Normal Macrophages Research Articles

Regulation of Lipid Metabolism and Cell Cycle Progression By PU.1 in Myeloid Progenitor Cells

PU.1 is a transcription factor essential for myeloid development. High PU.1 expression leads to myeloid differentiation and cell cycle arrest while low expression leads to increased proliferation and self-renewal. Reduced PU.1 can lead to the development of acute myeloid leukemia. PU.1 induces microRNAs targeting genes involved in cell cycle and lipid metabolism. Included in these genes are E2f1 and Acly that are associated with lipogenesis. E2F1 is directly involved in cell cycle activation and lipid metabolism. Acly encodes ATP citrate lyase (ACL) that is responsible for the production of acetyl-CoA from mitochondrial citrate. Acetyl-CoA is the first precursor molecule for lipid biosynthesis, and lipogenesis is essential for proliferation. Therefore, we hypothesized that PU.1 activates genes encoding microRNAs to downregulate E2F1 and ACL, leading to cell cycle arrest. To test this hypothesis, we utilized a PU.1 inducible cell line system that is derived from myeloid progenitor cells in the liver of fetal BN mice (Spi1BN/BN) that express PU.1 at 20% of normal levels (iBN cells). Following doxycycline administration, iBN cells can be induced to express high levels of PU.1, causing cell cycle arrest and differentiation of these myeloid progenitor cells into a macrophage like phenotype. MicroRNA (miR)-223 and miR-141 were identified as PU.1 inducible miRs that target E2f1 and Acly, respectively. Supplementation with acetyl-CoA or acetate significantly rescued cell cycle inhibition caused by PU.1 induction. Inhibition of ACL activity by the chemical inhibitor BMS303141 (BMS) was sufficient to reduce cell cycle progression in cultured iBN cells. Supplementation with acetyl-CoA or acetate significantly rescued cell cycle progression in iBN cells inhibited with BMS. To determine if ACL levels play a role during normal macrophage differentiation, we investigated mRNA transcript levels of Acly over the course of myeloid progenitor differentiation promoted by M-CSF or GM-CSF. We found that Acly mRNA transcript levels inversely correlated with Adgre1 (encoding F4/80) or Spi1 (encoding PU.1) during macrophage differentiation. We conclude that during myeloid differentiation, PU.1 regulates the expression of genes involved in lipid metabolism through miRs, and influences cell cycle progression by limiting fatty acid synthesis required for proliferation. Elucidation of the mechanisms by which lipid metabolism may control cell cycle in myeloid progenitor cells is an important issue for further study. DisclosuresNo relevant conflicts of interest to declare.

Starvation Stress in the Sbds -Null Zebrafish: Implications for Human Shwachman-Diamond Syndrome

