Mitochondria-derived vesicles (MDVs) and mitochondrial extracellular vesicles (mitoEVs) represent 2 related extensions of mitochondrial dynamics that link organelle maintenance to communication within and between cells. MDVs are small vesicles that bud directly from mitochondria, selectively packaging components of the outer membrane, inner membrane, or matrix. They serve as a localized quality control mechanism that removes oxidized or damaged material without engaging the entire mitophagic machinery. After budding, MDVs typically enter the endolysosomal pathway, where they can fuse with late endosomes or lysosomes for cargo degradation. A subset of MDVs also targets other organelles, particularly peroxisomes, contributing to organelle crosstalk, lipid metabolism, and redox balance. By contrast, mitoEVs released into the extracellular space contain intact functional mitochondria, mitochondrial contents (proteins, DNAs/RNAs, lipids, and so on), and nonmitochondrial cargo (ie, mRNAs, noncoding RNAs, and so on), which can be transferred to recipient cells and subsequently induce either pathogenic or beneficial outcomes. Therefore, mitoEVs have been implicated in metabolic cooperation, immune regulation, tissue remodeling, and aging. Accordingly, this review summarizes recent progress on the diverse mechanisms for the biogenesis of MDVs and mitoEVs, as well as available protocols for their isolation. The roles of MDVs and mitoEVs in mediating mitochondrial quality/quantity control and multiple layers of crosstalk between intracellular organelles and different cell types in health and disease are highlighted. Last, mitoEV-mediated pathogenic effects and therapeutic potential in cardiovascular disease are also discussed.

BackgroundHeart failure (HF) is a global health burden marked by high morbidity and limited treatment efficacy across subtypes. The lack of reliable molecular biomarkers for heart failure impedes personalized therapy. Emerging evidence suggests that macrophage-trained immunity drives chronic inflammation and cardiac remodeling, highlighting immune-related genes as promising biomarkers.MethodsWe integrated transcriptomic data from five independent HF cohorts and one macrophage-trained immunity model. Differentially expressed genes (DEGs) analysis, weighted gene co-expression network analysis (WGCNA), immune infiltration profiling, and six machine-learning algorithms were applied to screen immune-related candidate genes. Functional relevance was assessed by gene set enrichment analysis (GSEA) and single-cell RNA-seq of human cardiac tissue. Finally, we established a THP-1-derived macrophage trained immunity model to validate the paracrine effects of macrophage Maturin (MTURN) and Piezo-type mechanosensitive ion channel component 1 (PIEZO1) in cardiomyocytes.ResultsSeven hub genes were identified from HF-DEGs, the trained immunity transcriptional signature, and WGCNA co-expression modules. Among them, MTURN, an evolutionarily conserved regulator of differentiation and inflammation, emerged as the most robust candidate, showing consistent upregulation in HF samples across all cohorts with superior diagnostic performance. Importantly, GSEA linked MTURN to innate immune activation and adhesion/signaling pathways. Single-cell RNA-seq analyses of human cardiac tissue revealed MTURN enrichment in cardiac macrophages with a progressive increase along pseudotime. Experimentally, trained immunity macrophages displayed an elevation of glycolytic and inflammatory markers together with increased MTURN and PIEZO1. Accordingly, the conditioned medium collected from such trained macrophages could upregulate expression of HF markers (i.e., NPPA/B) in AC16 cardiomyocytes.ConclusionMulti-cohort, single-cell RNA-seq, and experimental data collectively suggest MTURN as a trained immunity-related biomarker for the diagnosis of heart failure with a potential link to PIEZO1-mediated cardiac remodeling.

