Classically-activated Macrophages Research Articles

P06.03.A IMMORTALIZED MACROPHAGES AS A TOOL TO STUDY PHAGOCYTOSIS AND POLARIZATION IN VITRO

Abstract BACKGROUND Macrophages, an important element of the tumour-microenvironment (TME), are classified based on polarization state: tumour inhibiting ‘classically-activated macrophages’ M1 and tumour promoting ‘alternatively-activated macrophages’ M2. It is wellknown that the glioblastoma (GBM) TME is dominated by immunosuppressive M2 macrophages contributing to tumor growth by promoting angiogenesis, invasion and immunosuppression. However, macrophage culture is difficult to maintain as the cells do not proliferate and expand, making co-culture studies challenging. In this work, we successfully immortalized and expanded bone marrow derived mouse macrophages (BMDMs) that can be used as a tool to study phagocytosis and polarization in co-culture with tumor cells. MATERIAL AND METHODS Primary macrophages, obtained from the bone marrow of C57/BL6 mice, were transduced with two lentiviral vectors: hTERT with antibiotic hygromycin (Hy) and Simian virus 40 (SV40) with mCherry selective markers. Viral-titers were prepared by co-transfecting HEK293T with hTERT and SV40 constructs and plasmids MDL-X (Gag/Pol):Vpx. Vpx is a virion-packaged accessory protein that induces degradation of SAMHD1, a GTP- activated dNTP hydrolase that inhibits lentiviral infection of macrophages. BMDMs transduced with hTERT were subjected to gradient Hy selection after 5 days infection and SV40.mCherry positive cells were sorted by flow cytometry. To assess phagocytotic capacity, BMDMs were incubated with apoptotic bodies of a murine GBM cell-line (GL261) which was treated with HSV-TK/ganciclovir (GCV) suicide gene therapy. Phagocytosis was measured by fluorescence microscopy and flow cytometry. Further, BMDMs were externally induced with either E. coli lipopolysaccharide (LPS) and interferon gamma (IFNγ) or interleukin 4 (IL4) to activate M1 and M2 polarization. Flow cytometry was used to detect iNOS, CD40 and CD86 markers for M1 phenotype and Arginase1 and CD206 for M2. RESULTS hTERT.MDL-X.Vpx.Hy BMDM showed no clear signs of immortalization with limited proliferation followed by cell death. In contrast, SV40.MDL-X.Vpx.mCherry BMDM exhibited proliferative expansion from single colony, efficient freeze-thaw cycles and were used for further studies. BMDMs showed successful phagocytosis of apoptotic bodies from HSV-tk/GCV treated GL261 cells with an increasing capacity upto 18 hours of application. The phagocytic capacity was higher compared to commercial cell lines such as BV2 and Raw 264.7. Immortalized BMDMs tend to have a M2 phenotype without external induction and it is possible to activate M1 phenotype by LPS and IFNγ induction. CONCLUSION Here, we successfully immortalized bone-marrow derived macrophages and showed that they can be used as a tool to study macrophage polarization and phagocytic capability in a tumor immunotherapy context.

The IRE1α-XBP1 Signaling Axis Promotes Glycolytic Reprogramming in Response to Inflammatory Stimuli.

Immune cells must be able to adjust their metabolic programs to effectively carry out their effector functions. Here, we show that the endoplasmic reticulum (ER) stress sensor Inositol-requiring enzyme 1 alpha (IRE1α) and its downstream transcription factor X box binding protein 1 (XBP1) enhance the upregulation of glycolysis in classically activated macrophages (CAMs). The IRE1α-XBP1 signaling axis supports this glycolytic switch in macrophages when activated by lipopolysaccharide (LPS) stimulation or infection with the intracellular bacterial pathogen Brucella abortus. Importantly, these different inflammatory stimuli have distinct mechanisms of IRE1α activation; while Toll-like receptor 4 (TLR4) supports glycolysis under both conditions, TLR4 is required for activation of IRE1α in response to LPS treatment but not B. abortus infection. Though IRE1α and XBP1 are necessary for maximal induction of glycolysis in CAMs, activation of this pathway is not sufficient to increase the glycolytic rate of macrophages, indicating that the cellular context in which this pathway is activated ultimately dictates the cell's metabolic response and that IRE1α activation may be a way to fine-tune metabolic reprogramming. IMPORTANCE The immune system must be able to tailor its response to different types of pathogens in order to eliminate them and protect the host. When confronted with bacterial pathogens, macrophages, frontline defenders in the immune system, switch to a glycolysis-driven metabolism to carry out their antibacterial functions. Here, we show that IRE1α, a sensor of ER stress, and its downstream transcription factor XBP1 support glycolysis in macrophages during infection with Brucella abortus or challenge with Salmonella LPS. Interestingly, these stimuli activate IRE1α by independent mechanisms. While the IRE1α-XBP1 signaling axis promotes the glycolytic switch, activation of this pathway is not sufficient to increase glycolysis in macrophages. This study furthers our understanding of the pathways that drive macrophage immunometabolism and highlights a new role for IRE1α and XBP1 in innate immunity.

