Normal Macrophages Research Articles

Self-replicating recombinant virus-like particles of lentivirus proliferating in glioblastoma cells and normal human macrophages

Introduction. Both attenuated and inactivated vaccines are used in disease control. Inactivated vaccines are very diverse and include whole cell and acellular vaccines containing protein target antigens or nucleic acids encoding target antigens. Immunity induced by inactivated vaccines is not believed to be long-lasting. It is very problematic to develop a vaccine against viruses that integrate into the genome of the host cell, as well as against persistent viruses that penetrate the central nervous system (CNS), which is typical for the human immunodeficiency virus type 1 (HIV-1). Aim of the study: to evaluate the possibility of forming HIV-1 recombinant virus-like particles (recVLPs) and HIV-1B recombinant VLPs and simian immunodeficiency virus (SIV) — SHIV89.6P based on self-replicating RNAs (srRNAs) producing target lentivirus antigens on the alphavirus replicon platform (Sindbis virus or Venezuelan equine encephalomyelitis virus (VEEV)), and also to evaluate the ability of HIV-1B and SHIV89.6P VLPs to infect glioblastoma cells and normal human macrophages. Materials and methods. BHK-21 cells were transfected with the srRNA mixture by electroporation. Recombinant virus-like particles (recVLP’s) in recVLP’s-infected cells were detected using the immunofluorescence assay (ELISA) and electron microscopy. recVLP’s were used to infect glioblastoma cells and normal macrophages from a healthy donor. Results. Based on the genomic RNA of the alphavirus, the plasmids were created, transcription from which makes it possible to obtain RNA that expresses lentiviral gene products in cells in quantities sufficient for the formation of mature VLPs. In BHK-21 cells infected with recVLP’s, virus-specific antigens are detected only in the cytoplasm, but not in the nucleus. Both glioblastoma cells (U87) and normal human macrophages containing CD4 receptor and SSR5 and CXR4 co-receptors give infectious progeny of HIV-1B and SHIV89.6P recVLP’s when infected with supernatant obtained after transfection of BHK-21 cells with srRNA. Discussion. The results obtained show the possibility of expressing lentivirus structural proteins in glioblastoma cells (U87) and in normal human macrophages and can be used in the future to study the presentation of antigens in native and functional conformations in appropriate model systems to study the possibility of suppressing HIV infection in viral reservoirs in the CNS.

Exosomal miR-1a-3p derived from glucocorticoid-stimulated M1 macrophages promotes the adipogenic differentiation of BMSCs in glucocorticoid-associated osteonecrosis of the femoral head by targeting Cebpz

BackgroundBy interacting with bone marrow mesenchymal stem cells (BMSCs) and regulating their function through exosomes, bone macrophages play crucial roles in various bone-related diseases. Research has highlighted a notable increase in the number of M1 macrophages in glucocorticoid-associated osteonecrosis of the femoral head (GA-ONFH). Nevertheless, the intricate crosstalk between M1 macrophages and BMSCs in the glucocorticoid-stimulated environment has not been fully elucidated, and the underlying regulatory mechanisms involved in the occurrence of GA-ONFH remain unclear.MethodsWe employed in vivo mouse models and clinical samples from GA-ONFH patients to investigate the interactions between M1 macrophages and BMSCs. Immunofluorescence staining was used to assess the colocalization of M1 macrophages and BMSCs. Flow cytometry and transcriptomic analysis were performed to evaluate the impact of exosomes derived from normal (n-M1) and glucocorticoid-stimulated M1 macrophages (GC-M1) on BMSC differentiation. Additionally, miR-1a-3p expression was altered in vitro and in vivo to assess its role in regulating adipogenic differentiation.ResultsIn vivo, the colocalization of M1 macrophages and BMSCs was observed, and an increase in M1 macrophage numbers and a decrease in bone repair capabilities were further confirmed in both GA-ONFH patients and mouse models. Both n-M1 and GC-M1 were identified as contributors to the inhibition of osteogenic differentiation in BMSCs to a certain extent via exosome secretion. More importantly, exosomes derived from GC-M1 macrophages exhibited a heightened capacity to regulate the adipogenic differentiation of BMSCs, which was mediated by miR-1a-3p. In vivo and in vitro, miR-1a-3p promoted the adipogenic differentiation of BMSCs by targeting Cebpz and played an important role in the onset and progression of GA-ONFH.ConclusionWe demonstrated that exosomes derived from GC-M1 macrophages disrupt the balance between osteogenic and adipogenic differentiation in BMSCs, contributing to the pathogenesis of GA-ONFH. Inhibiting miR-1a-3p expression, both in vitro and in vivo, significantly mitigates the preferential adipogenic differentiation of BMSCs, thus slowing the progression of GA-ONFH. These findings provide new insights into the regulatory mechanisms underlying GA-ONFH and highlight potential therapeutic targets for intervention.Graphical

Endogenous glucocorticoids are required for normal macrophage activation and gastric Helicobacter pylori immunity.