Shwachman-Diamond Syndrome (SDS) is an autosomal recessive disorder characterized by exocrine pancreatic insufficiency, bone marrow dysfunction, and an increase risk of acute myeloid leukemia. Patients with SDS exhibit abnormalities in other organs, including bone, liver and immunologic and endocrine systems. Almost all cases of SDS result from biallelic mutations in the SBDS gene1. SBDS is involved in ribosomal biogenesis and stabilization of mitotic spindle microtubules2,3. Recently, it has also been shown a reduction cell proliferation and mTOR and STAT3 expression, both with an important role in cellular growth and tumorigenesis, in SDS derived leukocytes.4Genetic ablation of Sbds results in early embryonic lethality (ED 7.5) in mice5. Zebrafish provide an attractive, alternative model organism to study SDS. Genome analysis of zebrafish (Danio rerio) revealed the presence of a single sbds gene, encoding a protein 90% identical to the human orthologue.We created sbds zebrafish knockout strains that phenocopies the human Shwachman-Diamond Syndrome. Survival rate of mutants was normal at 15 days post fertilization (dpf) (100%) but decline significantly at 21 dpf (4%). Western blot analysis showed a progressive decrease of Sbds during the first 8 dpf, with total absence at 10 dpf. These results suggest that larval viability is due to maternally deposited sbds transcript and corresponding protein, which was confirmed by RT-qPCR and immunoblot at earliest developmental stages. Zebrafish sbds mutants showed neutropenia at 5 and 15 days post fertilization (dpf) but normal macrophage number and hemoglobinization. Pancreas, liver, digestive tract, and eye showed histologic evidence of atrophy at 21 dpf. Moreover, we observed an important reduction in the intestinal epithelium at 15 dpf.To further characterize effects of Sbds loss, we tested expression of genes involved in lipid metabolism (acc, ppar α , ppar γ and srbp1) and pancreatic transcription factors (ptf1a and prox1) by qPCR. Our results showed that they were not affected at 10 dpf but upregulated at 18 dpf. After staining 15 dpf larvae with oil red we observed, in the mutants compared to their sibling wild type or heterozygous, significantly reduced accumulation of lipids. In addition, we found an increase of expression of genes involved in autophagy (atg5, atg7 and mTOR), and upregulation of casp9 and tp53 expression.With these results we thought that reduced viability was due to a problem in nutrient absorption and therefore starvation. We carried out two starvation experiments, a diet restriction from 10 to 15 dpf, and from 15 to 21 dpf. Unlike sbds mutants, mortality rates were not affected comparing to control groups in any of these experiments. However, genes related to lipid metabolism and autophagy were upregulated and histology showed a reduction in intestinal epithelium as well. Moreover, oil red staining showed lower lipid accumulation in starved fish as we observed in sbds mutants.With these results, we conclude sbds -null zebrafish showed signs of malnutrition and an effect in fatty acid-metabolism gene expression as seen in starved fish. Our results make it a highly relevant model to investigate new mechanistic hypotheses and develop new small molecule therapies for Shwachman-Diamond syndrome.1. Boocock GR, Morrison JA, Popovic M, et al. Mutations in SBDS are associated with Shwachman-Diamond syndrome. Nat Genet . 2003;33(1):97-101.2. Austin KM, Gupta ML, Jr., Coats SA, et al. Mitotic spindle destabilization and genomic instability in Shwachman-Diamond syndrome. J Clin Invest . 2008;118(4):1511-1518.3. Finch AJ, Hilcenko C, Basse N, et al. Uncoupling of GTP hydrolysis from eIF6 release on the ribosome causes Shwachman-Diamond syndrome. Genes Dev . 2011;25(9):917-929.4. Bezzerri V, Vella A, Calcaterra E, et al. New insights into the Shwachman-Diamond Syndrome-related haematological disorder: hyper-activation of mTOR and STAT3 in leukocytes. 2016;6:33165.5. Zhang S, Shi M, Hui CC, Rommens JM. Loss of the mouse ortholog of the Shwachman-Diamond syndrome gene (Sbds) results in early embryonic lethality. Mol Cell Biol . 2006;26(17):6656-6663. DisclosuresNo relevant conflicts of interest to declare.

Computational Fine-Tuning of Functional Single Nucleotide Polymorphisms Associated with ACP5 Gene to Characterize Missense Mutations

Acid phosphatase 5 (ACP5) gene plays a vibrant role in the synthesis of a tartrate-resistant acid phosphatase (TRAP5) enzyme. TRAP5 is ~35 KD glycosylated di-iron metalloenzyme responsible for the regulation of osteopontin (protein) activity. There are two isoforms of TRAP5, TRAP5a, and TRAP5b. TRAP5a functions with low enzymatic activity due to a loop interacting with the active site and the more active TRAP5b is generated upon proteolytic cleavage of this loop. TRAP5a works as a marker for systematic macrophage function and chronic inflammation activity, while TRAP5b for osteoclast activity. ACP5 is evolutionarily conserved in nature and acts as a multifunctional protein that involves generations of reactive oxygen species, normal bone development, macrophage function, and osteoblast regulation, affecting a series of pathways, as well as reflecting bone resorption and osteoclast activity. To understand its fundamental role, a functional investigation of missense mutations of the ACP5 gene was carried out through an in-silico approach. Two nsSNPs G109R and L201P were predicted to be deleterious using multiple computational tools like SIFT, Polyphen-1, PolyPhen-2, MAPP, SNAP, Predict SNP and PhD-SNP. Additionally, the structural analysis was performed. The result is that there was no similarity between the native and mutant structures. Therefore, these reported mutations in ACP5 modify the expression, function, and structure of a TRAP5 protein. These findings suggest that TRAP5 can be a therapeutic target in immunological disorders, cancer, and metabolic bone diseases. These deleterious mutations can be lethal to its function and may hamper its therapeutic strategy leading to various diseases such as autoimmune cytopenia, systemic lupus erythematosus (SLE) immune-osseous dysplasia, spasticity with leukodystrophy, moyamoya syndrome, and sjogren's syndrome.