Background: Extensive comorbidity between cardiovascular (CVD) and respiratory (RT) diseases is well-documented, yet the shared genetic mechanisms remain elusive. Genetic pleiotropy may play a pivotal role in understanding the intricate comorbidity patterns associated with cardiovascular and respiratory conditions. Methods: Our comprehensive analysis encompasses the largest available GWAS dataset of European ancestry covering six major CVDs (atrial fibrillation, coronary artery disease, venous thromboembolism, heart failure, peripheral arterial disease, and stroke) and four prevalent RTs (asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and sleep apnea). Initially, we aimed to unveil the common genetic basis of major CVDs, through genome-wide and local genetic correlations and polygenic overlap. Subsequently, the shared genetic mechanisms between RTs and CVDs was investigated in terms of both horizontal and vertical pleiotropy. From a horizontal pleiotropy perspective, cross-trait analysis was utilized to identify pleiotropic genetic determinants including genomic loci, single nucleotide polymorphisms (SNPs), genes, biological pathways, and protein targets. From a vertical pleiotropic perspective, Mendelian randomization was employed to evaluate potential causal relationships between CVDs and RTs. Results: Our study confirmed the significant existence of genetic correlations and overlaps between CVDs and RTs. Pleiotropy analysis under the composite null hypothesis identified 17,964 significant potential pleiotropic SNPs in 24 trait pairs, with 73 pleiotropic loci and 69 colocalized loci detected. Gene-based analysis revealed 59 candidate pleiotropic genes, highly enriched in unsaturated fatty acid biosynthetic processes and MHC class I-mediated antigen processing and presentation. Mendelian randomization analysis demonstrated a positive causal relationship only between chronic obstructive pulmonary disease and heart failure. Overall, the genetic basis between CVDs and RTs was inconsistent with vertical pleiotropy, suggesting the dramatic impact of horizontal pleiotropy. Conclusions: Our findings indicate widely distributed pleiotropic genetic determinants between RTs and CVDs across the genome. These results support a common genetic basis for RTs and CVDs and are important for intervention and therapeutic targets in comorbidities. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was supported by Shenzhen Pengcheng Peacock Plan (To Y.F), National Natural Science Foundation (Grant no. 82170339 and 82270241), NSFC Incubation Project of Guangdong Provincial People's Hospital (Grant no. KY0120220021), Natural Science Foundation of Guangdong Province (Grant no. 2023B1515020082) (To L.J.), National Institutes of Health R01 (Grant no. HL163148)(To W.H.) and Center for Computational Science and Engineering at Southern University of Science and Technology. The funder had no role in the design, implementation, analysis, interpretation of the data, approval of the manuscript, and decision to submit the manuscript for publication. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: GWAS summary statistics on Asthma and COPD are available from the Global Biobank Meta-Analysis Initiative (GBMI). Genetic information for OSA was obtained from the FinnGen consortium website: https://www.finngen.fi/en. GWAS summary statistics on IPF was obtained from International IPF Genetics Consortium. Genome-wide summary statistics on AF, HF, and Stroke are available at the GWAS Catalog (GCST90104539, GCST009541, and GCST90104539). GWAS summary statistics on CAD and PAD are publicly available for download at the Cardiovascular Disease Knowledge Portal (CVDKP) website: https://cvd.hugeamp.org/datasets.html. Genome-wide summary statistics on VTE are obtained from the deCODE genetics website: https://www.decode.com/summarydata/. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes GWAS summary statistics on Asthma and COPD are available from the Global Biobank Meta-Analysis Initiative (GBMI). Genetic information for OSA was obtained from the FinnGen consortium website: https://www.finngen.fi/en. GWAS summary statistics on IPF was obtained from International IPF Genetics Consortium. Genome-wide summary statistics on AF, HF, and Stroke are available at the GWAS Catalog (GCST90104539, GCST009541, and GCST90104539). GWAS summary statistics on CAD and PAD are publicly available for download at the Cardiovascular Disease Knowledge Portal (CVDKP) website: https://cvd.hugeamp.org/datasets.html. Genome-wide summary statistics on VTE are obtained from the deCODE genetics website: https://www.decode.com/summarydata/.

Junctophilin-2 (JP2) and junctin (JCN) are key proteins in maintaining calcium homeostasis in cardiomyocytes. Both are reduced in diseased hearts while overexpression of JP2 mitigates heart failure. This study demonstrates that JP2 and JCN are reduced in cardiomyocytes under stress, leading to intracellular calcium dysregulation and subsequent cell death. JP2 binds JCN, thereby blocking muscle ring finger protein-1 (MURF1)-JCN interaction and subsequently preventing MURF1-mediated JCN ubiquitination and degradation in cardiomyocytes. Thus, JP2 overexpression and MURF1 inhibition similarly preserve JCN protein and attenuate myocardial injury and remodeling, and improve myocardial function in preclinical animal models of lipid overload-induced cardiomyopathy and transverse aortic constriction-induced heart failure. Disruption of the JP2-JCN axis represents an important mechanism underlying heart disease and may serve as a potential therapeutic target for cardiac protection.

