Inflammatory M1 Macrophages Research Articles

Research Status and Progress of the Role of Macrophages in Rheumatoid Arthritis Inflammatory Response

Macrophages are essential immune cells that play a critical role in immune defense, immune homeostasis, and immune surveillance within the body. In rheumatoid arthritis (RA), an autoimmune disease, increased infiltration of synovial macrophages leads to heightened secretion of pro-inflammatory cytokines such as IL-6 and TNF-α, resulting in joint erosion. Macrophages have the ability to switch their functions through a process called macrophage polarization, giving rise to two main phenotypes: inflammatory macrophages (M1) and anti-inflammatory macrophages (M2). In RA, the balance between M1 and M2 phenotypes influences the disease’s pathogenesis and prognosis. M1 macrophages secrete pro-inflammatory cytokines, contributing to joint erosion, while M2 macrophages support tissue repair. Consequently, targeting the local inflammatory response initiated by M1 macrophages is crucial in RA treatment. Biological agents that block inflammatory factors and chemokines induced by macrophages are being developed to combat RA. Additionally, extracellular vesicles can guide macrophage reprogramming, promoting the transition from M1 to anti-inflammatory M2 macrophages and restoring tissue homeostasis.

Abstract 6789: The oncolytic virus VET3-TGI both blocks TGF-beta signaling and activates type 2 IFN responses, resulting in potent therapeutic responses in multiple mouse models

Abstract Background: TGFB1 mediated immune resistance is one of the major mechanisms of immune suppression utilized across multiple tumor types. Immune resistance imparted by TGFB1 is mediated through its pleiotropic effects on vasculature, fibrogenesis and regulatory/effector immune cells within the tumor microenvironment. Blockade of TGFB1 (TGFBi) will likely improve response to immunotherapy. IL-12 is a cytokine that through IFNg induction, promotes type 1 inflammatory response, M1 macrophages and effector CD8 T cell response. Combining TGFB1 blockade with IL-12 may maximize therapeutic benefits through simultaneously reducing immunosuppression and enhancing anti-tumor immune response. Current studies have developed a vaccinia-based immunotherapy, combining enhanced systemic virus delivery to CXCR3 ligand rich tumors and locally expressed IL-12 and TGFBi within the tumor microenvironment, for efficient control of multiple tumor models. Methods: An oncolytic vaccinia virus expressing CXCR3, IL-12 and a TGFB1 antagonizing mini-monomer was constructed (VET3-TGI) and the expression and function of the transgenes were confirmed. Using in vivo mouse RENCA, EMT-6 and MC38 tumor models, the functionality and therapeutic efficacy of VET-TGI were tested with comparison to control virus. Post-mortem analysis was used to analyze the impact of VET3-TGI on immune/stromal/endothelial milieu of the tumors and to determine toxicity profile. Results: VET3-TGI infected cells expressed CXCR3 and showed enhanced migration to CXCR3 ligands in vitro and improved systemic delivery to tumors expressing CXCR3 ligands in vivo, even in the face of pre-existing anti-viral immunity. IL-12 expression and TGFBi blockade of TGFB1 mediated suppression of CD8 T cell proliferation were confirmed in vitro. In vivo mouse studies using EMT6, RENCA and MC38 tumor models demonstrated potent therapeutic activity, including 100% CRs, even at doses several logs below equivalent clinical doses and in multiple models. Mechanism of activity studies suggested that the therapeutic efficacy of VET3-TGI is associated with considerable modification of the tumor microenvironment. In addition, preliminary toxicity studies demonstrated the safety of VET3-TGI in mouse models. Conclusions: VET3-TGI demonstrated an ability to reduce immunosuppression and dramatically enhance antitumor immune response leading to safe and potent therapeutic activity in multiple mouse tumor models. This data led to the selection of VET3-TGI as our lead clinical candidate. A human version of the virus is currently undergoing clinical manufacture and toxicology testing. Citation Format: Ravikumar Muthuswamy, Steve Thorne. The oncolytic virus VET3-TGI both blocks TGF-beta signaling and activates type 2 IFN responses, resulting in potent therapeutic responses in multiple mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6789.

