Gene Expression In Macrophages Research Articles

Expression of Network Medicine-Predicted Genes in Human Macrophages Infected with Leishmania major

Leishmania spp. commonly infects phagocytic cells of the immune system, particularly macrophages, employing various immune evasion strategies that enable their survival by altering the intracellular environment. In mammals, these parasites establish persistent infections by modulating gene expression in macrophages, thus interfering with immune signaling and response pathways, ultimately creating a favorable environment for the parasite’s survival and reproduction. In this study, our objective was to use data mining and subsequent filtering techniques to identify the genes that play a crucial role in the infection process of Leishmania spp. We aimed to pinpoint genes that have the potential to influence the progression of Leishmania infection. To achieve this, we exploited prior, curated knowledge from major databases and constructed 16 datasets of human molecular information consisting of coding genes and corresponding proteins. We obtained over 400 proteins, identifying approximately 200 genes. The proteins coded by these genes were subsequently used to build a network of protein–protein interactions, which enabled the identification of key players; we named this set Predicted Genes. Then, we selected approximately 10% of Predicted Genes for biological validation. THP-1 cells, a line of human macrophages, were infected with Leishmania major in vitro for the validation process. We observed that L. major has the capacity to impact crucial genes involved in the immune response, resulting in macrophage inactivation and creating a conducive environment for the survival of Leishmania parasites.

PARP7i Clinical Candidate RBN-2397 Exerts Antiviral Activity by Modulating Interferon-β Associated Innate Immune Response in Macrophages.

Polyadenosine diphosphate-ribose polymerase 7 (PARP7) acts as a suppressor of the type I interferon (IFN) signaling pathway via suppressing TANK-binding protein 1 (TBK1). Research study indicates that inhibition of PARP7 could potentially regulate tumor immunity. However, the effect of PARP7 inhibition on innate antiviral immunity in macrophages as well as the underlying mechanism have not been demonstrated else well. We report herein that PARP7 inhibitor clinical candidate RBN-2397 could augment type I interferon (IFN-I) production in macrophages by elevating retinoic acid-inducible gene I (RIG-I) and stimulator of interferon genes (STING) signaling pathways. Treatment with RBN-2397 leads to increased pattern recognition ligands-induced interferon-β production in primary bone marrow-derived macrophages (BMDM) and RAW264.7 cells. Additionally, RBN-2397 suppresses viral replication efficiency in macrophages infected by vesicular stomatitis virus (VSV) and amplifies the expression of interferon-stimulated chemokine genes (ISGs). Mechanistically, RBN-2397 promotes TBK1 phosphorylation, consequently leading to the amplified activation of RIG-I and STING signaling pathways. Furthermore, RBN-2397 enhances the phosphorylation of signal transducer and activator of transcription 1 (STAT1) and STAT2 induced by IFN-α/β and the expression of chemokine genes in macrophages in response to IFN stimulation. In vivo experiments demonstrated that RBN-2397 enhances innate antiviral immunity in mice infected with VSV, resulting in increased serum IFN-β levels, reduced viral loads, and alleviated pulmonary inflammatory responses of the VSV-infected mice. In conclusion, our findings highlight the potential of RBN-2397 as a promising antiviral therapeutic agent for enhancing the IFN-relative antiviral immune defense in host.

Possible involvement of Toll-like receptor 8-positive monocytes/macrophages in the pathogenesis of Sjögren's disease.

Sjögren's disease (SjD) is an autoimmune disease marked by lymphocytic infiltration of salivary and lacrimal glands, leading to glandular dysfunction, where CD4-positive helper T (Th) cells and their cytokines are crucial in the pathogenesis. Recent studies have demonstrated that Toll-like receptors (TLRs), particularly those recognizing immune complexes containing DNA and RNA, contribute to Th cell activation in various autoimmune diseases. This study explores the expression and function of these TLRs in SjD. DNA microarray analysis of salivary gland tissue from six SjD patients and real-time PCR (n = 32) was used to identify overexpressed TLRs. Single-cell RNA sequencing (scRNA-seq) was performed using tissue lesions and integrated with published scRNA-seq data from tissues and peripheral blood mononuclear cells to examine gene expression in macrophages and monocytes. Finally, multi-color immunofluorescence staining was conducted to confirm TLR8 expression and function in SjD lesions (n = 19). DNA microarray analysis revealed the up-regulation of TLR8, along with other TLRs and innate immune response genes in SjD. Real-time PCR showed significant up-regulation of TLR7 and TLR8. TLR8 up-regulated in both analyses. In scRNA-seq analysis, the TLR8-expressing cluster comprised macrophages and monocytes, which also produced T cell activation genes like CD86. TLR8-positive macrophages infiltrated inflammatory sites and frequently expressed CD86 in quantitative imaging approaches. These results suggest that infiltrating monocytes and macrophages may produce cytokines and chemokines through TLR8 stimulation, potentially enhancing B7 molecule expression, promoting the adaptive immune response, and contributing to SjD pathogenesis.

