Bone Marrow Macrophages Research Articles

(E, E)-farnesol and myristic acid-loaded lipid nanoparticles overcome colistin resistance in Acinetobacter baumannii

The rise of colistin-resistant Acinetobacter baumannii has severely limited treatment options for infections caused by this pathogen. While terpene alcohols and fatty acids have shown potential to enhance colistin’s efficacy, but their high lipophilicity limits their clinical application. To address this, we developed water-dispersible lipid nanoparticles (LNPs) in two sizes (40 nm and 130 nm), loaded with these compounds to act as colistin adjuvants. Among eleven LNP formulations, six significantly reduced colistin’s minimum inhibitory concentration (MIC) by 16- to 64-fold. The most effective, featuring (E,E)-farnesol and myristic acid, were further examined for bactericidal activity, membrane disruption, cytotoxicity, and in vivo efficacy in Galleria mellonella larvae. Time-kill studies demonstrated that at an adjuvant concentration of 60 mg/L, these LNPs eradicated bacteria when combined with 4 mg/L free colistin for resistant isolates (MIC = 128 mg/L) and 0.06 mg/L for susceptible isolates (MIC = 0.5 mg/L), without regrowth. Myristic acid-loaded LNPs combined with free colistin at 1/8 MIC resulted in a 4.2-fold higher mortality rate than the combination with (E,E)-farnesol-loaded LNPs in resistant strains. This result was correlated with a 45-fold faster increase in inner membrane permeability, measured by propidium iodide (PI) uptake, in the presence of myristic acid-loaded LNPs compared with a 13-fold faster increase with (E,E)-farnesol-loaded LNPs. DiSC3(5) assays revealed that LNPs alone depolarised the bacterial inner membrane, with enhanced effects when combined with colistin at 1/8 MIC, a result not observed with colistin alone at this concentration. As with PI uptake, this inner membrane depolarising effect was more pronounced with myristic acid-loaded LNPs than with (E,E)-farnesol-loaded LNPs in resistant strains, suggesting that the colistin adjuvant effect of these lipophilic compounds is due to their ability to help colistin destabilise the bacterial inner membrane. Cytotoxicity assays demonstrated no adverse effects on bone marrow macrophages after 6 h of exposure, although some toxicity was observed after 24 h. No mortality was observed in Galleria mellonella larvae over 7 days following three consecutive days of treatment with colistin and LNPs. Notably, the combination of (E,E)-farnesol-loaded LNPs and colistin significantly improved the survival of Galleria infected with A.baumannii. These results suggest that lipophilic-adjuvant-loaded LNPs may offer a promising strategy to enhance colistin efficacy and combat antibiotic-resistant A. baumannii infections.

Fibroblast growth factor 10 alleviates LPS-induced acute lung injury by promoting recruited macrophage M2 polarization.

Acute lung injury (ALI) is characterized by damage to the alveoli and an overabundance of inflammation. Representing a serious inflammatory condition, ALI lacks a precise treatment approach. Despite the recognized benefit impacts of Fibroblast growth factor-10 (FGF10) on ALI, the underlying mechanisms remain unelucidated. To study the role of FGF10 in ALI, C57BL/6J mice were intratracheally injected with 5mg/kg Lipopolysaccharide (LPS) with FGF10 (5mg/kg) or an equal volume of PBS. Inflammatory factors were quantified in bronchoalveolar lavage fluid (BALF) and plasma using ELISA. RNA sequencing of F4/80+Ly6G- macrophages in BALF explored changes in macrophage phenotype and potential mechanisms. Macrophage polarization in BALF was assessed using qRT-PCR, flow cytometry, and Western blot analysis. In vitro, a Transwell co-culture of mouse lung epithelial cells (MLE12) and bone marrow macrophages (BMDM) validated the role of FGF10 in modulating LPS-induced macrophage phenotypic changes. FGF10 ameliorated LPS-induced ALI by diminishing pro-inflammatory factors (IL-1β, TNF-α, and IL-6) and the neutrophil accumulation in BALF. FGF10 also increased the levels of anti-inflammatory factor IL-10. The FGF10 intervention group exhibited enhanced gene expression of macrophage arginine biosynthesis marker (ARG1), and expression of M2-type marker CD206 in monocytes and macrophages. In addition, phosphorylated STAT3 expression increased in isolated monocyte-derived macrophages. Experiments in vitro confirmed that FGF10 could elevate macrophage M2 marker ARG1 expression through the JAK2/STAT3 pathway. FGF10 ameliorates acute LPS-induced lung injury by modulating the polarization of monocyte-derived macrophages recruited in the alveolar space to the M2 type.

