role of maltreatment in altering intracellular and molecular markers of immune function, as well as implicate monocyte/macrophage functions as mechanisms through which childhood maltreatment may shape lifelong immune development and function.

Age-related macular degeneration (AMD) is invariably associated with the chronic accumulation of activated mononuclear phagocytes in the subretinal space. The mononuclear phagocytes are composed of microglial cells but also of monocyte-derived cells, which promote photoreceptor degeneration and choroidal neovascularization. Infiltrating blood monocytes can originate directly from bone marrow, but also from a splenic reservoir, where bone marrow monocytes develop into angiotensin II receptor (ATR1)+ splenic monocytes. The involvement of splenic monocytes in neurodegenerative diseases such as AMD is not well understood. Using acute inflammatory and well-phenotyped AMD models, we demonstrate that angiotensin II mobilizes ATR1+ splenic monocytes, which we show are defined by a transcriptional signature using single-cell RNA sequencing and differ functionally from bone marrow monocytes. Splenic monocytes participate in the chorio-retinal infiltration and their inhibition by ATR1 antagonist and splenectomy reduces the subretinal mononuclear phagocyte accumulation and pathological choroidal neovascularization formation. In aged AMD-risk ApoE2-expressing mice, a chronic AMD model, ATR1 antagonist and splenectomy also inhibit the chronic retinal inflammation and associated cone degeneration that characterizes these mice. Our observation of elevated levels of plasma angiotensin II in AMD patients, suggests that similar events take place in clinical disease and argue for the therapeutic potential of ATR1 antagonists to inhibit splenic monocytes for the treatment of blinding AMD.

Rheumatoid arthritis (RA) management leans toward achieving remission or low disease activity. In this study, we conducted single-cell RNA sequencing (scRNA-Seq) of peripheral blood mononuclear cells (PBMCs) from 36 individuals (18 patients with RA and 18 matched controls, accounting for age, sex, race, and ethnicity), to identify disease-relevant cell subsets and cell type-specific signatures associated with disease activity. Our analysis revealed 18 distinct PBMC subsets, including an IFN-induced transmembrane 3-overexpressing (IFITM3-overexpressing) IFN-activated monocyte subset. We observed an increase in CD4+ T effector memory cells in patients with moderate-high disease activity (DAS28-CRP ≥ 3.2) and a decrease in nonclassical monocytes in patients with low disease activity or remission (DAS28-CRP < 3.2). Pseudobulk analysis by cell type identified 168 differentially expressed genes between RA and matched controls, with a downregulation of proinflammatory genes in the γδ T cell subset, alteration of genes associated with RA predisposition in the IFN-activated subset, and nonclassical monocytes. Additionally, we identified a gene signature associated with moderate-high disease activity, characterized by upregulation of proinflammatory genes such as TNF, JUN, EGR1, IFIT2, MAFB, and G0S2 and downregulation of genes including HLA-DQB1, HLA-DRB5, and TNFSF13B. Notably, cell-cell communication analysis revealed an upregulation of signaling pathways, including VISTA, in both moderate-high and remission-low disease activity contexts. Our findings provide valuable insights into the systemic cellular and molecular mechanisms underlying RA disease activity.

IntroductionMaternal intervillous monocytes (MIMs) and fetal Hofbauer cells (HBCs) are myeloid-derived immune cells at the maternal-fetal interface. Maternal reproductive history is associated with differential risk of pregnancy complications. The molecular phenotypes and roles of these distinct monocyte/macrophage populations and the influence of gravidity on these phenotypes has not been systematically investigated.MethodsHere, we used RNA sequencing to study the transcriptional profiles of MIMs and HBCs in normal term pregnancies.ResultsOur analyses revealed distinct transcriptomes of MIMs and HBCs. Genes involved in differentiation and cell organization pathways were more highly expressed in MIMs vs. HBCs. In contrast, HBCs had higher expression of genes involved in inflammatory responses and cell surface receptor signaling. Maternal gravidity influenced monocyte programming, as expression of pro-inflammatory molecules was significantly higher in MIMs from multigravidae compared to primigravidae. In HBCs, multigravidae displayed enrichment of gene pathways involved in cell-cell signaling and differentiation.DiscussionOur results demonstrated that MIMs and HBCs have highly divergent transcriptional signatures, reflecting their distinct origins, locations, functions, and roles in inflammatory responses. Furthermore, maternal gravidity influences the gene signatures of MIMs and HBCs, potentially modulating the interplay between tolerance and trained immunity. The phenomenon of reproductive immune memory may play a novel role in the differential susceptibility of primigravidae to pregnancy complications.

In this randomized phase II clinical trial, we evaluated the effectiveness of adding the TLR agonists, poly-ICLC or resiquimod, to autologous tumor lysate-pulsed dendritic cell (ATL-DC) vaccination in patients with newly-diagnosed or recurrent WHO Grade III-IV malignant gliomas. The primary endpoints were to assess the most effective combination of vaccine and adjuvant in order to enhance the immune potency, along with safety. The combination of ATL-DC vaccination and TLR agonist was safe and found to enhance systemic immune responses, as indicated by increased interferon gene expression and changes in immune cell activation. Specifically, PD-1 expression increases on CD4+ T-cells, while CD38 and CD39 expression are reduced on CD8+ T cells, alongside an increase in monocytes. Poly-ICLC treatment amplifies the induction of interferon-induced genes in monocytes and T lymphocytes. Patients that exhibit higher interferon response gene expression demonstrate prolonged survival and delayed disease progression. These findings suggest that combining ATL-DC with poly-ICLC can induce a polarized interferon response in circulating monocytes and CD8+ T cells, which may represent an important blood biomarker for immunotherapy in this patient population.Trial Registration: ClinicalTrials.gov Identifier: NCT01204684.

BACKGROUND: Schistosomiasis is a common cause of pulmonary hypertension (PH) worldwide. Type 2 inflammation contributes to the development of Schistosoma-induced PH. Specifically, interstitial macrophages (IMs) derived from monocytes play a pivotal role by producing thrombospondin-1 (TSP-1), which in turn activates TGF-β, thereby driving the pathology of PH. Resident and recruited IM subpopulations have recently been identified. We hypothesized that in Schistosoma-PH, one IM subpopulation expresses monocyte recruitment factors, whereas recruited monocytes become a separate IM subpopulation that expresses TSP-1. METHODS: Mice were intraperitoneally sensitized and then intravenously challenged with S. mansoni eggs. Flow cytometry on lungs and blood was performed on wildtype and reporter mice to identify IM subpopulations and protein expression. Single-cell RNA sequencing (scRNAseq) was performed on flow-sorted IMs from unexposed and at day 1, 3 and 7 following Schistosoma exposure to complement flow cytometry based IM characterization and identify gene expression. RESULTS: Flow cytometry and scRNAseq both identified 3 IM subpopulations, characterized by CCR2, MHCII, and FOLR2 expression. Following Schistosoma exposure, the CCR2+ IM subpopulation expanded, suggestive of circulating monocyte recruitment. Schistosoma exposure caused increased monocyte-recruitment ligand CCL2 expression in the resident FOLR2+ IM subpopulation. In contrast, the vascular pathology-driving protein TSP-1 was greatest in the CCR2+ IM subpopulation. CONCLUSION: Schistosoma-induced PH involves crosstalk between IM subpopulations, with increased expression of monocyte recruitment ligands by resident FOLR2+ IMs, and the recruitment of CCR2+ IMs which express TSP-1 that activates TGF-β and causes PH.

