M2 MΦ Research Articles

Targeting and altering in vivo macrophage responses with modified polymer properties

Event Abstract Back to Event Targeting and altering in vivo macrophage responses with modified polymer properties Hannah C. Bygd1, Kiva D. Forsmark2 and Kaitlin M. Bratlie1, 2 1 Iowa State University, Materials Science & Engineering, United States 2 Iowa State University, Chemical & Biological Engineering, United States Introduction: Two pathways for activating MΦs exist. One of these routes is termed the classically activated M1 pathway and is achieved through exposure to lipopolysaccharide (LPS). M1 MΦs are known as pro-inflammatory cells. The other pathway is reached through interleukin-4 (IL-4) and is known as the alternatively activated M2 pathway. M2 MΦ produce pro-angiogenic factors. Here, we successfully use in vivo imaging and histological analysis to identify the MΦ response and activation. We demonstrate the ability to induce various MΦ phenotypes with a change in material functionality as well as identify certain materials parameters that seem to correlate with each phenotype. This suggests the potential to develop materials for specific applications and predict the outcome of MΦ activation in response to new surface chemistries. Experimental Methods: The poly(N-isopropylacrylamide) (pNIPAm) (~600nm) particles were synthesized using established methods and modified through conjugation with surface modifiers through carbodiimide chemistry. MΦs were polarized to the M1 and M2 phenotypes and internalization of functionalized pNIPAm particles were measured. These materials were also subcutaneously injected in SKH1-E mice. Arginase:iNOS profiles have been consistently used as a measure of MΦ phenotype. Arginine activity was measured indirectly through a urea assay in which the lysate was activated, exposed to arginine, and conversion to urea was quantified. Nitrites were measured through a Griess reagent assay to indirectly determine the levels of inducible nitric oxide synthase (iNOS). Material properties of the modified particles including water contact angle, melting temperature, and zeta-potential were also measured. Results: SKH1-E mice were injected with the polymer particles and imaged for cathepsin activity (Figure 1). A range in cathepsin activity was observed for the different surface modifiers. The tissue sections from the mice in Figure 1 were excised and homogenized. The arginase:iNOS displayed a spectrum between the M1 and M2 phenotypes for different surface modifiers as shown in Figure 2. Discussion: Distinct material parameters influence MΦ phenotypes. For example, increasing the hydrogen bonding of polymeric particles can polarize MΦs towards the M1 phenotype. The induction of multiple MΦ phenotypes confirms the ability to control MΦ activation in vivo and suggests the importance of chemical properties in the biocompatibility of materials. The results obtained suggest that changing properties can result in opposite MΦ phenotypes. Conclusion: With this work, useful insight was gained about in vivo MΦ response to various material functionalities. Overall, this in vivo study was successful at drawing conclusions about the biomaterial parameters that will allow for tuning MΦ polarization responses to biomaterials and targeting MΦs. National Science Foundation; Roy J. Carver Charitable Trust Keywords: in vivo, cell phenotype, Cell response, Cell modulation Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Biomaterials in immune response Citation: Bygd HC, Forsmark KD and Bratlie KM (2016). Targeting and altering in vivo macrophage responses with modified polymer properties. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.02096 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Hannah C Bygd Kiva D Forsmark Kaitlin M Bratlie Google Hannah C Bygd Kiva D Forsmark Kaitlin M Bratlie Google Scholar Hannah C Bygd Kiva D Forsmark Kaitlin M Bratlie PubMed Hannah C Bygd Kiva D Forsmark Kaitlin M Bratlie Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Abstract A30: Hypoxia induces macrophage polarization and re-education towards an M2 phenotype in glioblastoma