Significance of exosomes in osteosarcoma research: a systematic review and meta-analysis of a singular clinical investigation

BackgroundOsteosarcoma is the most prevalent among primary bone malignancies, and its standard intervention involves neoadjuvant chemotherapy - surgical adjuvant chemotherapy (MAP regimen) with adriamycin, cisplatin, and high-dose methotrexate. Early-stage osteosarcoma can be effectively treated with surgical resection along with chemotherapy or radiotherapy. However, as the cancer progresses, the efficacy of chemo- and radiotherapy decreases, and the associated problems increase. The current understanding of osteosarcoma development, diagnosis, and treatment does not meet clinical demands. More recently, there has been a significant increase in exosome-associated osteosarcoma research, potentially opening up novel possibilities for osteosarcoma research.PurposeWe comprehensively evaluated and analyzed the advancement of preclinical research related to exosome-osteosarcoma. We aimed to establish a practical, theoretical foundation for future research initiatives.Study designThe selected design was a systematic review and meta-analysis.MethodsScientific databases, such as PubMed, Embase, The Cochrane Library, and Web of Science, were extensively screened for exosome and osteosarcoma articles. Two highly trained investigators separately reviewed the literature, extracted relevant information, and assessed study quality. Subsequently, we conducted a meta-analysis using Review Manager 5.4.ResultsIn total, 25 animal-based randomized controlled trials (RCTs) were selected for analysis. Among them, 13 studies provided strong evidence of cellular exosomes regulating osteosarcoma development from bone marrow mesenchymal stem cells, osteosarcoma cells, and macrophages. In addition, 12 studies demonstrated the therapeutic potential of exosomes in managing osteosarcoma, among which 7 studies transplanted transfected exosomes directly into animals as drugs, and five studies employed exosomes as drug carriers, which were next transplanted into animals.ConclusionBased on our meta-analysis, macrophages strongly modulate osteosarcoma development, and engineered exosomes provide the most effective exosome-based osteosarcoma treatment.

Abstract 4120741: Estrogen Regulates Abdominal Aortic Aneurysm Tregs via CD45 + Fibroblast-derived IL-33

Background: Sexual dimorphisms manifest during the progression of abdominal aortic aneurysm (AAA). Regulatory T cells (Tregs) are a subset of T cells characterized by their immunosuppressive and tissue-repairing functions, therefore playing a protective role in AAA. Aims: We aim to elucidate the mechanism underlying sexual dimorphism in Tregs presented in AAA aortas, which is significant for unraveling the pathogenesis of AAA and developing gender-specific therapeutic approaches. Methods and Results: AAA mice were induced by receiving porcine pancreatic elastase. By flow cytometry, we observed that ovariectomy (OVX) increased the ratio of Tregs in female mice, while estrogen led to a decrease in aortic Tregs. Additionally, compared to littermate controls, estrogen receptor α (ERα) KO mice exhibited an increase in Treg ratio. Similar results were observed for the ST2 + Treg ratio. IL-33, as an alarming molecule, can promote the maintenance of ST2 + Tregs in tissues. Western blot revealed lower IL-33 expression levels in female mice than in male mice. By analyzing the IL-33 expression profiles of various cells in AAA tissue, we found that IL-33 mainly originates from fibroblasts. We found two subpopulations of fibroblasts, CD45 + and CD45 - fibroblasts. Immunofluorescence on the aortas verified the presence of cells co-expressing CD45 and PDGFRα in AAA fragments. Furthermore, IL-33 expression only showed gender differences in CD45 + fibroblasts. Adoptive transfer of CD45.1 + mouse bone marrow macrophages into CD45.2 + mice revealed that aortic CD45.1 + cells could regain the PDGFRα phenotype, with a lower proportion of female PDGFRα + cells in CD45.2 - CD45.1 + cells than in male mice. Lyz2(cre/+); Rosa26(tdtomato/+) mice showed the presence of lyz2-tdtomato + cells expressing PDGFRα in the aortic adventitia after AAA induction. This suggests that CD45 + fibroblasts are partially derived from bone marrow macrophages, which can migrate to aneurysms and acquire a PDGFRα phenotype. Conclusions: Female sex hormones negatively regulate CD45 + fibroblast formation and IL-33 secretion between female and male mice during AAA. This resulted in reduced Treg function in female mice compared to male mice.