HIF-1α regulates mitochondrial function in bone marrow-derived macrophages, but not in tissue-resident alveolar macrophages.

HIF-1α plays a critical role in shaping macrophage phenotype and effector function. We have previously shown that tissue-resident alveolar macrophages (TR-AMs) have extremely low glycolytic capacity at steady-state, but can shift toward glycolysis under hypoxic conditions. Here, using inducible HIF-1α knockout ( Hif1a -/- ) TR-AMs and bone marrow-derived macrophages (BMDMs) and show that TR-AM HIF-1α is required for the glycolytic shift under prolyl hydroxylase inhibition, but is dispensable at steady-state for inflammatory effector function. In contrast, HIF-1α deletion in BMDMs led to diminished glycolytic capacity at steady-state and reduced inflammatory capacity, but higher mitochondrial function. Gene set enrichment analysis revealed enhanced c-Myc transcriptional activity in Hif1a -/- BMDMs, and upregulation of gene pathways related to ribosomal biogenesis and cellular proliferation. The findings highlight the heterogeneity of HIF-1α function in distinct macrophage populations and provide new insight into how HIF-1α regulates gene expression, inflammation, and metabolism in macrophages.

Serotonin Availability Shapes the Effects of Phenelzine on Inflammatory Response and Gene Expression in Macrophages.

Background Serotonin (5-HT) is a neurotransmitter with extensive physiological influence in the central nervous system (CNS) and various behavioral and biological functions, including immune regulation through 5-HT receptors (5-HTRs) expressed by immune cells. A variety of serotonin-modulating drugs have been developed to treat neurological disorders. Phenelzine, a drug indicated for the management of treatment-resistant depression, is a potent, non-selectiveinhibitor of monoamine oxidase (MAO), the enzyme that metabolizes serotonin to 5-hydroxyindole acetic acid (5-HIAA). Given the emerging evidence of a bidirectional link between depression and inflammation, as well as the potential therapeutic applications of serotonin-modulating drugs in autoimmune diseases and cancer, our study investigated the pro-inflammatory and anti-inflammatory factors influenced by serotonin and phenelzine. Methodology We conducted experiments on RAW264.7 macrophages, exposing them to various combinations and concentrations of serotonin, 5-HIAA, and phenelzine. We assessed the relative gene expression of monoamine oxidase-A (MAO-A), CYP1B1, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) using real-time PCR and measured the production of IL-6, TNF-α, and IL-10 cytokines using enzyme-linked immunosorbent assays (ELISA). Results Our findings revealed that phenelzine can downregulate genes associated with the production of reactive oxygen and nitrogen species, reduce aryl hydrocarbon receptor (AHR)-related gene expression induced by serotonin, and enhance the production of inflammatory cytokines. These effects were significantly influenced by the concentration of available serotonin. Conclusions Our study demonstrates that various mechanisms, including AHR activation, modulation of reactive oxygen and nitrogen speciesproduction, and others, in addition to the increased availability of serotonin due to phenelzine treatment, can significantly influence the inflammatory state.

8729 Role of PARP1 in Macrophage-dependent Regulation of Adipose Tissue Inflammation, Adipogenesis, and Diet-induced Obesity