Abstract Introduction: Hypoxia, resulting in a decrease in tissue oxygen tension, is a main characteristic of glioblastoma (GBM) and is associated to a poor life expectancy of GBM patients (Spence et al., 2008). In the same time, it is now known that GBM are also inflammatory tumors and that macrophages (MΦ) are the most abundant immune cells found in these tumors (Badie et al., 2000). MΦ present in tissue can be polarized into at least two distinct phenotypes: the M1 phenotype (with antitumoral activity) and the M2 phenotype (with protumoral activity) (Mantovani et al., 2002), the most often phenotype found in patients with GBM (Lu-Emerson et al., 2013). However, factors responsible for their migration and phenotype acquisition in the context of GBM are not yet identified. The aim of this study was to evaluate, in vivo, the impact of hypoxia on MΦ tropism and polarization in a preclinical model of human GBM that progressively evolves towards severe hypoxia and, in vitro, the effect of hypoxia on MΦ polarization and re-education. Method: Animal models consisted of injection of human GBM U251 cells into the brain of immunodeficient rats. Hypoxia has been evaluated by pimonidazole and CAIX staining. MΦ have been detected on brain slices through a CD68 staining and polarization toward M1 or M2 phenotype with iNOS and Arginase1 staining, respectively. In parallel, murine primary cultures of MΦ have been freshly prepared from bone marrow. M0, M1 and M2 MΦ have been exposed to moderate (1% oxygen) or severe (0.2% oxygen) hypoxia for 24h and their phenotypes have been characterized by RT-qPCR and enzymatic assays. Results: In the U251 model, we have shown that MΦ migrated at the tumor site only when hypoxia takes place. We also demonstrated that the acquisition of the M2 phenotype was clearly a dynamic process with hypoxia as the major trigger for this transition. Furthermore, MΦ present at the shell of the tumor express M1 markers unlike MΦ found in hypoxic zones which exhibit M2 markers. In support of these in vivo findings, M0 but also M1 MΦ cultured in 1% or 0.2% oxygen displayed a phenotype close to that of M2 MΦ whose this phenotype was further reinforced by severe hypoxia. Conclusion: In this study, using a human preclinical GBM model which progressively develops severe hypoxia, we showed that the presence of MΦ in the tumor depends on presence or not of hypoxia. We have also shown that hypoxia polarizes and re-educates MΦ towards M2 MΦ but also that it fine-tunes the M2 phenotype. These results highlight the effect of hypoxia in the aggressiveness of GBM and may help to improve therapies for these tumors by taking in account that hypoxia drives MΦ towards a protumoral phenotype. Acknowledgements: Conseil Régional de Basse-Normandie, Université de Caen Basse-Normandie, Archade, CNRS, the European Regional Development Fund of Basse-Normandie (FEDER), “l'Europe s'engage en Basse Normandie” and the French Ministère de l'Enseignement Supérieur et de la Recherche (MESR). Citation Format: Marine M. Leblond, Aurélie N. Gérault, Aurélien Corroyer-Dulmont, Edwige Petit, Myriam Bernaudin, Samuel Valable. Hypoxia induces macrophage polarization and re-education towards an M2 phenotype in glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A30.

Engineering macrophages to control the inflammatory response and angiogenesis

Macrophage (MΦ) dysregulation is increasingly becoming recognized as a risk factor for a number of inflammatory complications including atherosclerosis, cancer, and the host response elicited by biomedical devices. It is still unclear what roles the pro-inflammatory (M1) MΦ and pro-healing (M2) MΦ phenotypes play during the healing process. However, it has been shown that a local overabundance of M1 MΦs can potentially lead to a chronically inflamed state of the tissue; while a local over-exuberant M2 MΦ response can lead to tissue fibrosis and even promote tumorigenesis. These notions strengthen the argument that the tight temporal regulation of this phenotype balance is necessary to promote inflammatory resolution that leads to tissue homeostasis. In this study, we have engineered pro-inflammatory MΦs, MΦ-cTLR4 cells, which can be activated to a M1-like MΦ phenotype with a small molecule, the chemical inducer of dimerization (CID) drug. The MΦ-cTLR4 cells when activated with the CID drug, express increased levels of TNFα, IL-6, and iNOS. Activated MΦ-cTLR4 cells stay stimulated for at least 48h; once the CID drug is withdrawn, the MΦ-cTLR4 cells return to baseline state within 18h. Further, in vitro CID-activated MΦ-cTLR4 cells induce upregulation of VCAM-1 and ICAM-1 on endothelial cells (EC) in a TNFα-dependent manner. With the ability to specifically modulate the MФ-cTLR4 cells with the presence or absence of a small molecule, we now have the tool necessary to observe a primarily M1 MФ response during inflammation. By isolating this phase of the wound healing response, it may be possible to determine conditions for ideal healing.

Tumor-associated macrophages in oral premalignant lesions coexpress CD163 and STAT1 in a Th1-dominated microenvironment

BackgroundTumor-associated macrophages (TAMs) are implicated in the growth, invasion and metastasis of various solid tumors. However, the phenotype of TAMs in premalignant lesions of solid tumors has not been clarified. In the present study, we identify the phenotype of TAMs in leukoplakia, an oral premalignant lesion, by immunohistochemical analysis and investigate the involvement of infiltrated T cells that participate in the polarization of TAMs.MethodsThe subjects included 30 patients with oral leukoplakia and 10 individuals with normal mucosa. Hematoxylin and eosin slides were examined for the histological grades, and immunohistochemical analysis was carried out using antibodies against CD68 (pan-MΦ), CD80 (M1 MΦ), CD163 (M2 MΦ), CD4 (helper T cells: Th), CD8 (cytotoxic T cells), CXCR3, CCR5 (Th1), CCR4 (Th2), signal transducer and activator of transcription (STAT1), phosphorylated STAT1 (pSTAT1) and chemokine CXCL9. The differences in the numbers of positively stained cells among the different histological grades were tested for statistical significance using the Kruskal-Wallis test. Correlations between different types of immune cells were determined using Spearman’s rank analysis.ResultsAn increase in the rate of CD163+ TAM infiltration was observed in mild and moderate epithelial dysplasia, which positively correlated with the rate of intraepithelial CD4+ Th cell infiltration. Although CCR4+ cells rarely infiltrated, CXCR3+ and CCR5+ cells were observed in these lesions. Cells positive for STAT1 and chemokine CXCL9, interferon- (IFN)-induced gene products, and pSTAT1 were also observed in the same lesions. Double immunofluorescence staining demonstrated that the cells that were positive for CD163 were also positive for STAT1.ConclusionsCD163+ TAMs in oral premalignant lesions coexpress CD163 and STAT1, suggesting that the TAMs in oral premalignant lesions possess an M1 phenotype in a Th1-dominated micromilieu.