Devouring Atherosclerotic Plaques: The Engineered Nanorobot Rousing Macrophage Efferocytosis by a Two‐Pronged Strategy

AbstractInvestigating the potential of macrophage efferocytosis has led to a growing interest in exploiting macrophage in immunotherapy. Macrophage‐specific nano‐medicines are currently being developed to stimulate the phagocytic clearance of apoptotic debris in atherosclerosis; however, these unnatural nanoparticles exhibit poor biocompatibility and limited efferocytosis activation ability. Here a pro‐efferocytosis biomimetic nanorobot UM‐EVLipo is developed based on nano‐motor and extracellular vesicles (EVs) hybridized with plaque double‐targeted liposomes. An elegant procedure with only 4 steps is integrated to prepare and purify these hybrid nanovesicles. It is first demonstrated that the EVs derived from bone marrow macrophages stimulated with IL‐4 can promote the transformation of M0 and M1 macrophages into M2, an efferocytosis phenotype. Furthermore, the EVs hybridized with SHP‐1 siRNA‐liposomes blocked the “don't eat me” signal from apoptotic debris by intervening CD47‐SIRPα‐SHP‐1 axis. This two‐pronged strategy of resetting phenotype and blocking checkpoints furthest activates macrophage efferocytosis. Importantly, the urease motor increased the swimming speed of nanovesicles in the blood by 12.4 times, making it easier for them to penetrate the endothelial barrier. The UM‐EVLipo accumulated within the atherosclerotic plaque, reactivate lesioned efferocytosis and reduce the plaque area in the carotid and coronary, which demonstrates the potential of a two‐pronged strategy to prevent atherosclerotic cardiovascular disease.

Effect and mechanism of Adipokine Omentin-1 on cardiac hypertrophy in heart failure with preserved ejection fraction

Abstract Background Omentin-1 secreted by epicardial adipose tissue can inhibit ventricular remodeling and improve early cardiac function after myocardial infarction. Our previous studies have identified the omentin-1 receptor as integrin receptors αvβ3 and αvβ5. However, the effect of omentin-1 on heart failure with preserved ejection fraction (HFpEF) remains poorly understood. Purpose The present study aims to study the role and mechanism of omentin-1 in cardiac function and myocardial cell structure in HFpEF. Methods and Results The influences of omentin-1 were investigated in HFpEF mice (HFD (60% calories from lard) and L-NAME (0.5 g/L in drinking water)), which were treated in vivo. Echocardiography showed better diastolic function in HFpEF mice treated with omentin-1 (5 ug/kg/h) subcutaneously for 4 weeks, and tissue staining showed alleviated myocardial fibrosis and myocardial hypertrophy. The RNA sequencing analysis revealed variations in the gene expression related to ketone body metabolism in HFpEF mice after omentin-1 treatment (600 ng/ml). The supernatant was obtained from mouse bone marrow macrophages treated with ultrapure endotoxin and ATP and was utilized as the macrophage-conditioned medium for culturing primary cardiomyocytes, which were stimulated with isoproterenol for 48 hours to construct the HFpEF cardiomyocyte model. In the heart tissue of HFpEF mice and HFpEF cardiomyocytes treated with omentin-1, there was a notable increase in the expression of a pivotal enzyme of ketone body metabolism- succinyl-CoA transferase (SCOT), and a decrease in the expression of hypertrophy signal. The myocardial hypertrophy model was established by stimulating the myocardial cell line H9C2 with isoproterenol. Following the inhibition of the integrin receptor and PI3K-AKT pathway, the impacts of omentin-1 on SCOT and myocardial hypertrophy signal expression in hypertrophic cardiomyocytes were eliminated. Conclusion This study provides evidence for the effects of omentin-1 on cardiac function and myocardial structure in HFpEF and proposes that omentin-1 may stimulate the downstream PI3K/AKT pathway via integrin receptors on cardiomyocytes, leading to increased expression of SCOT and improvement of cardiomyocyte hypertrophy.

Aging alters the effect of adiponectin receptor signaling on bone marrow-derived mesenchymal stem cells.