Glucocorticoids are steroid hormones well known for their potent anti-inflammatory effects. However, their immunomodulatory properties are multifaceted. Increasing evidence suggests that glucocorticoid signaling promotes effective immunity and that disruption of glucocorticoid signaling impairs immune function. In this study, we conditionally deleted the glucocorticoid receptor (GR) in the myeloid lineage using the LysM-Cre driver (myGRKO). We examined the impact on macrophage activation and gastric immune responses to Helicobacter pylori, the best-known risk factor of gastric cancer. Our results indicate that, compared with wild type (WT), glucocorticoid receptor knockout (GRKO) macrophages exhibited higher expression of proinflammatory genes in steroid-free conditions. However, when challenged in vivo, GRKO macrophages exhibited aberrant chromatin landscapes and impaired proinflammatory gene expression profiles. Moreover, gastric colonization with H. pylori revealed impaired gastric immune responses and reduced T cell recruitment in myGRKO mice. As a result, myGRKO mice were protected from atrophic gastritis and pyloric metaplasia development. These results demonstrate a dual role for glucocorticoid signaling in preparing macrophages to respond to bacterial infection but limiting their pathogenic activation. In addition, our results support that macrophages are critical for gastric H. pylori immunity.NEW & NOTEWORTHY Signaling by endogenous glucocorticoids primes macrophages toward more robust responses to pathogens. Disruption of glucocorticoid signaling caused dysregulation of the chromatin landscape, blunted proinflammatory gene activation upon bacterial challenge, and impaired the gastric inflammatory response to Helicobacter pylori infection.

Hyaluronic acid-coated polypeptide nanogel enhances specific distribution and therapy of tacrolimus in rheumatoid arthritis

Rheumatoid arthritis (RA) involves chronic inflammation, oxidative stress, and complex immune cell interactions, leading to joint destruction. Traditional treatments are often limited by off-target effects and systemic toxicity. This study introduces a novel therapeutic approach using hyaluronic acid (HA)-conjugated, redox-responsive polyamino acid nanogels (HA-NG) to deliver tacrolimus (TAC) specifically to inflamed joints. The nanogels’ disulfide bonds enable controlled TAC release in response to high intracellular glutathione (GSH) levels in activated macrophages, prevalent in RA-affected tissues. In vitro results demonstrated that HA-NG/TAC significantly reduced TAC toxicity to normal macrophages and showed high biocompatibility. In vivo, HA-NG/TAC accumulated more in inflamed joints compared to non-targeted NG/TAC, enhancing therapeutic efficacy and minimizing side effects. Therapeutic evaluation in collagen-induced arthritis (CIA) mice revealed HA-NG/TAC substantially reduced paw swelling, arthritis scores, synovial inflammation, and bone erosion while suppressing pro-inflammatory cytokine levels. These findings suggest that HA-NG/TAC represents a promising targeted drug delivery system for RA, offering potential for more effective and safer clinical applications.

Specnuezhenide ameliorates hepatic fibrosis via regulating SIRT6-Mediated inflammatory signaling cascades

Ethnopharmacological relevanceLigustrum lucidum W.T. Aiton is a traditional Chinese medicine that has long been used with high hepatoprotective therapeutic and condition value. Specnuezhenide (SP), the standard prominent secoiridoid compound of Fructus Ligustri Lucidi may ameliorate hepatic inflammation in chronic liver diseases. Aim of the studyRegulating inflammation through SIRT6-P2X7R axis has caused the emergence of novel molecular mechanism strategies for reversing hepatic fibrosis. This study focused on the mechanism of SP in modulating the liver inflammatory microenvironment in hepatic fibrosis. Materials and methodsC57BL/6 mice with hepatic fibrosis were stimulated with thioacetamide (TAA) prior to administration of SP. Hepatic stellate cells (HSCs) or normal mouse primary hepatocytes were exposed to transforming growth factor-β (TGF-β) treatment. Meanwhile, normal mouse bone marrow-derived macrophages (BMDMs) were treated with lipopolysaccharide/adenosine triphosphate (LPS/ATP), aiming to obtain the conditioned medium. HSCs and hepatocytes were transfected with SIRT6 knockdown vector (siRNA-SIRT6) to estimate the impact of SP on the SIRT6-P2X7R/NLRP3 signaling pathway. ResultsSP suppressed the HSCs extracellular matrix (ECM) deposition as well as pro-inflammatory cytokine levels induced by the medium of BMDMs or TGF-β. In addition, SP also significantly up-regulated SIRT6, inhibited P2X7R-NLRP3 inflammasome in HSCs and hepatocytes, and functioned as MDL-800 (a SIRT6 agonist). SP reduced the hepatocytes pyroptosis and further prevented the occurrence of inflammatory response in the liver. SP could inhibit the activation of BMDMs and impede IL-1β and IL-18 from entering extracellular regions. Moreover, deficiency of SIRT6 in HSCs or hepatocytes reduced SP's regulation of P2X7R suppression. For TAA-treated mice, SP mitigated histopathological changes, ECM accumulation, EMT process, and NETs formation in hepatic fibrosis. ConclusionsTherefore, SP decreased inflammatory response via SIRT6-P2X7R/NLRP3 pathway and suppressed fibrillogenesis. These findings supported SP as the novel candidate to treat hepatic fibrosis.

