Modulation Of Macrophage Phenotype Research Articles

Human macrophages are immunnoprofiled by pericardial fluid small extracellular vesicles modulating lipid metabolism mechanisms

Abstract Background Coronary artery disease (CAD) is the main cause of myocardial infarction and consequent ischemic heart disease (IHD). Macrophages are central to CAD. Small extracellular vesicles (sEVs) can shuttle microRNAs and other molecular cargos from cell to cell, mediating response in target cells. Recent evidence supports the sEVs role in modulating macrophage phenotype. The pericardial fluid (PF) contains sEVs and immune cells including cavity-like macrophages. Purpose This study investigates whether PF-sEVs regulate macrophages, contributing to a specific immunophenotype in CAD patients. Methods PF was collected from CAD patients undergoing coronary bypass surgery (CABG) and non-atherosclerotic patients operated for mitral valve repair (control group). sEVs were isolated and characterised for size (Nanosight tracking analysi), tetrasapanin content (Nanoview chips) and microRNA content (RNA seq analysis). Monocytes from healthy donors were isolated from buffy coats and differentiated into macrophages following established protocols. Macrophages were incubated with either CAD-sEVs or non-CAD sEVs for 24h at 37oC. The cells were collected and processed for mRNA analyses (qRT-PCR) and flow cytometry. Further bioinformatics were employed to understand functional pathways targeted by PF-sEVs-miRNA. Results In line with the human-PF macrophages profile, exposure to CAD-sEVs reduces the anti-inflammatory profile of human macrophages. CAD-sEVs treated macrophages showed a CCR2-, CD36+low, CD206+low profile. While non-CAD-sEVs did not statistically differ from PBS nor untouched groups, CAD-sEVs increased the mRNA level of IL1a, IL1b, TNFa and decreased MRC1. Bioinformatic analysis showed that 861 miRNAs were decreased in the PF-sEVs from CAD patients compared to non-CAD. miRNA targets prediction and pathway analyses reported that clusters of PF-miRNAs could regulate CD36, decreased in PF-macrophages. Conclusions We demonstrate, that sEVs isolated from PF-CAD reduce the anti-inflammatory profile of human macrophages, and target crucial lipid metabolism pathways. These clinically relevant results could drive to decipher improved therapeutics to modulate the epicardial/myocardial immune response in CAD patients.

The STAT3/SETDB2 axis dictates NF-κB-mediated inflammation in macrophages during wound repair.

Macrophage transition from an inflammatory to reparative phenotype after tissue injury is controlled by epigenetic enzymes that regulate inflammatory gene expression. We have previously identified that the histone methyltransferase SETDB2 in macrophages drives tissue repair by repressing NF-κB-mediated inflammation. Complementary ATAC-Seq and RNA-Seq of wound macrophages isolated from mice deficient in SETDB2 in myeloid cells revealed that SETDB2 suppresses the inflammatory gene program by inhibiting chromatin accessibility at NF-κB-dependent gene promoters. We found that STAT3 was required for SETDB2 expression in macrophages, yet paradoxically, it also functioned as a binding partner of SETDB2 where it repressed SETDB2 activity by inhibiting its interaction with the NF-κB component, RELA, leading to increased RELA/NF-κB-mediated inflammatory gene expression. Furthermore, RNA-Seq in wound macrophages from STAT3-deficient mice corroborated this and revealed STAT3 and SETDB2 transcriptionally coregulate overlapping genes. Finally, in diabetic wound macrophages, STAT3 expression and STAT3/SETDB2 binding were increased. We have identified what we believe to be a novel STAT3/SETDB2 axis that modulates macrophage phenotype during tissue repair and may be an important therapeutic target for nonhealing diabetic wounds.