Hypoxia induces macrophage polarization and re-education toward an M2 phenotype in U87 and U251 glioblastoma models

Hypoxia is a common feature of solid tumors, particularly in glioblastoma (GBM), and known to be a poor prognosis factor in GBM patients. The growth of GBM is also associated with a marked inflammation partially characterized by an accumulation of macrophage (MΦ) of the M2 phenotype. However, the transition between M1 MΦ (antitumoral) and M2 MΦ (protumoral) phenotypes is a dynamic process. We made the assumption that oxygen (O2) availability could be a major regulator of this transition and that the intratumoral O2 gradient is of importance. We evaluated, in vivo, the impact of hypoxia on MΦ tropism and polarization in two models of human GBM, well differentiated by their degree of hypoxia. MΦ migration in the tumor was more pronounced in the more hypoxic tumor of the two GBM models. In the more hypoxic of the models, we have shown that MΦ migrated at the tumor site only when hypoxia takes place. We also demonstrated that the acquisition of the M2 phenotype was clearly an evolving phenomenon with hypoxia as the major trigger for this transition. In support of these in vivo finding, M0 but also M1 MΦ cultured in moderate or severe hypoxia displayed a phenotype close to that of M2 MΦ whose phenotype was further reinforced by severe hypoxia. These results highlight the role of hypoxia in the aggressiveness of GBM, in part, by transforming MΦ such that a protumoral activity is expressed.

Mechanism by which commensal exopolysaccharides limit inflammation (MUC9P.744)

Abstract Trillions of bacteria live within the gastrointestinal tract and are critical for maintaining intestinal homeostasis; however, the mechanisms utilized by specific bacterial molecules to contribute to homeostasis are not well understood. We utilize a mouse model in which a single dose of the probiotic, B. subtilis, protects mice from acute colitis induced by the enteric pathogen C. rodentium. Our goal is to elucidate the mechanism by which B. subtilis prevents inflammation. We identified exopolysaccharides (EPS) to be the active molecule of B. subtilis, and a single dose of EPS protects mice from disease. EPS specifically binds F4/80+CD11b+ peritoneal macrophages (MΦ). Following EPS treatment, these cells increase expression of CD206+Arg1+YM-1+IL-4Rα+ and inhibit T cell activation and proliferation in vitro, indicative of M2 MΦ. CD4+ T cells drive much of the inflammation associated with C. rodentium infection, and we hypothesized that EPS inhibits CD4+ T cell responses in vivo. Accordingly, we measured levels of IFNγ (Th1), IL-17 (Th17), and IL-13 (Th2) in splenic T cells following EPS treatment and found decreased levels of these cytokines. Similarly, mesenteric lymph node from mice orally gavaged with B. subtilis spores produced decreased levels of T cell cytokines in an EPS dependent manner. We hypothesize that EPS-induced M2 MΦ mediate inhibition of CD4+ T cell responses in vivo and further, that these cells mediate protection from acute colitis induced by C. rodentium.

AhR-dependent secretion of PDGF-BB by human classically activated macrophages exposed to DEP extracts stimulates lung fibroblast proliferation

Lung diseases are aggravated by exposure to diesel exhaust particles (DEPs) found in air pollution. Macrophages are thought to play a crucial role in lung immune response to these pollutants, even if the mechanisms involved remain incompletely characterized. In the present study, we demonstrated that classically and alternative human macrophages (MΦ) exhibited increased secretion of PDGF-B in response to DEP extract (DEPe). This occurred via aryl hydrocarbon receptor (AhR)-activation because DEPe-induced PDGF-B overexpression was abrogated after AhR expression knock-down by RNA interference, in both M1 and M2 polarizing MΦ. In addition, TCDD and benzo(a)pyrene, two potent AhR ligands, also significantly increased mRNA expression of PDGF-B in M1 MΦ, whereas some weak ligands of AhR did not. We next evaluated the impact of conditioned media (CM) from MΦ culture exposed to DEPe or of recombinant PDGF-B onto lung fibroblast proliferation. The tyrosine kinase inhibitor, AG-1295, prevents phosphorylations of PDGF-Rβ, AKT and ERK1/2 and the proliferation of MRC-5 fibroblasts induced by recombinant PDGF-B and by CM from M1 polarizing MΦ, strongly suggesting that the PDGF-BB secreted by DEPe-exposed MΦ is sufficient to activate the PDGF-Rβ pathway of human lung fibroblasts. In conclusion, we demonstrated that human MΦ, whatever their polarization status, secrete PDGF-B in response to DEPe and that PDGF-B is a target gene of AhR. Therefore, induction of PDGF-B by DEP may participate in the deleterious effects towards human health triggered by such environmental urban contaminants.