Background Heart failure with preserved ejection fraction (HFpEF) is a heterogeneous syndrome associated with metabolic stress, hypertension, systemic inflammation, and microvascular dysfunction. Early cell-type-specific events and intercellular communication programs that accompany disease onset and progression remain poorly defined. Methods We performed a longitudinal study of HFpEF progression in high-fat diet (HFD)+L-NAME mice at control/baseline (0 weeks, 0w/Ctrl), early (1w), intermediate (4w), and established (8w) stages. Metabolic, hemodynamic, exercise, echocardiographic, and single-cardiomyocyte function were assessed. Cardiac non-cardiomyocytes (non-CMs) were profiled by single-cell RNA sequencing (scRNA-seq), with bulk RNA-seq for tissue-level comparison. Endothelial remodeling was assessed in an L-NAME-independent HFD plus mild transverse aortic constriction model (HFD+mTAC) and a published human HFpEF single-nucleus RNA-seq cohort. An endothelial–macrophage adhesion assay tested whether HFpEF-mimic stress promotes endothelial activation and macrophage adhesion. Results In the HFD+L-NAME model, metabolic dysfunction, hypertension, reduced exercise tolerance, abnormal diastolic filling with preserved ejection fraction, and altered cardiomyocyte calcium handling were detected by 1w and persisted through 8w. Bulk RNA-seq showed progressive remodeling, with limited change between 8w and 12w, guiding scRNA-seq timepoint selection. scRNA-seq of 94,848 cardiac non-CMs identified nine major populations with stage-dependent remodeling. Endothelial cells (ECs) were recovered in high proportion and showed an early, pronounced transcriptional response, with inflammatory, adhesion, interferon-response, migratory, and vascular-remodeling programs emerging by 1w. Related EC activation signatures were observed in HFD+mTAC and human HFpEF data. Functionally, HFpEF-mimic stress increased adhesion and chemokine expression in human ECs and enhanced macrophage adhesion. Fibroblast matrix remodeling occurred at later stages, while macrophages progressively shifted toward inflammatory states. CellChat suggested stage-dependent communication remodeling from early endothelial–immune interactions toward later macrophage–fibroblast crosstalk. Conclusion Time-resolved scRNA-seq reveals coordinated, stage-dependent remodeling of the cardiac microvascular and interstitial microenvironment during HFpEF progression. Early endothelial activation emerges before later fibroblast matrix remodeling and inflammatory macrophage remodeling, identifying candidate cell states and signaling pathways for future mechanistic investigation. Clinical Perspective What Is New? This study provides a time-resolved single-cell atlas of the cardiac non-cardiomyocyte compartment across baseline, early, intermediate, and established stages of HFpEF progression, rather than a single late-stage snapshot. Endothelial cells exhibit early inflammatory, adhesion, interferon-response, and vascular-remodeling programs within the first week of disease, preceding the later predominance of fibroblast matrix remodeling and inflammatory macrophage remodeling. This endothelial activation signature is supported across two mechanistically distinct HFpEF mouse models and aligns with endothelial inflammatory and vascular-remodeling programs in human HFpEF myocardium, supporting its translational relevance. What Are the Clinical Implications? Early endothelial activation may represent a targetable stage of HFpEF pathogenesis that arises before more established structural and fibrotic remodeling. Therapeutic strategies aimed at limiting endothelial inflammatory activation or endothelial–immune interactions may help attenuate downstream vascular, immune, and stromal remodeling in HFpEF. These findings provide a preclinical foundation for future longitudinal human studies testing whether early endothelial activation can serve as a biomarker, therapeutic target, or disease-staging feature in HFpEF.