Mesenchymal stem cells alleviate autoimmune thyroiditis by modulating macrophage phenotypes and through influencing the STING pathway

BackgroundHashimoto's thyroiditis is a chronic autoimmune inflammatory disease with a high prevalence and currently lacks effective treatment options. Previous preclinical and clinical trials have established mesenchymal stem cells (MSCs) as a promising therapeutic approach; however, there is limited research on MSC treatment for Hashimoto's thyroiditis, and the underlying molecular mechanisms remain unclear. MethodsMSCs isolated from 4 to 6-week-old Lewis rats were employed for thyroiditis treatment. The efficacy of MSCs was assessed through histological and serological parameters. Molecular mechanisms of MSC therapy for Hashimoto's thyroiditis were explored by examining macrophage presence within thyroid tissue and relevant pathways. ResultsIn this study, we observed elevated oxidative stress and endoplasmic reticulum stress within the thyroid tissue of Hashimoto's thyroiditis patients, and MSC therapy effectively mitigated this process. Furthermore, we found that the therapeutic potential of MSCs in the EAT model depended on the STING pathway. MSCs reduced endoplasmic reticulum stress and inflammasome levels within the thyroid tissue by modulating the STING pathway. Additionally, MSCs inhibited the expression of IRE1α in thyroid tissue macrophages, thereby reducing the polarization of M1-type macrophages ConclusionsThe STING pathway appears to be a crucial mechanism by which MSCs modulate macrophage polarization in thyroid tissue, offering a potential treatment for thyroiditis.

Eriocalyxin B ameliorated experimental autoimmune prostatitis via modulation of macrophage polarization through gut microbiota-mediated vitamin D3 alteration

BackgroundChronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a often heterogeneous condition in urology. Accumulating evidence suggests that the autoimmune response against prostate antigens is related to CP/CPPS. The gut microbiota may be a possible cause of a number of autoimmune diseases. Eriocalyxin B (EriB) is used as an anti-inflammatory treatment for autoimmune disorders. The underlying mechanism of fecal metabolome involved in CP/CPPS treatment by EriB remains unclear. MethodsThe experimental autoimmune prostatitis (EAP) mouse model was generated by subcutaneous immunization. Macrophages, inflammatory cytokines, intestinal microbiota, and fecal metabolome of the mice were analyzed. The alteration of the fecal metabolome was investigated in detail in EriB-treated EAP mice and confirmed by in vitro experiments. ResultsEriB ameliorated significantly decreased prostate inflammation in EAP mice and promoted macrophage phenotype polarizing from M1 to M2. The gut microbiome was altered, and intestinal barrier damage was improved by EriB treatment. Furthermore, the enrichment of vitamin digestion and absorption pathways in the fecal metabolome revealed that vitamin D3 was altered by EriB. In vitro experiments confirmed that macrophage polarization from M1 to M2 was promoted by vitamin D3. Finally, fecal transplantation from EriB-treated mice markedly reduced inflammatory indicators and the macrophage M1/M2 ratio in pseudogerm-free EAP mice. In our study, the immune state of macrophage regulated by gut microbiota-mediated vitamin D3 alteration was first time revealed in EAP treatment. ConclusionsEriB ameliorated in mice with EAP, the gut microbiota mediates vitamin D3 alterations to modulate macrophage phenotype polarizing from M1 to M2.

Update on Biomarkers of Chronic Inflammatory Processes Underlying Diabetic Neuropathy.

There is an increasing prevalence of diabetes mellitus (DM), particularly type 2 DM (T2DM), and its associated complications. T2DM is linked to insulin resistance, chronic inflammation, and oxidative stress, which can lead to both macrovascular and microvascular complications, including peripheral diabetic neuropathy (PDN). Inflammatory processes play a key role in the development and progression of T2DM and its complications, with specific markers like C-reactive protein (CRP), interleukins (ILs), and tumor necrosis factor (TNF)-α being associated with increased risk. Other key inflammatory markers such as nuclear factor kappa B (NF-κB) are activated under hyperglycemic and oxidative stress conditions and contribute to the aggravation of PDN by regulating inflammatory gene expression and enhancing endothelial dysfunction. Other important roles in the inflammatory processes are played by Toll-like receptors (TLRs), caveolin 1 (CAV1), and monocyte chemoattractant protein 1 (MCP1). There is a relationship between vitamin D deficiency and PDN, highlighting the critical role of vitamin D in regulating inflammation and immune responses. The involvement of macrophages in PDN is also suspected, emphasizing their role in chronic inflammation and nerve damage in diabetic patients. Vitamin D supplementation has been found to reduce neuropathy severity, decrease inflammatory markers, and improve glycemic control. These findings suggest that addressing vitamin D deficiency could offer therapeutic benefits for PDN. These molecular pathways are critical in understanding the pathogenesis of DM complications and may offer potential biomarkers or therapeutic targets including anti-inflammatory treatments, vitamin D supplementation, macrophage phenotype modulation, and lifestyle modifications, aimed at reducing inflammation and preventing PDN. Ongoing and more extensive clinical trials with the aim of investigating anti-inflammatory agents, TNF-α inhibitors, and antioxidants are needed to advance deeper into the understanding and treatment of painful diabetic neuropathy.