Adiponectin receptor signaling represents a promising therapeutic target for age-related conditions such as osteoporosis and diabetes. However, the literature presents conflicting evidence regarding the role of adiponectin signaling in bone homeostasis and fracture repair across different health states, ages, and disease conditions. These inconsistencies may arise from the complex endocrine and paracrine feedback mechanisms regulating adiponectin, as well as the variability in adiponectin isoforms and receptor expressions. In this study, we observed differential expression of adiponectin receptors in the bone marrow (BM) of aged mice, characterized by elevated levels of adiponectin receptor 2 and reduced levels of receptor 1, as corroborated by both single-cell sequencing and invivo staining. Additionally, circulating levels of adiponectin and its local expression were significantly higher in aged mice compared to younger counterparts. Treatment with adiponectin receptor agonist, AdipoRon, enhanced bone regeneration and repair in young mice by promoting osteogenesis and reducing osteoclastogenesis. Conversely, in aged mice, AdipoRon treatment led to cellular senescence, delayed bone repair, and inhibited osteogenic activity. Notably, the adiponectin receptor 1-Wnt and adiponectin receptor 2-MAPK and mTOR signaling pathways were differentially activated in AdipoRon-treated BM mesenchymal stem cells from young and aged mice. Additionally, the NF-κB, and AKT pathways were consistently downregulated in BM macrophages of both age groups following AdipoRon administration. In conclusion, aging significantly modulates the impact of adiponectin receptor signaling on BM mesenchymal stem cells. This modulation is potentially attributable to changes in receptor transcription and distribution, as well as differential activation of downstream signaling pathways.

Effective xanthine oxidase inhibitor urate lowering therapy in gout is linked to an emergent serum protein interactome of complement and inflammation modulators

Urate-lowering treatment (ULT) to target with xanthine oxidase inhibitors (XOIs) paradoxically causes early increase in gouty arthritis flares. Because delayed reduction in flare burden is mechanistically unclear, we tested for ULT inflammation responsiveness markers. Unbiased proteomics analyzed blood samples (baseline, 48 weeks ULT) in two, independent ULT out trial cohorts (n = 19, n = 30). STRING-db and multivariate analyses supplemented determinations of altered proteins via Wilcoxon matched pairs signed rank testing in XOI ULT responders. Mechanistic studies characterized proteomes of cultured XOI-treated murine bone marrow macrophages (BMDMs). At 48 weeks ULT, serum urate normalized in all gout patients, and flares declined in association with significantly altered proteins (p < 0.05) in clustering and proteome networks in sera and peripheral blood mononuclear cells. Sera demonstrated altered complement activation and regulatory gene ontology biologic processes. In both cohorts, a treatment-emergent serum interactome included key gouty inflammation mediators (C5, IL-1B, CXCL8, IL6). Last, febuxostat treatment decreased complement activation biologic process proteins in cultured BMDMs. Reduced gout flares are linked with a XOI treatment-emergent serum protein interactome that includes inflammation regulators, associated with altered complement activation and regulatory biologic processes. Serum and leukocyte proteomics could help identify when gouty inflammatory processes begin to subside in response to ULT.Trial Registration: ClinicalTrials.gov Identifier NCT02579096, posted October 19, 2015.

Osteoclastic ATP6AP2 maintains β-catenin levels to prevent hyper-osteoclastic activation and trabecular bone-loss.

Osteoclast (OC) formation and bone resorption are regulated by several factors, including V-ATPase, Wnt/β-Catenin, and RANKL/RANK signaling. ATP6AP2, also known as the prorenin receptor (PRR), is an accessory subunit of V-ATPase and a regulator of Wnt/β-Catenin signaling. While the V-ATPase subunit ATP6AP1 is essential for osteoclast formation and function, the role of ATP6AP2 in OC-lineage cells is less clear. Here, we provide evidence that ATP6AP2 plays a negative role in osteoclastogenesis and function, contrasting with the positive role of ATP6AP1. Mice with conditional knockout (cKO) of ATP6AP2 in OCs (Atp6ap2LysM) exhibit trabecular bone loss, likely due to the increased osteoclastogenesis and activity, since bone formation rates are comparable to control mice. In vitro assays using bone marrow macrophages (BMMs) show that Atp6ap2LysM cultures have more RANKL-induced TRAP+ OC-like cells and increased bone resorptive activity. Further studies reveal that while RANKL signaling and V-ATPase activity are normal, in ATP6AP2 KO OCs, but not BMMs, have reduced basal levels of Wnt/β-Catenin pathway proteins, such as LRP5/6 and β-Catenin, compared to controls. Wnt3A treatment induces β-Catenin and suppresses osteoclast formation in both control and ATP6AP2 KO OC-lineage cells, indicating that Wnt/β-Catenin signaling negatively regulates OC-formation and operates independently of ATP6AP2. Overall, these results suggest that ATP6AP2 is critical for maintaining basal levels of LRP5/6 receptors and β-Catenin in osteoclasts, thus acting as a negative regulator of osteoclastogenesis and activation.

