Monocytes In Response Research Articles

Airway and Blood Monocyte Transcriptomic Profiling Reveals an Antiviral Phenotype in Infants With Severe Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) infection is the primary cause of lower respiratory tract infections in children <5 years of age. Monocytes, especially in the respiratory tract, are suggested to contribute to RSV pathology, but their role is incompletely understood. With transcriptomic profiling of blood and airway monocytes, we describe the role of monocytes in severe RSV infection. Tracheobronchial aspirates and blood samples were collected from control patients (n = 9) and those infected with RSV (n = 14) who were admitted to the pediatric intensive care unit. Monocytes (CD14+) were sorted and analyzed by RNA sequencing for transcriptomic profiling. Peripheral blood and airway monocytes of patients with RSV demonstrated increased expression of antiviral and interferon-responsive genes as compared with controls. Cytokine signaling showed a shared response between blood and airway monocytes while displaying responses that were more pronounced according to the tissue of origin. Airway monocytes upregulated additional genes related to migration and inflammation. We found that the RSV-induced interferon response extends from the airways to the peripheral blood. Moreover, RSV induces a migration-promoting transcriptional program in monocytes. Unraveling the monocytic response and its role in the immune response to RSV infection could help the development of therapeutics to prevent severe disease.

Developmental Differences between Neonatal and Adult Platelets in Regard to Immune Protein Content and Regulation of Monocyte Inflammatory Responses

Preterm neonates frequently receive platelet transfusions (PTx), owing to their high incidence of thrombocytopenia and bleeding. However, a recent large randomized trial found a significantly higher incidence of death or major bleeding among more liberally transfused preterm neonates. The mechanisms underlying these deleterious effects are unknown, but it has been suggested that they are related to the developmental differences between adult and neonatal platelets, and to a potential “developmental mismatch” occurring when adult platelets (comparatively hyperreactive) are transfused into neonates. While most studies so far have focused on the hemostatic effects of PTx, it is now clear that platelets are key modulators of immune and inflammatory responses. We hypothesized that adult platelets would be more pro-inflammatory than neonatal platelets. To test this hypothesis, we first compared the proteome of resting cord blood (CB, n=9) and adult (n=7) platelets. Using data-independent acquisition mass spectrometry, we identified 4173 high-confidence proteins present in both adult and neonatal platelets. In a principal component analysis, neonatal and adult platelets clustered separately, and 359 proteins were present at significantly different relative abundances between the two groups (≥1.5-fold change, p&amp;lt;0.05). Pathway analysis of the 169 proteins significantly more abundant in the adult platelet proteome revealed enrichment for immune and inflammatory pathways, while neonatal platelets were enriched for pathways involved in metabolic processes. Next, we compared the effects of neonatal and adult platelets on the inflammatory response of human neonatal monocytes. Platelets and monocytes were isolated from healthy adult blood or full-term CB (n=6/group). Platelet P-selectin surface expression was determined by flow cytometry, and key platelet-derived immune regulators (RANTES, β-2 Microglobulin [β2M], and TGF-β) were quantified in the supernatant of resting and activated platelets by ELISA. After washing, neonatal and adult platelets had similarly low P-selectin surface expression levels. Following thrombin activation, P-selectin surface expression was significantly higher in adult than in neonatal platelets (MFI 4010±1645 vs. 1987±816, p=0.04). Thrombin activation increased RANTES and TGF-β release from adult and neonatal platelets to similar levels. In contrast, β2M levels were significantly higher in the supernatant from activated adult compared to neonatal platelets (37.9±10.2 vs 17.9±5.0 ng/mL, p=0.0004). Next, we co-cultured activated adult or neonatal platelets with neonatal monocytes and quantified the pro-inflammatory cytokines IL-8 and MCP-1 (CCL2) in the supernatants after 18 hours. The presence of activated platelets significantly increased IL-8 production from neonatal monocytes compared to monocytes alone, 2.9±1.2-fold when neonatal platelets were added and 4.2±1.8-fold with addition of adult platelets (p=0.01 and p=0.001, respectively). MCP-1 increased to similar levels with the addition of either neonatal or adult platelets. In a regression model, both P-selectin expression and platelet-released β2M levels were significantly associated with monocyte-produced IL-8 levels (p=0.001 and p=0.04, respectively). In summary, we found that proteins related to inflammatory and immune responses are more abundant in adult compared to neonatal platelets, and that activated adult platelets express more P-selectin and release more β2M than neonatal platelets, resulting in higher IL8 production by neonatal monocytes. These findings support the hypothesis that the pro-inflammatory effects of adult platelets might contribute to the increased morbidity and mortality associated with neonatal PTx.