Inflammatory or Reparative? Tuning Macrophage Polarization Using Anodized Anisotropic Nanoporous Titanium Implant Surfaces

Modulating macrophage phenotype based on implant surface characteristics, including topography and chemistry, has been employed to enhance osseointegration and long‐term functional outcomes for titanium (Ti)‐based implants. An excessive and/or prolonged M1 macrophage response can lead to damaging immune‐inflammatory reactions, negatively influencing the fate of the implant, and hence, modulating these responses via nanoscale implant surface modification is an emerging paradigm. Herein, an anodized titanium implant surface based on single‐step electrochemical anodization, with preserved underlying microfeatures and superimposed nanopores (50 and 70 nm), compared with irregular rough and microrough (machined‐like) surfaces, is investigated for its effect on the functions of primary macrophages in vitro. Significantly reduced macrophage proliferation and increased tissue‐reparative M2 phenotype polarization are confirmed for the nanopores, which are more pronounced for 70 nm diameter. Moreover, osteoclastogenesis is reduced while osteogenic differentiation of osteoblasts is enhanced for the nanopores (higher for 70 nm pores). Advanced nanoengineered Ti implants can enhance titanium implant tissue integration by modulating the inflammatory response at the implant–cell interface.

Metformin enhances osteogenic differentiation of BMSC by modulating macrophage M2 polarization

Bone marrow-derived mesenchymal stem cells (BMSCs) are capable of developing into osteoblastic cell lines in vitro and regenerating bone tissue in vivo, and they are considered to be a reliable source for bone regenerative medicine. In recent years, studies have shown that the immune microenvironment is important for osteogenesis, in which macrophages are an important component of innate immunity and coordinate with stem cells. Metformin (Met), a hypoglycemic drug that exerts a powerful effect on metabolic signaling, has been shown to modulate inflammatory responses and osteogenic activity. However, whether metformin modulates macrophage polarization and subsequently affects osteogenesis remains to be elucidated. In the present study, we investigated the potential immunomodulatory capacity of metformin on macrophage inflammatory responses and phenotypic switching, and the subsequent effects on osteogenic differentiation of BMSCs. Flow cytometry and qPCR were used to study the effects of metformin on macrophage phenotypic regulation. qPCR, ALP, ARS and calcium content measurement and ALP activity assay were used to determine the effects of macrophage-secreted activators on the osteogenic differentiation of BMSCs. Our study demonstrates that metformin can improve the immune microenvironment by modulating macrophage polarization towards an anti-inflammatory phenotype, promoting an increase in a range of anti-inflammatory factors and inhibiting pro-inflammatory factors. This was characterized by increased expression of IL-10 and CD206, Arg-1 and decreased expression of IL-1β, TNF-α, IL-6 and iNOS. In addition, metformin-modulated macrophage-conditioned medium promoted osteogenic differentiation of BMSCs, increased the expression levels of genes (ALP, Runx-2, OCN, and Col-1), enhanced ALP activity, and significantly formed mineralized nodules. In conclusion, our new study elucidates that metformin can promote osteogenic differentiation of BMSCs by modulating macrophage phenotype and thereby.

Substrate Curvature Influences Cytoskeletal Rearrangement and Modulates Macrophage Phenotype.

Inflammation serves as a critical defense mechanism against pathogens and tissue damage but can lead to chronic diseases, such as cardiovascular disease and diabetes, when dysregulated. Macrophages play a pivotal role in orchestrating inflammatory responses, transitioning from pro-inflammatory M1 to anti-inflammatory M2 phenotypes to resolve inflammation and promote tissue repair. Current approaches to modulate macrophage phenotype predominantly rely on biochemical cues, which may induce systemic side effects. Given the mechanosensitivity of macrophages, this study investigates biophysical cues, specifically substrate curvature, as a localized strategy to regulate macrophage phenotype and minimize systemic repercussions. We hypothesized that substrate curvature influences macrophage immunophenotype by modulating F-actin polymerization. To test this hypothesis, we fabricated spherical microgels with tunable curvatures and characterized their biophysical properties. Our findings indicate that macrophages adhere to microgel surfaces irrespective of curvature, but the curvature significantly alters F-actin dynamics. Furthermore, manipulating cytoskeletal dynamics via selective actin inhibition partially reversed curvature-induced changes in macrophage phenotype. These results underscore the pivotal role of substrate curvature in modulating macrophage behavior and immunophenotype. Overall, our study demonstrates that substrate curvature significantly influences macrophage cytoskeletal dynamics and resulting immunophenotype. This simple approach can be utilized as a localized immunomodulatory treatment for inflammatory diseases.