Abstract Background Diabetic vascular complications are driven by endothelial dysfunction, yet the role of 3D genome organization in this process is unknown. We sought to define the alterations in chromatin architecture in diabetic endothelium and identify the key regulators involved. Methods We generated a high-resolution 3D epigenomic atlas of diabetic endothelial cells from mouse models and human subjects using H3K27ac HiChIP, complemented by ChIP-seq, ATAC-seq, and RNA-seq. A human cohort was used to assess protein expression in diabetic versus non-diabetic endothelial cells. To identify JUNB-interacting proteins, we performed rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME), with protein-protein interaction validated by co-immunoprecipitation. Functional validation was performed using in vitro , ex vivo , and in vivo approaches, including endothelial-specific knockdown in a diabetic hindlimb ischemia model. Results Multi-omics profiling revealed extensive enhancer reprogramming in diabetic endothelium, with AP-1 binding motifs being consistently and selectively enriched in downregulated enhancers across three distinct diabetic models. Analysis of a human cohort confirmed significantly reduced JUNB protein levels in diabetic endothelial cells. We identified widespread disruption of JUNB-anchored enhancer-promoter interactions, which underlies transcriptional repression of key endothelial genes. RIME and co-immunoprecipitation established the E3 ubiquitin ligase RBBP6 as a direct JUNB interactor that promotes its polyubiquitination and proteasomal degradation in response to hyperglycemia. Human cohort analysis further showed reciprocal elevation of RBBP6 in diabetic endothelial cells. Either JUNB overexpression or RBBP6 knockdown restored enhancer-promoter connectivity, reactivated vasoprotective transcriptional programs, and rescued endothelial function. Critically, endothelial-specific knockdown of Rbbp6 in diabetic mice restored endothelium-dependent vasorelaxation and improved perfusion recovery after hindlimb ischemia, independent of systemic glucose levels. Conclusions Our study unveils a novel mechanism whereby hyperglycemia induces enhancer reprogramming and disrupts endothelial 3D genome architecture through RBBP6-mediated degradation of JUNB. The RBBP6-JUNB axis is established as a crucial link between metabolic stress and epigenomic reprogramming in vascular disease, presenting a promising therapeutic target for diabetic vasculopathy.

Myocardial ischemia/reperfusion (I/R) usually triggers a series of molecular and cellular changes, which yield excessive oxidative stress and massive cardiomyocyte death, leading to sterile inflammation, cardiac fibrosis, and, eventually, heart failure. Over the past two decades, numerous studies have demonstrated that noncoding RNAs (ncRNAs), including microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs), involve almost every aspect of adverse cardiac remodeling induced by I/R. They have emerged as key regulators in the process of cardiac cell death (i.e. apoptosis, necroptosis, ferroptosis, pyroptosis, and PANoptosis), fibrosis, angiogenesis, and immune responses during myocardial I/R. Herein, this review summarizes recent advancements on ncRNA-mediated regulation of cardiac cell death, cardiac angiogenesis, fibrosis, and macrophage function as well as intercellular communication following myocardial I/R. Finally, the therapeutic potential of ncRNAs for treating myocardial I/R injury and future research directions are also discussed.

Vascular leakage is a major cause of multiple organ failure and mortality in sepsis, and factors that regulate endothelial integrity could serve as promising biomarkers of septic shock development. Copine family members (CPNEs) are well-characterized as soluble membrane-binding proteins, whether CPNEs play a critical role in maintaining vascular integrity during sepsis, however, remains unclear. Human aorta single-nucleus RNA-sequencing data were analyzed for the expression profile of all Copine family members (CPNE1-9). Plasma levels of CPNE5, Ang-II, sICAM-1, and SDC-1 were measured in human sepsis patients at admission and healthy donors as well as in septic mice induced by injection i.p. with cecal slurry. The correlation of CPNE5 levels to other three factors (Ang-II, sICAM-1, SDC-1) were analyzed. CPNE5-knockdown endothelial cells (ECs) and global CPNE5-knockout (KO) mice were utilized to determine the critical role of CPNE5 in sepsis-triggered vascular leakage, organ damage and mortality. Among nine CPNEs, only CPNE5 is predominantly expressed in human aorta endothelial cells. In sepsis patients (n = 77), plasma levels of CPNE5 were significantly reduced, whereas plasma levels of Ang-II, sICAM-1, and SDC-1 were markedly elevated, compared to healthy donors (n = 44; p < 0.01). Similar findings were also observed in a murine sepsis model induced by cecal slurry (CS)-injection intraperitoneally. Furthermore, in the supernatants of cultured ECs treated with LPS or pro-inflammatory cytokine mixture (Cytomix: TNFα/IL-1β/IFNγ, each 10 ng/mL), the concentrations of CPNE5 were significantly lower, which was negatively correlated with the higher EC permeability, compared to the control group. Accordingly, siRNA-mediated knockdown of CPNE5 in ECs caused hyperpermeability upon stimulation with LPS or Cytomix. In vivo, we observed that loss of CPNE5 increased vascular leakage, leading to severe organ injury and higher mortality, compared to WT mice upon septic conditions. The initial mechanistic analysis showed that the reduction of CPNE5 in cardiac and pulmonary ECs was linked to the increased cleavage of membrane tight junctions and adherens junctions. These observations from human sepsis patients and a murine sepsis model suggest that reduced plasma levels of CPNE5 may contribute to sepsis-induced vascular leakage and mortality.

