Macrophage Endocytosis Research Articles

Hydrogen sulfide inhibits early development of atherosclerosis by modulating macrophage uptake of oxidized lipoproteins.

Atherosclerosis, a major cause of cardiovascular diseases, is characterized by the accumulation of oxidized lipoproteins (ox-LDL) within arterial walls, leading to inflammation and plaque formation. Hydrogen sulfide (H2S) has demonstrated anti-inflammatory and vascular protective properties, but its role in modulating macrophage endocytosis of ox-LDL and its impact on early atherosclerosis development remains unclear. Macrophage cultures were utilized for ox-LDL uptake experiments. Macrophages were pretreated with sodium hydrosulfide (NaHS) (50 μmol/L) or propargylglycine (PPG, 3 mmol/L) for 1 h, followed by incubation with DiI-ox-LDL (10 μg/mL) for an additional 2 h. DiI-ox-LDL uptake was visualized using live-cell imaging. The expression of scavenger receptors CD36 and SR-A was assessed through immunofluorescent staining and western blot analysis. To determine the intracellular signal transduction pathways involved, macrophages were pretreated with NF-κB pathway blocker pyrrolidine dithiocarbamate or MAPK inhibitor PD98059 before the addition of NaHS. NaHS significantly inhibited ox-LDL uptake by macrophages, while PPG treatment markedly increased this process. Immunocytochemistry and western blot analysis revealed that the expressions of CD36 and SR-A were induced by ox-LDL but inhibited by NaHS in a concentration- and time-dependent manner. Furthermore, H2S down-regulated ox-LDL receptors CD36 and SR-A through the NF-κB signal pathway. H2S inhibits early atherosclerosis development by modulating macrophage uptake of ox-LDL through the down-regulation of CD36 and SR-A receptors via the NF-κB signaling pathway. These findings provide new evidence for the role of H2S in atherosclerosis and its potential therapeutic value.

Macrophage-mediated controlled release of cysteine protease inhibitor from PLGA-PEG/hydroxyapatite microspheres for targeting cathepsin S in Alzheimer’s disease

The cause of Alzheimer’s disease (AD) remains unknown; however, studies have indicated the increased expression of cathepsin S (CTSS) in AD brains. Cathepsin S acts as a β-secretase, prompting the release of amyloid-β peptides and stimulating microglial cell activation, causing microglial migration, and contributing to neuroinflammation and disease progression. In this study, CT001, an inhibitor of the cysteine protease CTSS, was encapsulated in poly(lactic-co-glycolic acid)-polyethylene glycol microspheres combined with hydroxyapatite microspheres (CPPMs/HAMs) to achieve a constant and long-term drug release for AD. The results showed that CPPMs/HAMs had a uniform diameter of approximately 1 μm, enabling macrophage endocytosis and facilitating sustained CT001 release into the bloodstream, ultimately reaching the brain for AD treatment. The developed CPPMs/HAMs demonstrated excellent anti-inflammatory properties and mitigated CTSS expression in an in vitro inflammation microglial model. Intramuscular administration of CPPMs/HAMs improved learning and memory in AD mice during behavioral assessments. Furthermore, CPPMs/HAMs reduced CTSS expression, amyloid-beta accumulation, and deposition in vivo. This study demonstrated the potential of CT001 delivered via CPPMs/HAMs as a promising approach for managing AD in patients.

Allosteric inhibitor of SHP2 enhances macrophage endocytosis and bacteria elimination by increasing caveolae activation and protects against bacterial sepsis

The uncontrolled bacterial infection-induced cytokine storm and sequential immunosuppression are commonly observed in septic patients, which indicates that the activation of phagocytic cells and the efficient and timely elimination of bacteria are crucial for combating bacterial infections. However, the role of dysregulated immune cells and their disrupted function in sepsis remains unclear. Here, we found that macrophages exhibited the impaired endocytosis capabilities in sepsis by Single-cell RNA sequencing and bulk RNA sequencing. Caveolae protein Caveolin-1 (Cav-1) of macrophages was inactivated by SHP2 rapidly during Escherichia coli (E.coli) infection. Allosteric inhibitor of SHP2 effectively maintains Cav-1 phosphorylation to enhance macrophage to endocytose and eliminate bacteria. Additionally, TLR4 endocytosis of macrophage was also enhanced upon E.coli infection by SHP099, inducing an increased and rapidly resolved inflammatory response. In vivo, pretreatment or posttreatment with inhibitor of SHP2 significantly reduced the bacterial burden in organs and mortality of mice subjected E.coli infection or CLP-induced sepsis. The cotreatment of inhibitor of SHP2 with an antibiotic conferred complete protection against mortality in mice. Our findings suggest that Cav-1-mediated endocytosis and bacterial elimination may play a critical role in the pathogenesis of sepsis, highlighting inhibitor of SHP2 as a potential therapeutic agent for sepsis.