Modeling 3D Tumor Invasiveness to Modulate Macrophage Phenotype in a Human-Based Hydrogel Platform.

The immune system is a pivotal player in determining tumor fate, contributing to the immunosuppressive microenvironment that supports tumor progression. Considering the emergence of biomaterials as promising platforms to mimic the tumor microenvironment, human platelet lysate (PLMA)-based hydrogel beads are proposed as 3D platforms to recapitulate the tumor milieu and recreate the synergistic tumor-macrophage communication. Having characterized the biomaterial-mediated pro-regenerative macrophage phenotype, an osteosarcoma spheroid encapsulated into a PLMA hydrogel bead is explored to study macrophage immunomodulation through paracrine signaling. The culture of PLMA-Tumor beads on the top of a 2D monolayer of macrophages reveals that tumor cells triggered morphologic and metabolic adaptations in macrophages. The cytokine profile, coupled with the upregulation of gene and protein anti-inflammatory biomarkers clearly indicates macrophage polarization toward an M2-like phenotype. Moreover, the increased gene expression of chemokines identified as pro-tumoral environmental regulators suggest a tumor-associated macrophage phenotype, exclusively stimulated by tumor cells. This pro-tumoral microenvironment is also found to enhance tumor invasiveness ability and proliferation. Besides providing a robust in vitro immunomodulatory tumor model that faithfully recreates the tumor-macrophage interplay, this human-based platform has the potential to provide fundamental insights into immunosuppressive signaling and predict immune-targeted response.

Sustained Release of Hydrogen and Magnesium Ions Mediated by a Foamed Gelatin-Methacryloyl Hydrogel for the Repair of Bone Defects in Diabetes.

Diabetic bone defects, exacerbated by hyperglycemia-induced inflammation and oxidative stress, present significant therapeutic challenges. This study introduces a novel injectable scaffold, MgH2@PLGA/F-GM, consisting of foamed gelatin-methacryloyl (GelMA) and magnesium hydride (MgH2) microspheres encapsulated in poly(lactic-co-glycolic acid) (PLGA). This scaffold is uniquely suited for diabetic bone defects, conforming to complex shapes and fostering an environment conducive to tissue regeneration. As it degrades, Mg(OH)2 is released and dissolved by PLGA's acidic byproducts, releasing therapeutic Mg2+ ions. These ions are instrumental in macrophage phenotype modulation, inflammation reduction, and angiogenesis promotion, all vital for diabetic bone healing. Additionally, hydrogen (H2) released during degradation mitigates oxidative stress by diminishing reactive oxygen species (ROS). This multifaceted approach not only reduces ROS and inflammation but also enhances M2 macrophage polarization and cell migration, culminating in improved angiogenesis and bone repair. This scaffold presents an innovative strategy for addressing the complexities of diabetic bone defect treatment.

Extracellular vesicles from dental pulp mesenchymal stem cells modulate macrophage phenotype during acute and chronic cardiac inflammation in athymic nude rats with myocardial infarction