Exposure to diesel exhaust particle extracts (DEPe) impairs some polarization markers and functions of human macrophages through activation of AhR and Nrf2.

Macrophages (MΦ), well-known to play an important role in immune response, also respond to environmental toxic chemicals such as diesel exhaust particles (DEP). Potential effects of DEPs towards MΦ polarization, a key hall-mark of MΦ physiology, remain however poorly documented. This study was therefore designed to evaluate the effects of a reference DEP extract (DEPe) on human MΦ polarization. Human blood monocytes-derived MΦ were incubated with IFNγ+LPS or IL-4 to obtain M1 and M2 subtypes, respectively; a 24 h exposure of polarizing MΦ to 10 μg/ml DEPe was found to impair expression of some macrophagic M1 and M2 markers, without however overall inhibition of M1 and M2 polarization processes. Notably, DEPe treatment increased the secretion of the M1 marker IL-8 and the M2 marker IL-10 in both MΦ subtypes, whereas it reduced lipopolysaccharide-induced IL-6 and IL-12p40 secretion in M1 MΦ. In M2 MΦ, DEPe exposure led to a reduction of CD200R expression and of CCL17, CCL18 and CCL22 secretion, associated with a lower chemotaxis of CCR4-positive cells. DEPe activated the Nrf2 and AhR pathways and induced expression of their reference target genes such as Hmox-1 and cytochrome P-4501B1 in M1 and M2 MΦ. Nrf2 or AhR silencing through RNA interference prevented DEPe-related down-regulation of IL-6. AhR silencing also inhibited the down-secretion of IL-12p40 and CCL18 in M1- and M2-DEPe-exposed MΦ, respectively. DEPs are therefore likely to alter expression of some M1 and M2 markers in an AhR- and Nrf2-dependent manner; such regulations may contribute to deleterious immune effects of atmospheric DEP.

Prosurvival and Chemoprotective Effects of Tumor-Associated Macrophages Reversed By Anti-CSF-1R Monoclonal Antibody in B-Cell Lymphomas

The results of gene expression profiling (GEP) and immunohistochemical studies indicate that survival is worsened by macrophages (MΦ) in the tumor microenvironment of various B-cell lymphomas including follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). Tumor-associated macrophages (TAMs) are known to be different from other types of MΦ, but the effects of TAMs that worsen prognosis in B-cell lymphoma are essentially unknown, as are the mechanisms of these effects. Here, we determined the phenotype and effects of TAMs on tumor survival, proliferation, and drug resistance in B-cell lymphomas and evaluated strategies to reverse their effects.As compared to peripheral blood monocytes (Mo) from normal donors (ND), Mo from FL patients were differentiated less into M1 MΦ (defined as CD68+CD163loCD206loCD86hi) by culture with CSF-1 for 5 days followed by IFN-g + LPS for 2 days more. In contrast, Mo from FL patients and ND were differentiated similarly into M2 MΦ (defined as CD68+CD163hiCD206hiCD86lo) by culture with CSF-1 followed by IL-4. Consistent with this, MΦ gene signatures from FL tumors were more similar to previously-described signatures of M2 rather than M1 MΦ (Martinez et al, J Immunol, 2006, 177(10):7303-11).In co-culture, primary FL tumor cells and lymphoma cell lines (including RL, a transformed FL cell line; Granta 519, a mantle cell lymphoma (MCL) cell line; and Raji, a Burkitt lymphoma cell line) induced differentiation of Mo into MΦ. Differentiation could be prevented by CS4 monoclonal antibody (mAb), a fully human IgG1 anti-human CSF-1R mAb (ImClone/Eli Lilly), but not isotype control Ab. Elevated levels of CSF-1 in culture supernatants after addition of CS4 mAb and real-time PCR of tumor cells suggested secretion of CSF-1 by lymphoma cells. Spontaneous apoptosis of primary FL and MCL tumor cells, determined by Annexin V and propidium iodide staining, was significantly reduced by co-culture with ND Mo (p<0.01), whether pre-differentiated into MΦ with CSF-1 or not, but this protection could be reversed by CS4 mAb. Mo and/or pre-differentiated MΦ protected primary FL and MCL tumor cells from cytotoxic effects of doxorubicin and/or bendamustine (p<0.01), but CS4 mAb reversed this effect. To assess effects of MΦ on proliferation, lymphoma cell lines (RL, Granta 519, and Raji) were CFSE-labeled prior to co-culture with Mo and doxorubicin, and proliferation assessed by CFSE dilution by flow cytometry in the presence or absence of CS4 or isotype control mAbs. MΦ promoted proliferation of all three cell lines, but this effect could be reversed by CS4 mAb.To further understand the mechanism by which MΦ promote tumor survival and growth, we performed phosflow analysis and found increased phosphorylation of STAT3 in co-cultured lymphoma cells. Consistent with this, we observed a correlation between an 11-gene STAT3 activation signature, described by Huang et al in DLBCL tumors (J Clin Oncol, 2013, 52.8414), and a MΦ gene signature in whole genome GEP studies of 191 FL tumors (Pearson correlation co-efficient=0.396, p<0.001).In conclusion, our results suggest that Mo from FL patients are predisposed to differentiate into an M2-like MΦ state. The interaction between lymphoma cells and Mo/MΦ is reciprocal: a change in Mo (MΦ differentiation) induced by interaction with lymphoma tumor cells leads to a change in the tumor cells (promotion of survival, proliferation, and chemoresistance). More importantly, our results demonstrate that targeting TAMs using CS4, an anti-CSF-1R mAb, can be an effective strategy to overcome the adverse effects of TAMs and reverse chemoresistance. Further studies are needed to determine whether STAT3 activation contributes to the protumor effects of TAMs. This may provide novel insights into the molecular mechanisms related to TAMs and lymphoma cells and offers additional targets for therapeutic development. In the long term, strategies targeting TAMs is especially appealing, as they should be able to be combined with existing therapies including chemotherapy, other immunotherapy, and targeted therapy, potentially improving their efficacy without increasing toxicity for FL, DLBCL, and other B-cell malignancies. DisclosuresNo relevant conflicts of interest to declare.

