Macrophage Supernatants Research Articles

Loss of IDH1 and IDH2 in macrophages induces pathological phenotypes in cardiomyocytes

Abstract Introduction The clonal expansion of hematopoietic stem cells can be induced by a variety of age-related somatic mutations, a phenomenon known as "clonal hematopoiesis of indeterminate potential" (CHIP). CHIP is associated with an increased incidence and poor outcomes in patients with cardiovascular disease. The mechanisms underlying the crosstalk between CHIP cells and cardiac tissue are yet to be elucidated. Rare mutations in the metabolism-regulating isocitrate dehydrogenases encoding genes IDH1 and IDH2 have been identified as AML-like and cardiovascular high-risk CHIP-driving mutations in patient cohorts. However, the potential paracrine effects of these mutations in immune cells on cardiac tissue are unknown. Therefore, we assessed the impact of IDH1 and IDH2 mutations on the paracrine activities of macrophages. Methods and Results To gain first insights into the functional consequences of the IDH mutations, we used the AlphaMissense algorithm, which predicts the impact of mutations on protein function. IDH1 and IDH2 mutations identified in our patient cohorts were predicted to induce pathological alterations of protein function with a score >0.99. Therefore, we assessed the effects of silencing of the two genes in primary human CD14+ M0 macrophages on the paracrine activity on neonatal rat cardiomyocytes. Transfection with siRNA pools targeting IDH1 and IDH2 to mimic CHIP-driving conditions reduced mRNA abundance by 78% and 73%, respectively (both p<0.05). To assess the impact of IDH-silencing on cardiovascular cells, cardiomyocytes were treated with the supernatant of the macrophages. Interestingly, supernatants of IDH1 silenced macrophages significantly increased apoptosis of cardiomyocytes (8.6-fold increased expression of cleaved caspase-3; p<0.05) and reduced beating frequency. In addition, supernatants of IDH1 silenced macrophages induced a 1.5-fold increase in cardiomyocyte hypertrophy (p<0.01), whereas silencing of IDH2 showed no effect on cardiomyocyte hypertrophy and apoptosis. However, supernatants of both IDH1 and IDH2 silenced macrophages resulted in significantly increased numbers of bi- and multinucleated cardiomyocytes. Conclusion: Loss of isocitrate dehydrogenases in human macrophages affects cardiomyocyte functions in a paracrine fashion. Particularly silencing of IDH1 in macrophages introduced cardiomyocyte hypertrophy, apoptosis, and bi- and multinucleation, and reduced contractile function. Ongoing studies will address how IDH1 and IDH2 silencing-induced alterations in macrophages control their paracrine activity on cardiomyocytes.

Porphyromonas gingivalis Induces Chronic Kidney Disease through Crosstalk between the NF-κB/NLRP3 Pathway and Ferroptosis in GMCs.

Porphyromonas gingivalis (P.gingivalis) is a gram-negative bacterium found in the human oral cavity and is a recognized pathogenic bacterium associated with chronic periodontitis and systemic diseases, including chronic kidney disease (CKD), but the roles and molecular mechanism of P.gingivalis in CKD pathogenesis are unclear. In this study, an animal model of oral P.gingivalis administration and glomerular mesangial cells (GMCs) cocultured with M1-polarized macrophages and P.gingivalis supernatant were constructed. After seven weeks of P.gingivalis gavaged, peripheral blood was collected to detect the changes in renal function. By collecting the teeth and kidneys of mice, H&E staining and IHC were used to analyze the expression of periodontal inflammatory factors in mice, PAS staining was used to analyze glomerular lesions. The supernatant of macrophages was treated with 5% P.gingivalis supernatant. H&E staining, IHC, Western blot and RT-PCR were applied to analyze renal inflammatory factors, macrophage M1 polarization, NF-κB, NLRP3 and ferroptosis changes in vitro. We found that oral P.gingivalis administration induced CKD in mice. P.gingivalis supernatant induced macrophage polarization and inflammatory factor upregulation, which triggered the activation of the NF-κB/NLRP3 pathway and ferroptosis in GMCs. By inhibiting the NF-κB/NLRP3 pathway and ferroptosis in GMCs, cell viability and the inflammatory response were partially alleviated in vitro. We demonstrated that P.gingivalis induced CKD in mice by triggering crosstalk between the NF κB/NLRP3 pathway and ferroptosis in GMCs. Overall, our study suggested that periodontitis can promote the pathogenesis of CKD in mice, which provides evidence of the importance of periodontitis therapy in the prevention and treatment of CKD. P.gingivalis promotes ferroptosis in kidneys and accelerates the progression of CKD through NF-κB/NLRP3 signaling pathway.