Background/aimsExtracellular vesicles (EVs) derived from dental pulp mesenchymal stem cells (DP-MSCs) are a promising therapeutic option for the treatment of myocardial ischemia. The aim of this study is to determine whether MSC-EVs could promote a pro-resolving environment in the heart by modulating macrophage populations.MethodsEVs derived from three independent biopsies of DP-MSCs (MSC-EVs) were isolated by tangential flow-filtration and size exclusion chromatography and were characterized by omics analyses. Biological processes associated with these molecules were analyzed using String and GeneCodis platforms. The immunomodulatory capacity of MSC-EVs to polarize macrophages towards a pro-resolving or M2-like phenotype was assessed by evaluating surface markers, cytokine production, and efferocytosis. The therapeutic potential of MSC-EVs was evaluated in an acute myocardial infarction (AMI) model in nude rats. Infarct size and the distribution of macrophage populations in the infarct area were evaluated 7 and 21 days after intramyocardial injection of MSC-EVs.ResultsLipidomic, proteomic, and miRNA-seq analysis of MSC-EVs revealed their association with biological processes involved in tissue regeneration and regulation of the immune system, among others. MSC-EVs promoted the differentiation of pro-inflammatory macrophages towards a pro-resolving phenotype, as evidenced by increased expression of M2 markers and decreased secretion of pro-inflammatory cytokines. Administration of MSC-EVs in rats with AMI limited the extent of the infarcted area at 7 and 21 days post-infarction. MSC-EV treatment also reduced the number of pro-inflammatory macrophages within the infarct area, promoting the resolution of inflammation.ConclusionEVs derived from DP-MSCs exhibited similar characteristics at the omics level irrespective of the biopsy from which they were derived. All MSC-EVs exerted effective pro-resolving responses in a rat model of AMI, indicating their potential as therapeutic agents for the treatment of inflammation associated with AMI.

Modulating macrophage phenotype for accelerated wound healing with chlorogenic acid-loaded nanocomposite hydrogel

Wound healing involves distinct phases, including hemostasis, inflammation, proliferation, and remodeling, which is a complex and dynamic process. Conventional preparations often fail to meet multiple demands and provide prompt information about wound status. Here, a pH/ROS dual-responsive hydrogel (OHA-PP@Z-CA@EGF) was constructed based on oxidized hyaluronic acid (OHA), phenylboronic acid-grafted ε-polylysine (PP), chlorogenic acid (CA)-loaded ZIF-8 (Z-CA), and epidermal growth factor (EGF), which possesses intrinsic antibacterial, antioxidant, and angiogenic capacities. Due to the Schiff base and Phenylboronate ester bonds, the hydrogel exhibited excellent mechanical properties, strong adhesion, good biodegradability, high biocompatibility, stable rheological properties, and self-healing ability. Moreover, introducing Z-CA as an initiator and nanofiller led to the additional cross-linking of hydrogel through coordination bonds, which further improved the mechanical properties and antioxidant capabilities. Bleeding models of liver and tail amputations demonstrated rapid hemostatic properties of the hydrogel. Besides, the hydrogel regulated macrophage phenotypes via the NF-κB/JAK-STAT pathways, relieved oxidative stress, promoted cell migration and angiogenesis, and accelerated diabetic wound healing. The hydrogel also enabled real-time monitoring of the wound healing stages by colorimetric detection. This multifunctional hydrogel opens new avenues for the treatment and management of full-thickness diabetic wounds.

Effects of a Bioengineered Allogeneic Cellularized Construct (BACC) on Primary Human Macrophage Phenotype.

The mechanisms behind the pro-healing effects of multicellular, bioengineered allogeneic cellularized constructs (BACC) are not known. Macrophages are key regulators of every phase of the wound healing process and the primary cells that mediate the response to biomaterials. It is hypothesized that cells within the BACC modulate macrophage behavior, which may contribute to the mechanism by which BACC promotes healing. To probe the influence of cells within the BACC compared to effects of the underlying collagen substrate, primary human macrophages are cultured in direct or indirect contact with BACC or with the same collagen substrate used in the BACC manufacturing. Macrophage phenotype is characterized over time via multiplex gene expression, protein secretion, multidimensional flow cytometry, and functional assays with fibroblasts and endothelial cells. The BACC causes macrophages to exhibit a predominately reparative phenotype over time compared to relevant collagen substrate controls, with multiple subpopulations expressing both pro-inflammatory and reparative markers. Conditioned media from macrophage-BACC co-cultures causes distinct effects on fibroblast and endothelial cell proliferation, migration, and network formation. Given the critical role of the reparative macrophage phenotype in wound healing, these results suggest that modulation of macrophage phenotype may be a critical part of the mechanisms behind BACC's pro-healing effects.