Mechanistic/mammalian target of rapamycin complex 1 (mTORC1) signaling is involved in phagocytosis activation during THP-1 cell differentiation.

Mechanistic/mammalian target of rapamycin complex 1 (mTORC1) is a serine/threonine kinase that plays a major role in cell metabolism. Although mTORC1 inhibitors are known to exert immunosuppressive effects, their effects on immune cells are not fully understood. In the present study, we examined the involvement of mTORC1 in the differentiation and functions of macrophages using THP-1 cells, which are derived from human monocytic leukemia and differentiate into macrophage-like cells upon treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). We also examined the effects of two mTOR inhibitors, Torin 1 and rapamycin, on TPA-stimulated THP-1 cells. Although mTORC1 activation was observed upon TPA stimulation, mTOR inhibitors did not affect TPA-induced morphological changes or expression of the general macrophage marker, CD11b. In contrast, phagocytosis and fluid endocytosis were significantly impaired by the mTOR inhibitors. Endocytosis suppression was observed when mTOR inhibitors were added during differentiation, but not before or after differentiation, suggesting that endocytosis suppression altered the direction of differentiation. Furthermore, mTOR inhibitors altered the expression of M1/M2 polarization markers. These results suggest that the immunosuppressive effects of mTOR inhibitors may involve the suppression of macrophage endocytosis caused by abnormal cell differentiation.

Targeting macrophage endocytosis via platelet membrane coating for advanced osteoimmunomodulation

SummaryThe identification, uptake, and clearance of nanoparticles (NPs) by phagocytes are critical in NP-based therapeutics. The cell membrane coating technique has recently emerged as an ideal surface modification approach to help NP bypass phagocytosis. CD47, a regulatory protein for phagocytosis, is a cell surface glycoprotein expressed on all cell types, including platelets. Herein, we enclosed bioactive glass (BG) with a platelet membrane to bestow BG with unique cell surface functions for immune evasion and immunomodulation. Compared with the uncoated particles, platelet membrane-coated BG shows reduced cellular uptake and can generate an immune environment favorable for osteogenesis. This is evidenced by the triggering of robust osteogenic differentiation in bone mesenchymal stromal cells, suggesting the synergistic effect of platelet membrane and BG in bone regeneration. These collectively indicate that cell membrane coating is a promising approach to enhance the therapeutic efficacy of biomaterials and thus provide new insight into biomaterial-mediated bone regeneration.