PD04-06 IDENTIFYING NOVEL REGULATORS OF MACROPHAGE TUMOR CELL METABOLIC INTERACTIONS IN PROSTATE CANCER

You have accessJournal of UrologyCME1 Apr 2023PD04-06 IDENTIFYING NOVEL REGULATORS OF MACROPHAGE TUMOR CELL METABOLIC INTERACTIONS IN PROSTATE CANCER Asmaa Elkenawi, Ryan Putney, Amparo Serna, Jasreman Dhillon, and Kosj Yamoah Asmaa ElkenawiAsmaa Elkenawi More articles by this author , Ryan PutneyRyan Putney More articles by this author , Amparo SernaAmparo Serna More articles by this author , Jasreman DhillonJasreman Dhillon More articles by this author , and Kosj YamoahKosj Yamoah More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003228.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: African American men (AA) are more than twice as likely to die of prostate cancer (PCa) compared to European American men (EA). Among many contributing factors, unraveling the metabolic derangements in PCa of AA holds a promise in reducing health disparity. METHODS: We examined publicly available prostate cancer datasets for race dependent differential gene expression. We prospectively examined the differential gene expression of PCa from clinically matched AA and EA in the VANDAAM clinical trial. The VANDAAM study is a validation study of DecipherTM genomic testing in 240 men with localized PCa. We combined computational and experimental approaches to explore the molecular mechanisms of top candidate genes including these involved in metabolism. We then developed a novel metabolomic workflow that profiles patient- specific nutrient consumption to predict anti-cancer efficacy and validate novel biomarkers. RESULTS: We identified aminopeptidase N (ANPEP) as one of the top candidate genes to be overexpressed in AA. Aminopeptidase N is involved in endocytosis, cholesterol and amino acid transport and peptide hydrolysis. Unbiased computational analyses of the VANDAAM revealed that ANPEP correlates with signatures of cholesterol transport, estrogen and androgen receptor (AR) signaling. Based on our recent study demonstrating dominance of these signatures in macrophage rich PCa, we reasoned that ANPEP expression may be driven in part by high macrophage infiltration in AA. Thus, we compared immune cell repertoire in patients with high ANPEP and low ANPEP by deconvoluting immune cell content using the in silico approach, CIBERSORT. These analyses illustrated that only AA patients with high ANPEP expression significantly accumulated high content of M1 inflammatory macrophages. Immune phenotyping of prostate tumors demonstrated that ANPEP indeed represents a marker of M1 inflammatory macrophages. To unravel role of ANPEP in regulating cholesterol and amino acid transport, we developed a Liquid chromatography-high resolution mass spectrometry (LC-HRMS) -based approach to measure cholesterol and amino acids in explants derived from AA and EA prostate cancer patients. This innovative approach enables us to perform mechanistic studies by determining metabolic dependency on specific amino acids as a function ANPEP or macrophage infiltration. CONCLUSIONS: ANPEP represents a macrophage related protein in PCa with a potential role in cholesterol, amino acid transport and / or androgen signaling. Source of Funding: Prostate Cancer Foundation (AE), Urology Care Foundation (AE), Prostate Cancer Foundation (KY) and Department of Defense award CDMRP-PC181013 (KY) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e144 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Asmaa Elkenawi More articles by this author Ryan Putney More articles by this author Amparo Serna More articles by this author Jasreman Dhillon More articles by this author Kosj Yamoah More articles by this author Expand All Advertisement PDF downloadLoading ...

Granulosa cell-derived miR-379-5p regulates macrophage polarization in polycystic ovarian syndrome.

Polycystic ovarian syndrome (PCOS) is associated with hyperandrogenemia and ovarian antral follicle growth arrest. We have previously demonstrated that androgen-induced exosomal release of miR-379-5p (miR379) from preantral follicle granulosa cells increases the proliferation of target cells via phosphoinositide-dependent kinase 1 (PDK1) upregulation. Androgen also increases inflammatory M1 macrophage abundance, but reduces anti-inflammatory M2 polarization in rat antral and preovulatory follicles. However, the role of small extracellular vesicles (sEVs; also known as exosomes) secretion in determining the cellular content and function of miRNAs in exosome-receiving cells is largely unknown. Our objectives were to determine: 1) the regulatory role of granulosa cells (GC)-derived exosomal miR379 on macrophage polarization and ovarian inflammation; 2) whether miR379-induced M1 polarization regulates GC proliferation; and 3) if this regulated process is follicular stage-specific. Compared with non-PCOS subjects, PCOS subjects had a higher M1/M2 ratio, supporting the concept that PCOS is an inflammatory condition. Ovarian overexpression of miR379 increased the number of M1 macrophages and the M1/M2 ratio in preantral follicles specifically. Transfection of macrophages with a miR379 mimic reduced the cellular content of PDK1 and induced M0→M1 polarization; whereas its inhibitor polarized M0→M2. Conditioned media from macrophages transfected with miR379 mimic and follicular fluid from PCOS subjects had higher galectin-3 content, a pro-inflammatory cytokine which specifically suppresses human antral follicle GC proliferation. These results indicate that miR379 inhibits M2 macrophage polarization, a condition which suppresses GC proliferation in a follicle stage-dependent manner, as exhibited in PCOS.