Opposite Effects of M1 and M2 Macrophages on Hematopoietic Stem Cell Self-Renewal and Ex Vivo Expansion

Macrophages (MΦ) are professional phagocytes in the innate immune system. They are not only involved in regulation of various immune functions and inflammation, but also exhibit plasticity in modulation of tissue regeneration and repair after being polarized into M1 and M2 MΦ by different inflammatory cytokines. In addition, several recent studies show that MΦ are a new constituent of the hematopoietic stem cells (HSCs) niche and play a role in regulation of HSCs maintenance and mobilization in bone marrow (BM). However, it is not known whether MΦ can regulate HSCs self-renewal and whether the effects of MΦ on HSCs can be influenced by differential MΦ polarization. This was investigated using an ex vivo HSCs expansion model consisting of mouse bone marrow LSK (Lin-sca-1+c-Kit+) cells cultured with or without MΦ in a mouse HSCs expansion medium (StemSpanTM serum-free medium supplemented with 20ng/ml of stem cell factor [SCF] and thrombopoietin [TPO]). We found that LSK cells were expanded about 20-, 15-, and 30-fold after 6 days of co-culture with MΦ harvested from mouse BM, spleen, and peritoneal cavity, respectively, whereas there was no significant expansion after culture without MΦ or with BM Gr-1high or Gr-1low monocytes. In addition, we found that M1-MΦ polarized by INFγ were more effective than IL4-polarized M2-MΦ in promoting LSK cells expansion ex vivo (45-fold vs. 15-fold). However, the promotion of LSK cells expansion by M1-MΦ resulted in about 88% reduction in HSCs as judged by 5-week cobblestone area forming cell (CAFC) assay. In contrast, M2-MΦ significantly promoted HSCs expansion. A greater expansion of HSCs was achieved after LSK cells were co-cultured with M2-MΦ for 9 days than for 6 days (20-fold vs. 6-fold). These findings suggest that M1-MΦ are more effective than M2-MΦ in promoting LSK cells or hematopoietic progenitor cells (HPCs) expansion, at the expense of HSCs self-renewal, whereas M2-MΦ can promote HPCs expansion as well as HSCs self-renewal. This suggestion is supported by results of serial transplantation and competitive repopulation unit (CRU) assays. CRU assay showed that LT-HSCs (e.g. 4-month CRU) were increased about 13 folds relative to the starting numbers of CRU in the input after LSK cells were co-cultured with M2-MΦ for 9 days, but were barely detectable after the cells were cultured without MΦ or with M1-MΦ. The inhibitory effect of M1-MΦ on HSCs self-renewal and expansion was attenuated by inhibition of inducible nitric oxide synthase (iNOS) activity with an inhibitor or knockout iNOS. Inhibition of arginase and/or cyclooxygenase activities with an inhibitor attenuated the promotion of HSCs self-renewal and expansion by M2-MΦ. More importantly, we found that human CD34+ cells, 8-week CAFC, and SCID mice repopulating cells (SRCs) were increased 42±14, 8±2.1, and 4 folds over the input values, respectively, after human cord blood CD34+ cells were co-cultured with M2-MΦ generated from human cord blood CD34- cells for 7 days in a human HSCs expansion medium (StemSpanTM serum-free medium supplemented with 50 ng/ml of SCF, TPO, and FLT-3 ligand). These findings demonstrate that M1-MΦ and M2-MΦ have opposite effects on HSCs self-renewal, which may be important for regulation of hematopoiesis under various pathological conditions in which MΦ are differentially polarized to M1 or M2 by diverse inflammatory cytokines. In addition, M2-MΦ may be used to promote human cord blood HSCs ex vivo expansion to make human cord blood transplantation available to more patients. DisclosuresNo relevant conflicts of interest to declare.