Reprogramming tumor-associated macrophages and inhibiting tumor neovascularization by targeting MANF–HSF1–HSP70-1 pathway: An effective treatment for hepatocellular carcinoma

In advanced hepatocellular carcinoma (HCC) tissues, M2-like tumor-associated macrophages (TAMs) are in the majority and promotes HCC progression. Contrary to the pro-tumor effect of M2-like TAMs, M1-like TAMs account for a small proportion and have anti-tumor effects. Since TAMs can switch from one type to another, reprogramming TAMs may be an important treatment for HCC therapy. However, the mechanisms of phenotypic switch and reprogramming TAMs are still obscure. In this study, we analyzed differential genes in normal macrophages and TAMs, and found that loss of MANF in TAMs accompanied by high levels of downstream genes negatively regulated by MANF. MANF reprogrammed TAMs into M1 phenotype. Meanwhile, loss of MANF promoted HCC progression in HCC patients and mice HCC model, especially tumor neovascularization. Additionally, macrophages with MANF supplement suppressed HCC progression in mice, suggesting MANF supplement in macrophage was an effective treatment for HCC. Mechanistically, MANF enhanced the HSF1–HSP70-1 interaction, restricted HSF1 in the cytoplasm of macrophages, and decreased both mRNA and protein levels of HSP70-1, which in turn led to reprogramming TAMs, and suppressing neovascularization of HCC. Our study contributes to the exploration the mechanism of TAMs reprogramming, which may provide insights for future therapeutic exploitation of HCC neovascularization.

Synthesis and biological evaluation of orally active anti-Trypanosoma agents

In previous studies, we developed anti-trypanosome tubulin inhibitors with promising in vitro selectivity and activity against Human African Trypanosomiasis (HAT). However, for such agents, oral activity is crucial. This study focused on further optimizing these compounds to enhance their ligand efficiency, aiming to reduce bulkiness and hydrophobicity, which should improve solubility and, consequently, oral bioavailability. Using Trypanosoma brucei brucei cells as the parasite model and human normal kidney cells and mouse macrophage cells as the host model, we evaluated 30 new analogs synthesized through combinatorial chemistry. These analogs have fewer aromatic moieties and lower molecular weights than their predecessors. Several new analogs demonstrated IC50s in the low micromolar range, effectively inhibiting trypanosome cell growth without harming mammalian cells at the same concentration. We conducted a detailed structure–activity relationship (SAR) analysis and a docking study to assess the compounds’ binding affinity to trypanosome tubulin homolog. The results revealed a correlation between binding energy and anti-Trypanosoma activity. Importantly, compound 7 displayed significant oral activity, effectively inhibiting trypanosome cell proliferation in mice.

Abstract PO4-28-11: Murine Breast Cancer Cell Culture Supernatant Induces CAF-like and TAM-like Traits in Normal Cells

Abstract Several studies in the past have demonstrated the role of cell-to-cell interaction (crosstalk) between tumor cells and normal (nontumorigenic) cells in cancer progression and metastasis. Within the tumor microenvironment (TME), these interactions have a potential to transform the phenotypes and the behaviors of normal cells. Application of the 2D in-vitro cultures has been limited due to its inability to replicate the complex in-vivo TME. Conditioned Medium (CM) obtained from cultured cancer cells contains secreted growth factors that potentially regulate the phenotype and the functionality of normal cells. In this study, a culture of normal murine fibroblast NIH3T3 and macrophage RAW 264.7 cells with conditioned medium (CM) obtained from malignant mammary epithelial 4T1 cells (4T1CM) resulted in an altered phenotype with increased cell viability. 4T1CM treated NIH3T3 and RAW 264.7, in comparison with the respective control cells, resulted in an upregulation of the genes including- α-smooth muscle actin (αSMA), IL-10, CD206, and vascular endothelial growth factor (VEGF). In addition, 4T1.CM treated NIH3T3 showed an EMT phenotype as indicated by the regulation of EMT markers such as, E-cadherin, β-catenin, N-cadherin, and Vimentin. Interestingly, an upregulation of cyclooxygenase-2 (COX-2) and programmed death-ligand 1 (PDL-1) was observed in 4T1CM treated RAW 264.7, a tendency towards exhibiting an inhibitory immune response. Intriguingly, NIH3T3 cells conditioned with 4T1CM demonstrated an upregulation of stemness markers including- sex determining region Y-box 2, and Aldehyde dehydrogenase. Collectively, our study suggested a role for 4T1CM in transforming the normal NIH3T3 and RAW 264.7 into cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). RNAseq experiments are underway to map differentially expressed genes (DEGs) that potentially regulate the 4T1CM induced transformation of NIH3T3 and RAW 264.7 cells. Furthermore, invitro drug testing in 3D model of these transformed cells to target the pathway intermediates may provide novel therapeutic intervention strategies. Citation Format: Biplov Sapkota, Naveen Chintalaramulu, Abhishek Pandit, Shilpa Thota, Rizwana Begum, Joseph Francis. Murine Breast Cancer Cell Culture Supernatant Induces CAF-like and TAM-like Traits in Normal Cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-11.