Background: The gut microbiota may be involved in neuropathic pain. However, the causal association between gut microbiota and neuropathic pain remains unclear. Whether immune cells and inflammatory factors mediate the pathway from gut microbiota to neuropathic pain has not been elucidated. Methods: We obtained the summary data of 412 gut microbiota, 731 immune cells, 91 inflammatory factors, and five types of neuropathic pain (drug-induced neuropathy, postherpetic neuralgia, sciatica, trigeminal neuralgia, and unspecified neuralgia) from large-scale genome-wide association study (GWAS) datasets and the FinnGen database. We used bidirectional Mendelian randomization (MR) analysis to explore the causal association between gut microbiota and neuropathic pain. Additionally, we conducted a mediation analysis to identify whether immune cells and inflammatory factors act as mediators within these causal relationships. Results: Our study revealed 30 causal relationships between 26 gut bacterial taxa and five types of neuropathic pain, including four associated with drug-induced neuropathy, six with postherpetic neuralgia, five with sciatica, eight with trigeminal neuralgia, and seven with unspecified neuralgia. Moreover, we identified 35 gut bacterial pathway abundances causally involved in neuropathic pain. The reverse MR analysis showed no evidence of reverse causality from gut microbiota to neuropathic pain. Mediation analysis demonstrated that the immune cell phenotype "HLA-DR++ monocyte % leukocyte" mediated the causal relationship between p_Proteobacteria and sciatica with a mediation proportion of 36.15% (P = 0.038), whereas "CD11c on CD62L+ myeloid dendritic cell" mediated the causal pathway from assimilatory sulfate reduction to trigeminal neuralgia with a mediation proportion of 27.90% (P = 0.041). Conclusion: This study identified the causal relationships between several specific gut microbiota and various neuropathic pain subtypes. Additionally, two immune cells may act as potential mediators in the pathways from gut microbiota to neuropathic pain. [ABSTRACT FROM AUTHOR]

The role of type I interferon (IFN-I) in systemic lupus erythematosus (SLE) is well documented, but the role of interleukin (IL)-1β remains elusive. In this issue of Immunity , Caielli et al. identified an SLE monocyte population coproducing IL-1β and IFN-I and described how mitochondrial nucleic-acid-containing RBCs engage cGAS/STING, RIG-I, MDA5, and NLRP3 for unconventional IL-1β release. [ABSTRACT FROM AUTHOR]

Opsonization of red blood cells that retain mitochondria (Mito+ RBCs), a feature of systemic lupus erythematosus (SLE), triggers type I interferon (IFN) production in macrophages. We report that monocytes (Mos) co-produce IFN and mature interleukin-1β (mIL-1β) upon Mito+ RBC opsonization. IFN expression depended on cyclic GMP-AMP synthase (cGAS) and RIG-I-like receptors' (RLRs) sensing of Mito+ RBC-derived mitochondrial DNA (mtDNA) and mtRNA, respectively. Interleukin-1β (IL-1β) production was initiated by the RLR antiviral signaling adaptor (MAVS) pathway recognition of Mito+ RBC-derived mtRNA. This led to the cytosolic release of Mo mtDNA, which activated the inflammasome. Importantly, mIL-1β secretion was independent of gasdermin D (GSDMD) and pyroptosis but relied on IFN-inducible myxovirus-resistant protein 1 (MxA), which facilitated the incorporation of mIL-1β into a trans -Golgi network (TGN)-mediated secretory pathway. RBC internalization identified a subset of blood Mo expressing IFN-stimulated genes (ISGs) that released mIL-1β and expanded in SLE patients with active disease. [Display omitted] • Mos co-produce type I IFN and IL-1β upon Mito+ RBC opsonization • IFN and IL-1β expression rely on sensing RBC-derived mtDNA and mtRNA • IL-1β secretion is MxA mediated and independent of GSDMD and pyroptosis • GPA+ blood Mos expressing IFN and IL-1β are expanded in active SLE SLE is an interferonopathy, but blocking IFN does not benefit all patients. Caielli et al. found that SLE monocytes co-produce IFN and IL-1β, a phenotype recapitulated upon in vitro Mito+ RBC opsonization. Cytosolic sensing of mitochondrial nucleic acids induces MxA, which enables IL-1β secretion independently of GSDMD and pyroptosis. [ABSTRACT FROM AUTHOR]

S100A9, a multifunctional protein mainly expressed by neutrophils and monocytes, poses an immunological paradox. In virus infections or sterile inflammation, it functions as an alarmin attracting innate immune cells, as well as mediating proinflammatory effects through TLR4 signaling. However, in cancer, S100A9 levels have been shown to associate with poor prognosis and lack of response to immunotherapy. Its expression by myeloid cells has been related to an immune suppressive phenotype, the so-called myeloid derived suppressor cells (MDSCs). Targeting S100A9 in cancer has therefore been proposed as a potential way to relieve myeloid-mediated immune suppression. Surprisingly, we found that blocking the extracellular TLR4 signaling from S100A9 using the inhibitor Paquinimod, resulted in increased tumor growth and a detrimental effect on anti-PD-L1 efficacy in the CT26 tumor model. This effect was caused by a reduction in the tumor immune infiltration to about half of untreated controls, and the reduction was made up of a 5-fold decrease in Ly6Chigh monocytic cells. The suppressive Ly6G+ myeloid cells compartment was not reduced by Paquinimod treatment, suggesting alternative mechanisms by which S100A9 contributes to myeloid-mediated suppression. Intratumoral injection of recombinant S100A9 early after mice inoculation with CT26 cells had an anti-tumor effect. These findings indicate an important yet understudied role of S100A9 as an alarmin and immune stimulatory signal in cancer settings, and highlight the potential to exploit such signals to promote beneficial antitumor responses. [ABSTRACT FROM AUTHOR]

Neutrophils orchestrate a coordinated attack on bacteria, combining phagocytosis with a potent cocktail of oxidants, including the highly toxic hypochlorous acid (HOCl), renowned for its deleterious effects on proteins. Here, we examined the occurrence of lipid N -chloramines in vivo , their biological activity, and their neutralization. Using a chemical probe for N -chloramines, we demonstrate their formation in the membranes of bacteria and monocytic cells exposed to physiologically relevant concentrations of HOCl. N -chlorinated model membranes composed of phosphatidylethanolamine, the major membrane lipid in Escherichia coli and an important component of eukaryotic membranes, exhibited oxidative activity towards the redox-sensitive protein roGFP2, suggesting a role for lipid N -chloramines in protein oxidation. Conversely, glutathione a cellular antioxidant neutralized lipid N -chloramines by removing the chlorine moiety. In line with that, N -chloramine stability was drastically decreased in bacterial cells compared to model membranes. We propose that lipid N -chloramines, like protein N -chloramines, are involved in inflammation and accelerate the host immune response. [Display omitted] • HOCl forms lipid N -chloramines in bacterial and mammalian membranes. • Lipid N -chloramines oxidize protein thiols. • Stability of N -chloramines is drastically reduced in vivo compared to model membranes. [ABSTRACT FROM AUTHOR]

Objectives We sought to characterize the immunophenotype of acute myeloid leukemia (AML) with CBFB rearrangement and correlate the results with cytogenetic and molecular data. Methods Sixty-one cases of AML with CBFB rearrangement were evaluated. Results The sample population consisted of 33 men and 28 women, with a median age of 49 years. Flow cytometry immunophenotypic analysis showed that myeloblasts were positive for CD34 and CD117 in all cases, and myeloperoxidase was positive in 52 of 55 (95%) cases. The most common abnormalities included decreased CD38 in 90%, increased CD13 in 85%, increased CD123 in 84%, and decreased HLA-DR in 84% of cases. Monocytes were increased, with a mature immunophenotype, and accounted for 23.7% of total cells. Among 60 cases with available karyotype, inv(16)(p13.1q22) was most common in 50 (83%) cases, followed by t(16;16) (p13.1;q22) in 6 (10%). Type A CBFB::MYH11 transcript was most common, detected in 84% of cases. Mutational analysis showed mutations of NRAS in 37%, FLT3 in 25%, and KIT in 24% of cases. Comparing cases with type A vs non–type A transcripts, blasts in type A cases more frequently exhibited CD64 positivity and increased CD13 levels while showing a lower frequency of CD7 and CD56 expression. Trisomy 22 and mutations in KIT, NF1, and TET2 were identified only in cases with type A transcript. Conclusions Myeloblasts of AML with CBFB rearrangement are positive for CD34, CD117, and myeloperoxidase. These neoplasms most frequently carry inv(16)(p13.1q22) and type A fusion transcript. NRAS mutation was the most common mutation. Some immunophenotypic and genetic correlations occurred with different types of transcripts. [ABSTRACT FROM AUTHOR]

Background/Objectives: Chemical and carcinogenic risk in workplace are linked to an increase of the incidence of cancer in exposed workers. The aim of this observational study was to identify an association between exposure to chemical risk and changes in hematochemical parameters of workers exposed. Methods: The study examined blood count parameters performed on 4523 employees of Sapienza University of Rome from September 2019 to February 2020. A total of 1402 workers in research laboratories, exposed to chemical risk and carcinogens, was compared with a cohort of 3121 blood donors, in apparently good health, matched for age, sex, and period of blood collection. Results: Multivariate analysis demonstrated acrylamide is the most frequently associated chemical with significant changes between the groups. It is related to an increase in monocytes and MPV, a reduction of lymphocytes, platelets, and red series values, while MCH remained unchanged. Formaldehyde reveals the same changes as acrylamide, with the exception of the percentage of lymphocytes, red blood cells, and HB, which remain unchanged. Other chemicals and carcinogens show variations for few blood count parameters. Conclusions: The present study found out a significant change in blood count values in workers exposed to acrylamide and formaldehyde. It also showed an increase in the mean monocyte value in employees exposed to carcinogenic risk, compared to non-exposed, with the exception of Trypan Blue. The implementation of efficient health surveillance remains of critical importance. These data should be compared with those from follow-up studies. [ABSTRACT FROM AUTHOR]