Sickle Cell Hemoglobin Induces Autophagy in Human Macrophages

BACKGROUND: Autophagy plays an important role in multiple cell processes including elimination of misfolded proteins and damaged organelles, clearance of intracellular microbes and regulation of innate immunity. Autophagic activity is significantly up regulated in diseases characterized by proteins aggregation such as Alzheimer's, Huntington's, and Parkinson's diseases. Sickle cell anemia (SCA) is associated with E6V mutation in beta-globin gene that induces hemoglobin (Hb) polymerization under low oxygen or acidic conditions. SCA is characterized by chronic presence of low-level plasma Hb (3-10 µM). Hb is cleared from circulation by macrophage's endocytosis, resulting in degradation of Hb in lysosomes. Acidic lysosome environment may induce Hb polymerization, lysosome dysregulation and autophagy activation.HYPOTHESIS: We hypothesize that endocytosis of HbS induces autophagy in human macrophages.METHODS: The study was approved by Howard University review board (IRB) and all subjects provided written inform consent prior the sample collection. Whole blood samples were obtained from three SCA patients and healthy individuals. Human THP-1 promonocytic cells were differentiated into macrophages with 25 nM PMA for 72 hrs and treated with either purified HbS or HbA (5 µM, Sigma-Aldrich). Total RNA was depleted of cytoplasmic and mitochondrial ribosomal RNA, and strand-specific libraries were constructed using the Illumina ® TruSeq Stranded Total RNA Gold kit. Libraries were sequenced on an Illumina ® NextSeq 500 using 75 bp paired-end sequencing on two v2.5 150 cycle High-Output kits, generating 40-50 million paired-end reads per sample. The sequencing data were mapped to human reference genome using Dragen RNA v.3.8.4 software (Illumina). Comparisons were conducted using Dragen differential expression software v. 3.6.3 software (Illumina). Transmission electron microscopy (TEM) imaging was performed at Talos 200X transmission electron microscope (Thermo Fisher Scientific). CYTO-ID Autophagy detection kit (Enzo Life Science) was used for detection of autophagy in live cells by fluorescence microscopy, and fluorescent microplate assay.RESULTS: In THP-1-derived macrophages treated with HbS compared to HbA, 28362 genes were detected including 322 differentially expressed genes (1.1%, 1.5-fold difference, 187 down and 135 up) at 5% FDR. In THP-1-differentiated macrophages treated with SCD RBC lysate compared to control RBC lysate, 30840 genes were detected including 256 differentially expressed genes (0.83%, 1.5-fold difference, 117 down and 139 up) at 5% FDR.We focused our analysis on 33 autophagy-related genes. Among these 33 genes, 30 genes were up-regulated (1.2-1.7-fold) and only 3 genes were down-regulated in macrophages treated with HbS compared to HbA (Fig.1). These changes were statistically significant for 20 genes (p<0.01). No significant differences were found in expression of autophagy-related genes in macrophages treated either with HbS or SCD RBC lysate.Autophagy activation was validated by CYTO-ID Autophagy detection kit, demonstrating significant autophagy activation in HbS treated macrophages compared to HbA treatment (1.2-fold, p=0.0019). Transmission electron microscopy demonstrated accumulation of autophagosomes in the cells treated with HbS compared to non-treated cells and cells treated with HbA.CONCLUSION: Treatment of macrophages with sickle cell hemoglobin or lysate of red blood cells collected from SCD patients up-regulates expression of autophagy-related genes and increased autophagosome formation.ACKNOWLEDGMENTS: We thank Drs. Castle Raley and Keith Crandall, George Washington University SPH Genomics Core, for sequencing and guidance in data analysis; Dr. Christine Brantner, GW Nanofabrication and Imaging Center, for TEM imaging; and students Kavita Pandya, Gretchen Whitney High School, Cerritos, CA, and Tharindi Jayatilake, Montgomery Blair High School, Silver Spring, MD, for help in analysis of autophagy-related gene expression. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract PO021: Lung cancer cells and cancer-associated fibroblasts drive macrophage polarization in a co-culture model

Abstract Introduction: Macrophages are key architects of the immune landscape in the tumor microenvironment (TME), and the plasticity of macrophage phenotype correlates with prognosis in non-small cell lung cancer (NSCLC). M2-like macrophages promote immunosuppression and facilitate tumor progression, while M1-like macrophages may drive an inflammatory antitumor immune response. We investigated if NSCLC oncogenotype impacts macrophage phenotype in a novel tumor cell, cancer-associated fibroblast (CAF) and macrophage co-culture model. We hypothesize that macrophage-mediated immunosuppression is driven in part through tumor-secreted signaling molecules that, if identified and blocked, may re-sensitize cancer cells to immune surveillance and enhance antitumor immunity. Methods: We developed an in vitro co-culture system (patient-derived NSCLC cells, human CAFs, and mouse macrophages) to interrogate impact of NSCLC cells and CAFs on macrophage phenotype. Through species specific qPCR we measured the expression salient macrophage genes (i.e. Arginase, NOS2, IL-1β, IL-6, CHIL-3, SOCS3) in the co-cultures. We also performed whole genome RNA sequencing (RNAseq) in select cases and evaluated protein expression using cytokine arrays measuring expression of 40 inflammatory cytokines. Positive controls included stimulation of macrophages with LPS and IL-4. Results: We characterized the effect of over 70 NSCLC cell lines in the co-culture assay. Three highly reproducible clusters of macrophage phenotypes were identified: high Arginase (immunosuppressive), high IL-1β (inflammatory) or high SOCS3 (inflammatory, involved in JAK-STAT3 pathway). Major NSCLC oncogenotypes (i.e. KRAS, TP53, STK11, EGFR, BRAF mutation) did not correlate with macrophage phenotype. Analyses of differences between the 3 clusters is ongoing. We selected 10 exemplar NSCLC lines representing each of these 3 clusters for RNA sequencing (mouse genes) and cytokine array protein (human) profiling. Across all clusters, we found suppression of macrophage endocytosis pathways and activation of scavenger receptor A (SRA) signaling (M2-like phenotype) and increased expression of human IL-6, IL-8, and MCP1, which are implicated in suppression of innate immune sensing of tumor cells. RNAseq of CAF lines demonstrated mixed inflammatory and fibroblastic phenotype, with increased expression of genes associated with macrophage recruitment and activation including: IL-6, CSF-1, CXCL6, CCL2, and CCL7. Conclusions: We demonstrate in a novel co-culture model that patient-derived NSCLC cells reproducibly induce three major macrophage phenotypes. Cytokine release from NSCLC cells and CAFs are implicated in this process. This co-culture model provides a robust, physiologically consistent experimental platform to identify tumor cell and CAF features that drive macrophage phenotype. Citation Format: Josiah Flaming, Raghav Chandra, Luc Girard, Debolina Ganguly, Jason Toombs, John D. Minna, Rolf A. Brekken. Lung cancer cells and cancer-associated fibroblasts drive macrophage polarization in a co-culture model [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO021.