Vascular Endothelial Mitochondrial Oxidative Stress in Response to Placental Macrophages from Preeclamptic Pregnancies

Preeclampsia (PE), a hypertensive disorder of pregnancy characterized by maternal hypertension, fetal growth restriction (FGR), and chronic immune activation and inflammation, is a major cause of maternal and fetal morbidity and mortality worldwide. Prior clinical studies demonstrate that proinflammatory M1 macrophages are increased in women with PE. We do not understand the role that M1 macrophage polarization may play in PE pathophysiology. We set out to begin characterization of macrophages in patient samples obtained from the UMMC biobank and their ability to induce mitochondrial oxidative stress (ROS) in human umbilical vein endothelial cells (HUVECs). After obtaining informed consent, whole blood and placental tissues were collected from normal pregnant (n=5) and preeclamptic women (n=8) who delivered in the labor and delivery unit UMMC. Lymphocytes were isolated from blood on a Ficoll-Histopaque gradient and analyzed by flow cytometry for total and M1 macrophages. Total macrophages were isolated from digested placental tissue by magnetic bead separation using the Human Macrophage Isolation Kit (Miltenyi). Isolated macrophages were co-cultured with HUVECs overnight. After removal of culture inserts containing macrophages, HUVECs were serum-starved and incubated with MitoSox Red, collected, and analyzed on a flow cytometer. Total circulating macrophages were increased in placentas from PE women vs normal pregnant patients (10.8% PMBCs vs. 3.5% PMBCs, respectively, p<0.05). Furthermore, macrophage polarization to M1 macrophages was significantly higher in women with PE (15.02% of total macrophages) compared to NP women (6.23% of total macrophages, p<0.05 vs. PE). We also determined that mitochondrial ROS production was significantly elevated in HUVEC cells co-cultured with macrophages from PE pregnancies (15.37 A.U.) when compared to HUVECs co-cultured with normal pregnant control macrophages (7.788 A.U., p<0.05 vs. PE). Future studies will include enrollment of additional participants and determination of endothelial activation by measurement of adhesion molecules, growth factors, extracellular matrix proteins, and cytokines in HUVECs and conditioned media via Western Blot, ELISA, and multiplex analysis. These data show that PE placental macrophages induce mitochondrial ROS production in endothelial cells and may play a role to directly induce vascular dysfunction contributing to hypertension during a preeclamptic pregnancy. This work was supported by National Institutes of Health Grants T32HL105324 to C. A. Shields, and R01HL151407 to D. C. Cornelius. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Phenotypic comparison and the potential antitumor function of immortalized bone marrow-derived macrophages (iBMDMs).

Macrophages are an important component of innate immunity and involved in the immune regulation of multiple diseases. The functional diversity and plasticity make macrophages to exhibit different polarization phenotypes after different stimuli. During tumor progression, the M2-like polarized tumor-associated macrophages (TAMs) promote tumor progression by assisting immune escape, facilitating tumor cell metastasis, and switching tumor angiogenesis. Our previous studies demonstrated that functional remodeling of TAMs through engineered-modifying or gene-editing provides the potential immunotherapy for tumor. However, lack of proliferation capacity and maintained immune memory of infused macrophages restricts the application of macrophage-based therapeutic strategies in the repressive tumor immune microenvironment (TIME). Although J2 retrovirus infection enabled immortalization of bone marrow-derived macrophages (iBMDMs) and facilitated the mechanisms exploration and application, little is known about the phenotypic and functional differences among multi kinds of macrophages. HE staining was used to detect the biosafety of iBMDMs, and real-time quantitative PCR, immunofluorescence staining, and ELISA were used to detect the polarization response and expression of chemokines in iBMDMs. Flow cytometry, scratch assay, real-time quantitative PCR, and crystal violet staining were used to analyze its phagocytic function, as well as its impact on tumor cell migration, proliferation, and apoptosis. Not only that, the inhibitory effect of iBMDMs on tumor growth was detected through subcutaneous tumor loading, while the tumor tissue was paraffin sectioned and flow cytometry was used to detect its impact on the tumor microenvironment. In this study, we demonstrated iBMDMs exhibited the features of rapid proliferation and long-term survival. We also compared iBMDMs with RAW264.7 cell line and mouse primary BMDMs with in vitro and in vivo experiments, indicating that the iBMDMs could undergo the same polarization response as normal macrophages with no obvious cellular morphology changes after polarization. What's more, iBMDMs owned stronger phagocytosis and pro-apoptosis functions on tumor cells. In addition, M1-polarized iBMDMs could maintain the anti-tumor phenotypes and domesticated the recruited macrophages of receptor mice, which further improved the TIME and repressed tumor growth. iBMDMs can serve as a good object for the function and mechanism study of macrophages and the optional source of macrophage immunotherapy.