Selective Photothermal Therapy Based on Lipopolysaccharide Aptamer Functionalized MoS2 Nanosheet-Coated Gold Nanorods for Multidrug-Resistant Pseudomonas aeruginosa Infection.

Chronic wounds infected by multidrug-resistant gram-negative bacteria have evolved resistance to traditional antibiotic therapy, posing a threat to global public health in recent years. Herein, a selective therapeutic nanorod (MoS2 -AuNRs-apt) based on molybdenum disulfide (MoS2 ) nanosheets coated gold nanorods (AuNRs) targeting lipopolysaccharide (LPS) is presented. AuNRs have excellent photothermal conversion efficiency in 808nm laser-guided photothermal therapy (PTT), and the MoS2 nanosheets coating significantly enhances the biocompatibility of AuNRs. Furthermore, the conjugation of the nanorods with aptamer permits active targeting of LPS on the surface of gram-negative bacteria and a specific anti-inflammatory ability in the multidrug-resistant Pseudomonas aeruginosa (MRPA)-infected wound murine model. It is concluded that the antimicrobial effect of these nanorods is considerably more significant than non-targeted PTT. Moreover, they can precisely overcome MRPA bacteria by physical damage and effectively reduce excess M1 inflammatory macrophages to accelerate the healing of infected wounds. Overall, this molecular therapeutic strategy displays great potential as a prospective antimicrobial treatment for MRPA infections.

High-plasticity mineral trioxide aggregate and its effects on M1 and M2 macrophage viability and adherence, phagocyte activity, production of reactive oxygen species, and cytokines.

This study evaluated the effects of high-plasticity mineral trioxide aggregate (MTA-HP) on the activity of M1 and M2 macrophages, compared to white MTA (Angelus). Peritoneal inflammatory M1 (from C57BL/6 mice) and M2 (from BALB/c mice) macrophages were cultured in the presence of the tested materials. Cell viability (MTT and trypan blue assays), adhesion, phagocytosis, reactive oxygen species (ROS) production, and tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β production were evaluated. Parametric analysis of variance and the non-parametric Kruskal-Wallis test were used. Results were considered significant when p < 0.05. The MTT assay revealed a significant decrease in M1 metabolism with MTA-HP at 24 hours, and with MTA and MTA-HP later. The trypan blue assay showed significantly fewer live M1 at 48 hours and live M2 at 48 and 72 hours with MTA-HP, compared to MTA. M1 and M2 adherence and phagocytosis showed no significant differences compared to control for both materials. Zymosan A stimulated ROS production by macrophages. In the absence of interferon-γ, TNF-α production by M1 did not significantly differ between groups. For M2, both materials showed higher TNF-α production in the presence of the stimulus, but without significant between-group differences. Likewise, TGF-β production by M1 and M2 macrophages was not significantly different between the groups. M1 and M2 macrophages presented different viability in response to MTA and MTA-HP at different time points. Introducing a plasticizer into the MTA vehicle did not interfere with the activity of M1 and M2 macrophages.

Pachymic Acid Prevents Hemorrhagic Shock-Induced Cardiac Injury by Suppressing M1 Macrophage Polarization and NF-[Formula: see text]B Signaling Pathway.

Hemorrhagic shock (HS) is the leading cause of death in trauma patients. Inflammation following HS can lead to cardiac damage. Pachymic acid (PA), a triterpenoid extracted from Poria cocos, has been found to possess various biological activities, including anti-inflammatory and anti-apoptotic properties. Our research aims to investigate the protective effects of PA against HS-induced heart damage and the underlying mechanisms involved. Male Sprague-Dawley rats were intraperitoneally injected with PA (7.5 or 15[Formula: see text]mg/kg) daily for three days. Subsequently, we created a rat model of HS by drawing blood through a catheter inserted into the femoral artery followed by resuscitation. The results revealed that HS led to abnormalities in hemodynamics, serum cardiac enzyme levels, and cardiac structure, as well as induced cardiac apoptosis. However, pretreatment with PA effectively alleviated these effects. PA-pretreatment also suppressed mRNA and protein levels of interleukin (IL)-1[Formula: see text], IL-6, and tumor necrosis factor [Formula: see text] (TNF-[Formula: see text]) in the heart tissues of HS rats. Additionally, PA-pretreatment reduced inflammatory cell infiltration and M1 macrophage polarization while exaggerating M2 polarization in HS rat hearts. The study observed a decreased proportion of the expression of of M1 macrophages (CD86[Formula: see text]) and their marker (iNOS), along with an increased proportion of the expression of M2 macrophages (CD206[Formula: see text]) and their marker (Arg-1). Notably, PA-pretreatment suppressed NF-[Formula: see text]B pathway activation via inhibiting NF-[Formula: see text]B p65 phosphorylation and its nuclear translocation. In conclusion, PA-pretreatment ameliorates HS-induced cardiac injury, potentially through its inhibition of the NF-[Formula: see text]B pathway. Therefore, PA treatment holds promise as a strategy for mitigating cardiac damage in HS.