Amniotic extracelluar matrix microgels for attenuation of radiation-induced lung injury

Radiation-induced lung injury (RILI) is one of the major complications in patients exposed to accidental radiation and radiotherapy for thoracic malignancies. However, there is no reliable approach to effectively treat RILI. With favorable biological and physical characteristics, decellularized extracellular matrix (dECM) from amniotic membrane can not only act as a reactive oxygen species scavenger to attenuate the damage of oxidative stress, but also release bio-factors to polarize the macrophages from M1-like phenotype to M2-like phenotype, thus switching the microenvironment from pro-inflammation status to pro-healing status. Meanwhile, as a promising drug delivery system, microgels with a small size and high surface/volume ratio can not only be inhaled into lung tissues with a desired concentration, but also preserve the structural integrity and the functionalities of the contained therapeutics. Therefore, it will be a potent strategy to combine microgels and amniotic dECM for the treatment of RILI, in which the inhaled microgels show a high surface area to rapidly remove reactive oxygen species and release cargos to reduce the inflammation-mediated damage by modulating macrophage phenotypes. We hypothesize that amniotic dECM microgels will be a promising and effective biomaterial for the attenuation of RILI.

Extracellular vesicles derived from Lactobacillus johnsonii promote gut barrier homeostasis by enhancing M2 macrophage polarization

IntroductionDiarrheic disease is a common intestinal health problem worldwide, causing great suffering to humans and animals. Precise manipulation strategies based on probiotics to combat diarrheic diseases have not been fully developed. ObjectivesThe aim of this study was to investigate the molecular mechanisms by which probiotics manipulate macrophage against diarrheic disease. MethodsMetagenome reveals gut microbiome profiles of healthy and diarrheic piglets. Fecal microbial transplantation (FMT) was employed to explore the causal relationship between gut microbes and diarrhea. The protective role of probiotics and their derived extracellular vesicles (EVs) was investigated in ETEC K88-infected mice. Macrophage depletion was performed to assess the role of macrophages in EVs against diarrhea. Execution of in vitro cell co-culture and transcriptome analyses elucidated the molecular mechanisms by which EVs modulate the macrophage and intestinal epithelial barrier. ResultsEscherichia coli was enriched in weaned diarrheic piglets, while Lactobacillus johnsonii (L. john) showed a negative correlation with Escherichia coli. The transmission of diarrheic illness symptoms was achieved by transferring fecal microbiota, but not metabolites, from diarrheic pigs to germ-free (GF) mice. L. john's intervention prevented the transmission of disease phenotypes from diarrheic piglets to GF mice. L. john also reduces the gut inflammation induced by ETEC K88. The EVs secreted by L. john demonstrated enhanced efficacy in mitigating the adverse impacts induced by ETEC K88 through the modulation of macrophage phenotype. In vitro experiments have revealed that EVs activate M2 macrophages in a manner that shuts down ERK, thereby inhibiting NLRP3 activation in intestinal epithelial cells. ConclusionOur results reveal that intestinal microbiota drives the onset of diarrheic disease and that probiotic-derived EVs ameliorate diarrheic disease symptoms by modulating macrophage phenotypes. These findings can enhance the advancement of innovative therapeutic approaches for diarrheic conditions based on probiotic-derived EVs.

Identification of a novel, MSC-induced macrophage subtype via single-cell sequencing: implications for intervertebral disc degeneration therapy.

Intervertebral disc (IVD) degeneration is a common pathological condition associated with low back pain. Recent evidence suggests that mesenchymal signaling cells (MSCs) promote IVD regeneration, but underlying mechanisms remain poorly defined. One postulated mechanism is via modulation of macrophage phenotypes. In this manuscript, we tested the hypothesis that MSCs produce trophic factors that alter macrophage subsets. To this end, we collected conditioned medium from human, bone marrow-derived STRO3+ MSCs. We then cultured human bone marrow-derived macrophages in MSC conditioned medium (CM) and performed single cell RNA-sequencing. Comparative analyses between macrophages cultured in hypoxic and normoxic MSC CM showed large overlap between macrophage subsets; however, we identified a unique hypoxic MSC CM-induced macrophage cluster. To determine if factors from MSC CM simulated effects of the anti-inflammatory cytokine IL-4, we integrated the data from macrophages cultured in hypoxic MSC CM with and without IL-4 addition. Integration of these data sets showed considerable overlap, demonstrating that hypoxic MSC CM simulates the effects of IL-4. Interestingly, macrophages cultured in normoxic MSC CM in the absence of IL-4 did not significantly contribute to the unique cluster within our comparison analyses and showed differential TGF-β signaling; thus, normoxic conditions did not approximate IL-4. In addition, TGF-β neutralization partially limited the effects of MSC CM. In conclusion, our study identified a unique macrophage subset induced by MSCs within hypoxic conditions and supports that MSCs alter macrophage phenotypes through TGF-β-dependent mechanisms.