Matrix metalloproteinase-sensitive size-shrinkable liposomes targeting activated macrophages for the treatment of rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease characterized by inflammation, swelling, pain, and cartilage and bone destruction. Exploiting the pathophysiological characteristics of matrix metalloproteinases (MMPs), which are enzymes overexpressed at the inflammation site in RA, has resulted in the development of inflammation-responsive drug delivery vectors. We here prepared a multifunctional microenvironment-responsive liposome with MMP-responsive and particle-size-shrinking properties to target macrophages at inflammation sites. In this liposome, triptolide (TP) was encapsulated in a biofilm-like bilayer with mannose-conjugated DSPE-PEG2000 as the targeting molecule and the enzyme-sensitive peptide PVGLIG as the linker arm connecting DSPE-PEG5000, which formed a hydrated membrane envelope on the liposome surface, prolonging liposome circulation while preventing the active targeting molecule from binding to normal macrophages throughout the body and reducing drug accumulation in normal tissues, thereby alleviating efficacy and reducing toxic side effects. In the joint inflammation microenvironment, PVGLIG was cleaved by overexpressed MMPs, the hydration layer of PEG5000 detached, and the target ligand Man was exposed, thereby leading to Man uptake by activated macrophages and exerting an anti-RA effect. Through in vivo and in vitro evaluations, liposomes were found to effectively target inflammation sites and enrich activated macrophages. They also inhibited osteoclastogenesis and the release of inflammatory cytokines, reactive oxygen species, and MMPs. This offers a promising new strategy in which anti-inflammatory and osteoprotective effects are combined for comprehensive RA treatment.

Abstract 4219: Establishing CAF-like and TAM-like transformation induced by TNBC culture supernatant in 3D in-vitro culture as a model for targeted drug testing

Abstract Several previous studies have highlighted the significance of cell-to-cell communication, or crosstalk, between tumor cells and non-tumorigenic cells in the context of cancer progression and metastasis. Within the tumor microenvironment (TME), these interactions have the potential to alter the phenotypes and behaviors of normal cells. The use of 2D in-vitro cultures has been limited due to their inability to accurately replicate the intricate in-vivo TME. Conditioned medium (CM) derived from cultured cancer cells contains secreted factors that may influence the phenotype and functionality of normal cells. In our investigation, the exposure of normal murine fibroblast NIH3T3 and macrophage RAW 264.7 cells to conditioned medium (CM) obtained from malignant mammary epithelial 4T1 cells (4T1CM) resulted in a modified phenotype with enhanced cell viability. Treatment with 4T1CM led to the upregulation of genes, including α-smooth muscle actin (αSMA), IL-10, CD206, and vascular endothelial growth factor (VEGF), in NIH3T3 and RAW 264.7 cells compared to their respective control cells. Additionally, NIH3T3 cells treated with 4T1CM exhibited an epithelial-mesenchymal transition (EMT) phenotype, as indicated by the regulation of EMT markers such as E-cadherin, β-catenin, N-cadherin, and Vimentin. Notably, RAW 264.7 cells treated with 4T1CM showed an upregulation of cyclooxygenase-2 (COX-2) and programmed death-ligand 1 (PDL-1), suggesting a propensity for an inhibitory immune response. Moreover, NIH3T3 cells conditioned with 4T1CM demonstrated an upregulation of stemness markers, including sex determining region Y-box 2 and Aldehyde dehydrogenase. In summary, our study highlights the potential role of 4T1CM in transforming normal NIH3T3 and RAW 264.7 cells into cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). Ongoing proteomics analysis aims to map differentially expressed proteins potentially regulating the 4T1CM-induced transformation. To further investigate, a series of experiments using mono- and co-cultures of these cells are underway, specifically targeting the transformed macrophages and fibroblasts with the aim of either re-educating or eliminating them. This treatment involves drug combinations that specifically target the identified signaling pathway intermediates, offering novel therapeutic intervention strategies. Citation Format: Biplov Sapkota, Naveen Chintalaramulu, Abhishek Pandit, Shilpa Thota, Rizwana Begum, Amirsalar Mansouri, Jiri Adamec, Joseph Francis. Establishing CAF-like and TAM-like transformation induced by TNBC culture supernatant in 3D in-vitro culture as a model for targeted drug testing [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 4219.

Abstract 6627: Boosting macrophage-specific BCAA oxidation enhances immune activation within the tumor microenvironment and diminishes tumor growth in pancreatic cancer