Pdk3's role in RANKL-induced osteoclast differentiation: insights from a bone marrow macrophage model.

Osteoporosis (OP) is a chronic disease characterized by decreased bone mass, loss of skeletal structural integrity and increased susceptibility to fracture. Available studies have shown that the pyruvate dehydrogenase kinase (PDK) family is associated with osteoclastogenesis and bone loss, but the specific role of Pdk3 in bone pathology has not been systematically investigated. A cell OP model was established in receptor activator for nuclear factor-κB Ligand (RANKL)-induced bone marrow macrophages (BMMs). Hereafter, the expression levels of Pdk3 and osteoclastogenesis feature genes including nuclear factor of activated T cells 1 (Nfatc1), Cathepsin K (Ctsk), osteoclast associated Ig-like receptor (Oscar) in BMMs-derived osteoclasts were examined based on real-time quantitative PCR and western blotting methods. Further, the phosphorylation of ERK, P65 and JAK/STAT and their correlation was Pdk3 was gauged. In particular, changes in the activity of these signaling pathways were observed by silencing experiments of the Pdk3 gene (using small interfering RNA). Finally, the effects of Pdk3 gene silencing on signaling pathway activity, osteoclastogenesis, and related inflammatory and apoptotic indicators were observed by transfection with PDK3-specific siRNA. Following RANKL exposure, the levels of Pdk3 and osteoclastogenesis feature genes were all elevated, and a positive correlation between Pdk3 and osteoclastogenesis feature genes was seen. Meanwhile, ERK, P65 and JAK/STAT phosphorylation was increased by RANKL, and Pdk3 was confirmed to be positively correlated with the phosphorylation of ERK, P65 and JAK/STAT. Additionally, in RANKL-exposed osteoclasts, Pdk3 knockdown diminished the phosphorylation of ERK, P65 and JAK/STAT, reduced the expressions of osteoclastogenesis feature genes. Importantly, knockdown of Pdk3 also reduced the expression of inflammatory cytokines and resulted in elevated levels of Bax and Casp3 expression, as well as downregulation of Bcl2 expression. This study reveals for the first time the role of Pdk3 in RANKL-induced osteoclastogenesis and OP. These findings provide a foundation for future studies on the role of Pdk3 in other bone diseases and provide new ideas for the development of OP therapeutics targeting Pdk3.

Chronic microcystin-leucine-arginine exposure induces osteoporosis by breaking the balance of osteoblasts and osteoclasts

Microcystin-leucine-arginine (MC-LR) produced by cyanobacterial harmful algal blooms are hazardous materials. However, the toxicity and mechanisms of continuous exposure to MC-LR on the occurrence of osteoporosis remains poorly documented. In this study, to mimic the chronic influences of MC-LR on the bone tissues in humans, an animal model was constructed in which mice were treated with MC-LR through drinking water at an environmentally relevant level (1–30 μg/L) for 6 months. MC-LR was enriched in the skeletal system, leading to the destruction of bone microstructure, the decrease of bone trabecular number, the reduction of osteoblasts, the enhanced content of lipid droplets, and the activation of osteoclasts, which is the characteristic of osteoporosis. Herein, we revealed ferroptosis is a vital mechanism of osteoblast death in mouse models of MC-LR. MC-LR exposure activates AMPK/ULK1 signaling, further promotes ferritin selective autophagy, causes free iron release and lipid peroxidation deposition, and eventually leads to ferroptosis of osteoblasts. Importantly, the use of AMPK or ferroptosis inhibitors in vivo markedly reduced MC-LR-induced osteoblast death and impaired osteogenic differentiation. Interestingly, MC-LR exposure promotes iron uptake in bone marrow macrophages through the TF-TFR1 pathway, leading to its transformation to TRAP-positive pre-osteoclast cells, thereby promoting bone resorption. Overall, our data innovatively revealed the core mechanism of MC-LR-induced osteoporosis, providing the bi-directional regulation of MC-LR on osteoblast-osteoclast from the perspective of iron homeostasis imbalance.