Reduced/deficient expression of Protein Kinase C (PKC)ζ in Cord blood (CB) T cells is associated with allergy development in children and a propensity to maintain an immature T-helper (Th)2 cytokine profile. In addition, other PKC isozymes are also low in CBTCs. Since previous studies have reported that cord blood/neonatal monocyte and neutrophil functions are significantly lower than cells from adults, it was of interest to see if the CBTC PKC levels were reflected in CB monocytes and neutrophils. Compared to adult blood, CB expresses low levels of PKCα, β2, ε, θ, μ, ζ and λ/ι in monocytes and PKCα, β2, η, θ, μ, ζ and λ/ι in neutrophils. The T-cell PKCζ levels were positively correlated with levels in CB monocytes but not in neutrophils. However, neither the monocytes nor the neutrophil PKCζ were associated with T-cell development towards a Th1 or Th2 cytokine propensity, based on the production of interferon-gamma and interleukin-4 in response to phytohemagglutinin and phorbol myristate acetate. The results demonstrate that some newborn babies display a deficiency in PKC isozymes in monocytes and neutrophils, as reported for T cells. However, unlike T cells, the PKCζ levels of the phagocytes did not correlate with regulation of development towards a Th1 or Th2 cytokine phenotype. [ABSTRACT FROM AUTHOR]

We developed a flow cytometry (FC) assay enabling the rapid and accurate identification of bacterial and viral infections using whole blood samples. The streamlined flow cytometry assay is designed to be user-friendly, making it accessible even for operators with limited experience in FC techniques. The key components of the assay focus on the expression levels of specific surface markers—CD64 on polymorphonuclear neutrophils (PMN) as a marker for bacterial infection, and CD169 on monocytes (MO) for viral infection. The strong performance indicated by an area under the receiver operating characteristic (ROC) curve of 0.94 for both PMN CD64 positive predictive value (PPV) 97.96% and negative predictive value (NPV) 76.67%, and MO CD169 PPV 82.6% and NPV 86.9%, highlight the assay's robustness in differentiating between bacterial and viral infections accurately. The FC assay includes the assessment of immune system status through HLA-DR and IL-1R2 modulation in MO, providing a useful insight into the patients' immune response. The significant increase in the frequency of MO exhibiting reduced HLA-DR expression and elevated IL-1R2 levels in infected patients (compared to healthy controls) underscores the potential of these markers as indicators of infection severity. Although the overall correlation between HLA-DR and IL-1R2 expression levels was not significant across all patients, there was a trend in patients with more severe disease suggesting that these markers may have the potential to assist in stratifying patient risk. The present FC assay has the potential to become routine in the clinical microbiology laboratory community and to be helpful in guiding clinical decision making. [ABSTRACT FROM AUTHOR]

Objective: RBC transfusions (RBCT) are life-saving treatment for premature and critically ill infants. However, the procedure has been associated with the development of systemic inflammatory response syndrome (SIRS) and potentially multiple organ dysfunction syndrome (MODS) in neonates. The present study aimed to investigate the mechanisms of RBCT-related SIRS in severely anemic murine neonates. Methods: C57BL/6 (WT), TLR4−/− and myeloid-specific triggered myeloid receptor-1 (trem1)−/− mouse pups were studied in 4 groups (n = 6 each): (1) naïve controls, (2) transfused control, (3) anemic (hematocrit 20–24%) and (4) anemic with RBC transfused using our established murine model of phlebotomy-induced anemia (PIA) and RBC transfusion. Plasma was measured for quantifying inflammatory cytokines (IFN-γ, IL-1β, TNF-α, IL-6, MIP-1α, MIP-1β, MIP2 and LIX) using a Luminex assay. In vitro studies included (i) sensitization by exposing the cells to a low level of lipopolysaccharide (LPS; 500 ng/ml) and (ii) trem1-siRNA transfection with/without plasma supernatant from stored RBC to assess the acute inflammatory response through trem1 by qRT-PCR and immunoblotting. Results: Anemic murine pups developed cytokine storm within 2 h of receiving stored RBCs, which increased until 6 h post-transfusion, as compared to non-anemic mice receiving stored RBCTs ("transfusion controls"), in a TLR4-independent fashion. Nonetheless, severely anemic pups had elevated circulating endotoxin levels, thereby sensitizing circulating monocytes to presynthesize proinflammatory cytokines (IFN-γ, IL-1β, TNF-α, IL-6, MIP-1α, MIP-1β, MIP2, LIX) and express trem1. Silencing trem1 expression in Raw264.7 cells mitigated both endotoxin-associated presynthesis of proinflammatory cytokines and the RBCT-induced release of inflammatory cytokines. Indeed, myeloid-specific trem1-/- murine pups had significantly reduced evidence of SIRS following RBCTs. Conclusion: Severe anemia-associated low-grade inflammation sensitizes monocytes to enhance the synthesis of proinflammatory cytokines and trem1. In this setting, RBCTs further activate these monocytes, thereby inducing SIRS. Inhibiting trem1 in myeloid cells, including monocytes, alleviates the inflammatory response associated with the combined effects of anemia and RBCTs in murine neonates. [ABSTRACT FROM AUTHOR]

Background: Dysregulation of the complement system is involved in development of age-related macular degeneration (AMD). The complement cascade is regulated by membrane bound complement regulatory proteins (Cregs) on mononuclear leukocytes among others. This study aims to investigate systemic complement proteins and Cregs in AMD stages and their association with treatment response in neovascular AMD (nAMD). Methods: In this clinical prospective study, treatment-naïve patients with nAMD, intermediate AMD (iAMD) and healthy controls were recruited and systemic complement proteins C3, C3a and C5a were investigated with electrochemiluminescence immunoassays, and Creg expression (CD35, CD46 and CD59) on T cells (CD4 + and CD8+) and monocytes (classical, intermediate and non-classical) investigated with flow cytometry. Treatment response in nAMD patients was evaluated after loading dose and after one year, and categorized as good, partial or poor. Complement proteins and Creg expression levels were compared between healthy controls, iAMD and nAMD, as well as between good, partial and poor nAMD treatment response groups. Polymorphisms in the CFH and ARMS2 genes were analyzed and compared to complement proteins and Creg expression levels in nAMD patients. Results: One hundred patients with nAMD, 34 patients with iAMD and 61 healthy controls were included. 94 nAMD patients completed the 1-year follow-up. Distribution of treatment response in nAMD was 61 (65%) good, 26 (28%) partial, and 7 (7%) poor responders. The distribution of 1-year treatment response was 50 (53%) good, 33 (36%) partial, and 11 (11%) poor responders. The concentrations of systemic C3, C3a, and the C3a/C3-ratio were significantly increased in patients with nAMD compared to healthy controls (P < 0.001, P = 0.002, and P = 0.035, respectively). Systemic C3 was also increased in iAMD compared to healthy controls (P = 0.031). The proportion of CD46 + CD4 + T cells and CD59 + intermediate monocytes were significantly decreased in patients with nAMD compared to healthy controls (P = 0.018 and P = 0.042, respectively). The post-loading dose partial treatment response group had significantly lower concentrations of C3a and C5a compared to the good response group (P = 0.005 and P = 0.042, respectively). The proportion of CD35 + monocytes was significantly lower in the 1-year partial response group compared to the 1-year good response group (P = 0.039). High-risk CFH genotypes in nAMD patients was associated with increased C3a, C3a/C3-ratio, and expression levels of CD35 + CD8 + T cells and CD46 + classical monocytes, while expression level of CD46 + non-classical monocytes was decreased. Conclusion: Elevated concentrations of systemic complement proteins were found in patients with iAMD and nAMD. Decreased Creg expression levels were found in patients with nAMD. Partially responding nAMD patients had a dysregulated complement system and Cregs compared to good responders. [ABSTRACT FROM AUTHOR]