Abstract 12763: High Innate Aerobic Capacity Protects Against Angiotensin II-Induced Hypertension and Vascular Remodeling

Low aerobic capacity is associated with increased cardiovascular morbidity and mortality. A heterogeneous rat population selectively bred to segregate genes that sustain innate aerobic exercise capacity, low capacity runners (LCR) show impaired cardiac function in old age and premature death versus high capacity runners (HCR). Here we investigated whether high innate aerobic capacity offers early protection against cardiovascular disease by examining the hypertensive response of 7-month-old LCR and HCR rats to angiotensin II (Ang II, 150 μg/kg/min for 14 days). LCR and HCR rats showed no difference in basal blood pressure (105 mmHg) or vessel wall morphology, but in LCRs the mean arterial blood pressure increased to 145 mmHg versus 125 mmHg in HCRs. Congruently, LCR rats had increased aortic medial thickness and fibrosis. Aortic cytosolic and mitochondrial superoxide levels were also significantly increased in LCRs. Vascular reactivity experiments revealed impaired endothelium-dependent and -independent relaxation of mesenteric arteries of LCRs. Ang II infusion significantly increased the infiltration of macrophages into the aortic adventitia of LCRs as determined by CD68 and Mac-3 staining. Gene expression analysis by qPCR showed that markers of classically-activated macrophages CD86 and TNF-α were unchanged in rat strains but markers of alternatively-activated macrophages CD163 and Arg-1 were upregulated only in LCRs. Plasma IL-6, RANTES, CCL2 and Ang II and aortic VCAM1 levels were elevated only in LCRs whereas no difference in eNOS levels was observed between the rat strains. Aortic sections of LCRs showed increased senescence in adventitia whereas vascular smooth muscle cells (VSMC) isolated from LCR rats were more susceptible to Ang II-induced senescence. LCRs also showed increased cardiac hypertrophy and fibrosis. These data support the “outside-in” hypothesis, in which vascular inflammation initiated in adventitia progresses inwards to media and intima, causing VSMC and endothelial dysfunction, and that fibroblasts play a critical role in Ang II-induced hypertension and vascular remodeling under low innate aerobic capacity conditions, whereas high innate aerobic capacity is potentially protective against hypertension.

Specialized pro-resolving lipid mediators stimulate mitochondrial metabolism in macrophages

Abstract Chronic inflammation and sustained recruitment of classically activated macrophages (CAMs) with unchecked pro-inflammatory cytokine production is associated with disease development (e.g., atherosclerosis). Pro-inflammatory actions of CAMs are fueled by enhanced glycolysis and disruptions in the TCA cycle. Conversely, alternatively activated macrophages (AAMs) display an intact TCA cycle and mitochondrial oxidative phosphorylation (OXPHOS). Specialized pro-resolving lipid mediators (SPMs) stimulate resolution, in part, through actions on specific G-protein coupled receptors (GPCRs) in macrophages (e.g., RvD1□Fpr2). Whether SPMs exert pro-resolving effects by altering macrophage metabolism is unknown. We found that 1 hr treatment of SPMs (e.g., RvD1, RvD2, RvE1, and MaR1) enhanced basal (vehicle vs 1nM SPM; 6.11±0.1 vs RvD1 7.76±0.6 vs RvD2 9.48±0.9 vs RvE1 7.20±0.5 vs MaR1 8.73±0.9 pmol/min/μg protein; P<0.05; n=3), maximal (vehicle vs 0.1nM SPM; 9.06±1.1 vs RvD1 14.37±1.1 vs RvD2 16.54±2.7 vs RvE1 12.32±1.5 vs MaR1 13.28±1.3 pmol/min/μg protein; P<0.05; n=3), and ATP-linked respiration (vehicle vs 1nM SPM; 2.83±0.2 vs RvD1 4.50±0.3 vs RvD2 4.39±0.2 vs RvE1 4.71±0.3 vs MaR1 4.20±0.2 pmol/min/μg protein; P<0.05; n=3). Stimulation of mitochondrial metabolism with RvD1 was receptor mediated as Fpr2-deficient macrophages display no significant changes in metabolism following treatment (vehicle vs 1nM RvD1; basal 6.74±0.6 vs 6.52±0.2; maximal 11.54±1.2 vs 13.26±1.0; ATP-linked 5.84±0.8 vs 5.33±0.2 pmol/min/μg protein). Collectively, these data suggest that SPMs, despite having unique GPCRs, stimulate OXPHOS in macrophages, which could underlie their common pro-resolving properties.