Abstract B001: Macrophage adipocyte crosstalk as a node of intervention for pancreatic cancer-associated weight loss

Abstract Introduction: Up to 85% of patients with pancreatic ductal adenocarcinoma (PDAC) are afflicted with cachexia - a metabolic syndrome characterized by loss of skeletal muscle mass and adipose tissue wasting. Cachexia worsens patients’ quality of life, their ability to tolerate chemotherapy or surgical procedures, and, ultimately, their survival. Existing therapies often fail as the disease remains insufficiently understood. Drawing insights from obesity research, where changes in the local adipose immune environment play a crucial role in the pathophysiology, we explore the adipose tissue in the context of PDAC cachexia at single-cell resolution to uncover novel therapeutic avenues. Methods: We developed an ex vivo co-culture system to examine the effect of macrophages on adipocyte lipolysis by measuring the amount of released glycerol from adipocytes following incubation with macrophage supernatant. This robust platform can be used to interrogate factors directly affecting glycerol release from adipocytes. To identify candidate mediators of macrophage-adipocyte crosstalk, we profiled murine and human adipose tissue by flow cytometry and single-cell/nucleus RNA sequencing. The tissue samples were obtained from a cohort of genetically engineered KPC mice and a representative group of surgical patients. Patient adipose samples were collected intraoperatively without warm ischemic time at Heidelberg University Hospital. The cohort includes over 60 patients, divided into three groups: (1) PDAC with weight loss, (2) PDAC without weight loss, and (3) control patients without a history of malignancy. Of these, 25 samples are being used for single-nucleus RNA sequencing. The groups are balanced by sex and can be stratified into equally sized subgroups based on whether they had received neoadjuvant treatment. Results: Macrophage- conditioned media significantly elevated lipid release from murine adipocytes, demonstrating that macrophages may play a role in cancer-associated adipose wasting. Single-nucleus RNA sequencing data from our mice and from our human clinical cohort reveals distinct cell clusters with condition-specific subpopulations and differences unique to cancer-associated weight loss. The biggest phenotypic shifts were observed in macrophages and in adipocytes which showed signs of oxidative stress and altered metabolism associated with cachexia. Conclusion: We successfully established reliable workflows for processing and investigating murine and human AT in the context of PDAC cachexia. Our in vitro model highlights a distinct phenotype of murine macrophage supernatant inducing increased glycerol release from adipocytes. The in-depth analysis of our single-nucleus RNA sequencing data from our clinical cohort promises an unprecedentedly detailed and robust understanding of the human adipose immune environment, thereby enhancing our insights into PDAC cachexia. Citation Format:Felix P. Hambitzer, Farhad Faghihi, Brendan D. Parent, Michael Walsh, Susanne Roth, Andrew Aguirre, Christoph W. Michalski, Martin Loos, Stephanie K. Dougan, Evanna Mills, Max Heckler. Macrophage adipocyte crosstalk as a node of intervention for pancreatic cancer-associated weight loss [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B001.

Excretory/secretory antigens from Trichinella spiralis muscle larvae ameliorate HFD-induced non-alcoholic steatohepatitis via driving macrophage anti-inflammatory activity

No approved effective therapy for non-alcoholic steatohepatitis (NASH) is currently available. Trichinella spiralis (T. spiralis) infection and their products have positive impact on several metabolic diseases. Considering, we firstly investigated the effects of the T. spiralis-derived Excretory-Secretory antigens (ESA) on high fat diet (HFD)-induced NASH mouse models. To further elucidate the mechanism of action, HepG2 cells were incubated with palmitic acid (PA) to construct NASH-like cell model, and then the culture medium supernatant collected from ESA-treated macrophages was applied to intervene the cell model in vitro. In NASH mouse models, ESA significantly alleviated hepatic steatosis and hepatic inflammation, as reflected by reducing pro-inflammatory cytokines and inactivating TLR4/MYD88/NF-κB pathway and NLRP3 inflammasome. Meanwhile, the HFD-induced oxidative stress was restored by ESA through lessening the level of MDA, increasing the activity of T-SOD and enhancing Nrf2 signaling-related proteins, including p-Nrf2, NQO1, HO-1, GPX4, and p-AMPK. Notably, ESA preferentially promoted macrophages polarization toward M2 anti-inflammatory phenotype in vivo and vitro. Moreover, in vitro, intervention of PA-treated HepG2 cells with medium supernatant of ESA-treated macrophages attenuated lipid accumulation, inflammation, as well as oxidative stress. In conclusion, T. spiralis-derived ESA may serve as a novel promising candidate for the treatment of NASH via its properties of driving macrophage anti-inflammatory activity.

Potential active fractions and constituents in the flowers of Trollius chinensis Bunge responsible for treating acute pharyngitis