Patients with lung cancer have a high incidence of tumor recurrence even after curative surgical resection. Some reports indicated that immunosuppressive cells induced by surgical stress could contribute to tumor recurrence after surgery; however, the underlying mechanisms are not fully understood. In this study, we found that increased postoperative blood monocytes served as a risk factor for tumor recurrence in 192 patients with non-small cell lung cancer (NSCLC). We established the lung cancer recurrent mouse model after tumor resection and showed that the surgical stress immediately increased the level of serum monocyte chemoattractant protein-1 (MCP-1), which subsequently increased blood monocytes. These blood monocytes were rapidly recruited into distant micrometastases and became tumor growth-promoting tumor associated macrophages (TAMs). Furthermore, even after the blood MCP-1 and monocytes decreased enough 72 h after tumor resection, TAMs in micrometastases remained rich because the MCP-1 secreted by micrometastases themselves continued to recruit monocytes around the tumor. Consequently, tumor resection triggered the outgrowth of distant metastases via the MCP-1–Monocyte–TAM axis. When we administered the MCP-1 inhibitor to the lung cancer recurrent model mice, blood monocytes decreased after tumor resection, and TAMs in micrometastases also dramatically decreased. Finally, peri- and postoperative treatment with the MCP-1 inhibitor suppressed distant metastases after surgery. Targeting the MCP-1–Monocyte–TAM axis may inhibit surgical stress-induced NSCLC recurrence by attenuating postoperative immunosuppressive monocytes in micrometastases. [ABSTRACT FROM AUTHOR]

Several attempts have been made to elucidate the pathogenesis of drug-induced gingival overgrowth (DIGO), which is triggered by the chronic use of certain drugs that fall into three main categories: anticonvulsants, immunosuppressants, and calcium channel blockers. Previous research suggests that cytokines and impaired cellular functions play a role in DIGO. Of particular interest are macrophages, immune cells that can switch between M1 (pro-inflammatory) and M2 (anti-inflammatory) phenotypes in response to exogenous signals and stimuli. An imbalance between M1 and M2 macrophage populations may underlie DIGO. M1 may contribute to the initial tissue damage in DIGO, while M2 may then attempt to repair the damage with anti-inflammatory mechanisms. To test the hypothesis that drugs associated with DIGO could influence macrophage polarization, human monocytes (precursors of macrophages) were induced to differentiate into M0-naïve macrophages and then exposed to drugs: diphenylhydantoin, gabapentin, mycophenolate, and amlodipine. Quantitative real-time PCR amplification was used to measure the expression of specific genes associated with macrophage polarization. All of the drugs tested induced M0 macrophages to overexpress genes typical of the M1 phenotype, such as CCL5, CXCL10, and IDO1. This investigation provides the first evidence of a link between drugs that cause DIGO and M1 pro-inflammatory macrophage polarization. The knowledge gained from this research could be valuable for future DIGO treatment strategies. [ABSTRACT FROM AUTHOR]

Macrophage infiltration and accumulation in the atherosclerotic lesion are associated with plaque progression and instability. Depletion of macrophages from the lesion might provide valuable insights into plaque stabilization processes. Therefore, we assessed the effects of systemic and local macrophage depletion on atherogenesis. To deplete monocytes/macrophages we used atherosclerosis‐susceptible Apoe−/− mice, bearing a MaFIA (macrophage‐Fas‐induced‐apoptosis) suicide construct under control of the Csf1r (CD115) promotor, where selective apoptosis of Csf1r‐expressing cells was induced in a controlled manner, by administration of a drug, AP20187. Systemic induction of apoptosis resulted in a decrease in lesion macrophages and smooth‐muscle cells. Plaque size and necrotic core size remained unaffected. Two weeks after the systemic depletion of macrophages, we observed a replenishment of the myeloid compartment. Myelopoiesis was modulated resulting in an expansion of CSF1Rlo myeloid cells in the circulation and a shift from Ly6chi monocytes toward Ly6cint and Ly6clo populations in the spleen. Local apoptosis induction led to a decrease in plaque burden and macrophage content with marginal effects on the circulating myeloid cells. Local, but not systemic depletion of Csf1r+ myeloid cells resulted in decreased plaque burden. Systemic depletion led to CSF1Rlo‐monocyte expansion in blood, possibly explaining the lack of effects on plaque development. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii is an opportunistic Gram‐negative bacterium representing one of the leading causes of ventilator‐associated pneumonia. The development of pneumonia results from a complex interplay between pathogens and pulmonary innate mucosal immunity. Therefore, the knowledge of the host immune responses is pivotal for the development of effective therapeutics to treat A. baumannii infections. Previous studies were conducted using cell lines and animal models, but a comprehensive understanding of the interaction between A. baumannii and primary human immune cells is still lacking. To bridge this gap, we investigated the response of primary monocytes, macrophages, and dendritic cells to the A. baumannii‐type strain and an epidemic clinical isolate. We found that all immune cells trigger different responses when interacting with A. baumannii. In particular, macrophages and monocytes mediate bacterial clearance, whereas monocytes and dendritic cells activate a late response through the production of cytokines, chemokines, and the expression of co‐stimulatory molecules. The epidemic strain induces lower expression of interleukin‐10 and CD80 compared with the type strain, potentially constituting two immune evasion strategies. [ABSTRACT FROM AUTHOR]

Objective: To observe the protective effect and mechanism of hydroxyl safflower yellow A (HSYA) from myocardial ischemia-reperfusion injury on human umbilical vein endothelial cells (HUVECs). Methods: HUVECs were treated with oxygen-glucose deprivation reperfusion (OGD/R) to simulate the ischemia reperfusion model, and cell counting kit-8 was used to detect the protective effect of different concentrations (1.25–160 µ mol/L) of HSYA on HUVECs after OGD/R. HSYA 80 µ mol/L was used for follow-up experiments. The contents of inflammatory cytokines interleukin (IL)-18, IL-1 β, monocyte chemotactic protein 1 (MCP-1), tumor necrosis factor α (TNF-α) and IL-6 before and after administration were measured by enzyme-linked immunosorbent assay. The protein expressions of toll-like receptor, NOD-like receptor containing pyrin domain 3 (NLRP3), gasdermin D (GSDMD) and GSDMD-N-terminal domain (GSDMD-N) before and after administration were detected by Western blot. NLRP3 inflammasome inhibitor cytokine release inhibitory drug 3 sodium salt (CRID3 sodium salt, also known as MCC950) and agonist were added, and the changes of NLRP3, cysteine-aspartic acid protease 1 (Caspase-1), GSDMD and GSDMD-N protein expressions were detected by Western blot. Results: HSYA inhibited OGD/R-induced inflammation and significantly decreased the contents of inflammatory cytokines IL-18, IL-1 β, MCP-1, TNF-α and IL-6 (P<0.01 or P<0.05). At the same time, by inhibiting NLRP3/Caspase-1/GSDMD pathway, HSYA can reduce the occurrence of pyroptosis after OGD/R and reduce the expression of NLRP3, Caspase-1, GSDMD and GSDMD-N proteins (P<0.01). Conclusions: The protective effect of HSYA on HUVECs after OGD/R is related to down-regulating the expression of NLRP3 inflammasome and inhibiting pyroptosis. [ABSTRACT FROM AUTHOR]

Background: This study explores the significance of computed tomography (CT) radiomic features, along with inflammation and nutrition biomarkers, in the prognosis of postoperative patients with T3N0M0 esophageal squamous cell carcinoma (ESCC). The study aims to construct a related nomogram. Methods: A total of 114 patients were enrolled and randomly assigned to training and validation cohorts in a 7:3 ratio. Radiomic features were extracted from their preoperative chest‐enhanced CT arterial images of the primary tumor, and inflammatory and nutritional indices, including neutrophil‐to‐lymphocyte ratio (NLR), lymphocyte‐to‐monocyte ratio (LMR), platelet‐to‐lymphocyte ratio (PLR), systemic immune‐inflammation index (SII), and prognostic nutritional index (PNI), were calculated based on laboratory data from the 3 days before surgery. Intra‐class correlations coefficient (ICC) and least absolute shrinkage and selection operator (Lasso) were applied to screen valuable radiomics features predicting overall survival (OS), and the Rad‐score was calculated. In the training cohort, univariate and multivariate Cox regression analyses identified independent prognostic factors, which were adopted to establish the nomogram. Results: Eight radiomic features were selected for Rad‐score calculation. Multivariate Cox regression revealed Rad‐score, PNI, NLR, and PLR as independent prognostic factors for ESCC patients (p < 0.05). A nomogram was constructed based on these variables. The concordance index (C‐index) for the nomogram was 0.797 (95% CI: 0.726–0.868) in the training cohort and 0.796 (95% CI: 0.702–0.890) in the validation cohort. Calibration curves indicated good calibration ability, and the receiver operating characteristic (ROC) analysis demonstrated superior discriminative ability for the nomogram in comparison to the Rad‐score alone. Decision curve analysis (DCA) confirmed the clinical utility of the nomogram. Conclusion: We developed and validated a nomogram for predicting the OS of postoperative T3N0M0 ESCC patients, integrating nutritional, inflammatory markers, and radiomic signature. The combined nomogram can serve as a robust tool for risk stratification and clinical management. [ABSTRACT FROM AUTHOR]