Requirement of CRAMP for mouse macrophages to eliminate phagocytosed E. coli through an autophagy pathway.

ABSTRACTHost-derived antimicrobial peptides play an important role in the defense against extracellular bacterial infections. However, the capacity of antimicrobial peptides derived from macrophages as potential antibacterial effectors against intracellular pathogens remains unknown. In this study, we report that normal (wild-type, WT) mouse macrophages increased their expression of cathelin-related antimicrobial peptide (CRAMP, encoded by Camp) after infection by viable E. coli or stimulation with inactivated E. coli and its product lipopolysaccharide (LPS), a process involving activation of NF-κB followed by protease-dependent conversion of CRAMP from an inactive precursor to an active form. The active CRAMP was required by WT macrophages for elimination of phagocytosed E. coli, with participation of autophagy-related proteins ATG5, LC3-II and LAMP-1, as well as for aggregation of the bacteria with p62 (also known as SQSTM1). This process was impaired in CRAMP−/− macrophages, resulting in retention of intracellular bacteria and fragmentation of macrophages. These results indicate that CRAMP is a critical component in autophagy-mediated clearance of intracellular E. coli by mouse macrophages.

55270 The Histone Methyltransferase SETDB2 Regulates Inflammation in Normal and Diabetic Wound Repair

ABSTRACT IMPACT: Our data reveal a histone modifying enzyme involved in regulating inflammation that may be a novel target for treating non-healing diabetic wounds. OBJECTIVES/GOALS: We investigate molecular mechanisms that regulate the inflammatory phenotype of macrophages in normal and diabetic wound healing. Our goal is to identify novel pathways that may be used to better treat diabetic patients with non-healing wounds. METHODS/STUDY POPULATION: We utilize normal and transgenic murine models on standard chow or high-diet to identify chromatin modifying enzymes involved in regulating macrophage function during wound healing. We validate our murine studies with human blood monocytes or wound macrophages from diabetic patients undergoing limb amputation surgery. RESULTS/ANTICIPATED RESULTS: We have identified the histone methyltransferase SETDB2 as a regulator inflammation in normal and diabetic wound macrophages. We found that SETDB2 was dependent on IFNβ singaling and that both IFNβ and Setdb2 expression were impaired in diabetic wound macrophages. Further, we show that SETDB2 regulates inflammatory response and immune cell trafficking pathways. We also show that SETDB2 genomic localization is dependent on *NFκΒ deposition of the promoter. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our results indicate that SETDB2 is a regulator of macrophage plasticity and that SETDB2 expression is impaired in diabetic wound macrophages leading to hyper-inflammatory response and delayed wound healing. These data provide a novel potential therapeutic pathway for treating non-healing diabetic wounds.

Current concepts in granulomatous immune responses.

Persistent irritants that are resistant to innate and cognate immunity induce granulomas. These macrophage-dominated lesions that partially isolate the healthy tissue from the irritant and the irritant induced inflammation. Particles, toxins, autoantigens and infectious agents can induce granulomas. The corresponding lesions can be protective for the host but they can also cause damage and such damage has been associated with the pathology of more than a hundred human diseases. Recently, multiple molecular mechanisms underlying how normal macrophages transform into granuloma-inducing macrophages have been discovered and new information has been gathered, indicating how these lesions are initiated, spread and regulated. In this review, differences between the innate and cognate granuloma pathways are discussed by summarizing how the dendritic cell - T cell axis changes granulomatous immunity. Granuloma lesions are highly dynamic and depend on continuous cell replacement. This feature provides new therapeutic approaches to treat granulomatous diseases.

Interaction between Phagocytic Cells with Antiphagocytic Properties of Cryptococcus neoformans: When Love and Hate Collide

Global systemic fungal infection, including meningeal cryptococcosis caused by the encapsulated yeast Cryptococcus spp, continue to rise in number, especially among HIV infected individuals. Infection occur through inhalation of spore which is abundant in the environment. Initially this fungus stay in the lungs for a certain time without causing any symptoms and when the host’s cellular immune status is depleted, it can uses monocyte as a vehicle to take them to the brain, using a mechanism called Trojan Horse mechanism. Normal alveolar macrophage as the first line of innate immune system in the lungs are supposed to phagocytose, internalized and then destroy it inside an organelle named phagolysosome. But Cryptococcus spp seemed to have a built in antiphagocytic mechanism to avoid destruction and even can multiply therein. The interaction between this clever yeast and the host’s phagocytic cells determine the course of the disease.