Ethnopharmacological relevanceTrollius chinensis Bunge has a long history of use in China as traditional Chinese medicine and functional tea for the treatment of respiratory infections, such as pharyngitis, tonsillitis and bronchitis. Pharyngitis can impact the entire throat and adjacent lymphoid tissues, and may lead to significant systemic complications. However, the active components and mechanism of Trollius chinensis Bunge for treating acute pharyngitis remains unclear. Aim of the studyTrollius chinensis Bunge is recognized in China both as a medicinal herb and a functional tea. Research into its properties aimed to establish its effectiveness against pharyngitis and to pinpoint the active components and mechanism. Materials and methodsA 70% ethanol extract from the herb was prepared, which was refined using chromatography through a column containing D101 macroporous resin and varying ethanol solutions. The efficacy of the initial and refined extracts was tested using a rat model of ammonia-induced acute pharyngitis. Pathological examination, HE staining and ELISA were applied to screen activity fraction. The compounds were isolated by silica gel, sephadex LH-20 column chromatography and semi-preparative HPLC chromatography from active fraction. All of the isolated compounds were assessed for anti-inflammatory activity by acting on LPS-induced RAW 264.7 macrophage cells in vitro. Cytotoxicity of compounds was detected by CCK-8 assay. The Griess reaction was applied to evaluate the inhibitory effects of isolated compounds on NO production in RAW 264.7 cells induced by LPS. TNF-α, IL-6, IL-1β and PGE2 levels in macrophage supernatant were detected by ELISA. Molecular docking and western blot analysis were applied to study the anti-inflammatory mechanism of active compound. ResultsThe fraction extracted with 30% ethanol proved particularly effective, significantly reducing pharyngitis symptoms. This was evidenced by decreased levels of cytokines (TNF-α, IL-6, IL-1β and PGE2) and visible improvements in the pharyngeal tissue histology. In pursuit of pharyngitis treatments, 23 phenolic acids and 13 flavonoids were isolated from the 30% ethanol fraction and identified using spectral analysis. Of these, three were newly discovered compounds and eight were first-time isolates from the Trollius genus. These compounds were further investigated for their ability to suppress nitric oxide production in RAW 264.7 cells triggered by lipopolysaccharide. Compounds 3, 19, and 26 exhibited strong anti-inflammatory properties. HPLC analysis of the 30% ethanol fraction revealed that orientin was the predominant component, accounting for 44.4% of this fraction. Western blot analysis demonstrated that orientin reduced the expression levels of the protein p-p65 relative to p65, p-IκBα relative to IκBα and iNOS, indicating an anti-inflammatory effect potentially through the modulation of the NF-κB signaling pathway. ConclusionThe finding of this study provided strong support for the use of T. chinensis as a potential functional food for treating pharyngitis.

The role of progranulin in macrophages of a glioblastoma model.

Glioblastoma (GBM), characterized by astrocytic tumorigenesis, remains one of the most prognostically challenging tumor types. Targeting entire GBM microenvironment using novel therapeutic factors is currently desired investigation approach. In this study, we focused on progranulin (PGRN), a regulator of diverse cellular functions. Recent studies implicated PGRN in the poor prognostics of GBM patients. However, the specific role of PGRN in the GBM microenvironment remains elusive. We utilized public databases of GBM patient and previous single-cell RNA sequence to examine association between PGRN expression and patient survival/grade, and expression levels of PGRN in each cell constituting the tumor microenvironment. To clarify the role of PGRN in Tumor-associated macrophage (TAM), we examined cell proliferation and expression of some proteins in murine GBM cells when cell supernatants derived from TAM of PGRN knockout (Grn-/-) or wild type mice were treated with murine GBM cells. Our results reveal significant PGRN expression in macrophages within the GBM environment, suggesting an association between increased PGRN expression in macrophages and tumor malignancy. TAM induction led to PGRN expression enhancement. Treatment with Grn-/- mouse -derived bone marrow-derived macrophage (BMDM) supernatant resulted in diminished GBM cell proliferation and cell cycle- and mesenchymal GBM subtype-associated reduced protein expression. Furthermore, the Grn-/- mouse-derived BMDM supernatant treatment reduced the phosphorylated STAT3 expression in GBM cells, while the expression of IL-6 and IL-10, known STAT3 pathway activators, diminished in Grn-/- mouse-derived BMDMs. Our results suggest that macrophage-derived PGRN is pivotal for fostering malignant transformations within the tumor microenvironment.

In vitro and In vivo Activity of a New N-Oxide Derivative for Acne Vulgaris Treatment.

Furoxan and benzofuroxan are compounds containing an N-oxide function, known for their diverse pharmacological properties, including antimicrobial and antiinflammatory effects. This study aimed to investigate these activities using an in-house library of N-oxide compounds. Twenty compounds were tested against both Gram-positive and Gram-negative bacteria, including Cutibacterium acnes (C. acnes), a microorganism implicated in the development of acne vulgaris. One compound, (E)-4-(3-((2-(3-hydroxybenzoyl)hydrazone)methyl)phenoxy)-3- (phenylsulfonyl)-1,2,5-oxadiazol-2-N-oxide (compound 15), exhibited selective antimicrobial activity against C. acnes, with a Minimum Inhibitory Concentration (MIC) value of 2 μg/mL. Indirect measurement of Nitric Oxide (NO) release showed that compound 15 and isosorbide dinitrate, when treated with L-cysteine, produced nitrite levels of 20.1% and 9.95%, respectively. Using a NO scavenger (PTIO) in combination with compound 15 in a culture of C. acnes resulted in reduced antimicrobial activity, indicating that NO release is part of its mechanism of action. Cytotoxicity assessments using murine macrophages showed cellular viability above 70% at concentrations up to 0.78 μg/mL. Measurements of Interleukin-1 beta (IL1-β) and Tumor Necrosis Factor-alpha (TNF-α) indicated that compound 15 did not reduce the levels of these pro-inflammatory cytokines. Sustained NO production by inducible Nitric Oxide Synthase (iNOS) in macrophages or neutrophils has been found to be involved in the inflammatory process in acne vulgaris and lead to toxicity in surrounding tissues. Nitrite levels in the supernatant of murine macrophages were found to be decreased at a concentration of 0.78 μg/mL of compound 15, indicating an anti-inflammatory effect. In vivo studies were conducted using Balb/c nude mice inoculated subcutaneously with C. acnes. Cream and gel formulations of compound 15 were applied to treat the animals, along with commercially available anti-acne drugs, for 14 days. Animals treated with a cream base containing 5% of compound 15 exhibited less acanthosis with mild inflammatory infiltration compared to other groups, highlighting its anti-inflammatory properties. Similar results were observed in the benzoyl peroxide group, demonstrating that compound 15 presented comparable anti-inflammatory activity to the FDA-approved drug. These promising results suggest that compound 15 has a dual mechanism of action, with selective antimicrobial activity against C. acnes and notable anti-inflammatory properties, making it a potential prototype for developing new treatments for acne vulgaris.