Background: Intervertebral disc degeneration (IDD) is widely regarded as the primary contributor to low back pain(LBP). As an immune-privileged organ, upon the onset of IDD, various components of the nucleus pulposus (NP) are exposed to the host’s immune system, accumulating cytokines. Cytokines facilitate intercellular communication within the immune system, induce immune cells polarisation, and exacerbate oxidative stress in IDD. Methods: Machine learning was used to identify crucial immune cells. Subsequently, Immune Response Enrichment Analysis (IREA) was conducted on the key immune cells to determine their cytokine responses and polarisation states in IDD. “CellChat” package facilitated the analysis of cell-cell communication. Differential gene expression analysis, PPI network, GO and KEGG pathway enrichment analysis, GSVA, co-expressed gene analysis and key gene-related networks were also performed to explore hub genes and their associated functions. Lastly, the differential expression and functions of key genes were validated through in vitro and in vivo experiments. Results: Through multiple machine learning methods, monocytes were identified as the crucial immune cells in IDD, exhibiting significant differentiation capacity. IREA revealed that monocytes in IDD polarize into an IFN-a1 and IFN-b enriched Mono-a state, potentially intensifying inflammation. Cell–cell communication analysis uncovered alteration in ANNEXIN pathway and a reduction in CXCL signaling between macrophages and monocytes, suggesting immune response dysregulation. Furthermore, ten algorithms identified three hub genes. Both experiments conducted in vitro and in vivo have conclusively shown that IRF7 serves as a crucial target for the treatment of IDD, and its knockdown alleviates IDD. Eight small-molecule drugs were predicted to have therapeutic potential for IDD. Conclusion: These findings offer a multidimensional understanding of the pathogenesis of IDD, pinpointing monocytes and key genes as potential diagnostic and therapeutic targets. They provide novel insights into potential diagnostic and therapeutic targets for IDD. [ABSTRACT FROM AUTHOR]

Background: Severe acute pancreatitis (SAP) is characterized by inflammation, with inflammatory immune cells playing a pivotal role in disease progression. This study aims to understand variations in specific immune cell subtypes in SAP, uncover their mechanisms of action, and identify potential biological markers for predicting Acute Pancreatitis (AP) severity. Methods: We collected peripheral blood from 7 untreated SAP patients and employed single-cell RNA sequencing for the first time to construct a transcriptome atlas of peripheral blood mononuclear cells (PBMCs) in SAP. Integrating SAP transcriptomic data with 6 healthy controls from the GEO database facilitated the analysis of immune cell roles in SAP. We obtained comprehensive transcriptomic datasets from AP samples in the GEO database and identified potential biomarkers associated with AP severity using the “Scissor” tool in single-cell transcriptomic data. Results: This study presents the inaugural construction of a peripheral blood single-cell atlas for SAP patients, identifying 20 cell subtypes. Notably, there was a significant decrease in effector T cell subsets and a noteworthy increase in monocytes compared to healthy controls. Moreover, we identified a novel monocyte subpopulation expressing high levels of PPBP and PF4 which was significantly elevated in SAP. The proportion of monocyte subpopulations with high CCL3 expression was also markedly increased compared to healthy controls, as verified by flow cytometry. Additionally, cell communication analysis revealed insights into immune and inflammation-related signaling pathways in SAP patient monocytes. Finally, our findings suggest that the subpopulation with high CCL3 expression, along with upregulated proinflammatory genes such as S100A12, IL1B, and CCL3, holds promise as biomarkers for predicting AP severity. Conclusion: This study reveals monocytes’ crucial role in SAP initiation and progression, characterized by distinct pro-inflammatory features intricately linked to AP severity. A monocyte subpopulation with elevated PPBP and CCL3 levels emerges as a potential biomarker and therapeutic target [ABSTRACT FROM AUTHOR]

Background: Segregated-nucleus-containing atypical monocytes have recently been identified in mice. Segregated-nucleus-containing atypical monocytes are thought to originate from the bone marrow and induce fibrosis in the drug-injured lung. The Lyc6c− murine monocyte subset is the counterpart to human CD14−CD16++ non-classical monocytes; however, the human counterpart to murine segregated-nucleus-containing atypical monocytes has not yet been identified. Primary myelofibrosis is a well-known disease of progressive marrow fibrosis, and atypical megakaryocytes are thought to be closely related to fibrosis in primary myelofibrosis bone marrow. However, recently, monocytes have been reported to play an important role in marrow fibrosis in primary myelofibrosis. We speculated that, if there is a human counterpart to murine segregated-nucleus-containing atypical monocytes, it would present the same markers as murine segregated-nucleus-containing atypical monocytes, such as CD14−CD16+ macrophage-1 antigen (CD11b/CD18 complex)+, MSR1+, and CEACAM1+, and it might exist in the bone marrow of patients with primary myelofibrosis. Case presentation: A 74-year-old Japanese male visited our hospital for clinical follow-up after total prostatectomy for prostatic cancer. Anemia, thrombocytosis, and elevated lactate dehydrogenase were suddenly observed in a periodic examination. CALR mutation type 2 (p.K385fs*47) was observed. The histological features of the patient's bone marrow were consistent with fibrotic primary myelofibrosis. We immunohistochemically studied the bone marrow in an attempt to identify a human counterpart to murine segregated-nucleus-containing atypical monocytes. We detected a few CD16+MSR1+CEACAM1+ cells, but not CD14+MSR1+CEACAM1+ cells, by triple immunostaining. The patient is in a good condition and does not require treatment for primary myelofibrosis. Conclusion: There is a possibility that human segregated-nucleus-containing atypical monocytes exist in the bone marrow of primary myelofibrosis patients and might be related to marrow fibrosis. [ABSTRACT FROM AUTHOR]

Aging profoundly affects the immune system leading to an increased propensity for inflammation. Age-related dysregulation of immune cells is implicated in the development and progression of numerous age-related diseases such as: cardiovascular diseases, neurodegenerative disorders, and metabolic syndromes. Monocytes and monocyte-derived macrophages, being important players in the inflammatory response, significantly influence the aging process and the associated increase in inflammatory disease risk. Ischemic stroke is among age-related diseases where inflammation, particularly monocyte-derived macrophages, plays an important deteriorating role but could also strongly promote post-stroke recovery. Also, biological sex influences the incidence, presentation, and outcomes of ischemic stroke, reflecting both biological differences between men and women. Here, we studied whether human peripheral blood monocyte subtype (classical, intermediate, and non-classical) expression of genes implicated in stroke-related inflammation and post-stroke tissue regeneration depends on age and sex. A flow cytometry analysis of blood samples from 44 healthy volunteers (male and female, aged 28 to 98) showed that in contrast to other immune cells, the proportion of NK-cells increased in females. The proportion of B-cells decreased in both sexes with age. Gene expression analysis by qPCR identified several genes differentially correlating with age and sex within different monocyte subtypes. Interestingly, ANXA1 and CD36 showed a consistent increase with aging in all monocytes, specifically in intermediate (CD36) and intermediate and non-classical (ANXA1) subtypes. Other genes (IL-1β, S100A8, TNFα, CD64, CD33, TGFβ1, TLR8, CD91) were differentially changed in monocyte subtypes with increasing age. Most age-dependent gene changes were differentially expressed in female monocytes. Our data shed light on the nuanced interplay of age and sex in shaping the expression of inflammation- and regeneration-related genes within distinct monocyte subtypes. Understanding these dynamics could pave the way for targeted interventions and personalized approaches in post-stroke care, particularly for the aging population and individuals of different sexes. [ABSTRACT FROM AUTHOR]