Expression of Ki67 and CD68+ cells of red bone marrow monocyte sprout under triptorelin administration in the hypothalamic-pituitary-testis regulatory system: the experimental study

Background. Red bone marrow (RBM) is the main organ of human haemopoiesis. Monocytopoiesis plays an important role in the formation of transitional states: from normal to pathology and in the transformation of pathological processes from one stage to another. In modern urological practice, the substance triptorelin is widely used for androgen deprivation therapy, according to the recommendations of the European Association of Urology. Ki67, a commercially available monoclonal antibody that reacts with a nuclear antigen detected only in proliferating cells, is used to assess immunohistochemical changes. CD68 is a valuable cytochemical marker for immunostaining of monocytes/macrophages during histochemical analysis of tissues in inflammation, cancer and other immunohistopathological purposes. The purpose of the study is to evaluate the proliferative activity and differentiation of progenitor cells through the expression of Ki67 and CD68+ monocyte sprouting of RBM under chemical castration of central origin in male rats caused by the administration of triptorelin solution with quercetin addition to the diet for one year. Materials and methods. The study was conducted on 60 adult male white rats. They were divided into 3 groups: group I — control (n = 10), group II (n = 25) — subcutaneous injection of triptorelin, group III (n = 25) — subcutaneous injection of triptorelin acetate and quercetin. Immunohistochemical analysis of biopsy specimens was conducted following a standard protocol at the Department of Pathological Anatomy in Sumy State University, under the supervision of the Head of the Department, Prof. Romaniuk A.M. Results. The study evaluated Ki67 expression on microsections of rat red bone marrow through immunohistochemistry, which exclusively reacted with nuclear antigen in the monocyte sprout’s proliferating cells. Irregular changes were revealed depending on the experimental groups and time periods. Immunohistochemical analysis of RBM tissue using anti-CD68 antibodies in the experimental groups revealed a strong positive cytoplasmic response in monocytes and resident macrophages located in the monocyte sprout and surrounding environment. The data of the two experimental groups of RBM had a noticeable proliferating compartment, as evidenced by the high content of mitotically active DNA in them. These data correspond to the results obtained in the experiment with triptorelin, where we found a marked positivity of Ki67, depending on the timing of the experiment and the addition of the flavonoid quercetin. This discrepancy suggests that bone marrow cells that grow and proliferate under normal conditions are guided by natural control mechanisms and may lose their Ki67 expression after leaving the progenitor compartment and entering the differentiation compartment. Conclusions. Triptorelin administration induces hormonal imbalance in the hypothalamus-pituitary-testis-RBM system, resulting in quantitative and qualitative alterations in the cells of the RBM monocytic lineage. The level of cell proliferation, as measured by Ki67, is highest during the third month of observation. Cytoplasmic expression of CD68 is evident in two experimental groups from the third to the sixth month, suggesting activation of immunoreactive cells as they migrate from the progenitor compartment to the differentiation compartment.

Single-cell multi-omics analysis of COVID-19 patients with pre-existing autoimmune diseases shows aberrant immune responses to infection.

In COVID-19, hyperinflammatory and dysregulated immune responses contribute to severity. Patients with pre-existing autoimmune conditions can therefore be at increased risk of severe COVID-19 and/or associated sequelae, yet SARS-CoV-2 infection in this group has been little studied. Here, we performed single-cell analysis of peripheral blood mononuclear cells from patients with three major autoimmune diseases (rheumatoid arthritis, psoriasis, or multiple sclerosis) during SARS-CoV-2 infection. We observed compositional differences between the autoimmune disease groups coupled with altered patterns of gene expression, transcription factor activity, and cell-cell communication that substantially shape the immune response under SARS-CoV-2 infection. While enrichment of HLA-DRlow CD14+ monocytes was observed in all three autoimmune disease groups, type-I interferon signaling as well as inflammatory T cell and monocyte responses varied widely between the three groups of patients. Our results reveal disturbed immune responses to SARS-CoV-2 in patients with pre-existing autoimmunity, highlighting important considerations for disease treatment and follow-up.

TIM-3 Expression and M2 Polarization of Macrophages in the TGFβ-Activated Tumor Microenvironment in Colorectal Cancer.