Abstract Disclosure: J. Zhu: None. R. Gupte: None. T.S. Nandu: None. W.L. Kraus: None. D. Huang: None. Adipose tissue macrophages play a significant role in maintaining adipose tissue homeostasis, as well as promoting chronic low-grade inflammation during the development of obesity and metabolic dysfunction. However, the crosstalk between macrophages and adipocytes, especially how macrophages affect adipogenesis in obese adipose tissue, are still poorly understood. We have investigated the function of PARP1 in the control of adipogenesis by using an adipocyte precursor-specific Parp1 knockout mouse model. Here, to investigate the role of macrophage PARP1 in regulating adipogenesis, we deleted Parp1 in the myeloid lineage by crossing Parp1loxp/loxp mice with LysMCre mice (Parp1 cKOLysMCre). Macrophages-specific knockout of Parp1 dramatically reduced LPS-induced pro-inflammatory gene expression in bone marrow-derived macrophages (BMDMs). To explore whether macrophage-specific Parp1 knockout affects adipocyte differentiation, we treated the primary preadipocytes from stromal vascular fraction (SVF) of white adipose tissue with the conditioned medium from LPS-stimulated control or Parp1 cKOLysMCre BMDMs throughout a 6-day period of differentiation. We observed that exposure of preadipocytes to the conditioned medium of Parp1 cKOLysMCre macrophages promoted adipogenesis by Oil Red O staining, as well as by monitoring the expression of key proadipogenic marker genes (e.g., Adipoq and Fabp4) by RT-qPCR. To further investigate the role of macrophage-specific deletion of Parp1 in metabolic outcomes, we fed the Parp1 cKOLysMCre mice with a high fat diet for 12 weeks. The knockout mice exhibited a significant increase in body weight compared to the control mice. Interestingly, we observed a reduction of the total number of macrophages resident in the adipose tissue from macrophage-specific Parp1 knockout mice. To explore the underlying mechanism, we performed single cell RNA-seq for adipose tissue macrophages using sorted SVF cells collected from HFD-fed mice. The results demonstrated dramatic reductions in macrophage M1 marker genes and pro-inflammatory transcription factors in Parp1 cKOLysMCre mice. Taken together, our study suggests that Parp1 knockout in macrophages selectively affects the recruitment and pro-inflammatory activation of macrophages, as well as adipocyte differentiation in adipose tissue. Importantly, despite the decreased adipose tissue inflammation, macrophage-specific Parp1 knockout enhanced adipogenesis, which leads to obesity in mice.This work was supported by a grant from the NIH/NIDDK (R01 DK069710) and funds from the Cecil H. and Ida Green Center for Reproductive Biology Sciences Endowment, to W.L.K., and a grant from NSFC (82270929) to D.H. Presentation: 6/1/2024

Intracellular Methylglyoxal Accumulation in Classically Activated Mouse Macrophages is Mediated by HIF-1α.

Approximately one million cases of sepsis in the U.S.A. occur annually. The early phase of sepsis features dramatic changes in host metabolism and inflammation. While examining the effects of metabolic pathways on inflammation, we discovered that the highly reactive glycolytic metabolite, methylglyoxal (MG), accumulates intracellularly during classical activation of macrophages. Herein, we explored the role of glycolysis and the master regulator of glycolysis, Hypoxia-Inducing Factor-1α (HIF-1α), in inflammation and MG accumulation in mouse and human macrophages. To determine how HIF-1α regulates the inflammatory response of macrophages, we correlated HIF-1α stabilization with proinflammatory gene expression and MG-adduct accumulation in WT vs HIF1a-deficient macrophages treated with LPS or LPS+IFN-γ. A nearly complete loss of HIF-1α protein expression in response to the hypoxia mimetic, cobalt chloride, confirmed the phenotype of the HIF1a-deficient macrophages. Moreover, absence of HIF-1α was also associated with decreased MG accumulation. Increasing the glucose concentration in cultured macrophages was sufficient to cause accumulation of endogenous MG-adducts which correlated with increased Tnf and Il1b expression during classical activation. Use of the MG antagonist, aminoguanidine, led to a significant decrease in Tnf and Il1b expression in both mouse macrophages and in the THP-1 human macrophage cell line. Although off-target effects cannot be ruled out, these results are consistent with the possibility that MG regulates cytokine expression in classically activated macrophages. Collectively, this work suggests that HIF-1α stabilization is upstream of MG accumulation and that targeting the activity of HIF-1α in macrophages may be therapeutic during sepsis by limiting endogenous MG accumulation.