RNA editing enzyme APOBEC3A promotes pro-inflammatory M1 macrophage polarization

Pro-inflammatory M1 macrophage polarization is associated with microbicidal and antitumor responses. We recently described APOBEC3A-mediated cytosine-to-uracil (C > U) RNA editing during M1 polarization. However, the functional significance of this editing is unknown. Here we find that APOBEC3A-mediated cellular RNA editing can also be induced by influenza or Maraba virus infections in normal human macrophages, and by interferons in tumor-associated macrophages. Gene knockdown and RNA_Seq analyses show that APOBEC3A mediates C>U RNA editing of 209 exonic/UTR sites in 203 genes during M1 polarization. The highest level of nonsynonymous RNA editing alters a highly-conserved amino acid in THOC5, which encodes a nuclear mRNA export protein implicated in M-CSF-driven macrophage differentiation. Knockdown of APOBEC3A reduces IL6, IL23A and IL12B gene expression, CD86 surface protein expression, and TNF-α, IL-1β and IL-6 cytokine secretion, and increases glycolysis. These results show a key role of APOBEC3A cytidine deaminase in transcriptomic and functional polarization of M1 macrophages.

Transcriptional profiling of pediatric cholestatic livers identifies three distinct macrophage populations.

Background & aimsLimited understanding of the role for specific macrophage subsets in the pathogenesis of cholestatic liver injury is a barrier to advancing medical therapy. Macrophages have previously been implicated in both the mal-adaptive and protective responses in obstructive cholestasis. Recently two macrophage subsets were identified in non-diseased human liver; however, no studies to date fully define the heterogeneous macrophage subsets during the pathogenesis of cholestasis. Here, we aim to further characterize the transcriptional profile of macrophages in pediatric cholestatic liver disease.MethodsWe isolated live hepatic immune cells from patients with biliary atresia (BA), Alagille syndrome (ALGS), and non-cholestatic pediatric liver by fluorescence activated cell sorting. Through single-cell RNA sequencing analysis and immunofluorescence, we characterized cholestatic macrophages. We next compared the transcriptional profile of pediatric cholestatic and non-cholestatic macrophage populations to previously published data on normal adult hepatic macrophages.ResultsWe identified 3 distinct macrophage populations across cholestatic liver samples and annotated them as lipid-associated macrophages, monocyte-like macrophages, and adaptive macrophages based on their transcriptional profile. Immunofluorescence of liver tissue using markers for each subset confirmed their presence across BA (n = 6) and ALGS (n = 6) patients. Cholestatic macrophages demonstrated reduced expression of immune regulatory genes as compared to normal hepatic macrophages and were distinct from macrophage populations defined in either healthy adult or pediatric non-cholestatic liver.ConclusionsWe are the first to perform single-cell RNA sequencing on human pediatric cholestatic liver and identified three macrophage subsets with distinct transcriptional signatures from healthy liver macrophages. Further analyses will identify similarities and differences in these macrophage sub-populations across etiologies of cholestatic liver disease. Taken together, these findings may allow for future development of targeted therapeutic strategies to reprogram macrophages to an immune regulatory phenotype and reduce cholestatic liver injury.

Antimicrobial, Cytotoxic and Oxidative Stress Inhibitory Activities of Terpenoids and Flavonols from Senegalia nigrescens (Oliv.) P.J.H. Hurter.