In vivo blockade of mononuclear phagocyte system with solid nanoparticles: Efficiency and affecting factors

Smart nanomaterials, contrast nanoparticles and drug nanocarriers of advanced targeting architecture were designed for various biomedical applications. Most of such agents demonstrate poor pharmacokinetics in vivo due to rapid elimination from the bloodstream by cells of the mononuclear phagocyte system (MPS). One of the promising methods to prolong blood circulation of the nanoparticles without their modification is MPS blockade. The method temporarily decreases macrophage endocytosis in response to uptake of a low-toxic non-functional material. The effect of different factors on the efficiency of macrophage blockade in vivo induced by solid nanomaterials has been studied here. Those include: blocker nanoparticle size, ζ-potential, surface coating, dose, mice strain, presence of tumor or inflammation. We found that the blocker particle coating type had the strongest effect on MPS blockade efficiency, which allowed to prolong functional particle blood circulation half-life 18 times. The mechanisms capable of regulation of the MPS blockade have been demonstrated, which can promote application of this phenomenon in medicine for improving delivery of diagnostic and therapeutic nanomaterials.

Differentially expressed circular RNAs in a murine asthma model.

Allergic asthma is one of the most common allergic diseases; however, the mechanisms underlying its development have yet to be fully elucidated. Although allergic diseases are inheritable, genetic variance alone cannot explain the notable increase in the prevalence of allergic diseases over a short period of time in recent decades. Recently, research focus has been shifting to epigenetic factors, such as non-coding RNAs. Circular RNAs (circRNAs) are involved in the pathogenesis of various diseases. The aim of the present study was to further elucidate the etiology of allergic asthma by analyzing aberrantly expressed circRNAs in a murine asthma model. A mouse model of house dust mite allergen-induced asthma was established, and the qualified libraries were sequenced using next-generation sequencing. The expression levels of circRNAs were validated by reverse transcription-quantitative PCR (RT-qPCR) analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed for biological pathway classification and enrichment analysis of the aberrantly expressed circRNAs. In addition, the interaction network of the differentially expressed circRNAs and microRNAs (miRNAs) was constructed using Cytoscape. By next-generation sequencing, a total of 150 circRNAs were revealed to be upregulated and 130 were downregulated in the murine asthma model group compared with in the control group. GO and KEGG analyses demonstrated that the differentially expressed circRNAs were mainly involved in processes such as ‘autoimmune disease’, ‘cell adhesion molecules (CAMs)’ and ‘endocytosis’, among others. The expression levels of six circRNAs, namely three upregulated (circ_0000909, circ_0000629 and circ_0000455) and three downregulated (circ_0001454, circ_0000723 and circ_0001389) circRNAs, were validated by RT-qPCR. In conclusion, the analyses suggested that circRNAs performed critical functions via endocytosis (such as macrophage endocytosis), cell adhesion molecules and lipid metabolism in allergic asthma. The interaction network revealed that certain miRNAs that may serve a role in asthma could be regulated by the differentially expressed circRNAs.

Regulating Interactions Between Targeted Nanocarriers and Mononuclear Phagocyte System via an Esomeprazole-Based Preconditioning Strategy.

PurposeThe mononuclear phagocyte system (MPS) presents a formidable obstacle that hampers the delivery of various nanopreparations to tumors. Therefore, there is an urgent need to improve the off-MPS targeting ability of nanomedicines. In the present study, we present a novel preconditioning strategy to substantially increase the circulation times and tumor targeting of nanoparticles by regulating nanocarrier-MPS interactions.MethodsIn vitro, the effect of different vacuolar H+-ATPase inhibitors on macrophage uptake of targeted or nontargeted lipid vesicles was evaluated. Specifically, the clinically approved proton-pump inhibitor esomeprazole (ESO) was selected as a preconditioning agent. Then, we further investigated the blocking effect of ESO on the macrophage endocytosis of nanocarriers. In vivo, ESO was first intravenously administered into A549-tumor-bearing nude mice, and 24 h later, the c(RGDm7)-modified vesicles co-loaded with doxorubicin and gefitinib were intravenously injected.ResultsIn vitro, ESO was found to reduce the interactions between macrophages and c(RGDm7)-modified vesicles by interfering with the latter’s lysosomal trafficking. Studies conducted in vivo confirmed that ESO pretreatment greatly decreased the liver and spleen distribution of the targeted vesicles, enhanced their tumor accumulation, and improved the therapeutic outcome of the drug-loaded nanomedicines.ConclusionOur findings indicate that ESO can regulate the nanoparticle-MPS interaction, which provides a feasible option for enhancing the off-MPS targeting of nanomedicines.