Ultra-purification of Lipopolysaccharides reveals species-specific signalling bias of TLR4: importance in macrophage function

TLR4 location, and bacterial species-derived lipopolysaccharides, play a significant role in the downstream activation of transcription factors, accessory molecules, and products. Here, this is demonstrated through the use of classically-activated and alternatively-activated macrophages. We show that, when polarized, human macrophages differentially express and localize TLR4, resulting in biased recognition and subsequent signalling of LPS derived from Pseudomonas aeruginosa, Escherichia coli, and Salmonella enterica. Analysis of activation demonstrated that in classically activated macrophages, P. aeruginosa signals from the plasma membrane via TLR4 to p65 dependent on TAK1 and TBK1 signalling. E. coli signals dependent or independent of the endosome, utilizing both TAK1- and TBK1-signalling to induce P65 and IRF3 inducible genes and cytokines. S. enterica however, only induces P65 and IRF3 phosphorylation through signalling via the endosome. This finding outlines clear signalling mechanisms by which innate immune cells, such as macrophages, can distinguish between bacterial species and initiate specialized responses through TLR4.

IEX-1 deficiency in hematopoietic cells mitigates high fat diet-induced obesity and glucose intolerance

Abstract We previously showed that IEX-1 paucity in mice inhibited high fat diet (HFD)-induced obesity and insulin resistance. IEX-1 deficiency induced browning of white adipose tissue (WAT) by promoting alternative activation of macrophages (AAMs), increasing energy expenditure. We now hypothesize that IEX-1 deficiency in macrophages promotes alternative activation of adipose tissue macrophages (ATM) and induces browning to inhibit obesity. We performed reciprocal bone marrow (BM) transplantation between IEX-1 knockout (KO) and wild-type (WT) mice to generate four types of chimeras. We fed these mice with HFD for 20 weeks and monitored their body weight, glucose disposal, and determined ATM phenotype using flow cytometry. While WT+WT-BM control mice (WT mice received WT bone marrow) gained ~80% body weight on HFD, WT+KO-BM mice (WT mice received KO bone marrow) increased their body weight only by 50% (p<0.01). Conversely, KO+WT-BM mice (KO mice received WT bone marrow) exhibited increased weight gain on HFD as compared to KO+KO-BM control mice that remained lean (p<0.05). Likewise, WT+KO-BM mice displayed greater glucose disposal as compared to WT control, and KO+WT-BM mice became glucose intolerant as compared to KO control as determined by glucose tolerance test. Analysis of ATM phenotype in mice fed with HFD for 5 weeks showed that HFD increased the percentage of classically activated macrophages (CAMs) by 3–4-folds (p<0.01), and decreased AAMs by ~30% (P<0.05) in epidydmal and inguinal WAT of WT+WT-BM mice. However, these effects were markedly suppressed in WT+KO-BM mice. Collectively, these data suggest that IEX-1 in hematopoietic cells contributes to HFD-induced obesity and glucose intolerance via inhibiting AAMs polarization.

Hepcidin-mediated Iron Regulation in P19 Cells is Detectable by Magnetic Resonance Imaging

Magnetic resonance imaging can be used to track cellular activities in the body using iron-based contrast agents. However, multiple intrinsic cellular iron handling mechanisms may also influence the detection of magnetic resonance (MR) contrast: a need to differentiate among those mechanisms exists. In hepcidin-mediated inflammation, for example, downregulation of iron export in monocytes and macrophages involves post-translational degradation of ferroportin. We examined the influence of hepcidin endocrine activity on iron regulation and MR transverse relaxation rates in multi-potent P19 cells, which display high iron import and export activities, similar to alternatively-activated macrophages. Iron import and export were examined in cultured P19 cells in the presence and absence of iron-supplemented medium, respectively. Western blots indicated the levels of transferrin receptor, ferroportin and ubiquitin in the presence and absence of extracellular hepcidin. Total cellular iron was measured by inductively-coupled plasma mass spectrometry and correlated to transverse relaxation rates at 3 Tesla using a gelatin phantom. Under varying conditions of iron supplementation, the level of ferroportin in P19 cells responds to hepcidin regulation, consistent with degradation through a ubiquitin-mediated pathway. This response of P19 cells to hepcidin is similar to that of classically-activated macrophages. The correlation between total cellular iron content and MR transverse relaxation rates was different in hepcidin-treated and untreated P19 cells: slope, Pearson correlation coefficient and relaxation rate were all affected. These findings may provide a tool to non-invasively distinguish changes in endogenous iron contrast arising from hepcidin-ferroportin interactions, with potential utility in monitoring of different macrophage phenotypes involved in pro- and anti-inflammatory signaling. In addition, this work demonstrates that transverse relaxivity is not only influenced by the amount of cellular iron but also by its metabolism.