Senegalia nigrescens (knob thorn) is a deciduous tree distributed in savannah regions from Tanzania to South Africa used for timber but also medicinally for the treatment of convulsions, wounds, and skin problems. In this study, the biological activities of six phytocompounds, namely: 3β-hydroxy-20(29)-en-lupan-30-al (1), 30-hydroxylup-20(29)-en-3β-ol (2), ent-kaur-15-en-18,20-diol (3), melanoxetin (4), quercetin (5) and quercetin-3-O-methyl ether (6), isolated from S. nigrescens were investigated. The compounds were screened against two bacterial (Escherichia coli and Staphylococcus aureus) and one fungal (Candida albicans) strain and were also tested for their cytotoxicity on breast cancer (MDA-MB-231) and normal murine macrophage (RAW 264.7) cell line. Effects of the compounds on attenuating the lipopolysaccharide (LPS)-induced intracellular reactive oxygen species (ROS) production in RAW 264.7 cells were quantified with the H2DCF-DA assay. This study revealed that flavonols (5 and 6) had the strongest antibacterial and antifungal effects, both having MIC values of 62.5, 31.25 and 31.25 µg/mL on E. coli, S. aureus and C. albicans, respectively. Compounds 2, 3 and 6 were the most cytotoxic against the breast cancer cells with IC50 values of 11.86, 12.62 and 14.03 µg/mL, respectively, while the least toxicity towards normal cells were observed in compounds 2, 5 and 6. All compounds (1-6) significantly lowered ROS production in RAW264.7 cells. In conclusion, tested compounds represent potential promising candidates as antimicrobial, anticancer and antidotes for LPS-induced oxidative stress. This is the first report on the antifungal, cytotoxicity and antioxidative activities of the ent-kaurene diterpenoid, ent-kaur-15-en-18,20-diol (3).

E-cigarettes and health risks: more to the flavor than just the name.

The growing interest in regulating flavored E-liquids must incorporate understanding of the "flavoring profile" of each E-liquid-which flavorings (flavoring chemicals) are present and at what concentrations not just focusing on the flavor on the label. We investigated the flavoring profile of 10 different flavored E-liquids. We assessed bronchial epithelial cell viability and apoptosis, phagocytosis of bacteria and apoptotic cells by macrophages after exposure to E-cigarette vapor extract (EVE). We validated our data in normal human bronchial epithelial cells (NHBE) and alveolar macrophages (AM) from healthy donors. We also assessed cytokine release and validated in the saliva from E-cigarette users. Increased necrosis/apoptosis (16.1-64.5% apoptosis) in 16HBE cells was flavor dependent, and NHBEs showed an increased susceptibility to flavors. In THP-1 differentiated macrophages phagocytosis was also flavor dependent, with AM also showing increased susceptibility to flavors. Further, Banana and Chocolate were shown to reduce surface expression of phagocytic target recognition receptors on alveolar macrophages. Banana and Chocolate increased IL-8 secretion by NHBE, whereas all 4 flavors reduced AM IL-1β secretion, which was also reduced in the saliva of E-cigarette users compared with healthy controls. Flavorant profiles of E-liquids varied from simple 2 compound mixtures to complex mixtures containing over a dozen flavorants. E-liquids with high benzene content, complex flavoring profiles, high chemical concentration had the greatest impacts. The Flavorant profile of E-liquids is key to disruption of the airway status quo by increasing bronchial epithelial cell apoptosis, causing alveolar macrophage phagocytic dysfunction, and altering airway cytokines.

Systemic lupus erythematosus is associated with impaired autophagic degradation via interleukin-6 in macrophages

Systemic lupus erythematosus (SLE) is an autoimmune disease associated with dysregulated interleukin (IL)-6 and autophagy. Although such disturbances are increasingly recognized in patients with SLE and animal models of the disease, little is known about the specific role of IL-6 and autophagy in SLE macrophages. Here, we investigated alterations in the IL-6 axis and autophagy in macrophages derived from patients with SLE and determined whether IL-6 modulates autophagy using human macrophage models. Serum IL-6 detected by ELISA was higher in SLE patients (n = 19) than in normal controls (n = 19, p < 0.001). Levels of the IL-6 receptor (IL-6R) and autophagic markers LC3B and p62 in SLE and normal macrophages were assessed by real-time PCR, western blotting, and immunofluorescence. Compared with normal macrophages, SLE macrophages not only overexpressed IL-6Rs but also exhibited impaired autophagic degradation as evidenced by elevated levels of LC3B and p62. In vitro analyses using macrophage models revealed that prolonged exposure to exogenous recombinant human IL-6 induced a marked impairment of autophagic degradation indicated by elevated levels of LC3B and p62 in both primary macrophages and transformed macrophages. Pretreatment with tocilizumab, a humanized anti-IL-6R monoclonal antibody, restored autophagic degradation and reversed p62 accumulation in a paracrine manner in macrophages. These findings demonstrate that SLE involves IL-6-induced impairment of autophagic degradation through augmentation of IL-6R in human macrophages.