Zhiqiao Gancao decoction regulated JAK2/STAT3/ macrophage M1 polarization to ameliorate intervertebral disc degeneration

BackgroundZhiqiao Gancao decoction (ZQGCD) was created by Professor Gong Zhengfeng, a renowned Chinese medicine expert. Clinical studies have shown its efficacy in alleviating pain and enhancing lumbar function in intervertebral disc degeneration (IDD) patients. However, the precise mechanism of ZQGCD in treating IDD remains unclear. MethodsThe active components of ZQGCD were identified using Liquid chromatography-tandem mass spectrometry (LC-MS/MS). A rat model of intervertebral disc degeneration was established, and rats in each group received ZQGCD for three weeks. Assessment parameters included hyperalgesia status, observation of intervertebral disc tissue degeneration and macrophage infiltration, and analysis of JAK2/STAT3 pathway protein expression in the intervertebral disc. Primary macrophage M1 polarization was induced using LPS, with cells treated using the JAK2 inhibitor (AZD1480) and ZQGCD to evaluate macrophage polarization, cellular supernatant inflammatory factors, and JAK2/STAT3 pathway expression. Macrophage supernatant served as a conditioned medium to observe its effects on the proliferation of nucleus pulposus cells (NPCs) and the expression of collagen II and MMP3 proteins. ResultsA total of 81 active components were identified in ZQGCD. Following ZQGCD treatment, infiltrating macrophages in intervertebral disc tissues of model rats decreased, the content of M1 macrophages decreased, while the content of M2 macrophages increased, the expression of proinflammatory factors and pain-inducing factors in serum decreased, and the expression of substance P in intervertebral disc tissue decreased. Consequently, the intervertebral disc degeneration and hyperalgesia of rats were improved. In vitro studies revealed that LPS induced M1 macrophage polarization. By inhibiting the JAK2/STAT3 pathway, both JAK2 inhibitors and ZQGCD effectively suppressed M1 polarization, resulting in decreased levels of IL-1β, IL-6, TNF-α, and various other inflammatory factors. Consequently, this inhibition led to a delay in the degeneration of NPCs. ConclusionThere is macrophage infiltration in the intervertebral disc tissue of IDD rats, and JAK2/STAT3 pathway is activated, macrophages are polarized to M1 type, resulting in inflammatory microenvironment, leading to intervertebral disc degeneration and hyperalgesia. ZQGCD exhibited a delaying effect on IDD and improved hyperalgesia by inhibiting the JAK2/STAT3/macrophage M1 polarization pathway.

Unveiling the Cutting-Edge Impact of Polarized Macrophage-Derived Extracellular Vesicles and MiRNA Signatures on TGF-β Regulation within Lung Fibroblasts.

Depending on local cues, macrophages can polarize into classically activated (M1) or alternatively activated (M2) phenotypes. This study investigates the impact of polarized macrophage-derived Extracellular Vesicles (EVs) (M1 and M2) and their cargo of miRNA-19a-3p and miRNA-425-5p on TGF-β production in lung fibroblasts. EVs were isolated from supernatants of M0, M1, and M2 macrophages and quantified using nanoscale flow cytometry prior to fibroblast stimulation. The concentration of TGF-β in fibroblast supernatants was measured using ELISA assays. The expression levels of miRNA-19a-3p and miRNA-425-5p were assessed via TaqMan-qPCR. TGF-β production after stimulation with M0-derived EVs and with M1-derived EVs increased significantly compared to untreated fibroblasts. miRNA-425-5p, but not miRNA-19a-3p, was significantly upregulated in M2-derived EVs compared to M0- and M1-derived EVs. This study demonstrates that EVs derived from both M0 and M1 polarized macrophages induce the production of TGF-β in fibroblasts, with potential regulation by miRNA-425-5p.

Exosomes derived from periodontitis induce hepatic steatosis through the SCD-1/AMPK signaling pathway

AimTo investigate the impact of exosomes released by Porphyromonas gingivalis-Lipopolysaccharide activated THP-1 macrophages and human periodontal ligament fibroblasts on hepatocyte fat metabolism. ResultsThe liver of rats with experimental periodontitis showed obvious steatosis and inflammation compared with control rats. The culture supernatant of macrophages and human periodontal ligament fibroblasts (hPDLFs), when stimulated with Pg-LPS, induced lipogenesis in HepG2 cells. Furthermore, the lipid-promoting effect was effectively inhibited by the addition of the exosome inhibitor GW4869. Subsequently, we isolated exosomes from cells associated with periodontitis. Exosomes released by Pg-LPS-stimulated macrophages and hPDLFs are taken up by hepatocytes, causing mRNA expression related to fat synthesis, promoting triglyceride synthesis, and aggravating NAFLD progression. Finally, two sets of exosomes were injected into mice through the tail vein. In vivo experiments have also demonstrated that periodontitis-associated exosomes promote the development of hepatic injury and steatosis, upregulate SCD-1 expression and inhibit the AMPK signaling pathway. ConclusionsIn conclusion, we found that exosomes associated with periodontitis promote hepatocyte adipogenesis by increasing the expression of SCD-1 and suppressing the AMPK pathway, which indicates that close monitoring of the progression of stomatopathy associated extra-oral disorders is important and establishes a theoretical foundation for the prevention and management of fatty liver disease linked to periodontitis.