Standardized protocols for differentiation of THP-1 cells to macrophages with distinct M(IFNγ+LPS), M(IL-4) and M(IL-10) phenotypes

In vitro models of differing macrophage functions are useful since human monocyte–derived macrophages are short–lived, finite and vary from donor to donor. Published protocols using the promonocytic cell line THP-1 have tended to result in cells that closely resemble classically-activated macrophages, differentiated in IFNγ and LPS. However, no protocol, to date, has fully recapitulated polarization of THP-1 to the M(IL-4) or M(IL-10) macrophage phenotypes seen when human monocyte-derived macrophages are exposed to each cytokine. Here we present protocols that can be used to prepare M(IL-4) polarized THP-1 that transcribe CCL17, CCL26, CD200R and MRC1 and M(IL-10) cells which transcribe CD163, C1QA and SEPP1. We show that the inhibitory Fcγ Receptor IIb is preferentially expressed on the surface of M(IL-4) cells, altering the balance of activating to inhibitory Fcγ Receptors.Adoption of standardized experimental conditions for macrophage polarization will make it easier to compare downstream effector functions of different macrophage polarization states, where the impact of PMA exposure is minimized and rest periods and cytokine exposure have been optimized.

Innate immunity ascertained from blood and tracheal aspirates of preterm newborn provides new clues for assessing bronchopulmonary dysplasia.

AimThe study aimed to establish how granulocytes, monocytes and macrophages contribute to the development of bronchopulmonary dysplasia (BPD).Materials and methodsStudy A: samples of blood and tracheal aspirates (TAs) collected from preterm newborn infants during the first 3 days of life were investigated by flow cytometry, and testing for white blood cells (WBCs), neutrophils and neutrophil extracellular traps (NETs). Maternal blood samples were also collected. Study B: data from previously-tested samples of TAs collected from preterm newborn infants were re-analyzed in the light of the findings in the new cohort.ResultsStudy A: 39 preterm newborn infants were studied. A moderate correlation emerged between maternal WBCs and neutrophils and those of their newborn in the first 3 days of life. WBCs and neutrophils correlated in the newborn during the first 8 days of life. Decision rules based on birth weight (BW) and gestational age (GA) can be used to predict bronchopulmonary dysplasia (BPD). Neutrophil levels were lower in the TAs from the newborn with the lowest GAs and BWs. Study B: after removing the effect of GA on BPD development, previously-tested newborn were matched by GA. Monocyte phenotype 1 (Mon1) levels were lower in the blood of newborn with BPD, associated with a higher ratio of Monocyte phenotype 3 (Mon3) to Mon1. Newborn infants from mothers with histological chorioamnionitis (HCA) had lower levels of classically-activated macrophages (M1) and higher levels of alternatively-activated macrophages (M2) in their TAs than newborn infants from healthy mothers.ConclusionImmune cell behavior in preterm newborn infants was examined in detail. Surprisingly, neutrophil levels were lower in TAs from the newborn with the lowest GA and BW, and no correlation emerged between the neutrophil and NET levels in TAs and the other variables measured. Interestingly, monocyte phenotype seemed to influence the onset of BPD. The rise in the ratio of Mon 3 to Mon 1 could contribute to endothelial dysfunction in BPD.