Legumain protease-activated tuftsin-functionalized nanoparticles for dual-targeting TAMs and cancer chemotherapy

M2 tumor-associated macrophages (TAMs) play a pivotal role in cancer progression and therapy resistance. Inhibition of TAMs is of great significance to reshape the protumor environment to benefit therapeutic outcomes. In this work, we developed a novel TAMs and tumor cells dual-targeting nanoparticle (ATpep-NPs) system for cancer chemotherapy by integrating a docetaxel (DTX)-loaded nanocarrier and a multi-function peptide ATpep, which is composed of a phagocytosis-stimulating peptide-tuftsin (Tpep) fused with a substrate peptide-alanine-alanine-asparagine (AAN) of endoprotease legumain. In vitro protelytic and cellular uptake assays confirmed ATpep-NPs can be responsively activated into Tpep-NPs by cleavage of legumain that is overexpressed in both tumor cells and TAMs, which then promoted tumor cells internalization and TAMs phagocytosis through neuropilin-1/Fc receptor pathways. Due to AAN deactivation effect, ATpep-NPs can effectively decrease the Tpep-induced non-specific uptake by M1-polarized and normal macrophage during systemic circulation. Our results of in vivo experiments demonstrated ATpep-NPs outperformed Tpep-NPs in tumor and TAMs dual-targeting delivery efficiency with markedly enhanced efficacy against both tumor growth inhibition and TAMs depletion. Overall, this study offers a novel approach for development of multitargeted delivery vehicle for improved cancer chemotherapy.

Macrophage ubiquitin-specific protease 2 contributes to motility, hyperactivation, capacitation, and in vitro fertilization activity of mouse sperm.

Macrophages are innate immune cells that contribute to classical immune functions and tissue homeostasis. Ubiquitin-specific protease 2 (USP2) controls cytokine production in macrophages, but its organ-specific roles are still unknown. In this study, we generated myeloid-selectiveUsp2 knockout (msUsp2KO)miceand specifically explored the roles of testicular macrophage-derived USP2 in reproduction. The msUsp2KOmice exhibited normal macrophage characteristics in various tissues. In the testis, macrophage Usp2 deficiency negligibly affected testicular macrophage subpopulations, spermatogenesis, and testicular organogenesis. However, frozen-thawed sperm derived from msUsp2KO mice exhibited reduced motility, capacitation, and hyperactivation. In addition, macrophage Usp2 ablation led to a decrease in the sperm population exhibiting high intracellular pH, calcium influx, and mitochondrial membrane potential. Interrupted pronuclei formation in eggs was observed when using frozen-thawed sperm from msUsp2KO mice for in vitro fertilization. Administration of granulocyte macrophage-colony stimulating factor (GM-CSF), whose expression was decreased in testicular macrophages derived from msUsp2KO mice, restored mitochondrial membrane potential and total sperm motility. Our observations demonstrate a distinct role of the deubiquitinating enzyme in organ-specific macrophages that directly affect sperm function.

Wild-type huntingtin regulates human macrophage function

The huntingtin (HTT) protein in its mutant form is the cause of the inherited neurodegenerative disorder, Huntington’s disease. Beyond its effects in the central nervous system, disease-associated mutant HTT causes aberrant phenotypes in myeloid-lineage innate immune system cells, namely monocytes and macrophages. Whether the wild-type form of the protein, however, has a role in normal human macrophage function has not been determined. Here, the effects of lowering the expression of wild-type (wt)HTT on the function of primary monocyte-derived macrophages from healthy, non-disease human subjects were examined. This demonstrated a previously undescribed role for wtHTT in maintaining normal macrophage health and function. Lowered wtHTT expression was associated, for instance, with a diminished release of induced cytokines, elevated phagocytosis and increased vulnerability to cellular stress. These may well occur by mechanisms different to that associated with the mutant form of the protein, given an absence of any effect on the intracellular signalling pathway predominantly associated with macrophage dysfunction in Huntington’s disease.