TGFβ signaling in the tumor microenvironment (TME) drives immune evasion and is a negative predictor of immune checkpoint inhibitor (ICI) efficacy in colorectal cancer (CRC). TIM-3, an inhibitory receptor implicated in anti-tumor immune responses and ICI resistance, has emerged as an immunotherapeutic target. This study investigated TIM-3, M2 macrophages and the TGFβ-activated TME, in association with microsatellite instability (MSI) status and consensus molecular subtypes (CMSs). Transcriptomic cohorts of CRC tissues, organoids and xenografts were examined (n = 2240). TIM-3 and a TGFβ-inducible stromal protein, VCAN, were evaluated in CRC specimens using immunohistochemistry (n = 45). TIM-3 expression on monocytes and generated M2 macrophages was examined by flow cytometry. We found that the expression of HAVCR2 (TIM-3) significantly correlated with the transcriptional signatures of TGFβ, TGFβ-dependent stromal activation and M2 macrophage, each of which were co-upregulated in CMS4, CMS1 and MSI CRCs across all datasets. Tumor-infiltrating TIM-3+ immune cells accumulated in TGFβ-responsive cancer stroma. TIM-3 was increased on M2-polarized macrophages, and on monocytes in response to TGFβ treatment. In conclusion, we identified a close association between TIM-3 and M2-like polarization of macrophages in the TGFβ-rich TME. Our findings provide new insights into personalized immunotherapeutic strategies based on the TME for CRCs.

Immunoinflammatory Response to Acute Noise Stress in Male Rats Adapted with Different Exercise Training.

Noise pollution is a kind of stress that impairs various physiological functions. This study evaluated the effect of high-intensity interval training (HIIT) and moderate-intensity continuous training (MCT) on corticosterone, interleukin-6 (IL-6), and monocyte responses to acute noise stress in male rats. Forty-two male Wistar rats were divided into seven groups, including control which was assessed at the beginning, control time which was assessed simultaneously with experimental groups (CT), HIIT, MCT, HIIT followed by noise stress (HIIT+S), MCT followed by noise stress (MCT+S), and noise stress. HIIT and MCT were performed for 8 weeks. Noise stress was induced for one session. Blood samples were taken 48 hours after the last exercise session in training and CT groups and immediately after acute noise stress in stress groups of HIIT+S, MCT+S, and noise stress. In response to acute noise stress, MCT and HIIT adaptations increased corticosterone, while reduced monocytes compared to CT. MCT increased basal corticosterone and IL-6 and decreased monocytes; however, in response to acute noise stress, corticosterone was higher and monocyte count was lower in the HIIT+S group. Regarding the effect of training, corticosterone and monocytes in MCT were higher than in HIIT. The serum level of IL-6 was lower in MCT than CT group, while it was not significantly different between stress groups. In response to noise stress, previous exercise, especially HIIT, increased stress while did not increase inflammatory and innate immune response.

FDCA Attenuates Neuroinflammation and Brain Injury after Cerebral Ischemic Stroke.

Ischemic stroke is a deleterious cerebrovascular disease with few therapeutic options, and its functional recovery is highly associated with the integrity of the blood-brain barrier and neuroinflammation. The Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor fasudil (F) and the pyruvate dehydrogenase kinase (PDK) inhibitor dichloroacetate (DCA) have been demonstrated to exhibit neuroprotection in a series of neurological disorders. Hence, we synthesized and biologically examined the new salt fasudil dichloroacetate (FDCA) and validated that FDCA was eligible for attenuating ischemic volume and neurological deficits in the rat transient middle cerebral artery occlusion (tMCAO) model. Additionally, FDCA exerted superior effects than fasudil and dichloroacetate alone or in combination in reducing cerebral ischemic injury. Particularly, FDCA could maintain the blood-brain barrier (BBB) integrity by inhibiting matrix metalloproteinase 9 (MMP-9) protein expression and the degradation of zonula occludens (ZO-1) and Occludin protein. Meanwhile, FDCA could mitigate the neuroinflammation induced by microglia. The in vivo and in vitro experiments further demonstrated that FDCA disrupted the phosphorylations of myosin phosphatase target subunit 1 (MYPT1), mitogen-activated protein kinase (MAPK) cascade, including p38 and c-Jun N-terminal kinase (JNK), and pyruvate dehydrogenase (PDH) and limited excessive lactic acid metabolites, resulting in inhibition of BBB disruption and neuroinflammation. In addition, FDCA potently mitigated inflammatory response in human monocytes isolated from ischemic stroke patients, which provides the possibilities of a clinical translation perspective. Overall, these findings provided a therapeutic potential for FDCA as a candidate agent for ischemic stroke and other neurological diseases associated with BBB disruption and neuroinflammation.