Objectives: The study aimed to investigate the interaction of intraoperative stress hyperglycemia with monocyte functions and their impact on major adverse events (MAEs) in acute aortic dissection (AAD) patients who underwent open repair surgery. Methods: A total of 321 adults who underwent open surgery for AAD at two tertiary medical centers in China were enrolled in the study. The primary endpoint was defined as the incidence and characteristics of perioperative stress hyperglycemia. The secondary endpoints included the incidence of postoperative MAEs, postoperative monocyte counts and inflammatory cytokine expression. Multi-logistic, linear regression and receiver operating characteristic (ROC) curve analyses were used to establish relationships between intraoperative time-weighted average glucose (TWAG), day-one postoperative monocyte counts, serum inflammatory cytokines and postoperative outcomes. In addition, in vitro experiments were conducted to evaluate changes in the inflammatory features of monocytes under high glucose conditions. Results: Intraoperative hyperglycemia, as indicated by a TWAG level over 142 mg/dL, was associated with elevated postoperative monocyte counts and inflammatory cytokines, which correlated with extended intensive care unit (ICU) stays and worsened outcomes. In vitro, high glucose treatment induced mitochondrial impairment in monocytes, increased the release of inflammatory cytokines and the proportion of classical monocytes from AAD patients. Conclusions: Intraoperative stress hyperglycemia, in combination with day-one postoperative monocyte counts, were clinically significant for predicting adverse outcomes in AAD patients undergoing open repair surgery. Elevated glucose concentrations shaped the inflammatory features of monocytes in AAD by impairing mitochondrial functions. High glucose-driven mitochondrial functional impairment mediates a more pronounced monocytic inflammatory state in Stanford type-A AAD. [ABSTRACT FROM AUTHOR]

ABSTRACT Introduction Methods Results Conclusions Estimating patient risk before heart surgery (HS) is crucial. Perioperative inflammation is associated with several complications and mortality. This study investigated blood cell count inflammatory indices (BCCII) to predict risks, including neutrophil‐to‐lymphocyte ratio (NLR), derivate NLR (DNLR), neutrophil‐to‐platelet‐lymphocyte ratio (NLPR), lymphocyte‐to‐monocyte ratio, platelet‐to‐lymphocyte ratio (PLR), Systemic Inflammatory Index (SII), Systemic Inflammatory Reaction Index (SIRI), and Aggregate Index of Systemic Inflammation (AISI).Data from a cohort of patients undergoing on‐pump HS at a single center in Brazil were retrospectively analyzed. Data were obtained from medical records and a laboratory analyzer, and SPSS version 20.0 was used for index calculations and statistical analyses.In total, 444 surgeries were performed, and 40 in‐hospital deaths occurred. Except for PLR, all other indexes were independent predictors of death after multivariate adjustment (all p < 0.05). Discrimination performance was absent for PLR and AISI, and NLR, NLPR, and DNLR demonstrated the best area under the receiver operating characteristic curve (AUC > 0.7; all p < 0.0001). For survivors (n = 404), all indexes exhibited a correlation with the length of hospital stay (all p < 0.03), and NLR, NLPR, and DNLR were predictors (p < 0.026) of poor operative outcomes (acute myocardial infarction, cerebrovascular attack, cardiac arrest, low cardiac output, prolonged mechanical ventilation, renal failure, and sepsis).All BCCII scores were associated with length of hospital stay. Apart from PLR, all indexes were independent predictors of in‐hospital mortality. Accuracy was highest for NLR, NLPR, and DNLR; for survivors, these three factors were good predictors of poor operative outcomes. [ABSTRACT FROM AUTHOR]

Aberrant activation of the cGAS-STING signaling results in innate immune response induction. Herein, we report HSKB142, an orally bioavailable compound containing the 3H-pyrazolo [4,3-f]quinoline synthesized via a Povarov–Doebner MCR. HSKB142 is non-cytotoxic towards immune cells and suppresses type-1 interferon expression in human THP-1 monocytes upon treatment with 2′3′-cGAMP. [ABSTRACT FROM AUTHOR]

Inflammatory signals lead to recruitment of circulating monocytes and induce their differentiation into pro-inflammatory macrophages. Therefore, whether blocking inflammatory monocytes can mitigate disease progression is being actively evaluated. Here, we employ multiple lineage-tracing models and show that monocyte-derived macrophages (mo-mac) are the major population of immunosuppressive, liver metastasis-associated macrophages (LMAM), while the proportion of Kupffer cells (KC) as liver-resident macrophages is diminished in metastatic nodules. Paradoxically, genetic ablation of mo-macs results in only a marginal decrease in LMAMs. Using a proliferation-recording system and a KC-tracing model in a monocyte-deficient background, we find that LMAMs can be replenished either via increased local macrophage proliferation or by promoting KC infiltration. In the latter regard, KCs undergo transient proliferation and exhibit substantial phenotypic and functional alterations through epigenetic reprogramming following the vacating of macrophage niches by monocyte depletion. Our data thus suggest that a simultaneous blockade of monocyte recruitment and macrophage proliferation may effectively target immunosuppressive myelopoiesis and reprogram the microenvironment towards an immunostimulatory state. In liver metastasis of cancer, the inflammatory macrophage niche is dominated by both circulating monocytes and local expansion, but the nature and dynamic of this niche is still unclear. Here the authors use mouse genetic models and lineage-tracing tools to show that, under the condition of monocyte depletion, Kupffer cells pursue epigenetic reprogramming to contribute to this niche. [ABSTRACT FROM AUTHOR]

During inflammation, human neutrophils engage b2-integrins to migrate from the blood circulation to inflammatory sites with high cytokine but low oxygen concentrations. We tested the hypothesis that the inhibition of prolyl hydroxylase domain-containing enzymes (PHDs), cytokines, and b2-integrins cooperates in HIF pathway activation in neutrophils. Using either the PHD inhibitor roxadustat (ROX) (pseudohypoxia) or normobaric hypoxia to stabilize HIF, we observed HIF1a protein accumulation in adherent neutrophils. Several inflammatory mediators did not induce HIF1a protein but provided additive or even synergistic signals (e.g., GM-CSF) under pseudohypoxic and hypoxic conditions. Importantly, and in contrast to adherent neutrophils, HIF1a protein expression was not detected in strictly suspended neutrophils despite PHD enzyme inhibition and the presence of inflammatory mediators. Blocking b2- integrins in adherent and activating b2-integrins in suspension neutrophils established the indispensability of b2-integrins for increasing HIF1a protein. Using GM-CSF as an example, increased HIF1a mRNA transcription via JAK2- STAT3 was necessary but not sufficient for HIF1a protein upregulation. Importantly, we found that b2-integrins led to HIF1a mRNA translation through the phosphorylation of the essential translation initiation factors eIF4E and 4EBP1. Finally, pseudohypoxic and hypoxic conditions inducing HIF1a consistently delayed apoptosis in adherent neutrophils on fibronectin under low serum concentrations. Pharmacological HIF1a inhibition reversed delayed apoptosis, supporting the importance of this pathway for neutrophil survival under conditions mimicking extravascular sites. We describe a novel b2-integrincontrolled mechanism of HIF1a stabilization in human neutrophils. Conceivably, this mechanism restricts HIF1a activation in response to hypoxia and pharmacological PHD enzyme inhibitors to neutrophils migrating toward inflammatory sites. [ABSTRACT FROM AUTHOR]

Introduction: Macrophages play a key role in most of the inflammatory diseases such as Rheumatoid Arthritis (RA), but the mechanism underlying their pathogenesis is still under study. Among stem cells, human dental pulp stem cells (hDPSCs) have attracted attention due to their easy accessibility and immunomodulatory properties, making them a promising adjuvant therapy. In this study, we aimed to evaluate the capacity of hDPSCs to modulate the phenotypes of primary human macrophages. Additionally, we sought to observe the differences induced on macrophages when cultured directly with hDPSCs or through a cell culture insert, mimicking the paracrine communication pathway. Methods: Monocytes, isolated from buffy coats, were differentiated into proinflammatory M1 and anti-inflammatory M2 macrophages. Subsequently, they were cultured with hDPSCs either directly or via a cell-culture insert for 48 hours. Finally, they were analyzed for protein, gene expression, cytokines levels and immunofluorescence. Results: In our study, we have demonstrated that, hDPSCs, even without priming, can reduce TNFa levels and enhancing IL-10 release in pro-inflammatory macrophages, both through direct contact and paracrine signaling. Furthermore, we found that their effects are more pronounced when in cell-to-cell contact through the decrease of NF-kB and COX-2 expression and of CD80/PD-L1 colocalization. HDPSCs, when in contact with macrophages, showed enhanced expression of NF-kB, COX-2, ICAM-1, PD-L1, FAS-L, TNFa and IFNg. Conclusion: We showed that hDPSCs exert immunomodulatory effects on proinflammatory macrophages, with cell-to-cell contact yielding a more pronounced outcome compared to paracrine signaling. Our work highlights the immunomodulatory properties of hDPSCs on activated pro-inflammatory macrophages and the potential therapeutic role in inflamed tissue. [ABSTRACT FROM AUTHOR]