A new drug testing platform based on 3D tri-culture in lab-on-a-chip devices

Drug discovery has a 90% rate of failure because preclinical platforms for drug testing do not mimic the in vivo conditions. Doxorubicin (DOX) is a commonly used drug to treat breast cancer patients even though it has side effects. Lab-on-a-chip (LOC) devices provide spatial control at the micrometer scale and can thus emulate the cancer microenvironment. Here, using a multidisciplinary approach, a new drug testing platform based on 3D tri-culture in LOC devices was developed. Breast cancer cells alone or with normal mammary epithelial cells and macrophages were cultured in matrigel in LOC devices. The platform was used to test DOX and (R)-4′-methylklavuzon (KLA), which is a new anti-cancer drug candidate. Results showed that DOX and KLA were equally effective on breast cancer cells in 3D monoculture. KLA produced 26% less death for breast cancer cells than DOX in 3D tri-culture. More importantly, DOX was not selective between breast cancer cells and normal mammary epithelial cells in 3D tri- culture whereas KLA caused 56% less cell death than DOX for normal mammary epithelial cells. Results strongly recommend that 3D tri-culture in LOC devices be used for assessment of drug toxicity at the preclinical stage.

Antagonistic effect of N-acetyl-L-cysteine against cadmium-induced cytotoxicity and abnormal immune response on chicken peritoneal macrophages

Cadmium is a highly toxic metal threatening human and animal health. N-acetyl-L-cysteine (NAC) was reported to play a positive role in disease treatment and immune regulation. The present study aimed to explore the effect of NAC administration on Cd-induced cytotoxicity and abnormal immune response on chicken peritoneal macrophages. Peritoneal macrophages isolated from Isa Brown male chickens were exposed to CdCl2 (20 or 50 μM) and/or NAC (500 μM) for different time periods. Results showed that Cd caused dose-dependent damage on chicken peritoneal macrophages characterized by morphologic and ultrastructural alterations, increased cell apoptosis, reactive oxygen species accumulation and mitochondrial injury. Cd exposure inhibited phagocytic activity of chicken peritoneal macrophages, and promoted transcriptional status of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) in both unactivated macrophages and cells in response to lipopolysaccharide (LPS) stimuli. Pretreatment with 500 μM NAC did not affect growth of normal chicken peritoneal macrophages, while remarkably inhibiting Cd-caused cell death, oxidative stress, and mitochondrial membrane depolarization. NAC pretreatment significantly prevented intracellular Cd2+ accumulation in the Cd-exposed macrophages. Inhibitory effects of NAC on Cd-induced ROS accumulation and mitochondrial injury on chicken macrophages were confirmed in HD-11 macrophage cell line. In addition, NAC pretreatment promoted the phagocytic activity of Cd-exposed chicken peritoneal macrophages, and significantly inhibited expression of pro-inflammatory factors (IL-1β, IL-6 and TNF-α) in both Cd-exposed macrophages and Cd-treated cells in response to LPS stimuli. In conclusion, the present study firstly demonstrated the antagonistic effect of NAC against Cd-caused damage and abnormal immune response on chicken peritoneal macrophages. Protective effect of NAC on chicken macrophages was highly related to its suppression on Cd-induced ROS overproduction, pro-inflammatory reaction and intracellular Cd2+ accumulation.

Abstract 679: Selective targeting of immunosuppressive tumor macrophage from systemic administration