Abstract Treating pancreatic ductal adenocarcinoma (PDAC) poses a significant challenge, mainly due to therapy resistance linked to the immunosuppressive tumor microenvironment (TME) within PDAC tumors. This TME is characterized by a fibrotic stroma and an abundance of immunosuppressive cells, notably tumor-associated macrophages (TAMs). TAMs crucially hinder effector T cell activity and contribute to T cell exhaustion. Redirecting TAMs toward an immunostimulatory state has emerged as a promising approach in PDAC treatment. Recent investigations, including our own studies, reveal a profound influence of metabolic pathways on the immune phenotype of TAMs. Specifically, dysregulation in branched-chain amino acid (BCAA) metabolism has been observed in PDAC patients, manifesting as elevated circulating BCAA levels correlating with a doubled risk of PDAC development. However, the extent to which BCAA metabolism influences TAM phenotype and anti-tumor immune responses remains a largely unexplored territory, presenting a challenge for PDAC treatment. Our focus revolves around understanding how heightened BCAA levels in PDAC affect TAM phenotype, the immunosuppressive tumor environment, and tumor progression. Employing an orthotopic PDAC model and genetically modified mice, specifically BCKDKfl/fl BCKDK LysMcre/+ (mimicking increased BCAA oxidation), alongside a BCKDK-targeting pharmacological inhibitor (BT-2), multicolor flow cytometry, and in vitro models, our research has revealed crucial insights. We found a diminished expression of BCAA oxidation genes in TAMs compared to normal pancreatic macrophages. Interestingly, M1 and M2 macrophages seemed to utilize BCAAs differently, reflected in varying levels of TCA cycle intermediates derived from BCAA oxidation. Notably, boosting BCAA oxidation specifically in TAMs through genetic modifications resulted in reduced PDAC growth associated with an enhanced immunostimulatory TAM phenotype and activation of CD8+ T cells in the TME. Conversely, administering BCAAs induced an immunosuppressive TAM phenotype, fostering PDAC growth, suggesting a direct influence of BCAAs on TAM phenotype. Pharmacologically enhancing BCAA oxidation via BT-2 bolstered immune activation within the tumor environment and reduced PDAC growth. Intriguingly, our analysis of publicly available data unveiled a significant correlation between high BCAA oxidation and favorable responses to anti-PD1 therapy in cancer patients. Our findings emphasize the pivotal role of BCAA metabolism in shaping the immune landscape of PDAC, offering potential avenues for therapeutic interventions targeting TAMs and mitigating TME immunosuppression. Citation Format: Gauri Mirji, Sajad Ahmad Bhat, Zachary Schug, Ben Z. Stanger, Zoltan Arany, Rahul S. Shinde. Boosting macrophage-specific BCAA oxidation enhances immune activation within the tumor microenvironment and diminishes tumor growth in pancreatic cancer [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 6627.

Bioinspired Sonodynamic Nano Spray Accelerates Infected Wound Healing via Targeting and Disturbing Bacterial Metabolism

AbstractBacterial infections pose a major concern for the medical community, especially regarding wound healing. Traditional passive antibiotic therapies can be cytotoxic and lead to bacterial resistance, posing a continuing challenge to treat. Based on precision therapy, a novel targeted‐delivery nanosystem is developed to efficiently eliminate bacteria and promote bacteria‐infected wound healing. Macrophage membranes pre‐activated by Staphylococcus aureus (MMSa) are prepared. In doing so, Toll‐like receptors (TLRs), a typical pathogen‐associated molecular pattern (PAMP), are significantly higher than normal macrophage membranes (MM0). Subsequently, MMSa are coated onto ultrasound‐triggered piezocatalytic nano‐barium titanate (BaTiO3, BTO) surfaces, and these two components are assembled to form a novel targeting delivery nanosystem, namely BTO@MMSa. The in vitro and in vivo results demonstrate that the biocompatible BTO@MMSa nanosystem can target infected areas and rapidly generate reactive oxygen species (ROS) to kill bacteria under ultrasound (US) irradiation, as well as accelerate wound healing. Furthermore, prokaryotic RNA‐seq transcriptomics reveals that changes in bacterial membrane function and substance, and energy metabolism are responsible for the targeted antibacterial ability of BTO@MMSa with US. Compared to widely reported unselective antibacterial agents, this novel targeted delivery nanosystem has potential for precise bacterial infection treatment by using macrophage membrane functions.

A227 EXPLORING THE ROLE OF MICROBIAL AND GENETIC FACTORS IN DEFINING THE INTESTINAL IMMUNE SYSTEM USING A HUMANIZED GNOTOBIOTIC MOUSE MODEL