The effect of zwitterionic surface content on blood circulation time of nanocapsule.

Zwitterionic modification can prolong the blood circulation time of nanocarrier in vivo, but zwitterionic content will affect the functions of nanocarrier such as enzyme-responsive and intracellular or extracellular delivery. Therefore, it is necessary to explore the relationship between the zwitterionic content and circulation time of nanocarrier so as to figure out what content of zwitterion can enable the nanocarrier to obtain both the long blood circulation ability and other functions mentioned above. Herein, using nanocapsule as a research model, we investigated the nanocapsule modified with zwitterion of phosphorylcholine (PC) or carboxybetaine (CB) respectively, and through 1H-NMR quantification we determined the zwitterionic surface content, so as to study the effect of PC or CB surface content on blood circulation performance of nanocapsule. In vivo study showed that the nanocapsule possessed an optimal surface filling ratios range for blood circulation of 43-68% for PC and of 20-68% for CB, with the longest t1/2=37.35 h for PC-nanocapsule and t1/2=45.27 h for CB-nanocapsule. Furthermore, the protein adsorption and macrophage endocytosis experiments indicated that when the surface filling ratio reached 43% for PC-nanocapsule and 20% for CB-nanocapsule, it could effectively reduce the protein adsorption and weaken macrophage endocytosis, thus explaining the phenomenon of long circulation time of nanocapsules from the point of protein adsorption and interaction with immune cells. This study proposes a new direction for designing long-circulating nanocarrier, and provides basis for constructing enzyme-responsive and intracellular or extracellular delivery platform.

B31 Development of Multicell-Type Organoid Cultures for Preclinical Studies of Immunotherapeutics for Lung Cancer

Macrophages are key regulators of the immune landscape within the tumor microenvironment (TME). The plasticity of macrophage phenotypes in the TME has previously been correlated with prognosis within non-small cell lung cancer (NSCLC). Depending on their phenotype, macrophages in the TME can secrete protumor cytokine and chemokines, ultimately suppressing the function of other immune cells in the TME. The purpose of our study was to explore the ability of individual NSCLC preclinical models to alter macrophage phenotype in organoid cultures and to relate effects on macrophages to the molecular characteristics of different NSCLCs. We hypothesize that immune suppression occurs through tumor-secreted signaling molecules, and if blocked, macrophage suppression can be alleviated, resulting in a better antitumor immune response. We developed an in vitro organoid coculture system (NSCLC tumor cells, human cancer-associated fibroblasts, CAFs, and mouse macrophages) to interrogate cancer cell features causing heterogeneity of macrophage phenotypes across a panel of NSCLCs. We measured (with 4-7 replicates for each NSCLC): mRNA expression in mouse macrophages with a panel of qPCR probes for important macrophage-related genes (Arg, NOS2, IL1beta, IL-6, CHIL-3, SOCS3), and in selected cases whole-genome RNAseq; and protein expression using cytokine arrays measuring expression of 40 inflammatory cytokines. Positive controls were stimulation with LPS and IL-4. Using our platform, we characterized 70 NSCLC patient-derived lines by their ability to alter mouse macrophage phenotype. We found: 1. the macrophage phenotypes induced by any one NSCLC were highly reproducible; 2. three major clusters of cancer polarized macrophage phenotypes: high Arg (immune suppressive), high IL-1beta (inflammatory) or high SOCS3 (cGAS-STING pathway) expression; and 3. the major oncogenotypes (KRAS, TP53, STK11, EGFR, BRAF) have no correlation to the induced macrophage phenotype. We selected 7 NSCLC “exemplar” lines representing each of these 3 clusters for RNA sequencing (mouse genes) and cytokine array protein (human) profiling. Across all clusters we found: 1. suppression of macrophage endocytosis pathways and activation of scavenger receptor A (SRA) signaling (M2 immune suppressive phenotype); and 2. increased expression of human IL6, IL8, and MCP1 proteins, which have been implicated in suppressing innate immune tumor sensing. Analyses of differences between the 3 clusters is ongoing. Patient-derived NSCLC preclinical models have reproducible effects on altering macrophage phenotypes in organoid cultures. Three major classes of NSCLC initiated macrophage alteration, which are not linked to oncogenotype. Cytokines secreted by the NSCLCs appear responsible for these macrophage changes, and this system provides an experimental mechanism to systematically test each as potential therapeutic targets.