Monocytes directly contribute to atherosclerosis development by their recruitment to plaques in which they differentiate into macrophages. In the present study, we ask how modulating monocyte glucose metabolism could affect their homeostasis and their impact on atherosclerosis. Here we investigate how circulating metabolites control monocyte behavior in blood, bone marrow and peripheral tissues of mice. We find that serum glucose concentrations correlate with monocyte numbers. In diet-restricted mice, monocytes fail to metabolically reprogram from glycolysis to fatty acid oxidation, leading to reduced monocyte numbers in the blood. Mechanistically, Glut1-dependent glucose metabolism helps maintain CD115 membrane expression on monocytes and their progenitors, and regulates monocyte migratory capacity by modulating CCR2 expression. Results from genetic models and pharmacological inhibitors further depict the relative contribution of different metabolic pathways to the regulation of CD115 and CCR2 expression. Meanwhile, Glut1 inhibition does not impact atherosclerotic plaque development in mouse models despite dramatically reducing blood monocyte numbers, potentially due to the remaining monocytes having increased migratory capacity. Together, these data emphasize the role of glucose uptake and intracellular glucose metabolism in controlling monocyte homeostasis and functions. Monocytes participate in plaque formation, and adapt to metabolic changes to alter their functions. Here the authors show, using genetic mouse models and functional analyses, that Glut1-mediated glucose metabolism is important for regulating monocyte homeostasis and migration, but curiously has no impact on atherosclerotic plaque formation. [ABSTRACT FROM AUTHOR]

Background: For glioma patients, the long-term advantages of dendritic cells (DCs) immunization remain unknown. It is extremely important to develop new treatment strategies that enhance the immunotherapy effect of DC-based vaccines. DCs exposed to glioma stem cells (GSCs) are considered promising vaccines against glioma. Methods: Glioma stem cells were isolated from mouse glioma GL261 cells (GCs). Both were subjected to severe (47°C) and mild (42°C) heat shock to induce immunogenic cell death (ICD). Membrane mobilization of calreticulin (CRT) and release of heat shock proteins (HSPs) were detected by flow cytometry. Dendritic cells were then exposed to heat-inactivated cells and co-culturing of T cells tested for immunotherapeutic efficacy in vitro. In vivo, we investigated the GSC targeting effect of the GSC-DC vaccine combined with CD47 blockade. Results: Heat shock induced ICD in GCs and GSCs, as indicated by significant release of calreticulin, HSP70, and HSP90. Heat shock condition ICD lysates induce maturation and activation-associated marker expression on monocyte-derived DCs. Accordingly, DCs pulsed with GCs and GSCs inactivated reduced colony formation, sphere formation, migration, and invasion of glioma and GSCs in vitro. Glioma stem cell-DC vaccine in combination with anti-CD47 antibody significantly enhanced survival in mice with glioma, induced production of interferon (IFN)-γ, and enhanced T-cell expansion in vivo. Of note, DCs pulsed with inactivated GSCs were more effective to control tumor growth than DCs pulsed with inactive GCs. Conclusions: Severe heat shock induces ICD in vitro. These data showed that administration of anti-CD47 antibody combined with GSC-DC vaccine may represent an effective immunotherapeutic strategy for cancer patients in clinical. [ABSTRACT FROM AUTHOR]

Patients heterozygous for germline CBL loss-of-function (LOF) variants can develop myeloid malignancy, autoinflammation, or both, if some or all of their leukocytes become homozygous for these variants through somatic loss of heterozygosity (LOH) via uniparental isodisomy. We observed an upregulation of the inflammatory gene expression signature in whole blood from these patients, mimicking monogenic inborn errors underlying autoinflammation. Remarkably, these patients had constitutively activated monocytes that secreted 10 to 100 times more inflammatory cytokines than those of healthy individuals and CBL LOF heterozygotes without LOH. CBL-LOH hematopoietic stem and progenitor cells (HSPCs) outgrew the other cells, accounting for the persistence of peripheral monocytes homozygous for the CBL LOF variant. ERK pathway activation was required for the excessive production of cytokines by both resting and stimulated CBL-LOF monocytes, as shown in monocytic cell lines. Finally, we found that about 1 in 10,000 individuals in the UK Biobank were heterozygous for CBL LOF variants and that these carriers were at high risk of hematological and inflammatory conditions. [ABSTRACT FROM AUTHOR]

Migraine is a common and complex neurological disorder that has a high impact on quality of life. Recent advances with drugs that target the neuropeptide calcitonin gene-related peptide (CGRP) have helped, but treatment options remain insufficient. CGRP is released from trigeminal sensory fibers and contributes to peripheral sensitization, perhaps in part due to actions on immune cells in the trigeminovascular system. In this review, we will discuss the potential of cannabinoid targeting of immune cells as an innovative therapeutic target for migraine treatment. We will cover endogenous endocannabinoids, plant-derived phytocannabinoids and synthetically derived cannabinoids. The focus will be on six types of immune cells known to express multiple cannabinoid receptors: macrophages, monocytes, mast cells, dendritic cells, B cells, and T cells. These cells also contain receptors for CGRP and as such, cannabinoids might potentially modulate the efficacy of current CGRP-targeting drugs. Unfortunately, to date most studies on cannabinoids and immune cells have relied on cell cultures and only a single preclinical study has tested cannabinoid actions on immune cells in a migraine model. Encouragingly, in that study a synthetically created stable chiral analog of an endocannabinoid reduced meningeal mast cell degranulation. Likewise, clinical trials evaluating the safety and efficacy of cannabinoid-based therapies for migraine patients have been limited but are encouraging. Thus, the field is at its infancy and there are significant gaps in our understanding of the impact of cannabinoids on immune cells in migraine. Future research exploring the interactions between cannabinoids and immune cells could lead to more targeted and effective migraine treatments. [ABSTRACT FROM AUTHOR]

This study explores methods to isolate high-pure monocytes and optimize the best growth factor concentration to generate monocytes-derived dendritic cells (mo-DCs), subset DC1, which is crucial in immune responses. Three protocols for monocyte isolation from peripheral blood mononuclear cells (PBMCs) were evaluated: three-hour incubation on FBS-coated flasks; an overnight incubation on FBS-coated flasks; and Magnetic Activated Cell Sorting (MACS). Additionally, five different concentrations of human recombinant granulocyte-macrophage colony-stimulating factor (hrGM-CSF) and human recombinant interleukin-4 (hrIL-4) were compared. We used Flow cytometry to assess the isolation, purification, and generation of pure monocytes characterized as CD14+, and expression of mo-DC classical markers (HLA-DR, CD80, CD83, and CD86). The obtained results show that monocytes isolated with the second method (overnight incubation) had the highest purity (P < 0.0001) but the lowest yield (P > 0.05), balancing purity and cost-effectiveness. A combination of hrGM-CSF and hrIL-4 at 400 U/mL produced the most favorable outcomes, leading to the highest rate of mo-DC generation (P < 0.05). Notably, this concentration resulted in increasing expression of HLA-DR, CD80, and CD86 surface markers in the generated DCs (P < 0.0001), with no changes in CD83 expression levels. In conclusion, this study offers valuable insights into selecting the optimal approach for monocyte isolation and mo-DC generation in various research contexts, providing a foundation for more effective immunological studies. [ABSTRACT FROM AUTHOR]

Persistent inflammation, characterized by the intense interplay of inflammatory chemokine secretion and immune cell infiltration, is a hallmark of many skin disorders including diabetic wounds and psoriasis with inadequate therapeutic interventions. Monocyte chemotactic protein‐1 (MCP‐1) is an inflammatory chemokine that plays a key role in recruiting and polarizing monocytes into pro‐inflammatory macrophages to establish a vicious cycle that worsens the inflamed tissue microenvironment. Here, the sponge‐like microneedles (HPMN) technology is described to alleviate inflammatory skin disorders. Heparin/4‐arm PEG‐NH2 network crosslinked onto microneedle surface spatially attracted and sequestered multiple inflammatory chemokines including MCP‐1. Enrichment of MCP‐1 on microneedles recruits and traps inflammatory monocytes within the porous structure of microneedles. Subsequent removal of microneedles not only depletes inflammatory chemokine, MCP‐1, but also its cellular source. As a result, HPMN treatment facilitates 47.1% smaller open wound area in mice and 27.2% shorter wound length in pigs. To demonstrate the versatility of the HPMN technology, it is also shown that combining the method with standard‐of‐care immunosuppressants reduces 45.1% epidermis thickening and attenuated immune cell influx in a mouse psoriasis model. Overall, the HPMN technology is a novel demonstration of employing inflammatory chemokine and cell extraction to treat a broad range of inflammatory skin disorders. [ABSTRACT FROM AUTHOR]