ADENOSINE TO INOSINE RNA EDITING ASSOCIATED TUMOUR MICROENVIRONMENT AS A THERAPEUTIC TARGET IN GLIOBLASTOMA

Abstract AIMS Glioblastoma (GBM) remains incurable because of the high level of intratumor heterogeneity and the poor understanding of its complex genetics. We recently identified adenosine to inosine (A-to-I) RNA editing as a sex- specific prognostic factor in GBM. However, the underlying mechanism remains unclear. We sought to identify RNA editing-associated molecular effectors and assess their prognosis potential. METHOD We analyzed RNA-Seq data of 196 primary IDH wild-type GBMs from The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) cohorts. Genome-wide A-to-I RNA editing events were characterized and GBM tumours were split into two subtypes based on RNA editing signatures (high- and low-editing). Differential expression and tumour microenvironment analyses between the two subtypes were performed to identify molecular effectors. Survival impact of these effectors was determined using Cox proportional hazards model. RESULTS Genes more highly expressed in the high-editing subtype were enriched in immune-related pathways, while those more highly expressed in the low-editing subtype were enriched in cell proliferation and stemness path- ways. Consistent with differential expression analysis, high-editing GBMs exhibited higher infiltration levels of macrophages, monocytes and dendritic cells. Intriguingly, increased cell proliferation and elevated infiltration of dendritic cells were associated with poorer survival in men only, whereas enhanced CSF1 (colony stimulating factor 1) response and elevated infiltration of monocytes and Th1 cells conferred poorer survival outcomes in women only. CONCLUSIONS Our study suggests sex-specific impact of A-to-I RNA editing in the pathogenesis of glioblastoma, highlighting the importance of developing sex-specific treatments and opening a new avenue for the design of novel therapeutic targets.

Single-cell analysis of graft-infiltrating host cells identifies caspase-1 as a potential therapeutic target for heart transplant rejection.

Understanding the cellular mechanisms underlying early allograft rejection is crucial for the development of effective immunosuppressant strategies. This study aims to investigate the cellular composition of graft-infiltrating cells during the early rejection stage at a single-cell level and identify potential therapeutic targets. A heterotopic heart transplant model was established using enhanced green fluorescent protein (eGFP)-expressing mice as recipients of allogeneic or syngeneic grafts. At 3 days post-transplant, eGFP-positive cells infiltrating the grafts were sorted and subjected to single-cell RNA-seq analysis. Potential molecular targets were evaluated by assessing graft survival and functions following administration of various pharmacological inhibitors. A total of 27,053 cells recovered from syngrafts and allografts were classified into 20 clusters based on expression profiles and annotated with a reference dataset. Innate immune cells, including monocytes, macrophages, neutrophils, and dendritic cells, constituted the major infiltrating cell types (>90%) in the grafts. Lymphocytes, fibroblasts, and endothelial cells represented a smaller population. Allografts exhibited significantly increased proportions of monocyte-derived cells involved in antigen processing and presentation, as well as activated lymphocytes, as compared to syngrafts. Differential expression analysis revealed upregulation of interferon activation-related genes in the innate immune cells infiltrating allografts. Pro-inflammatory polarization gene signatures were also enriched in these infiltrating cells of allografts. Gene profiling and intercellular communication analysis identified natural killer cells as the primary source of interferon-γ signaling, activating inflammatory monocytes that displayed strong signals of major histocompatibility complexes and co-stimulatory molecules. The inflammatory response was also associated with promoted T cell proliferation and activation in allografts during the early transplant stages. Notably, caspase-1 exhibited specific upregulation in inflammatory monocytes in response to interferon signaling. The regulon analysis also revealed a significant enrichment of interferon-related motifs within the transcriptional regulatory network of downstream inflammatory genes including caspase-1. Remarkably, pharmacological inhibition of caspase-1 was shown to reduce immune infiltration, prevent acute graft rejection, and improve cardiac contractile function. The single-cell transcriptional profile highlighted the crucial role of caspase-1 in interferon-mediated inflammatory monocytes infiltrating heart transplants, suggesting its potential as a therapeutic target for attenuating rejection.

Sustained and intermittent hypoxia differentially modulate primary monocyte immunothrombotic responses to IL-1β stimulation.

Venous thromboembolism (VTE) is a leading cause of preventable deaths in hospitals, and its incidence is not decreasing despite extensive efforts in clinical and laboratory research. Venous thrombi are primarily formed in the valve pockets of deep veins, where activated monocytes play a crucial role in bridging innate immune activation and hemostatic pathways through the production of inflammatory cytokines, chemokines, and tissue factor (TF) - a principal initiator of coagulation. In the valve pocket inflammation and hypoxia (sustained/intermittent) coexist, however their combined effects on immunothrombotic processes are poorly understood. Inflammation is strongly associated with VTE, while the additional contribution of hypoxia remains largely unexplored. To investigate this, we modelled the intricate conditions of the venous valve pocket using a state-of-the-art hypoxia chamber with software-controlled oxygen cycling. We comprehensively studied the effects of sustained and intermittent hypoxia alone, and in combination with VTE-associated inflammatory stimuli on primary monocytes. TF expression and activity was measured in monocytes subjected to sustained and intermittent hypoxia alone, or in combination with IL-1β. Monocyte responses were further analyzed in detailed by RNA sequencing and validated by ELISA. Stimulation with IL-1β alone promoted both transcription and activity of TF. Interestingly, the stimulatory effect of IL-1β on TF was attenuated by sustained hypoxia, but not by intermittent hypoxia. Our transcriptome analysis further confirmed that sustained hypoxia limited the pro-inflammatory response induced by IL-1β, and triggered a metabolic shift in monocytes. Intermittent hypoxia alone had a modest effect on monocyte transcript. However, in combination with IL-1β intermittent hypoxia significantly altered the expression of 2207 genes and enhanced the IL-1β-stimulatory effects on several chemokine and interleukin genes (e.g., IL-19, IL-24, IL-32, MIF), as well as genes involved in coagulation (thrombomodulin) and fibrinolysis (VEGFA, MMP9, MMP14 and PAI-1). Increased production of CCL2, IL-6 and TNF following stimulation with intermittent hypoxia and IL-1β was confirmed by ELISA. Our findings provide valuable insights into how the different hypoxic profiles shape the immunothrombotic response of monocytes and shed new light on the early events in the pathogenesis of venous thrombosis.