An endosomal LAPF is required for macrophage endocytosis and elimination of bacteria

Macrophages can internalize the invading pathogens by raft/caveolae and/or clathrin-dependent endocytosis and elicit an immune response against infection. However, the molecular mechanism for macrophage endocytosis remains elusive. Here we report that LAPF (lysosome-associated and apoptosis-inducing protein containing PH and FYVE domains) is required for caveolae-mediated endocytosis. Lapf-deficient macrophages have impaired capacity to endocytose and eliminate bacteria. Macrophage-specific Lapf-deficient mice are more susceptible to Escherichia coli (E. coli) infection with higher bacterial loads. Moreover, Lapf deficiency impairs TLR4 endocytosis, resulting in attenuated production of TLR-triggered proinflammatory cytokines. LAPF is localized to early endosomes and interacts with caveolin-1. Phosphorylation of LAPF by the tyrosine kinase Src is required for LAPF-Src-Caveolin complex formation and endocytosis and elimination of bacteria. Collectively, our work demonstrates that LAPF is critical for endocytosis of bacteria and induction of inflammatory responses, suggesting that LAPF and Src could be potential targets for the control of infectious diseases.

Cathepsin B-Mediated Activation of Trypsinogen in Endocytosing Macrophages Increases Severity of Pancreatitis in Mice

BACKGROUND & AIMS:Acute pancreatitis is characterized by premature intracellular activation of digestive proteases within pancreatic acini and a consecutive systemic inflammatory response. We investigated how these processes interact during severe pancreatitis in mice.METHODS:Pancreatitis was induced in C57Bl/6 wild-type (control), cathepsin B (CTSB)- knockout, and cathepsin L-knockout mice by partial pancreatic duct ligation with supramaximal caerulein injection, or by repetitive supramaximal caerulein injections alone. Immune cells that infiltrated the pancreas were characterized by immunofluorescence detection of Ly6g, CD206, and CD68. Macrophages were isolated from bone marrow and incubated with bovine trypsinogen or isolated acinar cells; the macrophages were then transferred into pancreatitis control or cathepsin-knockout mice. Activities of proteases and nuclear factor (NF)-κB were determined using fluorogenic substrates and trypsin activity was blocked by nafamostat. Cytokine levels were measured using a cytometric bead array. We performed immunohistochemical analyses to detect trypsinogen, CD206, and CD68 in human chronic pancreatitis (n = 13) and acute necrotizing pancreatitis (n = 15) specimens.RESULTS:Macrophages were the predominant immune cell population that migrated into the pancreas during induction of pancreatitis in control mice. CD68-positive macrophages were found to phagocytose acinar cell components, including zymogen-containing vesicles, in pancreata from mice with pancreatitis, as well as human necrotic pancreatic tissues. Trypsinogen became activated in macrophages cultured with purified trypsinogen or co-cultured with pancreatic acini and in pancreata of mice with pancreatitis; trypsinogen activation required macrophage endocytosis and expression and activity of CTSB, and was sensitive to pH. Activation of trypsinogen in macrophages resulted in translocation of NF-kB and production of inflammatory cytokines; mice without trypsinogen activation (CTSB-knockout mice) in macrophages developed less severe pancreatitis compared with control mice. Transfer of macrophage from control mice to CTSB-knockout mice increased the severity of pancreatitis. Inhibition of trypsin activity in macrophages prevented translocation of NF-κB and production of inflammatory cytokines.CONCLUSIONS:Studying pancreatitis in mice, we found activation of digestive proteases to occur not only in acinar cells but also in macrophages that infiltrate pancreatic tissue. Activation of the proteases in macrophage occurs during endocytosis of zymogen-containing vesicles, and depends on pH and CTSB. This process involves macrophage activation via NF-κB-translocation, and contributes to systemic inflammation and severity of pancreatitis.

"Sweet" Architecture-Dependent Uptake of Glycocalyx-Mimicking Nanoparticles Based on Biodegradable Aliphatic Polyesters by Macrophages.