TanshinoneⅡA phenanthroimidazole derivative polarizes macrophage to improve metabolic homeostasis

Macrophages infiltrated in adipose tissue play a key role in obesity. Some traditional pharmaceutical compounds may shift the polarization of recruited macrophages to improve metabolic homeostasis. TanshinoneⅡA (TAN2A) is a major active component of Salvia miltiorrhiza, a traditional anti-inflammatory cardiovascular medicine. In our study, we firstly constructed a phenanthroimidazole derivative of TAN2A named TAN20 by chemical synthesis, then identified its structure by chromatography and hydrogen spectroscopy, and finally examined its effects on immunometabolic responses. We found that TAN20 significantly induced the alternatively-activated (M2) rather than the classically-activated macrophages (M1), mainly through releasing the type II cytokines. Such effects were more pronounced than that from TAN2A. Compared to TAN2A, TAN20 substantially reduced body weight, decreased serum free fatty acid and HOMA-IR, and increased insulin sensitivity in obesity-induced diabetic mice. These effects of TAN20 were further validated on diabetic cynomolgus monkeys, which are closer to human physiological conditions. Taken together, our findings explicitly showed that TAN20 significantly polarized the macrophage and improved metabolic homeostasis in obesity-induced diabetic models, suggesting that TAN20 may be a potential drug against diabetes and obesity.

Pan-caspase inhibitors induce necroptosis via ROS-mediated activation of mixed lineage kinase domain-like protein and p38 in classically activated macrophages

Classically activated macrophages (CAMs) play a crucial protective role in the host by killing the invading pathogens. However, excessive activation of CAMs causes chronic inflammation leading to host tissue damage. Thus, control of macrophage activity is necessary to prevent chronic inflammation. To date, regulation of CAMs in the development of chronic inflammatory diseases has not been elucidated. In this study, we investigated the effect of a pan-caspase inhibitor, zVAD-fmk, in cell death in lipopolysaccharide (LPS)-activated macrophages, CAMs. Necrostatin-1, an inhibitor of necroptosis, inhibited zVAD-fmk-induced cell death in CAMs. The expression of mixed lineage kinase domain-like protein (MLKL) involved in the necroptosis pathway was up-regulated by LPS in CAMs. zVAD-fmk enhanced the phosphorylation of MLKL in CAMs. Moreover, inhibition of activation of mitogen activated protein kinase p38 and generation of reactive oxygen species (ROS) reduced zVAD-fmk-induced cell death in CAMs. Inhibition of ROS generation decreased the activation of MLKL and p38 in zVAD-fmk-treated CAMs. These results, taken together, indicate that zVAD-fmk-induced cell death occurred by necroptosis through ROS-mediated activation of MLKL and p38 in CAMs. Elucidation of the molecular mechanism underlying zVAD-fmk-induced necroptosis in CAMs might help in better understanding its significance in chronic inflammatory diseases.

The impact of myogenic cell transplantation on collateral capillary arteriogenesis

Current treatments for peripheral arterial occlusive disease (PAOD), including angioplasty and bypass grafting fail in up to 25% of patients, or are not indicated in up to 50%, so there is a need to develop additional treatment options. Patients with native collaterals have a better prognosis, so a treatment such as cell transplantation that promotes collateral arteriogenesis is a potential PAOD treatment. Unfortunately, bone marrow derived stem cells, the main cell type that has been investigated, have limited clinical effectiveness in enhancing pre‐existing collateral growth. Therefore, delivering a stem cell type native to the collateral‐containing tissue, such as myogenic stem cells, could have improved outcomes. Additionally, about 20% of patients never develop collaterals, and no one, to our knowledge, has evaluated the effect of cell therapy on collateral capillary arteriogenesis (CCA), which can reperfuse ischemic tissue in animals without native arteriolar collaterals and therefore represents a potential therapeutic target in patients who lack native collaterals. To test the hypothesis that myogenic cells enhance CCA, we ligated the spinotrapezius feed artery in Balb/C mice to induce CCA, and delivered myogenic cells, thrombin (positive control), or vehicle to the muscle. Seven days post ligation, ‐‐‐‐arterialized collateral capillary (ACC) number and diameter were determined. As expected, thrombin increased ACC number, but myogenic cells had no effect, 9.3 ± 0.5 vs 7.1 ± 0.4, respectively. However, myogenic cells increased ACC diameter as compared to untreated (12.1 ± 0.8 μm vs 10.3 ± 0.3 μm, respectively) and both myogenic cells and thrombin increased the maximum ACC diameter (29.9 ± 3.4 μm and 27.9 ± 2.0 μm vs 18.8 ± 1.1 μm, respectively). These results are consistent with previous studies indicating that thrombin enhances arteriogenesis and suggest that myogenic cells have a similar effect, but likely without the deleterious pro‐inflammatory effects of thrombin. Because myogenic cells increase arteriogenesis and macrophages are an essential part of arteriogenesis, we also tested the hypothesis that myogenic cells increase macrophage content by performing CD68 staining. Although macrophage number increased following ligation, neither myoblasts nor thrombin further increased macrophage number. The lack of difference in macrophage number despite enhanced CCA could be because the day‐7 timepoint was too late, as macrophages peak around day‐3, or because a shift towards either classically‐activated macrophages or alternatively‐activated macrophages, which have a greater effect on arteriogenesis, would not be detected by the CD68 stain. Therefore, future studies may include a day‐3 timepoint and a stain for alternatively‐activated macrophages.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Epigenetic control of macrophage polarization: implications for targeting tumor-associated macrophages.