Mathematical models disentangle the role of IL-10 feedbacks in human monocytes upon proinflammatory activation

Inflammation is one of the vital mechanisms through which the immune system responds to harmful stimuli. During inflammation, proinflammatory and anti-inflammatory cytokines interplay to orchestrate fine-tuned and dynamic immune responses. The cytokine interplay governs switches in the inflammatory response and dictates the propagation and development of the inflammatory response. Molecular pathways underlying the interplay are complex, and time-resolved monitoring of mediators and cytokines is necessary as a basis to study them in detail. Our understanding can be advanced by mathematical models that enable to analyze the system of interactions and their dynamical interplay in detail. We, therefore, used a mathematical modeling approach to study the interplay between prominent proinflammatory and anti-inflammatory cytokines with a focus on tumor necrosis factor and interleukin 10 (IL-10) in lipopolysaccharide-primed primary human monocytes. Relevant time-resolved data were generated by experimentally adding or blocking IL-10 at different time points. The model was successfully trained and could predict independent validation data and was further used to perform simulations to disentangle the role of IL-10 feedbacks during an acute inflammatory event. We used the insight to obtain a reduced predictive model including only the necessary IL-10-mediated feedbacks. Finally, the validated reduced model was used to predict early IL-10-tumor necrosis factor switches in the inflammatory response. Overall, we gained detailed insights into fine-tuning of inflammatory responses in human monocytes and present a model for further use in studying the complex and dynamic process of cytokine-regulated acute inflammation.

Dichotomous roles of RIPK3 in regulating the IFN response and NLRP3 inflammasome in human monocytes.

Regulation of the profile and magnitude of toll-like receptor (TLR) responses is important for effective host defense against infections while minimizing inflammatory toxicity. The chemokine CXCL4 regulates the TLR8 response to amplify inflammatory gene and inflammasome activation while attenuating the interferon (IFN) response in primary monocytes. In this study, we describe an unexpected role for the kinase RIPK3 in suppressing the CXCL4 + TLR8-induced IFN response and providing signal 2 to activate the NLRP3 inflammasome and interleukin (IL)-1 production in primary human monocytes. RIPK3 also amplifies induction of inflammatory genes such as TNF, IL6, and IL1B while suppressing IL12B. Mechanistically, RIPK3 inhibits STAT1 activation and activates PI3K-Akt-dependent and XBP1- and NRF2-mediated stress responses to regulate downstream genes in a dichotomous manner. These findings identify new functions for RIPK3 in modulating TLR responses and provide potential mechanisms by which RIPK3 plays roles in inflammatory diseases and suggest targeting RIPK3 and XBP1- and NRF2-mediated stress responses as therapeutic strategies to suppress inflammation while preserving the IFN response for host defense.

Immediate-release niacin and a monounsaturated fatty acid-rich meal on postprandial inflammation and monocyte characteristics in men with metabolic syndrome