Glyconanoparticles made by self-assembled glycopolymers currently are practical and efficient mimics of the glycocalyx on cell surfaces. Considering the complexity of the glycocalyx, glyconanoparticles with different sugars on their coronas, i.e., mixed-shell glycomicelles, could be more valuable compared to homoshell micelles. In this paper, we explore the architectural effect of the glyconanoparticle corona on glyconanoparticle macrophage endocytosis and lectin-binding ability. A series of glyconanoparticles composed of a biodegradable polyester backbone functionalized with galactoside or mannoside pendants were designed and prepared. The different architectures explored were single-component (galactoside or mannoside) coronas, homogeneously mixed coronas (MG) made by galactoside-mannoside copolymer chains, and blend-mixed coronas (M/G) constructed from two homoglycopolymers. Nanoparticles with a mixed shell showed a higher efficiency in cellular uptake and lectin-binding than those with a single sugar component. Meanwhile, unexpectedly, MG presented a significantly higher efficiency than M/G, although they had the same particle size and ratio of mannoside to galactoside. We attributed this apparent architectural effect to the difference in the phase behavior between MG and M/G; i.e., the former having a homogeneous corona allowed more sugar-receptor interactions in the contact region, while the latter having phase separation limited the simultaneous interaction of the two kinds of sugar units with the cell receptors.

Monitoring in Vivo Behaviors of Protein Nanocages via Encapsulating an NIR-II Ag2S Quantum Dot

IntroductionProtein nanocages (PNCs) have been recognized as a promising platform for nanomedicine innovation. Real-time in vivo tracking of PNCs can provide critically important information for the development of PNC-based diagnostics and therapeutics. Here we demonstrate a strategy for monitoring the behaviors of PNCs in vivo by encapsulating a Ag2S quantum dot (QD) with fluorescence in the second near-infrared window (NIR-II, 1000-1700 nm) inside the PNC, using a simian virus 40 (SV40) PNC (PNCSV40) as a model. MethodsThe Ag2S QD was encapsulated into the PNCSV40 through controllable molecular self-assembly. The dynamic migration of Ag2S@PNCSV40 in living mouse was tracked in real time under an InGaAs-based shortwave infrared imaging system and was further corroborated by ex vivo imaging, inductively coupled plasma mass spectrometry analysis, and macrophage endocytosis assay. ResultsBenefitting from the high spatiotemporal resolution and deep tissue penetration of NIR-II fluorescence imaging, the dynamic distribution of the PNCSV40 in living mice was tracked in real time with high fidelity, revealing rapid clearance from bloodstream within 5min post-intravenous injection and selective accumulation in liver, spleen and bone marrow. Furthermore, adopting the PEGylation strategy, PEGylated PNCSV40 presents remarkably different behaviors in vivo with significantly prolonged blood circulation and much less uptake in the reticuloendothelial system (RES), leading to desirable pharmacokinetics and pharmacodynamics of PNC-based nanomedicines. DiscussionThis study represents the first evidence of real-time tracking of the intrinsic behaviors of PNCs in vivo without interference in PNC-host interactions by encapsulating nanoprobes inside, instead of conjugating nanoprobes onto the outer surface of PNCs. The as-described imaging strategy will facilitate the study of interactions between exogenously introduced PNCs and host body, prompting the development of future protein-based drugs, high-efficacy targeted delivery system, sensors, etc.

Extracellular Vesicles from Metastatic Rat Prostate Tumors Prime the Normal Prostate Tissue to Facilitate Tumor Growth.

Accumulating data indicates that tumor-derived extracellular vesicles (EVs) are responsible for tumor-promoting effects. However, if tumor EVs also prepare the tumor-bearing organ for subsequent tumor growth, and if this effect is different in low and high malignant tumors is not thoroughly explored. Here we used orthotopic rat Dunning R-3327 prostate tumors to compare the role of EVs from fast growing and metastatic MatLyLu (MLL) tumors with EVs from more indolent and non-metastatic Dunning G (G) tumors. Prostate tissue pre-conditioned with MLL-EVs in vivo facilitated G tumor establishment compared to G-EVs. MLL-EVs increased prostate epithelial proliferation and macrophage infiltration into the prostate compared to G-EVs. Both types of EVs increased macrophage endocytosis and the mRNA expression of genes associated with M2 polarization in vitro, with MLL-EVs giving the most pronounced effects. MLL-EVs also altered the mRNA expression of growth factors and cytokines in primary rat prostate fibroblasts compared to G-EVs, suggesting fibroblast activation. Our findings propose that EVs from metastatic tumors have the ability to prime the prostate tissue and enhance tumor growth to a higher extent than EVs from non-metastatic tumors. Identifying these differences could lead to novel therapeutic targets and potential prognostic markers for prostate cancer.