MicroRNA-324 mediates bone homeostasis and the regulation of osteoblast and osteoclast differentiation and activity

MicroRNAs (miRNAs) modulate the expression of other RNA molecules. One miRNA can target many transcripts, allowing each miRNA to play key roles in many biological pathways. Defects in bone homeostasis result in common age-related diseases including osteoporosis. Serum levels of miR-324-3p positively correlate with several features of bone maintenance. In contrast here, using in vivo micro-computed tomography and histology, global miR-324-null mice demonstrated increased bone mineral density and both trabecular and cortical thickness, with effect magnitudes increasing with age. The bone marrow of miR-324-null mice had reduced lipid content while TRAP staining revealed a decrease in osteoclasts, with histomorphometry demonstrating an increased rate of bone formation. Ex vivo assays showed that the high bone mass phenotype of miR-324-null mice resulted from both increased osteoblast activity and decreased osteoclastogenesis. RNA-seq analysis of osteoblasts, osteoclasts and bone marrow macrophages and target validation assays identified that the osteoclast fusion regulator Pin1 and the master osteogenic regulator Runx2 were targets of miR-324-5p in osteoclast lineage cells and osteoblasts, respectively. Indeed, in vitro Runx2 overexpression recapitulated the increased osteogenesis and decreased adipogenesis phenotype observed in vivo by the loss of miR-324. Overall, these data demonstrate the importance of miR-324 in bone homeostasis by regulating aspects of both bone formation and remodelling. Elucidation of pathways regulated by miR-324 offer promise for the treatment of bone diseases such as osteoporosis.

Mechanistic Exploration of Smilax glabra Roxb. in Osteoarthritis: Insights from Network Pharmacology, Molecular Docking, and In Vitro Validation.

Background: Arthritis, a debilitating joint disease, remains a significant global health burden. This study uncovers the therapeutic potential of the medicinal plant Smilax glabra Roxb. (SGR) in attenuating progression of disease by modulating immune responses. Methods: Through computational approaches, key bioactive compounds in SGR were identified by using freely available databases: TCMSP, TCMID, HIT2.0, HERB, and INPUT in order to elucidate their underlying mechanisms of action. Therapeutic targets for the disease have been retrieved by TTD, GeneCard, and OMIM databases. The STRING database was used to analyze the protein-protein interactions (PPI) of intersecting genes. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to reveal the functional roles of genes. Mcule was used for molecular docking and binding affinity of compounds and targets were evaluated by DeepPurpose model. ALP activity, cell viability assay, TRAP staining were also performed. Results: A total of 14 active SGR compounds with 59 common targets for arthritis have been identified. These targets have a major role in controlling biological processes such as wound healing, oxygen responses, and chemical stimuli. Molecular docking by Mcule platform demonstrated that quercetin and β-sitosterol showed higher binding energy affinities with TNF, TP53, PTGS2, and JUN as compared to other targets. To explore the complex relationship between compounds and targets, pre-trained Davis and KIBA models were used to predict the affinity values of selected compounds. In MC3T3-E1 cells, ALP activity was significantly increased and bone marrow macrophages (BMM) showed a low number of TRAP-positive cells in SGR-treated cells. Conclusions: Our findings demonstrate that SGR effectively inhibits/regulates inflammatory responses, prevents cartilage degradation, promotes bone regeneration, and can be used as a promising candidate for the development of novel arthritis treatment.

Myeloid beta-arrestin 2 depletion attenuates metabolic dysfunction-associated steatohepatitis via the metabolic reprogramming of macrophages

Macrophage-mediated inflammation has been implicated in the pathogenesis of metabolic dysfunction-associated steatohepatitis (MASH); however, the immunometabolic program underlying the regulation of macrophage activation remains unclear. Beta-arrestin 2, a multifunctional adaptor protein, is highly expressed in bone marrow tissues and macrophages and is involved in metabolism disorders. Here, we observed that β-arrestin 2 expression was significantly increased in the liver macrophages and circulating monocytes of patients with MASH compared with healthy controls and positively correlated with the severity of metabolic dysfunction-associated steatotic liver disease (MASLD). Global or myeloid Arrb2 deficiency prevented the development of MASH in mice. Further study showed that β-arrestin 2 acted as an adaptor protein and promoted ubiquitination of immune responsive gene 1 (IRG1) to prevent increased itaconate production in macrophages, which resulted in enhanced succinate dehydrogenase activity, thereby promoting the release of mitochondrial reactive oxygen species and M1 polarization. Myeloid β-arrestin 2 depletion may be a potential approach for MASH.