Targeted treatment of gouty arthritis by biomineralized metallic nanozyme-mediated oxidative stress-mitigating nanotherapy.

Gouty arthritis is characterized by chronic deposition of monosodium urate (MSU) crystals in the joints and other tissues, resulting in the production of excess reactive oxygen species (ROS) and proinflammatory cytokines that intensify synovial inflammation. This condition is mainly associated with inflammatory M1 macrophage activation and oxidative stress production. Hence, gout symptoms can often be resolved by eliminating M1 macrophage activation and scavenging oxidative stress in the inflamed areas. Herein, we developed M1-macrophage-targeting biomineralized metallic nanozymes (FALNZs) that deplete oxidative stress and reduce the M1 macrophage levels to mitigate gouty arthritis. Intra-articular injection of the FALNZs targets inflammatory macrophages and suppresses ROS levels in joints with MSU-crystal-induced arthritis. In addition, the FALNZs alleviate joint swelling, inflammatory cytokine production, and pathological features of the joints. Overall, the proposed therapeutic approach is biocompatible and is an effective ROS scavenger for the treatment of gouty pathogenesis.

Effects of substrate topography on the regulation of human fibroblasts and capsule formation via modulating macrophage polarization

The host-material interface is critical in determining the successful integration of medical devices into human tissue. The surface topography can regulate the fibrous capsule formation around implants through macrophage polarization, but the exact mechanism remains unclear. In this study, four types of microgrooves (10 or 50 µm in groove depths and 50 or 200 µm in groove widths) were fabricated on polydimethylsiloxane (PDMS) using lithography. The microgroove surfaces were characterized using the laser scanning confocal microscopy and fourier transform infrared spectroscopy. The effect of surface topography on macrophage phenotypes and conditioned medium (CM) collected from macrophages on human foreskin fibroblast 1 (HFF-1) were investigated. The result revealed that a deeper and narrower microgroove structure means a rougher surface. Macrophages tended to adhere and aggregate on group 50–50 surface (groove depths and widths of 50 µm). THP-1 cell polarized toward both inflammatory M1 and anti-inflammatory M2 macrophages on the surface of each group. Meanwhile, CM from macrophages culture on PDMS differentially up-regulated the proliferation, migration and fibrosis of HFF-1. Among them, the group 50–50 had the strongest promoting effect. In vivo, the inflammatory response and fibrotic capsule around the implants were observed at 1 week and 4 weeks. As time passed, the inflammatory response decreased, while the capsule thickness continued to increase. The rough material surface was more inclined to develop a severe fibrotic encapsulation. In conclusion, this finding further suggested a potential immunomodulatory effect of macrophages in mediating the fibrotic response to implants and facilitated the design of biomaterial interfaces for improving tissue integration.

Inflammatory Periodontal Ligament Stem Cells Drive M1 Macrophage Polarization via Exosomal miR-143-3p-Mediated Regulation of PI3K/AKT/NF-κB Signaling.

Macrophage polarization plays an important role in the progression of inflammation. Exosomes derived from stem cells are promising candidates for macrophage immunoregulation. However, how exosomes derived from periodontal ligament stem cells (PDLSCs) in an inflammatory environment influence macrophage polarization has yet to be fully elucidated. In this study, inflammatory PDLSCs were found to downregulate M2 macrophage polarization at the mRNA and protein levels in a Transwell coculture system of PDLSCs and THP-1-derived M0 macrophages. Furthermore, inflammatory PDLSC-derived exosomes shifted macrophages toward the M1 phenotype. The inhibition of inflammatory PDLSC-derived exosomes by GW4869 weakened inflammatory PDLSC-mediated M1 macrophage polarization. A miRNA microarray was used to determine the differential miRNAs shuttled by healthy and inflammatory PDLSC-derived exosomes. Compared with healthy exosomes, miR-143-3p was enriched in inflammatory PDLSC-derived exosomes, which targeted and inhibited the expression of PI3Kγ and promoted M1 macrophage polarization by suppressing PI3K/AKT signaling and activating NF-κB signaling, while an agonist of the PI3K pathway reversed this effect. Moreover, exosome-shuttled miR-143-3p from PDLSCs drove M1 macrophage polarization and aggravated periodontal inflammation in a mouse periodontitis model. In conclusion, these results demonstrate that inflammatory PDLSCs facilitate M1 macrophage polarization through the exosomal miR-143-3p-mediated regulation of PI3K/AKT/NF-κB signaling, providing a potential new target for periodontitis treatment.