Porphyromonas gingivalis lipopolysaccharide weakly activates M1 and M2 polarized mouse macrophages but induces inflammatory cytokines.

Porphyromonas gingivalis is associated with chronic periodontitis, an inflammatory disease of the tooth's supporting tissues. Macrophages are important in chronic inflammatory conditions, infiltrating tissue and becoming polarized to an M1 or M2 phenotype. As responses to stimuli differ between these phenotypes, we investigated the effect of P. gingivalis lipopolysaccharide (LPS) on M1 and M2 macrophages. M1 and M2 polarized macrophages were produced from murine bone marrow macrophages (BMMϕ) primed with gamma interferon (IFN-γ) or interleukin-4 (IL-4), respectively, and incubated with a low or high dose of P. gingivalis LPS or control TLR2 and TLR4 ligands. In M1-Mϕ, the high dose of P. gingivalis LPS (10 μg/ml) significantly increased the expression of CD40, CD86, inducible nitric oxide synthase, and nitric oxide secretion. The low dose of P. gingivalis LPS (10 ng/ml) did not induce costimulatory or antibacterial molecules but did increase the secretion of IL-1α, IL-6, IL-12p40, IL-12p70, and tumor necrosis factor alpha (TNF-α). P. gingivalis LPS marginally increased the expression of CD206 and YM-1, but it did enhance arginase expression by M2-Mϕ. Furthermore, the secretion of the chemokines KC, RANTES, eotaxin, and MCP-1 from M1, M2, and nonpolarized Mϕ was enhanced by P. gingivalis LPS. TLR2/4 knockout macrophages combined with the TLR activation assays indicated that TLR2 is the main activating receptor for P. gingivalis LPS and whole cells. In conclusion, although P. gingivalis LPS weakly activated M1-Mϕ or M2-Mϕ compared to control TLR ligands, it induced the secretion of inflammatory cytokines, particularly TNF-α from M1-Mϕ and IL-10 from M2-Mϕ, as well as chemotactic chemokines from polarized macrophages.

Ixazomib Citrate (MLN9708) Inhibits Kidney Allograft Infiltrating Macrophages in a CNI Minimizing Strategy.

Macrophages (MΦ) play an important role in acute rejection after kidney transplantation (Tx). Calcineurin inhibitor minimizing strategies may be associated with improved outcome after Tx. We sought to determine the role of a new protease inhibitor (PI, Ixazomib citrate, MLN9708) on MΦand MΦ associated genes in a CNI minimization strategy. Lewis (RT1l) recipients were transplanted with a full MHC mismatch Brown Norway (RT1n) rat kidney. Recipients were randomized into four groups: Control Brown Norway (C), Transplant with no treatment (Tx), transplant with full dose CsA (Tx, 10 mg/kg/d) or transplant with MLN9708 + half dose CsA (TxMLNCsA, 5 mg/kg/d). One week post-transplant, RNA was isolated from kidney for gene microarray analysis on Affymetrix Rat ST 2.0 chips. Differentially expressed gene (DEG) and pathway analyses were performed using iReport. Cellular analyses were performed using immunohistochemistry. Tx alone animals had a strong influx of MΦ into the kidney. Treatment with full dose CsA or low dose CsA + MLN9708 greatly reduced MΦ infiltration. The molecular signature associated with MΦ activation pathways was downregulated in animals treated with MLN9708 + ½ CsA, similar to full dose CsA.Table: No Caption available.These genes included IL-10, heme oxygenase 1 and biomarkers of M1 (iNOS) and M2 (arg1, CD163) MΦ phenotypes. These preclinical studies suggest that CNI minimization strategies with novel proteasome inhibitors may be effective in preventing macrophage-induced allograft injury. Further studies are needed to better characterize the significance and regulation of macrophage associated genes by proteasome inhibitors.