BACKGROUND:Doxorubicin (DOX), an effective chemotherapeutic drug for various cancers, has been demonstrated to induce cardiovascular toxicity in cancer survivors. Endothelial cell (EC) dysfunction is recognized to play a critical role in the onset and severity of cardiotoxicity associated with DOX. TFEB (transcription factor EB), a master regulator of autophagy and lysosome biogenesis, regulates cardiovascular homeostasis. In the present study, we aimed to test whether endothelial TFEB protects against EC damage and alleviates cardiac dysfunction induced by DOX treatment.METHODS:EC-specific TFEB transgenic mice, EC-specific TFEB knockout mice, and their corresponding littermate controls were administered DOX intravenously. Survival curves were generated, and cardiac functions were measured in mice. The effects of TFEB on mitochondrial reactive oxygen species production, autophagic flux, and apoptosis were evaluated in human and mouse cardiac microvascular ECs treated with DOX. RNA sequencing, single-cell RNA sequencing, and chromatin immunoprecipitation with quantitative polymerase chain reaction (ChIP-qPCR) was performed to dissect molecular mechanisms in DOX-treated ECs in vitro and in vivo. Mice with endothelium-specific deficiency of Dab2 gene (Disabled homolog 2) were subjected to measurement of cardiac function and fibrosarcoma growth under DOX treatment.RESULTS:EC-specific TFEB transgenic mice showed significantly reduced mortality and improved cardiac function, together with attenuation of perivascular fibrosis after DOX treatment. By contrast, EC-specific TFEB knockout exacerbated DOX-induced cardiac dysfunction in mice. Furthermore, we observed that TFEB enhanced autophagy and reduced oxidative stress in cardiac microvascular ECs treated with DOX. In addition, TFEB preserved EC barrier integrity, alleviated proinflammatory cytokine release from cardiac microvascular ECs, and maintained the EC-cardiomyocyte communication, contributing to the protective effects of EC TFEB on cardiomyocyte function. Mechanistically, DAB2, a clathrin- and cargo-binding endocytic adaptor protein, was identified as a TFEB target gene in ECs. Accordingly, DAB2 knockdown attenuated the inhibitory effects of TFEB on apoptosis and the secretion of proinflammatory cytokines from cardiac microvascular ECs. In vivo, EC-specific Dab2 deficiency abolished the protective effect of EC TFEB on DOX-induced cardiac dysfunction.CONCLUSIONS:Taken together, endothelial TFEB protects against EC damage and cardiac dysfunction, constituting a potential target for treating cardiotoxicity induced by DOX. Our study provides new mechanistic insights into cardiotoxicity associated with chemotherapy.