Both host and parasite non-coding RNAs co-ordinate the regulation of macrophage gene expression to reduce pro-inflammatory immune responses and promote tissue repair pathways during infection with fasciola hepatica

ABSTRACT Parasitic worms (helminths) establish chronic infection within mammalian hosts by strategically regulating their host’s immune responses. Deciphering the mechanisms by which host non-coding RNAs (ncRNA) co-ordinate the activation and regulation of immune cells is essential to understanding host immunity and immune-related pathology. It is also important to comprehend how pathogens secrete specific ncRNAs to manipulate gene expression of host immune cells and influence their response to infection. To investigate the contribution of both host and helminth derived ncRNAs to the activation and/or regulation of innate immune responses during a parasite infection, we examined ncRNA expression in the peritoneal macrophages from mice infected with Fasciola hepatica. We discovered the presence of several parasitic-derived miRNAs within host macrophages at 6 hrs and 18 hrs post infection. Target prediction analysis showed that these Fasciola miRNAs regulate host genes associated with the activation of host pro-inflammatory macrophages. Concomitantly, there was a distinct shift in host ncRNA expression, which was significant at 5 days post-infection. Prediction analysis suggested that these host ncRNAs target a different cohort of host genes compared to the parasite miRNAs, although the functional outcome was predicted to be similar i.e. reduced pro-inflammatory response and the promotion of a reparative/tolerant phenotype. Taken together, these observations uncover the interplay between host and parasitic ncRNAs and reveal a complementary regulation of the immune response that allows the parasite to evade immune detection and promote tissue repair for the host. These findings will provide a new understanding of the molecular interaction between parasites and host.

A Novel Non-Invasive Murine Model of Neonatal Hypoxic-Ischemic Encephalopathy Demonstrates Developmental Delay and Motor Deficits with Activation of Inflammatory Pathways in Monocytes.

Neonatal hypoxic-ischemic encephalopathy (HIE) occurs in 1.5 per 1000 live births, leaving affected children with long-term motor and cognitive deficits. Few animal models of HIE incorporate maternal immune activation (MIA) despite the significant risk MIA poses to HIE incidence and diagnosis. Our non-invasive model of HIE pairs late gestation MIA with postnatal hypoxia. HIE pups exhibited a trend toward smaller overall brain size and delays in the ontogeny of several developmental milestones. In adulthood, HIE animals had reduced strength and gait deficits, but no difference in speed. Surprisingly, HIE animals performed better on the rotarod, an assessment of motor coordination. There was significant upregulation of inflammatory genes in microglia 24 h after hypoxia. Single-cell RNA sequencing (scRNAseq) revealed two microglia subclusters of interest following HIE. Pseudobulk analysis revealed increased microglia motility gene expression and upregulation of epigenetic machinery and neurodevelopmental genes in macrophages following HIE. No sex differences were found in any measures. These results support a two-hit noninvasive model pairing MIA and hypoxia as a model for HIE in humans. This model results in a milder phenotype compared to established HIE models; however, HIE is a clinically heterogeneous injury resulting in a variety of outcomes in humans. The pathways identified in our model of HIE may reveal novel targets for therapy for neonates with HIE.

Metformin modulates microbiota and improves blood pressure and cardiac remodeling in a rat model of hypertension.

Metformin has been attributed to cardiovascular protection even in the absence of diabetes. Recent observations suggest that metformin influences the gut microbiome. We aimed to investigate the influence of metformin on the gut microbiota and hypertensive target organ damage in hypertensive rats. Male double transgenic rats overexpressing the human renin and angiotensinogen genes (dTGR), a model of angiotensin II-dependent hypertension, were treated with metformin (300 mg/kg/day) or vehicle from 4 to 7 weeks of age. We assessed gut microbiome composition and function using shotgun metagenomic sequencing and measured blood pressure via radiotelemetry. Cardiac and renal organ damage and inflammation were evaluated by echocardiography, histology, and flow cytometry. Metformin treatment increased the production of short-chain fatty acids (SCFA) acetate and propionate in feces without altering microbial composition and diversity. It significantly reduced systolic and diastolic blood pressure and improved cardiac function, as measured by end-diastolic volume, E/A, and stroke volume despite increased cardiac hypertrophy. Metformin reduced cardiac inflammation by lowering macrophage infiltration and shifting macrophage subpopulations towards a less inflammatory phenotype. The observed improvements in blood pressure, cardiac function, and inflammation correlated with fecal SCFA levels in dTGR. Invitro, acetate and propionate altered M1-like gene expression in macrophages, reinforcing anti-inflammatory effects. Metformin did not affect hypertensive renal damage or microvascular structure. Metformin modulated the gut microbiome, increased SCFA production, and ameliorated blood pressure and cardiac remodeling in dTGR. Our findings confirm the protective effects of metformin in the absence of diabetes, highlighting SCFA as a potential mediators.