Identification of a novel epigenetic code, histone H1 fucosylation, and its roles in macrophage plasticity in rheumatoid arthritis (IRM7P.491)

Abstract Macrophages (MΦ) are key players in rheumatoid arthritis (RA). High plasticity renders MΦ an attractive tool for studying epigenetic reprogramming. We have observed a highly positive correlation between TNFα and fucosylation catalyzing enzymes, fucosyltransferases (FUTs) 1, 2, 3, 4, 5, 6, 7, 9, 10, 11 (p=0.0001) in human RA synovia; these FUTs are predominantly detected in M1 inflammatory MΦ but not M2 MΦ and T cells from RA synovial fluid. A fucosylation inhibitor, 2-Deoxy-D-galactose (2-D-gal) precluded collagen II-induced arthritis in DBA/1J mice (scores 9.5±1.7 vs 0.5± 0.3, p&amp;lt;0.01) with reduced M1 MΦ in draining LN, decreased TNF-α and anti-CII in the serum (p&amp;lt;0.05).Ulex Europaeus Agglutinin I (UEA 1) affinity chromatography coupled to mass spectrometry indicated that the linker Histone H1, but not core histone H 2-4, is the major protein that modified by a fucose-α-(1-2)-gal moiety in M1 MΦ. Disrupting this moiety by 2-D-gal skewed the differentiation of M1 MΦ toward M2 anti-inflammatory phenotype with a reprogrammed gene expression, including i) robustly upregulated Il10, Arg1, and Megf8 (p&amp;lt;0.01) (M2 markers); ii) downregulated Ifnγ and Il6 (p&amp;lt;0.05) (M1 markers); iii) inhibited Actin and its associated molecules including Ahnak and Cap1, and suppressed the antigen presentation of M1 MΦ phenotype (p&amp;lt;0.01). We uncovered Histone 1 fucosylation as a novel epigenetic code that orchestrates MΦ transcriptome reprogramming and renders it an attractive target for RA therapy.

The role of a Stat4 deficiency in Atherosclerosis (CAM1P.237)

Abstract Atherosclerosis is a chronic inflammatory process that leads to plaque formation in large and medium size vessels. Th1 cells constitute a majority of plaque T cells and play a pro-atherogenic role. Signal transducer and activator of transcription 4 (Stat4) is a transcription factor involved in Th1 cell differentiation. To test a role of Stat4 in atherogenesis, we bred Stat4-deficient (Stat4-/-) and atherosclerosis-prone Apolipoprotein E (Apoe-/-) mice. Stat4 deficiency resulted in a 70% reduction in plaque burden in Stat4-/-Apoe-/- vs Apoe-/- mice (p&amp;lt;0.01). Surprisingly, reduced atherogenesis was not due to attenuated IFNγ production suggesting an IFNγ-independent pro-atherogenic role of Stat4. Stat4 is highly expressed in T cells, but also detected in macrophages (MΦ). To further test an impact of Stat4 deficiency in myeloid cells, we analyzed activation status of MΦ. Stat4-/-Apoe-/- CD11b+ MΦ expressed low levels of CD69, MHCII, and CD86 when pulsed with LPS vs Apoe-/- MΦ. Differentiated in vitro Stat4-/-Apoe-/- M1 or M2 MΦ also had reduced expression of CCL2 and CXCL10 vs Apoe-/- M1 and M2 MΦ. Importantly, the percentage of aortic Ly6C+ (p&amp;lt;0.02) and CD68+ (p&amp;lt;0.06) cells was reduced in Stat4-/-Apoe-/- vs Apoe-/- mice. The data provides evidence for a functional role of Stat4 in atherosclerosis by elevating levels of MΦ activation and aortic MΦ content, and thus promoting the localized aortic pro-inflammatory immune response. Supported by R01 HL112605 and Suppl.Grant 02S1.

Unique Macrophages Different from M1/M2 Macrophages Inhibit T Cell Mitogenesis while Upregulating Th17 Polarization

Mycobacterial infection induces suppressor macrophages (MΦs), causing disease exacerbation. There are two major MΦ subsets (M1 and M2 MΦs) that are phenotypically and functionally different. Here, we examined which of the MΦ subsets the mycobacterial infection-induced suppressor MΦs (MIS-MΦs) belong to. MIS-MΦs down-regulated T cell production of Th1 and Th2 cytokines but markedly increased production of interleukin (IL)-17A and IL-22 through up-regulation of Th17 cell expansion. In this phenomenon, a novel MΦ population, which is functionally distinguishable from M1 and M2 MΦ subsets and possesses unique phenotypes (IL-12+, IL-1βhigh, IL-6+, tumor necrosis factor (TNF)-α+, nitric oxide synthase (NOS) 2+, CCR7high, IL-10high, arginase (Arg)-1−, mannose receptor (MR)low, Ym1high, Fizzlow, and CD163high), played central roles through the action of IL-6 and transforming growth factor (TGF)-β but not IL-21 and IL-23. This new type of MΦ population was induced in infected mice and actively supported the in vivo expansion of Th17 cells.