Background: Myocardial ischemia/reperfusion (I/R) injury is mainly ascribed to excessive sterile inflammation resulting from the inefficient clearance of dead cardiomyocytes by macrophages (termed efferocytosis). Bacterium-released extracellular vesicles (bEVs) have been shown as a critical mediator in macrophage (MΦ) function and modulating inflammation. However, their possible role in MΦ efferocytosis during I/R has not been investigated. Methods: BEVs were isolated from the culture supernatants of three probiotic strains include Lactobacillus rhamnosus GG (LGG), Bifidobacterium BB-12, and Escherichia coli Nissle 1917 (EcN). The collected EVs were then added to bone marrow–derived MΦs (BMDMs), followed by incubation with Deep Red (APC)-labelled dead H9c2 cells for determining MΦ efferocytosis, assessed by flow cytometry. For the in vivo experiments, EVs were administered via tail vein injection into cardiac I/R-operated mCherry-transgenic mice. Subsequently, cardiac MΦ efferocytosis and cardiac remodeling will be analyzed. The mechanism underlying bEV-mediated efferocytosis will be assessed by RNA-sequencing and bioinformatics assays. Results: We observed that LGG-EVs were the best among these three bEVs in stimulating MΦs to engulf dead H9c2 cells. Using mCherry-Tg mice to undergo the ligation of left anterior descending artery for 45 min, LGG-bEVs (2 µg/g) were injected into mice via the tail vein just prior to reperfusion. One day 4 post-I/R, LGG-bEVs-treated mice exhibited a higher capacity of cardiac MΦ efferocytosis, lower levels of cardiac cells death, inflammatory cytokines (IL-6, TNF-α, MCP-1), and inflammatory cell infiltration, compared to PBS-treated I/R mice (n = 6, p < 0.05). Accordingly, at 1-month post-I/R, LGG-bEV-treated mice showed a marked improvement in cardiac function, along with reduced cardiac fibrosis (n = 8, p < 0.05). Mechanistically, RNA-seq and bioinformatic assays identified that LGG-bEVs contained higher levels of 5sRNA-derived short-RNA fragments, which interacted with coding regions of Ddx5, Ywhaz, and Frmd4a genes, three genes known to promote MΦ efferocytosis. Treatment of MΦs with LGG-bEVs greatly upregulated the expression of Ddx5, Ywhaz, and Frmd4a, which was further validated by co-transfection of these gene-expression plasmids with this short-RNA fragment. Conclusions: This study suggests that LGG-EVs have therapeutic effects against I/R-induced cardiac injury through promoting MΦ efferocytosis.

DNA damage-inducible transcript 3 (DDIT3) is a well-known transcription factor that regulates the expression of apoptosis-related genes for promoting apoptosis during endoplasmic reticulum stress. Here, we report an unrecognized role of DDIT3 in facilitating necroptosis. DDIT3 directly binds and competitively prevents the p38 MAPK-MK2 interaction and thereby blocking MK2 activation while stimulating p38 MAPK activation. This blockage of MK2 activation initially prevents RIPK1 phosphorylation at Ser320 (inactivation), subsequently relieving its suppression of RIPK1 activation. Consequently, p38 MAPK facilitates RIPK1 phosphorylation at Ser166 (activation) through DDIT3 phosphorylation-related mechanisms, leading to necroptosis. Mechanistically, a 10-amino acid segment (Glu19-Val28) within DDIT3’s N-terminus is identified to account for its pro-necroptotic function. In vivo studies demonstrate that forced expression of DDIT3 induces necroptosis, whereas deletion of DDIT3 alleviates necroptosis in mouse hearts under stress. These findings shed light on a novel regulatory mechanism by which DDIT3 promotes RIPK1 activation and subsequent necroptosis.