The identification of key molecules and pathways in the crosstalk of calcium oxalate-treated TCMK-1 cells and macrophage via exosomes

The interplay between crystals and epithelial cells forms the cornerstone of kidney stone development, communication between epithelial cells and macrophages emerging as a pivotal role in this process. We conducted next-generation sequencing on the secreted exosomes of TCMK-1 cells treated with calcium oxalate monohydrate (OX_EXO) or controls (NC_EXO), and on the macrophage cell line RAW264.7 stimulated with OX_EXO or NC_EXO, followed by validation of differentially expressed target proteins and miRNAs through Western blot and PCR. UPSET plots were employed to identify genes co-targeted by exosomal miRNAs. Various bioinformatic analyses were employed to predict potential mechanisms of the dysregulated genes. We integrated sequencing data from the GEO database, and validated findings using clinical patient urine and kidney tissues. We identified 665 differentially expressed exosomal miRNAs between OX_EXO and NC_EXO. Among the top 10 down-regulated miRNAs, the most targeted genes were AAK1 and NUFIP2, whereas PLCB1 was significantly targeted among the top 10 up-regulated miRNAs. In clinical specimens, we confirmed the differential expressions of five homologous miRNAs, as well as CNOT3, CNCNA1C, APEX1, and TMEM199. In conclusion, treatment of TCMK-1 cells with calcium oxalate significantly alerted the expression profile of exosomal miRNAs, subsequently influencing gene expression in macrophages, thereby modulating the processes of kidney stone formation.

Multigenic family 110 (1L-5-6L) of African swine fever virus modulate cytokine genes expression in vitro.

African swine fever (ASF) is a viral disease that affects pigs and wild boars providing economic burden in swine industry. In this study, we investigated the effect of deleting the ASFV multigene family 110 (MGF110) fragment (1L-5-6L) on apoptosis modulation and the expression of proinflammatory cytokines. Gene expression in swine peripheral blood macrophages infected with either the parental "Volgograd/14c" strain or the gene-deleted "Volgograd/D(1L-5-6L) MGF110" strain was analyzed. Caspase-3 activity was 1.15 times higher in macrophages infected with the parental ASFV strain compared to the gene-deleted strain. Gene expression analysis of Caspase-3 (Cas-3), Interferon-A (IFN-A), Tumor Necrosis Factor A (TNF-A), B-cell Lymphoma-2 (Bcl-2), Nuclear Factor Kappa B (NF-kB), Interleukin-12 (IL-12), and Heat Shock Protein-70 (HSP-70) using RT-qPCR at various time points after infection revealed significant differences in expression profiles between the strains. The peak expression of cytokines (except NF-kB) occurred at 24h post-infection with the "Volgograd/D(1L-5-6L) MGF110" strain. In samples infected with the ASFV "Volgograd/14c" strain, the most intense expression was observed at 72 and 96h, except for Bcl-2 and NF-kB, which peaked at 6h post-infection. The cytokine expression trend for the "Volgograd/D(1L-5-6L) MGF110" strain was more stable with higher expression values. The expression trend for the parental strain increased over time, reaching maximum values at 72 and 96h post-infection, but the overall expression level was lower than that of the gene-deleted strain. These findings suggest that deleting the multigene family 110 members (1L-5-6L) contributes to ASFV attenuation without affecting virus replication kinetics.

Perioperative systemic IL-6 and immune-adipose- metabolism transcription in tumour and tumour adjacent breast cancer.