LPS-induced senescence of macrophages aggravates calcification and senescence of vascular smooth muscle cells via IFITM3

Background Cellular senescence, macrophages infiltration, and vascular smooth muscle cells (VSMCs) osteogenic transdifferentiation participate in the pathophysiology of vascular calcification in chronic kidney disease (CKD). Senescent macrophages are involved in the regulation of inflammation in pathological diseases. In addition, senescent cells spread senescence to neighboring cells via Interferon-induced transmembrane protein3 (IFITM3). However, the role of senescent macrophages and IFITM3 in VSMCs calcification remains unexplored. Aims To explore the hypothesis that senescent macrophages contribute to the calcification and senescence of VSMCs via IFITM3. Methods Here, the macrophage senescence model was established using Lipopolysaccharides (LPS). The VSMCs were subjected to supernatants from macrophages (MCFS) or LPS-induced macrophages (LPS-MCFS) in the presence or absence of calcifying media (CM). Senescence-associated β-galactosidase (SA-β-gal), Alizarin red (AR), immunofluorescent staining, and western blot were used to identify cell senescence and calcification. Results The expression of IFITM3 was significantly increased in LPS-induced macrophages and the supernatants. The VSMCs transdifferentiated into osteogenic phenotype, expressing higher osteogenic differentiation markers (RUNX2) and lower VSMCs constructive makers (SM22α) when cultured with senescent macrophages supernatants. Also, senescence markers (p16 and p21) in VSMCs were significantly increased by senescent macrophages supernatants treated. However, IFITM3 knockdown inhibited this process. Conclusions Our study showed that LPS-induced senescence of macrophages accelerated the calcification of VSMCs via IFITM3. These data provide a new perspective linking VC and aging, which may provide clues for diagnosing and treating accelerated vascular aging in patients with CKD.

Disruption of pulmonary microvascular endothelial barrier by dysregulated claudin-8 and claudin-4: uncovered mechanisms in porcine reproductive and respiratory syndrome virus infection

The pulmonary endothelium is a dynamic and metabolically active monolayer of endothelial cells. Dysfunction of the pulmonary endothelial barrier plays a crucial role in the acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), frequently observed in the context of viral pneumonia. Dysregulation of tight junction proteins can lead to the disruption of the endothelial barrier and subsequent leakage. Here, the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) served as an ideal model for studying ALI and ARDS. The alveolar lavage fluid of pigs infected with HP-PRRSV, and the supernatant of HP-PRRSV infected pulmonary alveolar macrophages were respectively collected to treat the pulmonary microvascular endothelial cells (PMVECs) in Transwell culture system to explore the mechanism of pulmonary microvascular endothelial barrier leakage caused by viral infection. Cytokine screening, addition and blocking experiments revealed that proinflammatory cytokines IL-1β and TNF-α, secreted by HP-PRRSV-infected macrophages, disrupt the pulmonary microvascular endothelial barrier by downregulating claudin-8 and upregulating claudin-4 synergistically. Additionally, three transcription factors interleukin enhancer binding factor 2 (ILF2), general transcription factor III C subunit 2 (GTF3C2), and thyroid hormone receptor-associated protein 3 (THRAP3), were identified to accumulate in the nucleus of PMVECs, regulating the transcription of claudin-8 and claudin-4. Meanwhile, the upregulation of ssc-miR-185 was found to suppress claudin-8 expression via post-transcriptional inhibition. This study not only reveals the molecular mechanisms by which HP-PRRSV infection causes endothelial barrier leakage in acute lung injury, but also provides novel insights into the function and regulation of tight junctions in vascular homeostasis.

Abstract 2141: An IL12/IL23 Positive Feedback Loop Modulates Smooth Muscle Differentiation Via Setd4 During Macrophage-mediated Inflammation