Abstract Background Sepsis-induced organ failure and high mortality are largely ascribed to the failure of bacterial clearance from the infected tissues. Recently, probiotic bacteria-released extracellular vesicles (BEVs) have been implicated as critical mediators of intercellular communication which are widely involved in the regulation of the inflammatory response. However, their functional role in macrophage phagocytosis during sepsis has never been explored. Methods BEVs were collected from three different strains of probiotics including Lactiplantibacillus plantarum WCFS1 (LP WCFS1), Lactobacillus rhamnosus Gorbach-Goldin (LGG), and Escherichia coli Nissle 1917 (EcN), or from LGG cultured under three pH conditions (pH5-acid, pH6.5-standard, pH8-akaline) through differential centrifugation, filtration, and ultracentrifugation of their culture supernatants. In vitro phagocytosis was measured in Raw264.7 cells and bone marrow-derived macrophages using pHrodo red E. coli BioParticles. The in vivo therapeutic effects of BEVs were tested using a feces-injection-in-peritoneum (FIP) model of polymicrobial sepsis. Results LGG-derived EVs (BEVLGG) were the best among these three probiotics BEVs in stimulating macrophages to take up bacteria. Furthermore, BEVLGG collected from pH8 culture condition (BEVpH8) exhibited the strongest capacity of phagocytosis, compared with BEVpH5 and BEVpH6.5. Treatment of septic mice with BEVpH8 significantly prolonged animal survival; increased bacterial clearance from the blood, peritoneal lavage fluid, and multiple organs; and decreased serum levels of pro-inflammatory cytokines/chemokines, as well as reduced multiple organ injuries, in comparison with control-treated septic mice. Mechanistically, RNA-seq and bioinformatic analysis identified that the FPR1/2 signaling was remarkably activated, along with its downstream pathways (PI3K-Akt-MARCO and NADPH-ROS) in BEVpH8-treated macrophages, compared with control cells. Accordingly, pre-addition of Boc2, a specific antagonist of FPR1/FPR2, to macrophages significantly attenuated BEVpH8-mediated phagocytosis, compared to controls. Conclusions This study demonstrates that LGG-derived BEVs may have therapeutic effects against sepsis-induced organ injury and mortality through enhancing FPR1/2-mediated macrophage phagocytosis.

Introduction: Efficient clearance of dead/dying cells by macrophages (MΦs) (termed efferocytosis) is critical for timely repairing the injured heart after ischemia/reperfusion (I/R). Prior work by us and others shows that Lipocalin 10 (Lcn10), a secreted protein of lipocalin family, can regulate MΦ polarization and is greatly downregulated in cardiac tissue of patients with heart failure. However, the potential role of Lcn10 in MΦ efferocytosis during cardiac I/R has never been investigated. Methods: Wild-type (WT) and Lcn10-knockout (KO) mice were subjected to 45 minutes of ischemia via the ligation of left anterior descending artery (LAD) followed by reperfusion. Cardiac function was measured by echocardiography. Flow cytometry was utilized to assess MΦ efferocytosis. Bone marrow-derived MΦs (BMDMs) were used to test efferocytosis in vitro and dissect mechanism. Dead cells were prepared by treating red-dye-labelled H9C2 cells with H 2 O 2 (1mM, 2h). Results: After co-culturing BMDMs with dead H9C2 cells for 2h, we observed a 25% reduction of efferocytosis in Lcn10-KO BMDMs, compared to WTs. Using Lcn10-KO mCherry mouse model (cardiomyocyte-specific overexpression of mCherry in Lcn10-KO background), we consistently found that cardiac MΦs in Lcn10-KO mice taken up fewer dead mCherry-myocytes at 4 days post-I/R surgery, leading to an increased accumulation of cardiac dead cells, higher serum levels of troponin I, greater myocardial fibrosis, and dramatically impaired cardiac function, compared to WT mCherry controls. In contrast, pre-treatment of BMDMs with recombinant Lcn10 protein (rLcn10) enhanced efferocytosis by 50%. Mechanistically, RNA-seq analysis revealed that many efferocytosis-related genes were dysregulated ( e.g., downregulation of Id 1, Id 3, Itga 8, Itga v, Cd 44, Cx3cr 1, Msr 1, and upregulation of Sirp α) in Lcn10-KO BMDMs, compared to WTs. Interestingly, most of these dysregulated genes are controlled by transcription factors Id1/3. Further studies showed that rLcn10 interacted with Cd44 and activated the expression of its downstream Id1/3, leading to enhanced MΦ efferocytosis. Importantly, rLcn10-mediated efferocytosis was greatly impaired by pre-treatment of MΦs with either anti-CD44 blocking antibody (KM201, 10µg/ml) or Id1/3 inhibitor (AGX51, 25µM). Conclusion: Our data indicate that Lcn10 is essential for MΦ efficient efferocytosis to repair I/R-induced cardiac damage though affecting the Cd44-Id1/3 signaling and its related MΦ surface receptors.