When considered separately, long-term immediate-release niacin and fatty meals enriched in monounsaturated fatty acids (MUFA) decrease postprandial triglycerides, but their effects on postprandial inflammation, which is common in individuals with metabolic syndrome, are less known. Moreover, successful combination is lacking and its impact on acute disorders of the innate immune cells in the metabolic syndrome remains unclear. Here, we aimed to establish the effects from combination with niacin of different fats [butter, enriched in saturated fatty acids (SFA), olive oil, enriched in MUFA, and olive oil supplemented with eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids] on plasma inflammatory markers and circulating monocyte subsets, activation and priming at the postprandial period in individuals with metabolic syndrome. A random-order within-subject crossover experiment was performed, in which 16 individuals with metabolic syndrome and 16 age-matched healthy volunteers took 2g immediate-release niacin together with the corresponding fatty meal or a meal with no fat as control. In total, 128 postprandial curves were analysed. We sampled hourly over 6h for plasma concentrations of soluble inflammatory markers and triglycerides. Circulating monocyte subsets (CD14/CD16 balance), activation (CCL2/CCR2 axis) and priming (M1/M2-like phenotype) at the time of postprandial hypertriglyceridemic peak were also addressed. Dietary SFA (combined with niacin) promote postprandial excursions of circulating IL-6, IL-1β, TNF-α and CD14/CCR2-rich monocytes with a pro-inflammatory M1-like phenotype, particularly in individuals with metabolic syndrome. In contrast, dietary MUFA (combined with niacin) postprandially increased circulating CD16-rich monocytes with an anti-inflammatory M2-like phenotype. Omega-3 PUFA did not add to the effects of MUFA. The co-administration of a single-dose of immediate-release niacin with a fatty meal rich in MUFA, in contrast to SFA, suppresses postprandial inflammation at the levels of both secretory profile and monocyte response in individuals with metabolic syndrome. These findings highlight a potential role of combining niacin and dietary MUFA for the homeostatic control of inflammation and the innate immune system, identifying a new search direction for the management of disorders associated with the metabolic syndrome.

Cytocompatibility Evaluation of PEG-Methylsulfone Hydrogels.

Methylsulfone derivatized poly(ethylene) glycol (PEG) macromers can be biofunctionalized with thiolated ligands and cross-linked with thiol-based cross-linkers to obtain bioactive PEG hydrogels for in situ cell encapsulation. Methylsulfonyl-thiol (MS-SH) reactions present several advantages for this purpose when compared to other thiol-based cross-linking systems. They proceed with adequate and tunable kinetics for encapsulation, they reach a high conversion degree with good selectivity, and they generate stable reaction products. Our previous work demonstrated the cytocompatibility of cross-linked PEG-MS/thiol hydrogels in contact with fibroblasts. However, the cytocompatibility of the in situ MS-SH cross-linking reaction itself, which generates methylsulfinic acid as byproduct at the cross-linked site, remains to be evaluated. These studies are necessary to evaluate the potential of these systems for in vivo applications. Here we perform an extensive cytocompatibility study of PEG hydrogels during in situ cross-linking by the methylsulfonyl-thiol reaction. We compare these results with maleimide-thiol cross-linked PEGs which are well established for cell culture and in vivo experiments and do not involve the release of a byproduct. We show that fibroblasts and endothelial cells remain viable after in situ polymerization of methylsulfonyl-thiol gels on the top of the cell layers. Cell viability seems better than after in situ cross-linking hydrogels with maleimide-thiol chemistry. The endothelial cell proinflammatory phenotype is low and similar to the one obtained by the maleimide-thiol reaction. Finally, no activation of monocytes is observed. All in all, these results demonstrate that the methylsulfonyl-thiol chemistry is cytocompatible and does not trigger high pro-inflammatory responses in endothelial cells and monocytes. These results make methylsulfonyl-thiol chemistries eligible for in vivo testing and eventually clinical application in the future.

CFLIPS regulates alternative NLRP3 inflammasome activation in human monocytes.

The innate immune responses, including inflammasome activation, are paramount for host defense against pathogen infection. In contrast to canonical and noncanonical inflammasome activation, in this study, heat-killed gram-negative bacteria (HK bacteria) were identified as single-step stimulators of the NLRP3 inflammasome in human monocytes, and they caused a moderate amount of IL-1β to be released from cells. Time course experiments showed that this alternative inflammasome response was finished within a few hours. Further analysis showed that the intrinsically limited NLRP3 inflammasome activation response was due to the negative regulation of caspase-8 by the short isoform of cFLIP (cFLIPs), which was activated by NF-κB. In contrast, overexpressed cFLIPS, but not overexpressed cFLIPL, inhibited the activation of caspase-8 and the release of IL-1β in response to HK bacteria infection in human monocytes. Furthermore, we demonstrated that TAK1 activity mediated the expression of cFLIPs and was upstream and essential for the caspase-8 cleavage induced by HK bacteria in human monocytes. The functional specificity of cFLIPs and TAK1 revealed unique responses of human monocytes to a noninvasive pathogen, providing novel insights into an alternative regulatory pathway of NLRP3 inflammasome activation.

TLR7 activation at epithelial barriers promotes emergency myelopoiesis and lung antiviral immunity.