P38 MAPK-dependent phosphorylation of TFEB promotes monocyte-to-macrophage differentiation.

The transcription factor EB (TFEB) regulates energy homeostasis and cellular response to a wide variety of stress conditions, including nutrient deprivation, oxidative stress, organelle damage, and pathogens. Here we identify S401 as a novel phosphorylation site within the TFEB proline-rich domain. Phosphorylation of S401 increases significantly in response to oxidative stress, UVC light, growth factors, and LPS, whereas this increase is prevented by p38 MAPK inhibition or depletion, revealing a new role for p38 MAPK in TFEB regulation. Mutation of S401 in THP1 cells demonstrates that the p38 MAPK/TFEB pathway plays a particularly relevant role during monocyte differentiation into macrophages. TFEB-S401A monocytes fail to upregulate the expression of multiple immune genes in response to PMA-induced differentiation, including critical cytokines, chemokines, and growth factors. Polarization of M0 macrophages into M1 inflammatory macrophages is also aberrant in TFEB-S401A cells. These results indicate that TFEB-S401 phosphorylation links differentiation signals to the transcriptional control of monocyte differentiation.

Mesenchymal stromal/stem cells: breaking the deadlock in the treatment of multiple organ dysfunction syndrome

Mesenchymal stromal/stem cells: breaking the deadlock in the treatment of multiple organ dysfunction syndrome

Single-cell transcriptome reveals immunopathological cell composition of skin lesions in subacute cutaneous lupus erythematosus

Subacute cutaneous lupus erythematosus (SCLE) is a clinical subtype of cutaneous lupus erythematosus with psoriatic-like or annular papules with scaly erythemas, the pathological mechanism of which is poorly understood. To investigate the immune pathogenesis of SCLE, we performed single-cell RNA sequencing (scRNA-seq) of SCLE skin lesions and integrated the scRNA-seq data from skin tissues of healthy controls (HCs). Our results identified expanded fibroblasts and keratinocytes subtypes, abnormally activated lymphocyte and inflammatory M1 macrophages in SCLE. In SCLE, stromal cells, such as keratinocytes and fibroblasts, showed enhanced chemotactic functions for recruiting immune cells. Importantly, interferon-related genes were identified as key intermediate genes in the potential trajectory of fibroblasts, keratinocytes, and B cells from HCs to SCLE. Our investigation provides a comprehensive description of cell composition in SCLE and highlights several important clues for understanding the pathogenesis of SCLE.

RF05 | PSUN358 ANPEP: A potential regulator of tumor cell - macrophage metabolic interactions in prostate cancer of African American men

Abstract African American men (AA) are more than twice as likely to die of prostate cancer (PCa) compared to European American men (EA). Among many contributing factors, unraveling the metabolic derangements in PCa of AA holds a promise in reducing health disparity. We recently discovered that aminopeptidase N (ANPEP, CD13) represents one of the differentially expressed genes in PCa of AA compared with EA. While ANPEP plays regulatory and/or modulatory functions in many immune and metabolic pathological conditions, its role in PCa remains unknown. We sought to investigate the metabolic functions of ANPEP in PCa development and exploit its role as a therapeutic vulnerability, particularly in AA men. Accordingly, we prospectively examined the differential gene expression of PCa from clinically matched AA and EA in the VANDAAM clinical trial. The VANDAAM study is a validation study of DecipherTM genomic testing in 240 men with localized PCa. Our findings indicate that ANPEP is the top differentially expressed gene between AA and EA men. To explore the molecular mechanisms of ANPEP in PCa in unbiased fashion, we combined computational and experimental approaches. Our preliminary computational analyses revealed that ANPEP correlates with signatures of cholesterol transport, estrogen and androgen receptor (AR) signaling. Based on our recent study demonstrating dominance of these signatures in macrophage-rich PCa, we reasoned that ANPEP expression may be driven in part by high macrophage infiltration in AA. Thus, we compared immune cell repertoire in patients with high ANPEP and low ANPEP by deconvoluting immune cell content using the in silico approach, CIBERSORT. These analyses illustrated that only AA patients with high ANPEP expression significantly accumulated high content of M1 inflammatory macrophages. Immune phenotyping of prostate tumors demonstrated that ANPEP indeed represents a marker of M1 inflammatory macrophages and tumor-associated macrophages. In conclusion, these findings suggest that ANPEP is a macrophage related protein in PCa with a potential role in cholesterol transport and / or androgen signaling. Future work will focus on the functional role of ANPEP activity in the tumor immune microenvironment using Liquid chromatography-high resolution mass spectrometry (LC-HRMS) and explants derived from AA and EA prostate cancer patients. Presentation: Saturday, June 11, 2022 1:00 p.m. - 1:05 p.m., Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Modulation of inflammatory M1-macrophages phenotype by valvular interstitial cells