Diabetic patients have increased risk of vascular disease (e.g., atherosclerosis), which is driven by macrophage-mediated inflammation. In diseases such as atherosclerosis, smooth muscle cells (SMC) undergo phenotypic modulation from a mature, contractile to a synthetic phenotype, thus leading to increased neointima formation. We hypothesize that pro-inflammatory macrophages as in the setting of diabetes epigenetically pattern SMC during vascular disease. In this study, we use single cell-RNA sequencing (scRNA-seq) of human arteries, loss of function experiments, and mouse models of disease to identify epigenetic mechanisms that regulate SMC phenotype. We identified the H3K4 and H3K9 histone lysine methyltransferase Setd4, which was increased in aortic SMC from diabetic mice and in our scRNA-seq dataset from human atherosclerotic arteries. siRNA mediated knockdown of Setd4 in mouse aortic SMC increased expression of the early SMC differentiation markers, Acta2 and Tagln (p<0.05). To model the pro-inflammatory environment in vitro, supernatant from LPS-stimulated macrophages increased Setd4 expression and inhibited smooth muscle gene expression ( Acta2, Tagln, Cnn1, Myh11 ), which was reversed by siRNA-mediated knockdown of Setd4 in SMC (p<0.05). IL23 increased Setd4 expression in SMC and enrichment at smooth muscle gene promoters, while deceasing SMC-specific gene expression (p<0.05). Treatment of SMC with an IL23 receptor neutralizing antibody prevented downregulation of SMC gene expression by macrophage supernatant (p<0.05). Intriguingly, ELISA of mouse aortic SMC supernatant revealed that IL23 increased SMC secretion of IL12, which also inhibited SMC gene expression via Setd4 (p<0.05). In conclusion, we demonstrate that pro-inflammatory macrophage IL23 represses smooth muscle gene expression via SETD4, thereby shifting the SMC to a synthetic, diseased phenotype. IL23 also stimulates vascular SMC secretion of IL12, which further increases production of inflammatory cytokines from macrophages and leads to additional repression of smooth muscle gene expression. Thus, targeting the IL23-SETD4 axis in SMC may be a potential therapy for the treatment of atherosclerosis, especially in patients who also have diabetes.

Anti-inflammatory Effect of the Hydroethanolic Extract from Dacryodes kukachkana Leaves in Mice: Role of Macrophage Nitric Oxide

Aims: This study aimed to characterize and evaluate the effect of the hydroethanolic extract from D. kukachkana leaves (HEDkL) in mice models of acute inflammation and in isolated macrophages stimulated with carrageenan, along with possible adverse effects in the central nervous system. Study Design: After characterization of phenolic constituents, HEDkL was injected in the animals 60 min before the administration of inflammatory stimulis. The anti-inflammatory effect of HEDkL was evaluated in the models of paw edema, peritonitis and air pouch and in peritoneal macrophages stimulated with carrageenan in vitro. The per se effect of HEDkL was evaluated for behavioral alterations. Place and Duration of Study: Higher Institute of Biomedical Sciences and Animal Health Research Center, State University of Ceara; Physiopharmacology Laboratory, Federal University of Acre; between March 2021 and March 2023. Methodology: The characterization of phenolic compounds was performed by HPLC-DAD. Swiss mice (25 - 35 g) received HEDkL (25 - 200 mg/kg; per oral) 60 min before carrageenan. Macrophages were incubated with HEDkL (100 - 200 µg/mL) before being stimulated. The anti-inflammatory effect was assessed for edema, hypernociception, vascular permeability, leukocyte infiltrate, oxidative stress and inflammatory. Behavioral alterations (exploratory, anxiolytic, depressant activities) were evaluated in the animals treated with HEDkL (50 mg/kg). Results: The HPLC-DAD revealed the presence of ellagic acid, epicatechin and gallic acid. In the paw edema model, HEDkL (50 mg/kg) inhibited the edema late phase (2 - 5h) by 45% and the activity of the enzyme myeloperoxidase by 48%. In the peritonitis model, HEDkL (50 mg/kg) reduced leukocyte migration (45%), hypernociception (73%), total proteins (20%) and NO2-/NO3- (23%). In the air pouch model, HEDkL (50 mg/kg) reduced the migration of total leukocytes (76%) and neutrophils (88%). In vitro HEDkL (200 µg/mL) reduced by 31% the NO2-/NO3- in the supernatant of macrophages. HEDkL did not alter the animals exploratory, anxiolytic, or depressant behaviors. Conclusion: HEDkL containing flavonoids and phenols presents anti-inflammatory effect in vivo and in vitro involving nitric oxide inhibition, being devoid of behavioral alterations.

Abstract 4241: Development of stroma cells-rich oral cancer organoid for functional analysis of tumor microenvironment

Abstract Background: Tumor microenvironment (TME), composed of cancer cells, endothelial cells, fibroblast, immune cells, and etc, plays crucial roles in tumor progression and treatment resistance. Thus, it is extremely important to find its role involved in the interaction of TME. However, it is more difficult to examine the biological activities of those cells in vitro assay. The purpose of this study is to develop stroma cells-incorporating preclinical model, termed stroma cells-rich oral cancer organoid, for functional analysis of TME. Materials and Methods: We have first developed the stroma cells-rich oral cancer organoid by selecting the types of stroma cells such as endothelial cells, fibroblast, ratio of those cells, culture method, 3-D methods, culture media, and supplement of growth factors. Then, growth rate, morphology, radiosensitivity, and drug response were analyzed to investigate the effect of TME. Finally, the function of tumor-associated macrophage was determined using this organoid model. The growth rate of cancer cells and drug response were investigated using the organoid treated with supernatant of M1 and M2 macrophages polarized from THP-1 monocyte. Results: Oral cancer cells were co-cultured with endothelial cells (ECs) and mesenchymal stem cells (MSCs) in a particular ratio in 3D-culture, and the formed organoid became sphere. The vascular formation in the organoid was confirmed by immunofluorescent analysis. The organoid also showed higher tumorigenicity in nude mice when inoculated, compared to cancer cells alone or mixed cells with ECs and MSCs. Cell viability of luciferase-expressing tongue cancer cells (OSC-19-luc) in the organoid was assessed after treatment with cisplatin using the IVIS system showing that OSC-19-luc organoid was resistance to cisplatin compared to 3D-cultured OSC-19-luc alone or OSC-19-luc co-cultured with ECs/MSCs. The supernatant of M1 or M2 macrophage had no effect on cancer cell growth in the organoid. However, when the organoid was treated with cisplatin in the presence of M2 macrophage supernatant, the drug sensitivity was low compared to those of M1 suggesting that secretory factor from M2 macrophage may lead to drug resistance of oral cancer cells in the presence of TME. Conclusions: We have successfully established stroma cells-incorporating preclinical model that recreates the tumor microenvironment by co-culturing stromal cells. This will serve as a platform that can recapitulate the tumor biology and drug response profiles of cancer cells derived from individual oral cancer patients. This will also enable us to elucidate the role of other factors in TME and to identify novel targets for the treatment of chemo-resistant oral cancer. Citation Format: Ami Shimoda, Shizuka Shiiya, Haruka Yoshii, Kenji Mitsudo, Mitomu Kioi. Development of stroma cells-rich oral cancer organoid for functional analysis of tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4241.