Objective To investigate the value of monocyte/high-density lipoprotein cholesterol (HDL-C) ratio (MHR) in the differential diagnosis of benign and malignant thyroid nodules in reproductive-aged women. Methods A total of 186 reproductive-aged women with single thyroid nodule were selected as the observation group, and were divided into the malignant subgroup (23 cases) and benign subgroup (163 cases) according to surgical and pathological results； in the same period，93 healthy subjects who came to the hospital for physical examination were used as the control group. The clinical data of the malignant subgroup and benign subgroup were collected and compared. The peripheral blood mononuclear cell and HDL-C were detected to calculate the MHR in each group. Multivariate Logistic regression was used to analyze the effect of MHR on the thyroid nodules in reproductive-aged women. Hosmer-Lemeshow goodness of fit test was used to determine the fit of the model. Receiver operating characteristic curve (ROC curve) was used to analyze the diagnostic efficacy of MHR in preoperative thyroid nodules in reproductive-aged women. Results The MHR of observation group was higher than that of control group, and the MHR of malignant subgroup was higher than that of benign subgroup (both P< 0. 05). The proportion of tea drinking, education level, sleep time, frequency of physical exercise, frequency of eating aquatic products/seafood and eggs in the malignant subgroup were lower than those in the benign subgroup, and the proportion of diabetes, uterine fibroids, life and work stress, anxiety and depression, anger frequency, and depression in the past 1 year were higher than those in the benign subgroup (all P<0. 05). Multilevel Logistic regression analysis was conducted. The results showed that when MHR was not included in model 1, life and work pressure, anxiety and depression, and anger frequency were independent risk factors for malignant thyroid nodules in reproductive-aged women (all P< 0. 05). When MHR was included in model 2, life and work pressure, anger frequency, and MHR were independent risk factors for malignant thyroid nodules in reproductive-aged women (all P<0. 05). Using the predicted probability of the Logistic regression analysis as the independent test variable, when MHR was not included, the AUC value of logit (P1) for differential diagnosis of thyroid nodules in reproductive-aged women was 0. 745； when MHR was included, the AUC value of logit (P2) for differential diagnosis of thyroid nodules in reproductive-aged women was 0. 925, which was greater than that without MHR (P<0. 01). Conclusion The MHR of malignant thyroid nodules increases in reproductive-aged women, MHR is an independent risk factor for malignant thyroid nodules in reproductive-aged women, and detection of MHR is helpful to distinguish benign and malignant thyroid nodules in reproductive-aged women. [ABSTRACT FROM AUTHOR]

BACKGROUND: Activation of the immune system contributes to cardiovascular diseases. The role of human-specific long noncoding RNAs in cardioimmunology is poorly understood. METHODS: Single-cell sequencing in peripheral blood mononuclear cells revealed a novel human-specific long noncoding RNA called HEAT4 (heart failure-associated transcript 4). HEAT4 expression was assessed in several in vitro and ex vivo models of immune cell activation, as well as in the blood of patients with heart failure (HF), acute myocardial infarction, or cardiogenic shock. The transcriptional regulation of HEAT4 was verified through cytokine treatment and single-cell sequencing. Loss-offunction and gain-of-function studies and multiple RNA-protein interaction assays uncovered a mechanistic role of HEAT4 in the monocyte anti-inflammatory gene program. HEAT4 expression and function was characterized in a vascular injury model in NOD.CB17-Prkdc scid/Rj mice. RESULTS: HEAT4 expression was increased in the blood of patients with HF, acute myocardial infarction, or cardiogenic shock. HEAT4 levels distinguished patients with HF from people without HF and predicted all-cause mortality in a cohort of patients with HF over 7 years of follow-up. Monocytes, particularly anti-inflammatory CD16+ monocytes, which are increased in patients with HF, are the primary source of HEAT4 expression in the blood. HEAT4 is transcriptionally activated by treatment with anti-inflammatory interleukin-10. HEAT4 activates anti-inflammatory and inhibits proinflammatory gene expression. Increased HEAT4 levels result in a shift toward more CD16+ monocytes. HEAT4 binds to S100A9, causing a monocyte subtype switch, thereby reducing inflammation. As a result, HEAT4 improves endothelial barrier integrity during inflammation and promotes vascular healing after injury in mice. CONCLUSIONS: These results characterize a novel endogenous anti-inflammatory pathway that involves the conversion of monocyte subtypes into anti-inflammatory CD16+ monocytes. The data identify a novel function for the class of long noncoding RNAs by preventing protein secretion and suggest long noncoding RNAs as potential targets for interventions in the field of cardioimmunology. [ABSTRACT FROM AUTHOR]

Stress may play a key role in alopecia areata (AA), though the exact interactions of stress with AA remain undefined. Corticotropin‐releasing hormone (CRH), the proximal regulator of the stress axis, has been recognized as an immunomodulatory factor in tissues and peripheral blood mononuclear cells (PBMCs). We used multicolour flow cytometry to identify receptor CRHR1 expression on PBMC subsets in AA patients (n = 54) and controls (n = 66). We found that CRHR1 was primarily expressed by circulating monocytes. CRHR1 expression on monocytes was enhanced in AA compared with controls (3.17% vs. 1.44%, p = 0.002, chi‐squared test). AA incidence was correlated to elevated CD14+ monocyte numbers (R = 0.092, p = 0.036) and markedly independently correlated with increased CRHR1 expression (R = 0.215, p = 0.027). High CRHR1 expression was significantly related to chronic AA (disease duration >1 year; p = 0.003, chi‐squared test), and large lesion area (AA area >25%; p = 0.049, chi‐squared test). We also observed enhanced percentages of active monocytes and reduced CD16+ CD3− NK cell numbers in AA patients' PBMCs (p = 0.010; 0.025, respectively). In vitro CRH treatment of PBMCs and human monocyte cell line THP‐1 promoted CD86 upregulation. The findings imply that stress‐related factors CRH and CRHR1 contribute to AA development and progression where higher CRHR1 expression is associated with chronic AA and larger lesions. [ABSTRACT FROM AUTHOR]

tRNA-derived fragments function as markers in addition to playing the key role of signalling molecules in a number of disorders. It is known that the repertoire of these molecules differs greatly in different cell types and varies depending on the physiological condition. The aim of our research was to compare the pattern of tRF expression in the main blood cell types and to determine how the composition of these molecules changes during COVID-19-induced cytokine storms. Erythrocytes, monocytes, lymphocytes, neutrophils, basophils and eosinophils from control donors and patients with severe COVID-19 were obtained by fluorescence sorting. We extracted RNA from FACS-sorted cells and performed NGS of short RNAs. The composition of tRNA-derived fragments was analysed by applying a semi-custom bioinformatic pipeline. In this study, we assessed the length and type distribution of tRFs and reported the 150 most prevalent tRF sequences across all cell types. Additionally, we demonstrated a significant (p < 0.05, fold change >16) change in the pattern of tRFs in erythrocytes (21 downregulated, 12 upregulated), monocytes (53 downregulated, 38 upregulated) and lymphocytes (49 upregulated) in patients with severe COVID-19. Thus, different blood cell types exhibit a significant variety of tRFs and react to the cytokine storm by dramatically changing their differential expression patterns. We suppose that the observed phenomenon occurs due to the regulation of nucleotide modifications and alterations in activity of various Rnases. [ABSTRACT FROM AUTHOR]

Human monocytes can be subdivided into phenotypically and functionally different classical, intermediate and non-classical monocytes according to the cell surface expression of CD14 and CD16. A precise identification and characterisation of monocyte subsets is necessary to unravel their role in inflammatory diseases. Here, we compared three different flow cytometric strategies (A–C) and found that strategy C, which included staining against CD11b, HLA-DR, CD14 and CD16, followed by several gating steps, most reliably identified monocyte subtypes in blood samples from healthy volunteers and from patients with stable coronary heart disease (CHD) or ST-elevation myocardial infarction (STEMI). Additionally, we established a fixation and permeabilisation protocol to enable the analysis of intracellular markers. We investigated the phagocytosis of lipid nanoparticles, the uptake of 2-NBD-glucose and the intracellular levels of CD74 and HLA-DM. This revealed that classical and intermediate monocytes from patients with STEMI showed the highest uptake of 2-NBD-glucose, whereas classical and intermediate monocytes from patients with CHD took up the largest amounts of lipid nanoparticles. Interestingly, intermediate monocytes had the highest expression level of HLA-DM. Taken together, we present a robust flow cytometric approach for the identification and functional characterisation of monocyte subtypes in healthy humans and patients with diseases. [ABSTRACT FROM AUTHOR]