BackgroundAortic valve stenosis involves inflammation, excess deposition of a collagen-rich extracellular matrix, and calcification. Recent studies have shown that M1 or inflammatory macrophages derived from infiltrating monocytes promote calcification of valvular interstitial cells, the most prevalent cell type of the aortic valve. We hypothesized that valvular interstitial cells could modulate inflammatory macrophages phenotype. MethodsWe first assessed macrophage phenotype in human aortic valve stenosis and control aortic valves from donors. Then, we examined profibrotic and inflammatory-related gene expression in valves and valvular interstitial cells. Finally, we investigated whether valvular interstitial cells can modify the phenotype of inflammatory macrophages. ResultsCirculating monocytes and plasma transforming growth factor beta-1 levels of patients with aortic valve stenosis were significantly higher compared with patients without aortic valve stenosis. Histologic analysis of thickened spongiosa of the aortic valve from patients with aortic valve stenosis showed a high macrophage infiltration but a low matrix metalloproteinase-9 expression compared with control aortic valves. On the other hand, valvular interstitial cell culture of aortic valve stenosis exhibited a profibrotic phenotype with a high expression of transforming growth factor beta-1 and transforming growth factor beta-1/transforming growth factor beta-3 ratio but a decreased expression of the peroxisome proliferator-activated receptor gamma nuclear receptor. Valvular interstitial cell–conditioned media of aortic valve stenosis led to a decrease in enzymatic activity of matrix metalloproteinase-9 and an increase in production of collagen in inflammatory macrophages compared with valvular interstitial cell–conditioned media from control aortic valve donors. ConclusionsThese findings indicate that profibrotic valvular interstitial cells promote the imbalance of extracellular matrix remodeling by reducing matrix metalloproteinase-9 production on inflammatory macrophages that lead to excessive collagen deposition observed in aortic valve stenosis. Further investigation is needed to clarify the role of transforming growth factor beta-1/proliferator-activated receptor gamma nuclear receptor/matrix metalloproteinase-9 in aortic valve stenosis.

Protective effect of α7 nicotinic acetylcholine receptor activation on experimental colitis and its mechanism

BackgroundInflammatory bowel disease (IBD) is a common chronic remitting disease with no satisfactory treatment. The aim of this study was to investigate the protective effect of α7 nicotinic acetylcholine receptor (α7nAChR), and to determine the underlying mechanism of its activity.MethodsThe expression and distribution of α7nAChR in the intestinal tissue of patients with ulcerative colitis and Crohn’s disease were analyzed. The effects of vagal excitation on murine experimental colitis were investigated. The colitis model was induced in C57BL/6 mice by the administration of 3% dextran sulfate sodium (DSS). The therapeutic group received treatment with the α7nAChR agonist PNU-282987 by intraperitoneal injection.ResultsOur results showed that there was significantly increased expression of α7nAChR in colitis and Crohn’s disease intestinal tissue, and its expression was mainly located in macrophages and neutrophils, which were extensively infiltrated in the disease status. Treatment with an α7nAChR agonist potently ameliorated the DSS-induced illness state, including weight loss, stool consistency, bleeding, colon shortening, and colon histological injury. α7nAChR agonist exerted anti-inflammatory effects in DSS colitis mice by suppressing the secretion of multiple types of proinflammatory factors, such as IL6, TNFα, and IL1β, and it also inhibited the colonic infiltration of inflammatory cells by blocking the DSS-induced overactivation of the NF-κB and MAPK signaling pathways. Mechanistically, activation of α7nAChR decreased the number of infiltrated M1 macrophages in the colitis intestine and inhibited the phagocytosis ability of macrophages, which were activated in response to LPS stimulation.ConclusionThus, an α7nAChR agonist ameliorated colonic pathology and inflammation in DSS-induced colitis mice by blocking the activation of inflammatory M1 macrophages.