Monocytes are heterogeneous innate effector leukocytes generated in the bone marrow and released into circulation in a CCR2-dependent manner. During infection or inflammation, myelopoiesis is modulated to rapidly meet the demand for more effector cells. Danger signals from peripheral tissues can influence this process. Herein we demonstrate that repetitive TLR7 stimulation via the epithelial barriers drove a potent emergency bone marrow monocyte response in mice. This process was unique to TLR7 activation and occurred independently of the canonical CCR2 and CX3CR1 axes or prototypical cytokines. The monocytes egressing the bone marrow had an immature Ly6C-high profile and differentiated into vascular Ly6C-low monocytes and tissue macrophages in multiple organs. They displayed a blunted cytokine response to further TLR7 stimulation and reduced lung viral load after RSV and influenza virus infection. These data provide insights into the emergency myelopoiesis likely to occur in response to the encounter of single-stranded RNA viruses at barrier sites.

Air pollution induces pyroptosis of human monocytes through activation of inflammasomes and Caspase-3-dependent pathways

According to the World Health Organization (WHO), air pollution is one of the most serious threats for our planet. Despite a growing public awareness of the harmful effects of air pollution on human health, the specific influence of particulate matter (PM) on human immune cells remains poorly understood. In this study, we investigated the effect of PM on peripheral blood monocytes in vitro. Monocytes from healthy donors (HD) were exposed to two types of PM: NIST (SRM 1648a, standard urban particulate matter from the US National Institute for Standards and Technology) and LAP (SRM 1648a with the organic fraction removed). The exposure to PM-induced mitochondrial ROS production followed by the decrease of mitochondrial membrane potential and activation of apoptotic protease activating factor 1 (Apaf-1), Caspase-9, and Caspase-3, leading to the cleavage of Gasdermin E (GSDME), and initiation of pyroptosis. Further analysis showed a simultaneous PM-dependent activation of inflammasomes, including NLRP3 (nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3) and Caspase-1, followed by cleavage of Gasdermin D (GSDMD) and secretion of IL-1β. These observations suggest that PM-treated monocytes die by pyroptosis activated by two parallel signaling pathways, related to the inorganic and organic PM components. The release of IL-1β and expression of danger-associated molecular patterns (DAMPs) by pyroptotic cells further activated the remnant viable monocytes to produce inflammatory cytokines (TNF-α, IL-6, IL-8) and protected them from death induced by the second challenge with PM.In summary, our report shows that PM exposure significantly impacts monocyte function and induces their death by pyroptosis. Our observations indicate that the composition of PM plays a crucial role in this process—the inorganic fraction of PM is responsible for the induction of the Caspase-3-dependent pyroptotic pathway. At the same time, the canonical inflammasome path is activated by the organic components of PM, including LPS (Lipopolysaccharide/endotoxin).Graphical PM-induced pyroptosis of human monocytes. Particulate matter (PM) treatment affects monocytes viability already after 15 min of their exposure to NIST or LAP in vitro. The remnant viable monocytes in response to danger-associated molecular patterns (DAMPs) release pro-inflammatory cytokines and activate Th1 and Th17 cells. The mechanism of PM-induced cell death includes the increase of reactive oxygen species (ROS) production followed by collapse of mitochondrial membrane potential (ΔΨm), activation of Apaf-1, Caspase-9 and Caspase-3, leading to activation of Caspase-3-dependent pyroptotic pathway, where Caspase-3 cleaves Gasdermin E (GSDME) to produce a N-terminal fragment responsible for the switch from apoptosis to pyroptosis. At the same time, PM activates the canonical inflammasome pathway, where activated Caspase-1 cleaves the cytosolic Gasdermin D (GSDMD) to produce N-terminal domain allowing IL-1β secretion. As a result, PM-treated monocytes die by pyroptosis activated by two parallel pathways—Caspase-3-dependent pathway related to the inorganic fraction of PM and the canonical inflammasome pathway dependent on the organic components of PM.

Abstract P3082: Loss Of Myeloid Cell-specific Beta2-adrenergic Receptor Improves Cardiac Function After Acute Ischemia