Surgical resection is the primary treatment approach for patients with breast cancer. Despite optimal multimodal treatment, metastatic recurrence remains a risk. Surgery-mediated systemic inflammation and local tissue inflammation generate an immunosuppressive and wound-healing environment that may accelerate cancer recurrence and metastasis post-operatively. Investigating the impact of surgery on local and systemic inflammation may provide knowledge for improvement of patient prognosis and treatment opportunities. Systemic cytokines were quantified in the blood plasma of patients with breast cancer pre-operatively, early post-operatively, and late post-operatively. Early post-operative levels of IL-6 were significantly elevated in patients who underwent mastectomy compared with wide local excision. Post-operative IL-6 levels correlate with clinicopathological features (age and BMI). The transcriptomes of local matched tumour and normal tumour adjacent (normal) breast tissue, from patients with breast cancer, were analysed by RNA-Seq. Elevated gene expressions of IL6, ADIPOQ, FABP4, LPL, PPARG, and CD36 in normal tissue were associated with worse overall survival of patients with ER-positive breast cancer. In tissue with higher expression of IL6 and ADIPOQ, a higher abundance of M2-like macrophage gene expression was identified. This study revealed perioperative systemic dynamics of inflammatory mediators and identified local immune-adipose-metabolism gene expression in tumour-adjacent tissue associated with pro-tumour function.

Eicosatetraynoic Acid Regulates Pro-Fibrotic Pathways in an Induced Pluripotent Stem Cell Derived Macrophage:Human Intestinal Organoid Model of Crohn's Disease.

We previously identified small molecules predicted to reverse an ileal gene signature for future Crohn's Disease (CD) strictures. Here we used a new human intestinal organoid (HIO) model system containing macrophages to test a lead candidate, eicosatetraynoic acid (ETYA). Induced pluripotent stem cell lines (iPSC) were derived from CD patients and differentiated into macrophages and HIOs. Macrophages and macrophage:HIO co-cultures were exposed to lipopolysaccharide (LPS) with and without ETYA pre-treatment. Cytospin and flow cytometry characterized macrophage morphology and activation markers, and RNA sequencing defined the global pattern of macrophage gene expression. TaqMan Low Density Array, Luminex multiplex assay, immunohistologic staining, and sirius red polarized light microscopy were performed to measure macrophage cytokine production and HIO pro-fibrotic gene expression and collagen content. iPSC-derived macrophages exhibited morphology similar to primary macrophages and expressed inflammatory macrophage cell surface markers including CD64 and CD68. LPS-stimulated macrophages expressed a global pattern of gene expression enriched in CD ileal inflammatory macrophages and matrisome secreted products, and produced cytokines and chemokines including CCL2, IL1B, and OSM implicated in refractory disease. ETYA suppressed CD64 abundance and pro-fibrotic gene expression pathways in LPS stimulated macrophages. Co-culture of LPS-primed macrophages with HIO led to up-regulation of fibroblast activation genes including ACTA2 and COL1A1, and an increase in HIO collagen content. ETYA pre-treatment prevented pro-fibrotic effects of LPS-primed macrophages. ETYA inhibits pro-fibrotic effects of LPS-primed macrophages upon co-cultured HIO. This model may be used in future untargeted screens for small molecules to treat refractory CD.

Single-nuclei sequencing of uterine serous carcinoma reveals racial differences in immune signaling

Significant racial disparities exist between Black and White patients with uterine serous carcinoma (USC). While the reasons for these disparities are unclear, several studies have demonstrated significantly different rates of driver mutations between racial groups, including TP53. However, limited research has investigated the transcriptional differences of tumors or the composition of the tumor microenvironment (TME) between these groups. Here, we report the single-nuclei RNA-sequencing profiles of primary USC tumors from diverse racial backgrounds. We find that there are significant differences between the tumors of Black and White patients. Tumors from Black patients exhibited higher expression of specific genes associated with aggressiveness, such as PAX8, and axon guidance and synaptic signaling pathways. We also demonstrated that T cell populations are reduced in the tumor tissue compared to matched benign, while anti-inflammatory macrophage populations are retained within the TME. Furthermore, we investigated the connection between PAX8 overexpression and immunosuppression in USC through regulation of several cytokines and chemokines. Notably, we show that PAX8 activity can influence macrophage gene expression and protein secretion. These studies provide a detailed understanding of the USC transcriptome and TME, and identify differences in tumor biology from patients of different racial backgrounds.