Non-covalent pomegranate (Punica granatum) hydrolyzable tannin-protein complexes modulate antigen uptake, processing and presentation by a T-cell hybridoma line co-cultured with murine peritoneal macrophages

In this work we characterize the interaction of pomegranate hydrolyzable tannins (HT) with hen egg-white lysozyme (HEL) and determine the effects of non-covalent tannin-protein complexes on macrophage endocytosis, processing and presentation of antigen. We isolated HT from pomegranate and complex to HEL, the resulting non-covalent tannin-protein complex was characterized by gel electrophoresis and MALDI-TOF MS. Finally, cell culture studies and confocal microscopy imaging were conducted on the non-covalent pomegranate HT-HEL protein complexes to evaluate its effect on macrophage antigen uptake, processing and presentation to T-cell hybridomas. Our results indicate that non-covalent pomegranate HT-HEL protein complexes modulate uptake, processing and antigen presentation by mouse peritoneal macrophages. After 4 h of pre-incubation, only trace amounts of IL-2 were detected in the co-cultures treated with HEL alone, whereas a non-covalent pomegranate HT-HEL complex had already reached maximum IL-2 expression. Pomegranate HT may increase rate of endocytose of HEL and subsequent expression of IL-2 by the T-cell hybridomas.

The Rab5A gene of marine fish, large yellow croaker (Larimichthys crocea), and its response to the infection of Cryptocaryon irritans.

Rab GTPases, members of the Ras superfamily, encode monomeric G-proteins. Rab proteins regulate key steps in membrane traffic transport and endocytic pathway of host immune responses. Rab5A is involved in immune regulation, particularly in T cell migration and macrophage endocytosis in higher vertebrates. However, little is known of the molecular structure of Rab5A gene in marine teleost fish species and its expression profile during the parasite infection. In this study, the full-length cDNA sequence and genomic structure of Rab5A gene of the large yellow croaker (Larimichthys crocea) (LycRab5A), one of the most economical marine fishes, were identified and characterized. The LycRab5A protein, containing the ATPase/GTPase binding motifs and the effector molecules binding motifs, was highly homologous to that of other animals. The expression plasmid containing LycRab5A cDNA fused with GST was engineered and transformed into Escherichia coli to produce recombinant protein GST-LycRab5A, which was purified to prepare a polyclonal antibody specifically against LycRab5A. Subcellular localization revealed that LycRab5A expressed in the membrane and cytoplasm. Based on real-time PCR and Western blot analysis, we found that both mRNA and protein of LycRab5A were expressed in all tissues we examined; especially it was highly expressed in blood and gill. Interestingly, both mRNA and protein of LycRab5A were substantially up-regulated when parasitic ciliate protozoan (Cryptocaryon irritans) was infected. The expression of LycRab5A was reached to the maximal level at 24h after infection. The line of evidence suggested that LycRab5A might play an important role in large yellow croaker defense against parasite infection. Moreover, on the basis of protein interaction, it was found that the LycRab5A interacted with myosin light chain (designated as LycMLC), a crucial protein in the process of phagocytosis. This discovery might contribute better understanding to the molecular events involved in fish immune responses.

Soluble cluster of differentiation 36 concentrations are not associated with cardiovascular risk factors in middle-aged subjects.

Cluster of differentiation 36 (CD36) is involved in the development of atherosclerosis by enhancing macrophage endocytosis of oxidized low-density lipoproteins and foam cell formation. Soluble CD36 (sCD36) was found to be elevated in type 2 diabetic patients and possibly acted as a marker of insulin resistance and atherosclerosis. In young subjects, sCD36 was associated with cardiovascular risk factors including obesity and hypertriglyceridemia. The present study was conducted to further investigate the association between plasma sCD36 and cardiovascular risk factors among middle-aged patients with metabolic syndrome (MetS) and healthy controls. sCD36 concentrations were determined by enzyme-linked immunosorbent assays (ELISA) for 41 patients with MetS and 36 healthy controls. Data for other variables were obtained from patient medical records. sCD36 concentrations were relatively low compared to the majority of other studies and were not significantly different between the MetS group and controls (P=0.17). sCD36 was also not correlated with age, body mass index, glucose, lipid profile, serum electrolytes and blood counts. sCD36 was not significantly different between subjects with obesity, hyperglycemia, dyslipidemia, hypertension or cardiovascular disease, and those without these abnormalities (P>0.05). The inconsistency between results reported in the present study and other studies may be unique to the study population or be a result of the lack of a reliable standardized method for determining absolute sCD36 concentrations. However, further investigations are required to assess CD36 tissue expression in the study population and to assess the accuracy of various commercially available sCD36 ELISA kits. Thus, the availability of a standardized simple sCD36 ELISA that could be performed in any basic laboratory would be more favorable to the specialized flow cytometry methods that detect CD36+ microparticles if it was to be used as a biomarker.