Inflammation environment-adaptive matrix confinement for three-dimensional modulation of macrophages.

The balance of macrophages in immune reactions is crucial for tissue repair. Despite some studies on responsive surfaces for immunomodulation regulation of macrophage phenotypes via external stimuli, 2D and manual interventions are limited. Herein, to address these limitations, we developed an inflammation environment-responsive macrophage-laden hydrogel-filled scaffold for investigating the impact of matrix confinement on macrophage phenotypes adaptively. We fabricated gelatin scaffolds with a controllable pore size and found that macrophages within smaller pores tended to have an anti-inflammation phenotype. We prepared poly(vinyl alcohol) (PVA)-based hydrogels crosslinked with phenylboronic acid (PBA)-based linkers. The hydrogels possessed shear-thinning, cell-loading, and ROS-sensitive-degradation abilities. Subsequently, a macrophage-laden hydrogel-filled scaffold was fabricated by filling the hydrogels into the porous scaffold under vacuum. With the degradation of the hydrogels under the overexpression of ROS in an inflammation environment, the macrophages were transferred from a state with strong matrix confinement to that with a weaker one. Meanwhile, with the change in matrix confinement, the macrophages upregulated the expressions of Arg-1 and IL-10 and downregulated the expressions of IL-1β, TNF-α, and IL-6, indicating polarization toward the anti-inflammatory phenotype. The inflammation environment-adaptive modulation of macrophage phenotypes in 3D provides a smart and biomimetic strategy for immunomodulation and regenerative medicine.

The potential role of hydrogen sulfide in regulating macrophage phenotypic changes via PINK1/parkin-mediated mitophagy in sepsis-related cardiorenal syndrome

Objective Sepsis is one of major reasons of cardiorenal syndrome type 5 (CRS-5), resulting in irreversible tissue damage and organ dysfunction. Macrophage has been demonstrated to play key role in the pathophysiology of sepsis, highlighting the need to identify therapeutic targets for modulating macrophage phenotype in sepsis. Methods and Results In this study, a rapid-releasing hydrogen sulfide (H2S) donor NaSH, and a slow-releasing H2S compound S-propargyl-cysteine (SPRC) which is derived from garlic, have been studied for the immune-regulatory effects on macrophages. The NaSH and SPRC showed the potential to protect the heart and kidney from tissue injury induced by LPS. The immunohistochemistry of F4/80+ revealed that the infiltration of macrophages in the heart and kidney tissues of LPS-treated mice was reduced by NaSH and SPRC. In addition, in the LPS-triggered inflammatory cascade of RAW264.7 macrophage cells, NaSH and SPRC exhibited significantly inhibitory effects on the secretion of inflammatory cytokines, production of reactive oxygen species (ROS), and regulation of the macrophage phenotype from M1-like to M2-like. Moreover, autophagy, a crucial process involved in the elimination of impaired proteins and organelles during oxidative stress and immune response, was induced by NaSH and SPRC in the presence of LPS stimulation. Consequently, there was an increase in the number of mitochondria and an improvement in mitochondrial membrane potential. This process was mainly mediated by PINK1/Parkin pathway mediated mitophagy. Discussion These results demonstrated that the immunoregulatory effects of H2S donors were through the PINK1/Parkin-mediated mitophagy pathway. Overall, our study provided a new therapeutic direction in LPS-induced cardiorenal injury.