P17.06: Escape From Macrophage-Mediated Rejection by Human Surfactant Protein (SP)-A

Introduction: Macrophage-mediated xenogeneic rejection is one of the important immunological obstructions that need to be overcome. Because macrophage is one of the main sources of proinflammatory cytokines and the fact that proinflammatory cytokines orchestrate a variety of inflammatory responses, the regulation of macrophages could result in solving the problem of xenogeneic rejection. We recently reported that membrane-type surfactant D (SP-D) on swine endothelial cells (SECs) suppresses macrophage-mediated rejection. Similar to SP-D, the carbohydrate recognition domain of surfactant protein-A (SP-A) induces inhibitory signals in effector cells. In this study, we examined the suppressive effect of SP-A on macrophage-mediated xenogeneic rejection. Methods: Naïve SEC and SP-A-transfected SEC (SEC/SP-A) were co-cultured with THP-1 cells and cytotoxicity was evaluated. To investigate the effect on phagocytosis, human macrophages were co-cultured with SEC or SEC/SP-A, and the extent of phagocytosis and production of reactive oxygen species (ROS) were assessed by flow cytometry. The mRNA expression of inflammatory cytokines in macrophages was determined using RT-PCR. In addition, THP-1 cell with a luciferase reporter gene driven by an NF-kB response element (THP-1 Luc NF-kB) cells was used to verify their effects on the NF-κB transcription factors. Results: The cytotoxicity of SEC/SP-A was significantly reduced compared to those of naïve SEC (40.7 vs 22.9%, p=0.025). The phagocytosis by macrophages against SEC was also significantly suppressed by SP-A on SEC (75.8 vs 33.0%, n=5, p=0.0010). Co-culture of human macrophages with SEC/SP-A decreased ROS production (%MFI: 99.9 vs 79.5, p=0.013). The mRNA expression of TNFα was reduced in macrophages (1.160 vs 0.009, p=0.0022), whereas that of IL-10 was increased (1.357 vs 10.20, p=0.0007). The balance between iNOS and Arg-1 was significantly suppressed by CL-SPA (1.002 vs 0.4792, p = 0.0027), indicating that CL-SPA on porcine cells inhibits the differentiation of peripheral blood monocytes into inflammatory M1 macrophages. The NF-κB transcription was decreased in THP-1 Luc NF-kB which was co-cultured with SEC/SP-A compared to that in THP-1 Luc NF-kB with SEC (RLU: 1160 vs 983.8, p=0.0258). Conclusion: The ectopic expression of human SP-A in porcine cells represents an attractive method for suppressing macrophage-mediated cytotoxicity. To further investigate the effects of SP-A on xenogeneic innate immune response in more detail, in vivo studies should be performed in the future.

Evidence for Monocyte Reprogramming in a Long-Term Postsepsis Study.

OBJECTIVES:This study sought to identify monocyte alterations from septic patients after hospital discharge by evaluating gene expression of inflammatory mediators and monocyte polarization markers. It was hypothesized that sepsis reprograms the inflammatory state of monocytes, causing effects that persist after hospital discharge and influencing patient outcomes.DESIGN:The gene expression patterns of inflammatory receptors, M1 and M2 macrophage polarization markers, NLRP3 inflammasome components, and pro- and anti-inflammatory cytokines in monocytes were assessed.PATIENTS:Thirty-four patients from the University of São Paulo Hospital, during the acute sepsis phase (phase A), immediately after ICU discharge (phase B), and 3 months (phase C), 6 months (phase D), 1 year (phase E), and 3 years (phase F) after discharge, were included. Patients that died during phases A and B were grouped separately, and the remaining patients were collectively termed the survivor group.MEASUREMENTS AND MAIN RESULTS:The gene expression of toll-like receptor (TLR)2 and TLR4 (inflammatory receptors), NLRP3, NFκB1, adaptor molecule apoptosis-associated speck-like protein containing a CARD, caspase 1, caspase 11, and caspase 12 (NLRP3 inflammasome components), interleukin-1α, interleukin-1β, interleukin-18, and high-mobility group box 1 protein (proinflammatory cytokines), interleukin-10 (anti-inflammatory cytokine), C-X-C motif chemokine ligand 10, C-X-C motif chemokine ligand 11, and interleukin-12p35 (M1 inflammatory polarization markers), and C-C motif chemokine ligand 14, C-C motif chemokine ligand 22, transforming growth factor-beta (TGF-β), SR-B1, and peroxisome proliferator-activated receptor γ (M2 anti-inflammatory polarization and tissue repair markers) was upregulated in monocytes from phase A until phase E compared with the control group.CONCLUSIONS:Sepsis reprograms the inflammatory state of monocytes, probably contributing to postsepsis syndrome development and mortality.