Antioxidant enzyme Prdx1 inhibits osteoclastogenesis via suppressing ROS and NFATc1 signaling pathways.

Bone is a dynamic organ which continuously undergoes remodeling throughout one's lifetime. Cellular production of reactive oxygen species (ROS) is essential for regulating bone homeostasis. Osteoclasts, multinucleated giant cells differentiated from macrophage lineage, are responsible for osteolytic bone conditions which are closely linked to ROS signaling pathways. In this study, an anti-ROS enzyme, peroxiredoxin 1 (Prdx1) was found to be expressed both in bone marrow macrophages and osteoclasts. Recombinant Prdx1 protein was found to dose-dependently inhibit ROS production and osteoclast differentiation. Mechanistically, Prdx1 protein also attenuated NFATc1 activation as well as the expression of C-Fos, V-ATPase-d2, Cathepsin K, and Integrin αV. Collectively, Prdx1 is a negative regulator on osteoclast formation via inhibiting RANKL-mediated ROS activity, thus suggesting its potential application for treating osteoclast related disorders.

Aged bone marrow macrophages drive systemic aging and age-related dysfunction via extracellular vesicle-mediated induction of paracrine senescence.

The accumulation and systemic propagation of senescent cells contributes to physiological aging and age-related pathology. However, which cell types are most susceptible to the aged milieu and could be responsible for the propagation of senescence has remained unclear. Here we found that physiologically aged bone marrow monocytes/macrophages (BMMs) propagate senescence to multiple tissues, through extracellular vesicles (EVs), and drive age-associated dysfunction in mice. We identified peroxisome proliferator-activated receptor α (PPARα) as a target of microRNAs within aged BMM-EVs that regulates downstream effects on senescence and age-related dysfunction. Demonstrating therapeutic potential, we report that treatment with the PPARα agonist fenofibrate effectively restores tissue homeostasis in aged mice. Suggesting conservation to humans, in a cohort study of 7,986 participants, we found that fenofibrate use is associated with a reduced risk of age-related chronic disease and higher life expectancy. Together, our findings establish that BMMs can propagate senescence to distant tissues and cause age-related dysfunction, and they provide supportive evidence for fenofibrate to extend healthy lifespan.

Pseudomonas aeruginosa Mediates Host Necroptosis through Rhl-Pqs Quorum Sensing Interaction.

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen that can cause serious infections in immunocompromised patients. Quorum sensing (QS), a communication system evolved by P. aeruginosa to survey its density, is well acknowledged to be involved in various activities during bacterial infection. Recent studies have revealed the link between P. aeruginosa QS and host innate immune response. Previous evidence suggests that programmed cell death exists in response to P. aeruginosa infection. However, it remains unclear whether QS plays a role in the host programmed cell death process during the infection. In this study, we found that the deficiency of one of QS subsystems, rhl, markedly increased mouse bone marrow macrophage cell death induced by P. aeruginosa, which was accompanied by elevated phosphorylation of RIPK3 and MLKL. This highly increased necroptosis activation was caused by the upregulation of another QS subsystem, pqs, because the deletion of pqs in rhl-deficient P. aeruginosa abolished macrophage necroptosis invitro and invivo. In sum, our data highlight the cross-talk between P. aeruginosa QS and host necroptosis, which is executed through the rhl-pqs axis.

Excessive glucocorticoids combined with RANKL promote the differentiation of bone marrow macrophages (BMM) into osteoclasts and accelerate the progression of osteoporosis by activating the SYK/SHP2/NF-κB signaling pathway.