Carboxymethylated and sulfated Cyclocarya paliurus polysaccharides inhibited colon cancer cells growth via PI3K/AKT-MAPKs/NF-κB pathways and immunomodulation

Our previous research revealed that carboxymethylation modification (CM-CP) and sulfation modification (S-CP) could enhance the antitumor activity of Cyclocarya paliurus polysaccharides (CP), however, the antitumor mechanisms of CM-CP and S-CP are not fully clarified and remain largely unexplored. The aims of the present study were to investigate the dual anti-tumor function of CM-CP and S-CP in inhibiting cancer cells and activating macrophages to exert immunomodulation. Results revealed that CM-CP and S-CP preferentially inhibited the proliferation of colon cancer CT-26 cells in dose-dependent and time-dependent ways, without toxicity to normal cell lines. Flow cytometry values further established that CM-CP and S-CP interventions elicited acidification in the cells, arrested the cell cycle in S stage, raised the amount of reactive oxygen species and activated oxidative stress. CM-CP and S-CP treatment elicited the depolarization of mitochondrial membranous potentials and Ca2+ superload, and disrupted the equilibrium of Na+/K+-ATPase and Ca2+-ATPase. Simultaneously, apoptotic proteins expression assays revealed that CM-CP and S-CP promoted CT-26 cell apoptosis through PI3K/AKT-MAPKs/NF-κB signal pathways. In parallel, CM-CP and S-CP dramatically promoted the proliferation and secretory activity (TNF-α and NO) of RAW264.7 cells. Co-culture of cancer cells with CM-CP and S-CP-stimulated macrophage supernatants resulted in more pronounced suppression of cancer cell viability. These findings demonstrated that CM-CP and CM-CP not only inhibit cancer cell growth directly, but also activate macrophages to exert immunomodulatory effects to suppress cancer cells. Furthermore, CM-CP and CM-CP make a contribution to the development of potential functional foods or supplements for the prevention or treatments of colorectal cancer.

Exosomal miR-223 promotes ARDS by targeting insulin-like growth factor 1 receptor: A cell communication study

Background Acute respiratory distress syndrome (ARDS) is a respiratory failure syndrome characterized by hypoxemia and changes in the respiratory system. ARDS is the most common cause of death in COVID-19 deaths was ARDS. In this study, we explored the role of miR-223 in exosomes in ARDS. Methods Exosomes were purified from the supernatants of macrophages. qPCR was used to detect relative mRNA levels. A luciferase reporter assay was performed to verify the miRNA target genes. Western blotting was used to detect the activation of inflammatory pathways. Flow cytometry was performed to assess apoptosis. An LPS-induced ARDS mouse model was used to assess the function of miR-223 in ARDS. Results Exosomes secreted by macrophages promoted apoptosis in A549 cells. Macrophages and exosomes contain high levels of miR-223. Exogenous miR-223 can decrease the expression of insulin-like growth factor 1 receptor (IGF-1R) in A549 and promote the apoptosis of A549.Transfection of anti-miR223 antisense nucleotides effectively reduced the level of miR-223 in macrophages and exosomes and eliminated the pro-apoptotic effect of A549. In vivo, LPS stimulation increased inflammatory cell infiltration in the lungs of mice, whereas knockdown of miR-223 in mice resulted in significantly reduced eosinophil infiltration. Conclusions Macrophages can secrete exosomes containing miR-223 and promote apoptosis by targeting the IGF-1R/Akt/mTOR signaling pathway in A549 cells and mouse models, suggesting that miR-223 is a potential target for treating COVID-19 induced ARDS.