Abstract 243: Berberine eases inflammation in the anaplastic thyroid cancer microenvironment

Abstract Only 2% of thyroid cancer (TC) cases are anaplastic thyroid cancer (ATC), but this rare, undifferentiated cancer with a highly inflamed tumor microenvironment (TME) presents universally as stage IV with a 5-year survival rate of 3%. Due to its loss of differentiation, ATC is resistant to radioiodine therapy, refractory to external beam radiation, and insufficiently treated by chemotherapy or immunotherapy. Berberine (BBR), a natural plant alkaloid, has vast pharmacological activities, including anti-inflammatory and anti-cancer roles. BBR blocks proliferation, induces cell cycle arrest, promotes apoptosis, and hinders invasion and metastasis. BBR may also inhibit cell proliferation through miRNA interactions and regulate the TME through its anti-inflammatory and antioxidant properties. Research in ATC using BBR is limited, specifically investigation on how BBR reprograms the inflammatory TME via altering miRNA cargo of secretory exosomes and polarization of tumor associated macrophages (TAMs). ATC tissues have extensive infiltration of a mixed TAM population. In ATC, infiltration with classically anti-tumorigenic, inflammatory M1 macrophages has a pro-tumorigenic role. We assessed the secretory profile of ATC cells and found a high expression of pro-inflammatory cytokines, such as TNFα, IL-6, IL-1, MCP-1/2, and MIP-1α/β in ATC conditioned media (CM), supporting the existence of an inflammatory TME. As this chronic inflammation is partially mediated by the presence of M1 TAMs in the TME, we activated monocyte cell line U937 with TPA and polarized with IFNγ and LPS into an M1 phenotype. In the presence of BBR at the activation and polarization stages, inflammatory mediators IL-1β, IL-6, IL-6R, CXCL9, MIP-1α/β, and TNFR1/2 were downregulated in the CM compared to M1 macrophages activated and polarized without BBR. BBR also appeared to prevent some level of activation and polarization of these cells into the M1 phenotype altogether. BBR may have an essential role in lessening the burden of inflammation in ATC through remodeling its tumor immune infiltrates, potentially priming it for greater success in combination therapy. Intervention prior to metastasis warrants secretome analysis to define ATC disease progression. Exosomal cargo, particularly miRNA, secreted by ATC cells was evaluated as exosomes have potential as biomarkers to detect ATC earlier improving prognosis, guide treatment, and avoid unneeded surgeries. Distinct miRNA expression from ATC-secreted exosomes compared to papillary thyroid cancer (PTC)-secreted exosomes revealed six miRNAs specifically downregulated in ATC-secreted exosomes that are known tumor suppressors. We are currently evaluating how BBR alters the miRNA profile of ATC- and PTC-secreted exosomes. The ability of BBR to alleviate inflammation in ATC and remodel its TME releases its chronic hold on ATC progression and may prime the tumor for increased responsiveness to therapy. Citation Format: Tara Jarboe, Kaci Kopec, Nicole R. DeSouza, Sarnath Singh, Augustine Moscatello, Jan Geliebter, Xiu-Min Li, Raj K. Tiwari. Berberine eases inflammation in the anaplastic thyroid cancer microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 243.

Biofabricated macrophage and fibroblast membranes synergistically promote skin wound healing.

Effective skin wound healing is a complex process involving anti‐inflammation, fibrosis, matrix reconstruction, and angiogenesis. This work aimed to integrate the macrophage‐mediated anti‐inflammation and fibroblast‐assisted matrix reconstruction for enhanced skin wound healing. Herein, we utilized the cytomembranes derived from repolarized M2 macrophages and fibroblasts to prepare the natural biologics. Results showed that the inflammatory M1 macrophages were repolarized to M2 phenotype by the M2 macrophage cytomembranes. As a consequence, the cytomembranes of M2 macrophage could facilitate the wound closure in mice. Furthermore, the addition of fibroblast membranes to the macrophage cytomembranes contributed to a better matrix reconstruction, neovascularization and angiogenesis. Next, we used a transforming growth factor‐β (TGF‐β) inhibitor to attenuate cutaneous scar formation. Therefore, our modality could promote skin wound healing and effectively suppress scar formation in the preclinical murine skin wounds. The cytomembrane biologics might provide a biocompatible and versatile tool for wound healing.