Extracellular vesicles derived from endothelial cells modulate macrophage phenotype in vitro

Extracellular vesicles (EVs) mediate cell-to-cell communication by horizontally transferring biological materials from host cells to target cells. During exposure to pathogens, pathogen-associated molecular patterns (e.g., lipopolysaccharide, LPS) get in contact with endothelial cells and stimulate the secretion of endothelial cell-derived EVs (E-EVs). The triggered EVs secretion is known to have a modulating influence on the EVs-receiving cells. Macrophages, a major component of innate immunity, are polarized upon receiving external inflammatory stimuli, in which toll-like receptor4 (TLR4)—nuclear factor kappa B (NFκB) pathway plays a key role. However, the functions of LPS-induced E-EVs (ELPS-EVs) in modulating macrophage phenotype and activation remain elusive. We collected the EVs from quiescent endothelial cells (ENor-EVs) and ELPS-EVs to detect their stimulatory role on NR8383 macrophages. Isolated EVs were characterized by transmission electron microscopy (TEM), western blot assay, and nanoparticle tracking analysis (NTA). NR8383 macrophages were stimulated with ELPS-EVs, ENor-EVs, or PBS for 24 h. Hereafter, the uptake of EVs by the macrophages was investigated. Upon EVs stimulation, cellular viability was determined by MTT assay, while macrophage phenotype was analyzed by flow cytometry and immunofluorescence analysis. Furthermore, a western blot assay was conducted to evaluate the potentially involved TLR4–NFκB pathway. Interestingly, upon exposure to LPS, endothelial cells secreted significantly higher amounts of EVs (i.e., ELPS-EVs) when compared to quiescent cells or cells in PBS. The ELPS-EVs were also better internalized by NR8383 macrophages than ENor-EVs. The cellular viability of ELPS-EVs-treated macrophages was 1.2 times higher than those in the ENor-EVs and PBS groups. In addition, ELPS-EVs modulated NR8383 macrophages towards a proinflammatory macrophage M1-like phenotype. This was indicated by the significantly upregulated expressions of proinflammatory macrophage biomarkers CD86 and inducible nitric oxide synthase (iNOS) observed in ELPS-EVs-treated macrophages. The TLR4–NFκB signaling pathway was substantially activated in ELPS-EVs-treated macrophages, indicated by the elevated expressions of makers TLR4 and phosphorylated form of nuclear factor kappa B p65 subunit (p-NFκBp65). Overall, our results indicate that E-EVs play a crucial role in macrophage phenotype modulation under inflammatory conditions.

Abstract 17622: CCN3 Deficiency in Myeloid Cells Exacerbates Aortic Dissection by Governing a Proinflammatory Program in Macrophages

Introduction: Aortic dissection (AD) is a fatal disease. Macrophages are known central regulators of aortic inflammation and elastic fiber disruption, two major hallmarks of AD. Cellular communication network factor 3 (CCN3/NOV) is a matricellular protein associated with various biological cellular functions in cardiovascular disease and cancers. Emerging evidence strongly implicates a protective role of CCN3 against several types of vascular pathologies, however its role in AD remains unknown. Hypothesis: CCN3 deficiency in myeloid cells promotes AD development in murine models through the modulation of macrophage phenotype. Aims: We investigated the causal role of CCN3 loss in AD pathogenesis, focusing on its role in macrophages. Methods: 3-4-week-old male myeloid-specific CCN3 knockout mice (CCN3F/F;LysMCre) and control mice (LysMCre) were given BAPN (β-aminopropionitrile monofumarate; 0.2%) in their drinking water for 4 weeks for AD modeling. Mouse survival rate and histological changes of the aorta were assessed. Single cell RNA sequencing analysis was performed to recapitulate cellular transcriptome profile changes. Finally, gain and loss of function studies in bone marrow-derived macrophages (BMDMs) were performed to decipher the underlying mechanism by which CCN3 controls macrophage plasticity. Results: Following BAPN administration, myeloid-specific CCN3 knockout mice had a higher mortality rate due to AD formation and aortic rupture. In accordance with this phenotype, vascular media thickness was markedly reduced, and elastic fiber fragmentation and macrophage infiltration were elevated in myeloid-specific CCN3 knockout mice. Using single-cell RNA-seq, we found that myeloid-deficiency of CCN3 was associated with a pathogenic pro-inflammatory phenotype of perivascular tissue-resident macrophages, which promoted deleterious aortic wall remodeling at early stages. Furthermore, CCN3 deficient BMDMs are prone to M1 macrophage polarization, an effect dependent on the activation of the JAK/STAT1 signaling pathway. Conclusions: Myeloid-deficiency of CCN3 drives vascular macrophages toward an M1-like, pathogenic phenotype and promotes dissecting aortic aneurysm.