Human liver sinusoidal endothelial cells support the development of functional human pluripotent stem cell-derived Kupffer cells

In mice, the first liver-resident macrophages, known as Kupffer cells (KCs), are thought to derive from yolk sac (YS) hematopoietic progenitors that are specified prior to the emergence of the hematopoietic stem cell (HSC). To investigate human KC development, we recapitulated YS-like hematopoiesis from human pluripotent stem cells (hPSCs) and transplanted derivative macrophage progenitors into NSG mice previously humanized with hPSC-liver sinusoidal endothelial cells (LSECs). We demonstrate that hPSC-LSECs facilitate stable hPSC-YS-macrophage engraftment for at least 7weeks. Single-cell RNA sequencing (scRNA-seq) of engrafted YS-macrophages revealed a homogeneous MARCO-expressing KC gene signature and low expression of monocyte-like macrophage genes. In contrast, human cord blood (CB)-derived macrophage progenitors generated grafts that contain multiple hematopoietic lineages in addition to KCs. Functional analyses showed that the engrafted KCs actively perform phagocytosis and erythrophagocytosis invivo. Taken together, these findings demonstrate that it is possible to generate human KCs from hPSC-derived, YS-like progenitors.

Ferroptosis-Related Gene Signatures in Epilepsy: Diagnostic and Immune Insights.

Epilepsy is characterized by a multifaceted aetiology. Ferroptosis has recently been implicated in seizure pathophysiology, although its mechanistic role in epilepsy remains obscure. We examined the roles of ferroptosis-related genes (FRGs) in epilepsy cohorts from the GSE143272 dataset. We investigated the associations between gene expression and the immune response by performing CIBERSORT and MCP-counter analyses. By employing unsupervised consensus clustering and weighted gene coexpression network analysis (WGCNA), we delineated robust gene clusters across cohorts. Single-cell RNA sequencing data from the GSE201048 dataset provided insights into the interactions between pivotal ferroptosis-related genes and immune cells. Additionally, we employed qRT‒PCR technology to measure the levels of these central genes in the tissues of epileptic patients and mice. Our findings revealed seven pivotal genes (TFRC, POR, PTGS2, RELA, PGD, TRIM21, and QSOX1) at the forefront in epilepsy specimens. A diagnostic model harnessing these genes exhibited substantial efficacy (AUC = 0.913). Similarly, the qRT‒PCR analysis of samples from epileptic patients and mouse epileptic brain tissues substantiated these findings. Stratification of 91 patients with epilepsy via WGCNA, based on gene expression, revealed distinct immunological profiles. The scRNA-seq data further indicated increased expression of central genes in macrophages and microglia. Notably, these cells and those with elevated ferroptosis scores were significantly enriched in inflammation-related pathways. These findings support the strong involvement of FRGs in the pathogenesis of epilepsy, particularly neuroinflammation. These central genes hold promise as novel diagnostic biomarkers for epilepsy.

Fat Phagocytosis Promotes Anti-Inflammatory Responses of Macrophages in a Mouse Model of Osteonecrosis.

Osteonecrosis (ON) of the femoral head (ONFH) is a devastating bone disease affecting over 20 million people worldwide. ONFH is caused by a disruption of the blood supply, leading to necrotic cell death and increased inflammation. Macrophages are the key cells mediating the inflammatory responses in ON. It is unclear what the dynamic phenotypes of macrophages are and what mechanisms may affect macrophage polarization and, therefore, the healing process. In our preliminary study, we found that there is an invasion of macrophages into the repair tissue during ON healing. Interestingly, in both ONFH patients and a mouse ON model, fat was co-labeled within macrophages using immunofluorescence staining, indicating the phagocytosis of fat by macrophages. To study the effects of fat phagocytosis on the macrophage phenotype, we set up an in vitro macrophage and fat co-culture system. We found that fat phagocytosis significantly decreased M1 marker expression, such as IL1β and iNOS, in macrophages, whereas the expression of the M2 marker Arg1 was significantly increased with fat phagocytosis. To investigate whether the polarization change is indeed mediated by phagocytosis, we treated the cells with Latrunculin A (LA, which inhibits actin polymerization and phagocytosis). LA supplementation significantly reversed the polarization marker gene changes induced by fat phagocytosis. To provide an unbiased transcriptional gene analysis, we submitted the RNA for bulk RNA sequencing. Differential gene expression (DGE) analysis revealed that the top upregulated genes were related to anti-inflammatory responses, while proinflammatory genes were significantly downregulated. Additionally, using pathway enrichment and network analyses (Metascape), we confirmed that gene-enriched categories related to proinflammatory responses were significantly downregulated in macrophages with fat phagocytosis. Finally, we validated the similar macrophage phenotype changes in vivo. To summarize, we discovered that fat phagocytosis occurs in both ONFH patients and an ON mouse model, which inhibits proinflammatory responses with increased anabolic gene expression in macrophages. This fat-phagocytosis-induced macrophage phenotype is consistent with the in vivo changes shown in the ON mouse model. Our study reveals a novel phagocytosis-mediated macrophage polarization mechanism in ON, which fills in our knowledge gaps of macrophage functions and provides new concepts in macrophage immunomodulation as a promising treatment for ON.