Abstract Background Studies of the gut microbial ecosystem and its role in disease are often complicated by the intricate network of interactions between its microbial members and the host. Therefore, mouse models in which the microbiota diversity can be strictly controlled, and the functional potential be easily assessed, are needed. Aims Establishing a taxonomically and functionally diverse human gnotobiotic microbiota that recapitulates many microbe-host interactions of the complex human microbiome. To validate the effects of the defined bacterial gut community on the host, we will assess host physiology, intestinal immunity and the response to inflammatory stimuli. Methods Germ-free (GF) mice were inoculated with a defined bacterial community isolated from the stool of a healthy human donor. Stability of colonization across multiple generations and spatial organization of the bacterial community in the intestine were assessed using 16S rRNA gene sequencing and species-specific quantitative PCR. Immune cells were isolated from the colonic lamina propria and characterized by flow cytometry. Results Inoculated GF mice were reproducibly colonized with 8 bacterial species, collectively termed Human Defined Community 1 (HDC1). The HDC1 is vertically transmitted from mothers to offspring and the relative abundances remain stable in the colony over an extended period in a gnotobiotic isolator. We observed spatial stratification of the HDC1 microbiota between the small intestine and colon or cecum. HDC1 mice form a colonic inner mucus layer comparable in thickness to SPF mice suggesting physiological spatial organization of the bacterial community over the cross-sectional axis of the colon. Characterization of colonic lamina propria immune cells in HDC1 mice revealed myeloid and lymphoid cell abundances closely resembling those of mice harboring a complex microbiota (specific pathogen-free [SPF] mice), including normal macrophage and dendritic cell counts and normal proportions of CD4+ cells (regulatory T, Th1 and Th17 cells). Finally, we showed that HDC1 mice can be transplanted with human gut commensals and Crohn's disease (CD)-associated bacteria, demonstrating the tractability of this human defined microbiota. Conclusions In summary, HDC1 gnotobiotic mice are stably colonized with a human defined bacterial community and harbor an intestinal innate and adaptive immune system similar to SPF mice. Therefore, HDC1 mice represent a reliable and tractable tool for host-microbe interaction studies. Ultimately, we will employ the HDC1 model to decipher the role of Nod2 – the strongest genetic risk factor for CD development – in shaping both the microbiota and host immunity. Funding Agencies CIHROGS

Enhanced Sensitivity of A549 Cells to Doxorubicin with WS2 and WSe2 Nanosheets via the Induction of Autophagy.

The excellent physicochemical properties of two-dimensional transition-metal dichalcogenides (2D TMDCs) such as WS2 and WSe2 provide potential benefits for biomedical applications, such as drug delivery, photothermal therapy, and bioimaging. WS2 and WSe2 have recently been used as chemosensitizers; however, the detailed molecular basis underlying WS2- and WSe2-induced sensitization remains elusive. Our recent findings showed that 2D TMDCs with different thicknesses and different element compositions induced autophagy in normal human bronchial epithelial cells and mouse alveolar macrophages at sublethal concentrations. Here, we explored the mechanism by which WS2 and WSe2 act as sensitizers to increase lung cancer cell susceptibility to chemotherapeutic agents. The results showed that WS2 and WSe2 enhanced autophagy flux in A549 lung cancer cells at sublethal concentrations without causing significant cell death. Through the autophagy-specific RT2 Profiler PCR Array, we identified the genes significantly affected by WS2 and WSe2 treatment. Furthermore, the key genes that play central roles in regulating autophagy were identified by constructing a molecular interaction network. A mechanism investigation uncovered that WS2 and WSe2 activated autophagy-related signaling pathways by interacting with different cell surface proteins or cytoplasmic proteins. By utilizing this mechanism, the efficacy of the chemotherapeutic agent doxorubicin was enhanced by WS2 and WSe2 pre-treatment in A549 lung cancer cells. This study revealed a feature of WS2 and WSe2 in cancer therapy, in which they eliminate the resistance of A549 lung cancer cells against doxorubicin, at least partially, by inducing autophagy.

Gelatin-decorated Graphene oxide: A nanocarrier for delivering pH-responsive drug for improving therapeutic efficacy against atherosclerotic plaque

The progressive inflammatory disease atherosclerosis promotes myocardial infarction, stroke, and heart attack. Anti-inflammatory drugs treat severe atherosclerosis. They are inadequate bioavailability and cause adverse effects at higher doses. A new nanomaterial coupled pH-apperceptive drug delivery system for atherosclerotic plaque is outlined here. We have synthesized a Graphene Oxide-Gelatin-Atorvastatin (GO-Gel-ATR) nanodrug characterized by spectroscopic and imaging techniques. The encapsulation efficiency of GO-Gel-ATR (79.2%) in the loading process is observed to be better than GO-ATR (66.8%). The internal milieu of the plaque cells has a pH of 6.8. The GO-Gel-ATR displays sustained and cumulative release profile at pH 6.8 compared to ATR and GO-ATR. Our proposed nanocomposite demonstrated high cytocompatibility up to 100μg/mL in foam cells induced by Oxidized-Low Density Lipoprotein (Ox-LDL) and Lipopolysaccharides (LPS) compared to normal macrophages for 24 and 48 h. The uptake efficacy of the nanodrugs is shown to be enhanced in foam cells compared to normal macrophage. Oil red O staining of foam cells with and without drugs confirmed therapeutic efficacy. Foam cells treated with nanocomposite had more lipids efflux than ATR. The finding of the in-vitro study reveals that the GO-Gel-ATR nanocomposite carriers have the potential to deliver anti-atherosclerotic drugs effectively and inhibit atherosclerotic plaque progression.

Cell Membrane-Coated Nanotherapeutics for the Targeted Treatment of Acute and Chronic Colitis.