Heart failure (HF) is one of the leading causes of global mortality, which is often caused by acute myocardial infarction (MI). This type of acute cardiac injury promotes robust inflammatory cascades by regulating spatiotemporal myeloid cell (neutrophil, monocyte and macrophage) responses in the infarct area, which are essential in the orchestration of post-infarct tissue remodeling and repair. We previously reported that deletion of β2-adrenergic receptor (β2AR) in all cells of hematopoietic origin resulted in generalized disruption of immune cell responsiveness to injury, but with unknown impact of myeloid cells in particular. To investigate this, we crossed floxed β2AR (β2 f/f ) mice with myeloid cell-expressing Cre (LysM-Cre) mice to generate myeloid cell-specific β2AR knockout mice (LB2) and subjected them to MI. M-mode echocardiography indicated a preservation of ejection fraction (%EF) and fractional shortening (%FS) in the LB2 mice versus either β2 f/f or LysM-Cre mice at 3 days post-MI, which was maintained at 28 days post-MI, concurrent with reduced infarct size and interstitial fibrosis, suggesting a cardioprotective role of myeloid cell-specific β2-AR deletion. While immune cell profiling by FACS analysis showed similar CD45 + CD11b + Ly6C + monocyte, CD45 + CD11b + Ly6G + neutrophil and CD45 + CD11b + F4/80 + macrophage accumulation in LB2 and β2 f/f mouse hearts at 1 day post-MI, neutrophil frequency was significantly reduced in LB2 mouse hearts by 4 days post-MI, suggesting LB2 hearts have enhanced capacity for efferocytosis of dead cells. Notably, RT-qPCR analysis revealed a significant upregulation of pro-efferocytosis genes in LB2 bone marrow cells, including annexin A1 (ANXA1). Additionally, ANXA1 protein expression was shown to be upregulated in LB2 versus β2 f/f cardiac CD11b + myeloid cells, including macrophages, at 1-day post-MI. In vitro efferocytosis assay data suggested that there was significant increment in %-efferocytosis of LB2 neutrophils as compared to β2 f/f . Taken together, our data suggest that β2AR is a crucial regulator of myeloid cell-dependent efferocytosis in the heart following injury and suggests a crucial timepoint at which β2AR-selective therapeutics may promote the clearance of dead cells to promote repair.

COVID-19 inactivated and non-replicating viral vector vaccines induce regulatory training phenotype in human monocytes under epigenetic control.

Trained immunity is the enhanced innate immune response resulting from exposure to pathogens or vaccines against an unrelated pathogen stimulus. Certain vaccines induce a memory like response in monocytes and NK cells, leading to modulation in cytokine production, metabolic changes, and modifications in histone patterns. Here, we hypothesized that vaccination against SARS-CoV-2 could induce the training of monocytes in addition to stimulating the adaptive immune response. Therefore, we aimed to investigate the immunophenotyping, cytokine and metabolic profile of monocytes from individuals who were completely immunized with two doses of inactivated COVID-19 vaccine or non-replicating viral vector vaccine. Subsequently, we investigated the epigenetic mechanisms underlying monocyte immune training. As a model of inflammatorychallenge, to understand if the monocytes were trained by vaccination and how they were trained, cells were stimulated in vitro with the endotoxin LPS, an unrelated stimulus that would provoke the effects of training. When challenged in vitro, monocytes from vaccinated individuals produced less TNF-α and those who received inactivated vaccine produced less IL-6, whereas vaccination with non-replicating viral vector vaccine induced more IL-10. Inactivated vaccine increased classical monocyte frequency, and both groups showed higher CD163 expression, a hallmark of trained immunity. We observed increased expression of genes involved in glycolysis and reduced IRG1 expression in vaccinated subjects, a gene associated with the tolerance phenotype in monocytes. We observed that both vaccines reduced the chromatin accessibility of genes associated with the inflammatory response, the inactivated COVID-19 vaccine trained monocytes to a regulatory phenotype mediated by histone modifications in the IL6 and IL10 genes, while the non-replicating viral vector COVID-19 vaccine trained monocytes to a regulatory phenotype, mediated by histone modifications in the IL6, IL10, TNF, and CCL2 genes. Our findings support the recognized importance of adopting vaccination against SARS CoV-2, which has been shown to be effective in enhancing the adaptive immune response against the virus and reducing mortality and morbidity rates. Here, we provide evidence that vaccination also modulates the innate immune response by controlling the detrimental inflammatory response to unrelated pathogen stimulation.

ADP as a novel stimulus for NLRP3-inflammasome activation in mice fails to translate to humans

The NLRP3-inflammasome is a cytosolic multiprotein complex that triggers an inflammatory response to certain danger signals. Recently adenosine diphosphate (ADP) was found to activate the NLRP3-inflammasome in murine macrophages via the P2Y1 receptor. Blockade of this signaling pathway reduced disease severity in a murine colitis-model. However, the role of the ADP/P2Y1-axis has not yet been studied in humans. This present study confirmed ADP-dependent NLRP3-inflammasome activation in murine macrophages, but found no evidence for a role of ADP in inflammasome activation in humans. We investigated the THP1 cell line as well as primary monocytes and further looked at macrophages. Although all cells express the three human ADP-receptors P2Y1, P2Y12 and P2Y13, independent of priming, neither increased ASC-speck formation could be detected with flow cytometry nor additional IL-1β release be found in the culture supernatant of ADP stimulated cells. We now show for the first time that the responsiveness of monocytes and macrophages to ADP as well as the regulation of its purinergic receptors is very much dependent on the species. Therefore the signaling pathway found to contribute to colitis in mice is likely not applicable to humans.