The progression of cancer is a result of not only the growth of the malignant cells but also the behavior of other components of the tumor microenvironment (TME). Tumor-associated macrophages (TAMs) are key components of the TME that influence tumor growth and disease progression. TAMs can either inhibit or support tumor growth depending on their polarization to classically-activated macrophages (M1s) or alternatively-activated macrophages (M2s), respectively. Epigenetic regulation plays a significant role in determining this polarization and manipulating the epigenetic regulation in macrophages would provide a means for selectively targeting M2s thereby eliminating tumor-supporting TAMs while sparing tumor-inhibiting M1 TAMs. Many pharmacologic modulators of epigenetic enzymes are currently used clinically and could be repurposed for treating tumors with high TAM infiltrate. While much research involving epigenetic enzymes and their modulators has been performed in M1s, significantly less is known about the epigenetic regulation of M2s. This review highlights the field’s current knowledge of key epigenetic enzymes and their pharmacologic modulators known to influence macrophage polarization.

Neutrophil Microvesicles from Healthy Control and Rheumatoid Arthritis Patients Prevent the Inflammatory Activation of Macrophages

Microvesicles (MVs) are emerging as a novel means to enact cell-to-cell communication in inflammation. Here, we aimed to ascertain the ability of neutrophil-derived MVs to modulate target cell behaviour, the focus being the macrophage.MVs were generated in response to tumour necrosis factor-α, from healthy control neutrophils or those from rheumatoid arthritis patients. MVs were used to stimulate human monocyte-derived macrophages in vitro, or administered intra-articularly in the K/BxN mouse model of arthritis. A macrophage/fibroblast-like synoviocyte co-culture system was used to study the effects of vesicles on the crosstalk between these cells.We demonstrate a direct role for phosphatidylserine and annexin-A1 exposed by the MVs to counteract classical activation of the macrophages, and promote the release of transforming growth factor-β, respectively. Classically-activated macrophages exposed to neutrophil MVs no longer activated fibroblast-like synoviocytes in subsequent co-culture settings. Finally, intra-articular administration of neutrophil MVs from rheumatoid arthritis patients in arthritic mice affected the phenotype of joint macrophages.Altogether these data, with the identification of specific MV determinants, open new opportunities to modulate on-going inflammation in the synovia – mainly by affecting macrophage polarization and potentially also fibroblast-like synoviocytes - through the delivery of autologous or heterologous MVs produced from neutrophils.

Selective upregulation of TNFα expression in classically-activated human monocyte-derived macrophages (M1) through pharmacological interference with V-ATPase

Pharmacological interference with vacuolar-type H(+)-ATPase (V-ATPase), a proton-translocating enzyme involved in protein transport and pH regulation of cell organelles, is considered a potential strategy for cancer therapy. Macrophages are critically involved in tumor progression and may occur as pro-tumoral M2 phenotype, whereas classically-activated M1 can inhibit tumor development for example by releasing tumor-suppressing molecules, including tumor necrosis factor (TNF)α. Here, we show that targeting V-ATPase by selective inhibitors such as archazolid upregulates the expression and secretion of TNFα in lipopolysaccharide (LPS)- or LPS/interferon (INF)γ-activated M1-like macrophages derived from human blood monocytes. In contrast, archazolid failed to elevate TNFα production from uncommitted (M0) or interleukin (IL)-4-treated M2-like macrophages. Secretion of other relevant cytokines (i.e., IL-1β, IL-6, IL-10) or chemokines (i.e. IL-8 and monocyte chemotactic protein-1) from M1 was not affected by archazolid. Though V-ATPase inhibitors elevated the lysosomal pH in M1 comparable to chloroquine or ammonium chloride, the latter agents suppressed TNFα secretion. Archazolid selectively increased TNFα mRNA levels, which was abolished by dexamethasone. Interestingly, archazolid enhanced the phosphorylation and nuclear translocation of the p65 subunit of NFκB and stimulated phosphorylation of SAPK/JNK. In a microfluidically-supported human tumor biochip model, archazolid-treated M1 significantly reduced tumor cell viability. Together, our data show that V-ATPase inhibition selectively upregulates TNFα production in classically-activated macrophages along with NFκB and SAPK/JNK activation. Such increased TNFα release caused by V-ATPase inhibitors may contribute to tumor suppression in addition to direct targeting cancer cells.