The primary objective of this study was to explore the extensive implications and complex molecular interactions arising from the confluence of excessive glucocorticoids and RANKL on the differentiation process of BMM into osteoclasts, profoundly impacting osteoporosis development. The methodology encompassed X-ray analysis and HE staining for evaluating bone loss in mice, while immunohistochemical staining was utilized to observe phosphorylated SHP2 (p-SHP2) expression. The assessment of several phosphorylated and total protein expression levels, including NF-κB, SHP2, SYK, JAK2, TAK1, NFATC1, c-fos, and Cathepsin K, was conducted via Western blotting. Additional experiments, involving CCK8 and monoclonal proliferation assays, were undertaken to determine BMM proliferation capacity. Immunofluorescence staining facilitated the quantification of TRAP fluorescence intensity. In vivo analysis revealed that glucocorticoid surplus triggers SHP2 signaling pathway activation, accelerating osteoporosis progression. Western blot results demonstrated that SHP2 inhibition could decrease the expression of specific proteins such as p-NF-κB and p-SHP2, with minimal effects on p-SYK levels. In vitro findings indicated that glucocorticoid and RANKL interaction activates the SHP2 pathway through NF-κB and SYK pathways, enhancing expressions of p-JAK2, p-TAK1, NFATC1, c-fos, and Cathepsin K, thereby promoting BMM to osteoclast transformation. Conclusion: Excessive glucocorticoids and RANKL interaction advance osteoclast differentiation from BMM by activating the SYK/SHP2/NF-κB signaling pathway, expediting osteoporosis progression.

Upregulation of Mitochondrial Sirt3 and Alleviation of the Inflammatory Phenotype in Macrophages by Estrogen.

Aging and comorbidities like type 2 diabetes and obesity contribute to the development of chronic systemic inflammation, which impacts the development of heart failure and vascular disease. Increasing evidence suggests a role of pro-inflammatory M1 macrophages in chronic inflammation. A shift of metabolism from mitochondrial oxidation to glycolysis is essential for the activation of the pro-inflammatory M1 phenotype. Thus, reprogramming the macrophage metabolism may alleviate the pro-inflammatory phenotype and protect against cardiovascular diseases. In the present study, we hypothesized that the activation of estrogen receptors leads to the elevation of the mitochondrial deacetylase Sirt3, which supports mitochondrial function and mitigates the pro-inflammatory phenotype in macrophages. Experiments were performed using the mouse macrophage cell line RAW264.7, as well as primary male or female murine bone marrow macrophages (BMMs). Macrophages were treated for 24 h with estradiol (E2) or vehicle (dextrin). The effect of E2 on Sirt3 expression was investigated in pro-inflammatory M1, anti-inflammatory/immunoregulatory M2, and naïve M0 macrophages. Mitochondrial respiration was measured by Seahorse assay, and protein expression and acetylation were determined by western blotting. E2 treatment upregulated mitochondrial Sirt3, reduced mitochondrial protein acetylation, and increased basal mitochondrial respiration in naïve RAW264.7 macrophages. Similar effects on Sirt3 expression and mitochondrial protein acetylation were observed in primary female but not in male murine BMMs. Although E2 upregulated Sirt3 in naïve M0, pro-inflammatory M1, and anti-inflammatory/immunoregulatory M2 macrophages, it reduced superoxide dismutase 2 acetylation and suppressed mitochondrial reactive oxygen species formation only in pro-inflammatory M1 macrophages. E2 alleviated the pro-inflammatory phenotype in M1 RAW264.7 cells. The study suggests that E2 treatment upregulates Sirt3 expression in macrophages. In primary BMMs, female-specific Sirt3 upregulation was observed. The Sirt3 upregulation was accompanied by mitochondrial protein deacetylation and the alleviation of the oxidative and pro-inflammatory phenotype in M1 macrophages. Thus, the E2-Sirt3 axis might be used in a therapeutic strategy to fight chronic systemic inflammation and prevent the development of inflammation-linked diseases.

Development of the PD9-9 Monoclonal Antibody for Identifying Porcine Bone Marrow-Derived Dendritic Cells.

The purpose of this study was to develop a monoclonal antibody (mAb) that can identify porcine dendritic cells (DCs) that have differentiated from bone marrow progenitor cells. Hybridoma technology was used to obtain mAbs, and bone marrow-derived DCs (BMDCs) were employed as immunogens for producing antibodies. The generated PD9-9 mAbs exhibited considerable reactivity towards porcine BMDCs with applications in flow cytometry and immunostaining. The antibody was composed of heavy immunoglobulin gamma-1 chains and light kappa chains. The PD9-9 mAb recognized fully differentiated porcine BMDCs and cells undergoing DC differentiation. In contrast, bone marrow cells and macrophages were not recognized by PD9-9. In addition, the PD9-9 mAb promoted porcine DC proliferation. Consequently, the PD9-9 mAb may be a biomarker for porcine DCs and will be advantageous for investigating porcine DC biology.