Bruton's tyrosine kinase (BTK) inhibitors alter blood glucose and insulin in obese mice but reduce inflammation independent of BTK.

Obesity is associated with metabolic inflammation, which can contribute to insulin resistance, higher blood glucose, and higher insulin indicative of prediabetes progression. The nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is a metabolic danger sensor implicated in metabolic inflammation. Many features of metabolic disease can activate the NLRP3 inflammasome; however, it is not yet clear which upstream triggers to target, and there are no clinically approved NLRP3 inflammasome inhibitors for metabolic disease. Bruton's tyrosine kinase (BTK) mediates activation of the NLRP3 inflammasome. Ibrutinib is the most-studied pharmacological inhibitor of BTK, and it can improve blood glucose control in obese mice. However, inhibitors of tyrosine kinases are permissive, and it is unknown if BTK inhibitors require BTK to alter endocrine control of metabolism or metabolic inflammation. We tested whether ibrutinib and acalabrutinib, a new generation BTK inhibitor with higher selectivity, require BTK to inhibit the NLRP3 inflammasome, metabolic inflammation, and blood glucose in obese mice. Chronic ibrutinib administration lowered fasting blood glucose and improved glycemia, whereas acalabrutinib increased fasting insulin levels and increased markers of insulin resistance in high-fat diet-fed CBA/J mice with intact Btk. These metabolic effects of BTK inhibitors were absent in CBA/CaHN-Btkxid/J mice with mutant Btk. However, ibrutinib and acalabrutinib reduced NF-κB activity, proinflammatory gene expression, and NLRP3 inflammasome activation in macrophages with and without functional BTK. These data highlight that the BTK inhibitors can have divergent effects on metabolism and separate effects on metabolic inflammation that can occur independently of actions on BTK.NEW & NOTEWORTHY Bruton's tyrosine kinase (BTK) is involved in immune function. It was thought that BTK inhibitors improve characteristics of obesity-related metabolic disease by lowering metabolic inflammation. However, tyrosine kinase inhibitors are permissive, and it was not known if different BTK inhibitors alter host metabolism or immunity through actions on BTK. We found that two BTK inhibitors had divergent effects on blood glucose and insulin via BTK, but inhibition of metabolic inflammation occurred independently of BTK in obese mice.

Glutamine metabolism improves left ventricular function but not macrophage-mediated inflammation following myocardial infarction.

Glutamine is a critical amino acid that serves as an energy source, building block, and signaling molecule for the heart tissue and the immune system. However, the role of glutamine metabolism in regulating cardiac remodeling following myocardial infarction (MI) is unknown. In this study, we show in adult male mice that glutamine metabolism is altered both in the remote (contractile) area and in infiltrating macrophages in the infarct area after permanent left anterior descending artery occlusion. We found that metabolites related to glutamine metabolism were differentially altered in macrophages at days 1, 3, and 7 after MI using untargeted metabolomics. Glutamine metabolism in live cells was increased after MI relative to no MI controls. Gene expression in the remote area of the heart indicated a loss of glutamine metabolism. Glutamine administration improved left ventricle (LV) function at days 1, 3, and 7 after MI, which was associated with improved contractile and metabolic gene expression. Conversely, administration of BPTES, a pharmacological inhibitor of glutaminase-1, worsened LV function after MI. Neither glutamine nor BPTES administration impacted gene expression or bioenergetics of macrophages isolated from the infarct area. Our results indicate that glutamine metabolism plays a critical role in maintaining LV contractile function following MI and that glutamine administration improves LV function. Glutamine metabolism may also play a role in regulating macrophage function, but macrophages are not responsive to exogenous pharmacological manipulation of glutamine metabolism.NEW & NOTEWORTHY Glutamine metabolism is altered in both infarct macrophages and the remote left ventricle (LV) following myocardial infarction (MI). Supplemental glutamine improves LV function following MI while inhibiting glutamine metabolism with BPTES worsens LV function. Supplemental glutamine or BPTES does not impact macrophage immunometabolic phenotypes after MI.