Elimination of classically-activated macrophages in tumor-conditioned medium by alternatively-activated macrophages.

ABSTRACTCellular interactions are critical during development, tissue fitness and epithelial tumor development. The expression levels of specific genes confer to tumoral cells a survival advantage versus the normal neighboring cells. As a consequence, cells surrounding tumors are eliminated and engulfed by macrophages. We propose a novel scenario in which circulating cells facing a tumor can reproduce these cellular interactions. In vitro cultured macrophages from murine bone marrow were used to investigate this hypothesis. M1 macrophages in tumoral medium upregulated markers of a suboptimal condition, such as Sparc and TyrRS, and undergo apoptosis. However, M2 macrophages display higher Myc expression levels and proliferate at the expense of M1. Resulting M1 apoptotic debris is engulfed by M2 in a Sparc- and TyrRS-dependent manner. These findings suggest that tumor-dependent macrophage elimination could deplete immune response against tumors. This possibility could be relevant for macrophage based anti-tumoral strategies.

Silencing MicroRNA-155 Attenuates Cardiac Injury and Dysfunction in Viral Myocarditis via Promotion of M2 Phenotype Polarization of Macrophages

Macrophage infiltration is a hallmark feature of viral myocarditis. As studies have shown that microRNA-155 regulates the differentiation of macrophages, we aimed to investigate the role of microRNA-155 in VM. We report that silencing microRNA-155 protects mice from coxsackievirus B3 induced myocarditis. We found that microRNA-155 expression was upregulated and localized primarily in heart-infiltrating macrophages and CD4+ T lymphocytes during acute myocarditis. In contrast with wildtype (WT) mice, microRNA-155−/− mice developed attenuated viral myocarditis, which was characterized by decreased cardiac inflammation and decreased intracardiac CD45+ leukocytes. Hearts of microRNA-155−/− mice expressed decreased levels of the IFN-γ and increased levels of the cytokines IL-4 and IL-13. Although total CD4+ and regulatory T cells were unchanged in miR-155−/− spleen proportionally, the activation of T cells and CD4+ T cell proliferation in miR-155−/− mice were significantly decreased. Beyond the acute phase, microRNA-155−/− mice had reduced mortality and improved cardiac function during 5 weeks of follow-up. Moreover, silencing microRNA-155 led to increased levels of alternatively-activated macrophages (M2) and decreased levels of classically-activated macrophages (M1) in the heart. Combined, our studies suggest that microRNA-155 confers susceptibility to viral myocarditis by affecting macrophage polarization, and thus may be a potential therapeutic target for viral myocarditis.

The interaction of human macrophage subsets with silicone as a biomaterial

Silicones are widely used as biomaterials for medical devices such as extracorporeal equipments. However, there is often conflicting evidence about their supposed cell- and histocompatibility. Macrophages could mediate silicone-induced adverse responses such as foreign body reaction and fibrous encapsulation. The polarization behaviour of macrophages could determine the clinical outcome after implantation of biomaterials. Induction of classically activated macrophages (CAM) may induce and support uncontrolled inflammatory responses and undesired material degradation. In contrast, polarization into alternatively activated macrophages (AAM) is assumed to support healing processes and implant integration.This study compared the interaction of non-polarized macrophages (M0), CAM, and AAM with commercially available tissue culture polystyrene (TCP) and a medical grade silicone-based biomaterial, regarding the secretion of inflammatory mediators such as cytokines and chemokines. Firstly, by using the Limulus amoebocyte lysate (LAL) test the silicone films were shown to be free of soluble endotoxins, which is the prerequisite to investigate their interaction with primary immune cells. Primary human monocyte-derived macrophages (M0) were polarized into CAM and AAM by addition of suitable differentiation factors. These macrophage subsets were incubated on the materials for 24 hours and their viability and cytokine secretion was assessed. In comparison to TCP, cell adhesion was lower on silicone after 24 hours for all three macrophage subsets. However, compared to TCP, silicone induced higher levels of certain inflammatory and chemotactic cytokines in M0, CAM, and AAM macrophage subsets.Conclusively, it was shown that silicone has the ability to induce a pro-inflammatory state to different magnitudes dependent on the macrophage subsets. This priming of the macrophage phenotype by silicone could explain the incidence of severe foreign body complications observed in vivo.