Integrin α4β1 and α4β7 are overexpressed in macrophages and leukocytes and play important roles in mediating cell homing and recruitment to inflammatory tissues. Herein, to enhance the targeting ability of nanotherapeutics for inflammatory bowel disease (IBD) treatment, cyclosporine A-loaded nanoparticles (CsA NPs) were coated with macrophage membranes (MM-CsA NPs) or leukocyte membranes (LM-CsA NPs). Invitro experiments demonstrated that the physicochemical properties of the nanotherapeutics (e.g., size, zeta potential, polymer dispersity index, and drug release profiles) did not obviously change after cell membrane coating. However, integrin α4β1 and α4β7 were expressed in MM-CsA NPs and LM-CsA NPs, respectively, which significantly inhibited normal macrophage phagocytosis and obviously increased uptake by proinflammatory macrophages and endothelial cells. Invivo experiments verified that cell membrane-coated nanotherapeutics have longer retention times in inflammatory intestinal tissues. Importantly, LM-CsA NPs significantly mitigated weight loss, alleviated colon shortening, decreased disease activity indices (DAIs), and promoted colon tissue repair in acute and chronic colitis model mice. Furthermore, LM-CsA NPs significantly decreased the expression of inflammatory factors such as TNF-α and IL-6 and increased the expression of gut barrier-related proteins such as E-cadherin, ZO-1, and occludin protein in colitis mice.

SNHG17 alters anaerobic glycolysis by resetting phosphorylation modification of PGK1 to foster pro-tumor macrophage formation in pancreatic ductal adenocarcinoma

BackgroundWithin the tumor immune microenvironment (TME), tumor-associated macrophages (TAMs) are crucial in modulating polarization states to influence cancer development through metabolic reprogramming. While long non-coding RNAs (lncRNAs) have been shown to play a pivotal role in the progression of various cancers, the underlying mechanisms by which lncRNAs alter M2 polarization through macrophage metabolism remodeling remain unelucidated.MethodsRNA sequencing was used to screen for differentially expressed lncRNAs in TAMs and normal tissue-resident macrophages (NTRMs) isolated from pancreatic ductal adenocarcinoma (PDAC) tissues, whilst RT-qPCR and FISH were employed to detect the expression level of SNHG17. Moreover, a series of in vivo and in vitro experiments were conducted to assess the functions of SNHG17 from TAMs in the polarization and glycolysis of M2-like macrophages and in the proliferation and metastasis of pancreatic cancer cells (PCs). Furthermore, Western blotting, RNA pull-down, mass spectrometry, RIP, and dual-luciferase assays were utilized to explore the underlying mechanism through which SNHG17 induces pro-tumor macrophage formation.ResultsSNHG17 was substantially enriched in TAMs and was positively correlated with a worse prognosis in PDAC. Meanwhile, functional assays determined that SNHG17 promoted the malignant progression of PCs by enhancing M2 macrophage polarization and anaerobic glycolysis. Mechanistically, SNHG17 could sponge miR-628-5p to release PGK1 mRNA and concurrently interact with the PGK1 protein, activating the pro-tumorigenic function of PGK1 by enhancing phosphorylation at the T168A site of PGK1 through ERK1/2 recruitment. Lastly, SNHG17 knockdown could reverse the polarization status of macrophages in PDAC.ConclusionsThe present study illustrated the essential role of SNHG17 and its molecular mechanism in TAMs derived from PDAC, indicating that SNHG17 might be a viable target for PDAC immunotherapy.

Alveolar macrophage-derived gVPLA2 promotes ventilator-induced lung injury via the cPLA2/PGE2 pathway

BackgroundVentilator-induced lung injury (VILI) is a clinical complication of mechanical ventilation observed in patients with acute respiratory distress syndrome. It is characterized by inflammation mediated by inflammatory cells and their secreted mediators.MethodsTo investigate the mechanisms underlying VILI, a C57BL/6J mouse model was induced using high tidal volume (HTV) mechanical ventilation. Mice were pretreated with Clodronate liposomes to deplete alveolar macrophages or administered normal bone marrow-derived macrophages or Group V phospholipase A2 (gVPLA2) intratracheally to inhibit bone marrow-derived macrophages. Lung tissue and bronchoalveolar lavage fluid (BALF) were collected to assess lung injury and measure Ca2 + concentration, gVPLA2, downstream phosphorylated cytoplasmic phospholipase A2 (p-cPLA2), prostaglandin E2 (PGE2), protein expression related to mitochondrial dynamics and mitochondrial damage. Cellular experiments were performed to complement the animal studies.ResultsDepletion of alveolar macrophages attenuated HTV-induced lung injury and reduced gVPLA2 levels in alveolar lavage fluid. Similarly, inhibition of alveolar macrophage-derived gVPLA2 had a similar effect. Activation of the cPLA2/PGE2/Ca2 + pathway in alveolar epithelial cells by gVPLA2 derived from alveolar macrophages led to disturbances in mitochondrial dynamics and mitochondrial dysfunction. The findings from cellular experiments were consistent with those of animal experiments.ConclusionsHTV mechanical ventilation induces the secretion of gVPLA2 by alveolar macrophages, which activates the cPLA2/PGE2/Ca2 + pathway, resulting in mitochondrial dysfunction. These findings provide insights into the pathogenesis of VILI and may contribute to the development of therapeutic strategies for preventing or treating VILI.