Abstract Macrophage in tumors in the M2 immunosuppressive state have been shown to play a role in reducing the effectiveness of immunotherapy and facilitating metastases formation. Many current approaches to targeting macrophage are not selective to the tumor microenvironment potentially resulting in systemic proinflammatory responses. Hydroxyl dendrimers without targeting moieties have been shown to be selectively endocytosed by reactive macrophages in solid tumors but not by normal quiescent macrophages. To evaluate the effect of hydroxyl dendrimer size and circulation time on targeting M2 tumor associated macrophages (TAMs), we generated fluorescently tagged hydroxyl dendrimers of two types, Generation 4 dendrimer (~14,000 Da, 4 nm) conjugated with Cy5 (D4-Cy5) and Generation 6 dendrimer (~58,000 Da, 7 nm) conjugated with VivoTag 680 (D6-V). A novel CSF1R tyrosine kinase inhibitor was also conjugated to the G6 hydroxyl dendrimer along with VivoTag 680 (C-D6-V). The syngeneic murine colon cancer line, MC38, was subcutaneously injected in C57BL/6 mice (n=10/group) and tumors were allowed to grow to a minimum average size of 80-120 mm3. After tumor establishment, either D4-Cy5, D6-V, or C-D6-V was injected IV (55 mg/kg, 10 mL/kg) and mice were sacrificed 48 hrs post-dose (D4 and D6 are systemically cleared within 48 hr). Tumors were analyzed for total radiant fluorescence, FACS analysis for immune cell subpopulations, and immunohistochemistry. Analysis of total fluorescence indicated a greater tumor uptake of D6-V compared to D4-Cy5 consistent with previous studies. Selective uptake and retention was observed in M2 macrophage, M1 macrophage, and mMDSCs. Tumors consisted of ~56% CD45+ cells of which ~20-25% were M2 macrophage, ~13% were M1 macrophage, and ~8% were mMDSCs. 34 ± 4 % of all M2 macrophage, 6.5% ± 1.7% of all M1 macrophage, and 8.1 ± 1.6% of all mMDSCs contained D4-Cy5 after the single IV dose. The fraction of dendrimer in other immune cell populations was less than 5%. The C-D6-V had greater tumor uptake than D6-V suggesting that CSF1R binding may further enhance tumor targeting as well as potentially impact M2 TAMs. These results indicate the potential to selectively target M2 TAMs and other tumor resident immune cells after systemic administration. Hydroxyl dendrimers may provide a novel carrier for delivery of immune modulators to tumors while minimizing their systemic toxicity. Efficacy studies are ongoing to evaluate CSF1R inhibitors and other therapeutics conjugated to the hydroxyl dendrimers. Citation Format: Jeffrey L. Cleland, Rishi Sharma, Santiago Appiani, Emily O'Koren, Lindsay Usher, Kannan Rangaramanujam. Selective targeting of immunosuppressive tumor macrophage from systemic administration [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 679.

Stimulatory effect of gastroesophageal reflux disease (GERD) on pulmonary fibroblast differentiation

Epidemiological studies indicate that prolonged micro-aspiration of gastric fluid is associated in gastroesophageal reflux disease with the development of chronic respiratory diseases, possibly caused by inflammation-related immunomodulation. Therefore, we sought to ascertain the effect of gastric fluid exposure on pulmonary residential cells. The expression of α-smooth muscle actin as a fibrotic marker was increased in both normal human pulmonary fibroblast cells and mouse macrophages. Gastric fluid enhanced the proliferation and migration of HFL-1 cells and stimulated the expression of inflammatory cytokines in an antibody assay. Elevated expression of the Rho signaling pathway was noted in fibroblast cells stimulated with gastric fluid or conditioned media. These results indicate that gastric fluid alone, or the mixture of proinflammatory mediators induced by gastric fluid in the pulmonary context, can stimulate pulmonary fibroblast cell inflammation, migration, and differentiation, suggesting that a wound healing process is initiated. Subsequent aberrant repair in pulmonary residential cells may lead to pulmonary fibroblast differentiation and fibrotic progression. The results point to a stimulatory effect of chronic GERD on pulmonary fibroblast differentiation, and this may promote the development of chronic pulmonary diseases in the long term.

The Effect of Bovine Viral Diarrhea Virus (BVDV) Strains and the Corresponding Infected-Macrophages' Supernatant on Macrophage Inflammatory Function and Lymphocyte Apoptosis.

Bovine viral diarrhea virus (BVDV) is an important viral disease of cattle that causes immune dysfunction. Macrophages are the key cells for the initiation of the innate immunity and play an important role in viral pathogenesis. In this in vitro study, we studied the effect of the supernatant of BVDV-infected macrophage on immune dysfunction. We infected bovine monocyte-derived macrophages (MDM) with high or low virulence strains of BVDV. The supernatant recovered from BVDV-infected MDM was used to examine the functional activity and surface marker expression of normal macrophages as well as lymphocyte apoptosis. Supernatants from the highly virulent 1373-infected MDM reduced phagocytosis, bactericidal activity and downregulated MHC II and CD14 expression of macrophages. Supernatants from 1373-infected MDM induced apoptosis in MDBK cells, lymphocytes or BL-3 cells. By protein electrophoresis, several protein bands were unique for high-virulence, 1373-infected MDM supernatant. There was no significant difference in the apoptosis-related cytokine mRNA (IL-1beta, IL-6 and TNF-a) of infected MDM. These data suggest that BVDV has an indirect negative effect on macrophage functions that is strain-specific. Further studies are required to determine the identity and mechanism of action of these virulence factors present in the supernatant of the infected macrophages.