Periodontal Ligament Stem Cell-Derived Exosomes Regulate Muc5ac Expression in Rat Conjunctival Goblet Cells via Regulating Macrophages Toward an Anti-Inflammatory Phenotype

ABSTRACT Background Several studies have reported the protective effects of mesenchymal stem cell-derived exosomes (MSC-Exos) in reducing inflammation and decreasing conjunctival goblet cell (CGC) loss in dry eye disease. However, whether MSC-Exos provide anti-inflammatory profiles in macrophages, thus contributing to CGC protection, has remained elusive. Methods Macrophages were incubated with PKH26‐labeled periodontal ligament mesenchymal stem cell-derived exosomes (PDLSC-Exos) for 12 h, and uptake of PDLSC-Exos by macrophages was observed by a confocal fluorescence microscope. The mRNA expression of TNF-α, IL-10, and Arg1 was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression of TNF-α and IL-10 were quantified using western blotting. Then, CGCs were exposed to different macrophage supernatants and qRT-PCR was used to detect the Muc5ac mRNA expression of CGCs in response to or absence of cholinergic stimulation. ELISA was used to determine the Muc5ac secretion of CGCs in response to cholinergic stimulation. Results The uptake of PDLSC-Exos by M1 macrophages facilitates M2 macrophage polarization with the elevated expressions of IL-10 and Arg1. In macrophage supernatant-treated CGCs systems, PDLSC-Exo-treated M1 macrophage supernatant significantly enhanced the Muc5ac expression of CGCs in response to, or in the absence of, cholinergic stimulation, while the addition of PDLSC-Exos to the control macrophage supernatant did not generate a change in Muc5ac expression. Conversely, the addition of PDLSC-Exos to the diluted control macrophage supernatant induced a significant increase in Muc5ac expression. Conclusion PDLSC-Exos could protect CGCs against M1 macrophage-mediated inflammation, and the protective effects of PDLSC-Exos are partly attributable to their effects on M1 macrophages.

GTS-21 attenuates ACE/ACE2 ratio and glycocalyx shedding in lipopolysaccharide-induced acute lung injury by targeting macrophage polarization derived ADAM-17

Acute lung injury (ALI) has received considerable attention in intensive care owing to its high mortality rate. It has been demonstrated that the selective alpha7 nicotinic acetylcholine receptor agonist Gainesville Tokushima scientists (GTS)-21 is promising for treating ALI caused by lipopolysaccharides (LPS). However, the precise underlying mechanism remains unknown. This study aimed to investigate the potential efficacy of GTS-21 in the treatment of ALI. We developed mouse models of ALI and alveolar epithelial type II cells (AT2s) injury following treatment with LPS and different polarized macrophage supernatants, respectively. Pathological changes, pulmonary edema, and lung compliance were assessed. Inflammatory cells count, protein content, and pro-inflammatory cytokine levels were analysed in the bronchoalveolar lavage fluid. The expression of angiotensin-converting enzyme (ACE), ACE2, syndecan-1 (SDC-1), heparan sulphate (HS), heparanase (HPA), exostosin (EXT)-1, and NF-κB were tested in lung tissues and cells. GTS-21-induced changes in macrophage polarization were verified in vivo and in vitro. Polarized macrophage supernatants with or without recombination a disintegrin and metalloproteinase-17 (ADAM-17) and small interfering (si)RNA ADAM-17 were used to verify the role of ADAM-17 in AT2 injury. By reducing pathological alterations, lung permeability, inflammatory response, ACE/ACE2 ratio, and glycocalyx shedding, as well as by downregulating the HPA and NF-κB pathways and upregulating EXT1 expression in vivo, GTS-21 significantly diminished LPS-induced ALI compared to that of the LPS group. GTS-21 significantly attenuated macrophage M1 polarization and augmented M2 polarization in vitro and in vivo. The destructive effects of M1 polarization supernatant can be inhibited by GTS-21 and siRNA ADAM-17. GTS-21 exerted a protective effect against LPS-induced ALI, which was reversed by recombinant ADAM-17. Collectively, GTS-21 alleviates LPS-induced ALI by attenuating AT2s ACE/ACE2 ratio and glycocalyx shedding through the inhibition of macrophage M1 polarization derived ADAM-17.

Macrophage exosomes modified by miR-365-2-5p promoted osteoblast osteogenic differentiation by targeting OLFML1.

In the bone immune microenvironment, immune cells can regulate osteoblasts through a complex communication network. Macrophages play a central role in mediating immune osteogenesis, exosomes derived from them have osteogenic regulation and can be used as carriers in bone tissue engineering. However, there are problems with exosomal therapy alone, such as poor targeting, and the content of loaded molecules cannot reach the therapeutic concentration. In this study, macrophage-derived exosomes modified with miR-365-2-5p were developed to accelerate bone healing. MC3T3-E1 cells were incubated with the culture supernatants of M0, M1 and M2 macrophages, and it was found that the culture medium of M2 macrophages had the most significant effects in contributing to osteogenesis. High-throughput sequencing identified that miR-365-2-5p was significantly expressed in exosomes derived from M2 macrophages. We incubated MC3T3-E1 with exosomes overexpressing or knocking down miR-365-2-5p to examine the biological function of exosome miR-365-2-5p on MC3T3-E1 differentiation. These findings suggested that miR-365-2-5p secreted by exosomes increased the osteogenesis of MC3T3-E1. Moreover, miR-365-2-5p had a direct influence over osteogenesis for MC3T3-E1. Sequencing analysis combined with dual luciferase detection indicated that miR-365-2-5p binded to the 3'-UTR of OLFML1. In summary, exosomes secreted by M2 macrophages targeted OLFML1 through miR-365-2-5p to facilitate osteogenesis.