Abstract 3355: Are cancer cells redefining macrophages’ aggressiveness

Abstract Breast cancer is one of the deadliest diseases in the United States. One in 5 women will be diagnosed with breast cancer during their lifetime and about 1 in 8 will develop invasive breast cancer. It has been shown that breast tumor's microenvironment contains large amounts of immune cells. In some cases macrophages account for up to 50% of the tumor mass. Macrophages belong to the innate immune response, and are mainly responsible for initiating the inflammatory response and engulfing foreign particles and cellular debris. They respond to multiple signals, which means they can perform a variety of functions ranging from phagocytosis to tissue repair. Classically-activated macrophages, referred as M1, are responsible for a pro-inflammatory response. Alternatively activated macrophages or M2 macrophages secrete anti-inflammatory cytokines, help in tissue repair, and are often involved in angiogenesis. In this project, we studied the effects of MCF-7 and MDA-MB-231 breast cancer cell lines on macrophages. We stimulated U937 cells with phorbol 12-myristate 13-acetate (PMA) for 24 hours. We checked for differentiation, morphology changes, and adhesion at 24 hours. We incubated 500,000 differentiated U937 cells with 500,000 cells (either MCF-7 or MDA-MB-231) or 500uL spent media from either MCF-7 or MDA-MB-231 cells for 48 hours. After the 48 hour incubation period, fluorescent microspheres were added to the macrophages and incubated for 1 hour to allow for phagocytosis. Macrophages were ran in a flow cytometer to quantify microsphere engulfment. We found about 50% decrease in engulfment activity in macrophages exposed to MCF-7 or MDA-MB-231 cells or media. When we analyzed the population of macrophages that engulfed 3+ beads, for MDA-MB-231 treatment, we found a 57% and 79% engulfment reduction in macrophages for media and cells respectively. For the MCF-7 treatment, the population of macrophages that engulfed 3+ beads was reduced 54% and 65.4% for media and cells respectively. We also studied gene expression of these macrophages to account for macrophage phenotype (M1 or M2). We found up-regulation on IL-10, down-regulation on TNF-α, and down-regulation on IL-12 suggesting that macrophages show a M2-like phenotype. This proposes that the tumor microenvironment (including cells, cytokines, cell vesicles, etc.) may be influencing the decrease in macrophage aggressiveness. Further studies will allows us to learn more in depth all of the interaction of cancer cells, their microenvironment, and macrophages. Citation Format: Elias Inga Jaco, Evita G. Weagel, Nicholas B. Anderson, Wei Meng, Joshua J. Davis, Richard A. Robinson, Kim L. O'Neill. Are cancer cells redefining macrophages’ aggressiveness. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3355. doi:10.1158/1538-7445.AM2015-3355

Expression pattern analysis and activity determination of matrix metalloproteinase derived from human macrophage subsets.

The polarization behavior of macrophages determines the clinical outcome after implantation of biomaterials. Formation of classically activated macrophages (CAM) may result in cell fusion to form foreign body giant cells, which induce and support uncontrolled inflammatory responses and can cause undesired material degradation. In contrast, polarization into alternatively activated macrophages (AAM) is assumed to support healing processes and implant integration. The expression of matrix metalloproteinases (MMP) by the different macrophage subsets might play a crucial role for inflammatory and wound healing processes and may subsequently influence the implant integration. Therefore, it is of importance to characterize the MMP expression pattern by the different macrophage subsets. This knowledge could support the design of biomaterials in which specific MMP cleavage sites are incorporated allowing a controlled cell-mediated degradation of the material. However, it needs to be considered that the pure expression levels may not correlate with the enzymatic activity of the MMP, which depends on a variety of different parameters such as additional co-factors. For this reason, the differential MMP expression levels and the overall enzymatic activity of in vitro generated human non-polarized macrophages (M0), CAM, and AAM are analyzed in this study. While MMP-1, MMP-3, and MMP-10 showed the highest expression levels in CAM, MMP-12 was most strongly expressed by AAM. Interestingly, although various MMP were expressed at high levels in CAM, the enzymatic MMP activity was increased in supernatants of AAM cultures. The data presented here illustrate the importance to combine the measurement of MMP expression levels with the analysis of the enzymatic activity. The observed MMP-12 expression in combination with the higher enzymatic activity detected in AAM supernatants might motivate the design of biomaterials, whose structure could be modified by MMP-12 catalyzed reactions leading to interactive polymers.