Virulence and genotype-associated infectivity of interferon-treated macrophages by porcine reproductive and respiratory syndrome viruses

The polarization into M1 and M2 macrophages (MΦ) is essential to understand MΦ function. Consequently, the aim of this study was to determine the impact of IFN-γ (M1), IL-4 (M2) and IFN-β activation of MΦ on the susceptibility to genotype 1 and 2 porcine reproductive respiratory syndrome (PRRS) virus (PRRSV) strains varying in virulence. To this end, monocyte-derived MΦ were generated by culture during 72h and polarization was induced for another 24h by addition of IFN-γ, IL-4 or IFN-β. MΦ were infected with a collection of PRRSV isolates belonging to genotype 1 and genotype 2. Undifferentiated and M2 MΦ were highly susceptible to all PRRSV isolates. In contrast, M1 and IFN-β activated MΦ were resistant to low pathogenic genotype 1 PRRSV but not or only partially to genotype 2 PRRSV strains. Interestingly, highly virulent PRRSV isolates of both genotypes showed particularly high levels of infection compared with the prototype viruses in both M1 and IFN-β-treated MΦ (P<0.05). This was seen at the level of nucleocapsid expression, viral titres and virus-induced cell death. In conclusion, by using IFN-γ and IFN-β stimulated MΦ it is possible to discriminate between PRRSV varying in genotype and virulence. Genotype 2 PRRSV strains are more efficient at escaping the intrinsic antiviral effects induced by type I and II IFNs. Our in vitro model will help to identify viral genetic elements responsible for virulence, an information important not only to understand PRRS pathogenesis but also for a rational vaccine design. Our results also suggest that monocyte-derived MΦ can be used as a PRRSV infection model instead of alveolar MΦ, avoiding the killing of pigs.

Cytokine Induced Phenotypic and Epigenetic Signatures Are Key to Establishing Specific Macrophage Phenotypes

Macrophages (MΦ) play an essential role in innate immune responses and can either display a pro-inflammatory, classically activated phenotype (M1) or undergo an alternative activation program (M2) promoting immune regulation. M-CSF is used to differentiate monocytes into MΦ and IFN-γ or IL-4+IL-13 to further polarize these cells towards M1 or M2, respectively. Recently, differentiation using only GM-CSF or M-CSF has been described to induce a M1- or M2-like phenotype, respectively. In this study, we combined both approaches by differentiating human MΦ in GM-CSF or M-CSF followed by polarization with either IFN-γ or IL-4+IL-13. We describe the phenotypic differences between CD14hi CD163hi CD206int FOLR2-expressing M-CSF MΦ and CD14lo CD163lo CD206hi GM-CSF MΦ but show that both macrophage populations reacted similarly to further polarization with IFN-γ or IL-4+IL-13 with up- and down-regulation of common M1 and M2 marker genes. We also show that high expression of the mannose receptor (CD206), a marker of alternative activation, is a distinct feature of GM-CSF MΦ. Changes of the chromatin structure carried out by chromatin modification enzymes (CME) have been shown to regulate myeloid differentiation. We analyzed the expression patterns of CME during MΦ polarization and show that M1 up-regulate the histone methyltransferase MLL and demethylase KDM6B, while resting and M2 MΦ were characterized by DNA methyltransferases and histone deacetylases. We demonstrate that MLL regulates CXCL10 expression and that this effect could be abrogated using a MLL-Menin inhibitor. Taken together we describe the distinct phenotypic differences of GM-CSF or M-CSF MΦ and demonstrate that MΦ polarization is regulated by specific epigenetic mechanisms. In addition, we describe a novel role for MLL as marker for classical activation. Our findings provide new insights into MΦ polarization that could be helpful to distinguish MΦ activation states.

Fucosylation is a hallmark of inflammatory macrophages and a novel therapeutic target in rheumatoid arthritis (P5137)

Abstract Inflammatory macrophages (MΦ) play an important role in rheumatoid arthritis (RA). Fucosylation is a regulator of inflammation and we have observed a highly positive correlation between Fucosyltransferases (Fut)1 or Fut3 and Tnfα (p=0.0001) in RA synovial tissues. In sorted cells from human RA synovial fluid, Fut1 and Fut3 was highly expressed in M1 inflammatory MΦ (CD68+CD80+), but not in M2 MΦ (CD68+CD80-), synovial fibroblasts, Th1, Th17, and other T cells (p&amp;lt;0.01, n=5). Fucosylation inhibitor, 2-Deoxy-D-galactose (2-D-gal), precluded the differentiation of M1 MΦ derived by GM-CSF from mouse bone marrow or human PBMC monocytes, strongly indicating the indispensable role of fucosylation in M1 MΦ differentiation. Additionally, phalloidin staining showed 2-D-gal disrupted MΦ actin, suggesting a potential effect of fucosylation in antigen processing and presentation. Indeed, 2-D-gal treatment of fully differentiated M1 MΦ for 2 days dramatically reduced their uptake, processing and presentation of GFP-Eα (from I-Edα) and FITC-CII antigens in a dose dependent manner (p&amp;lt;0.01). In vivo, 2-D-gal dramatically blocked the bovine CII-induced arthritis in DBA/1J mice (scores 9.5±1.7 vs 0.5± 0.3, p&amp;lt;0.01) with reduced inflammatory MΦ in draining LN (1.3±0.3% vs 0.5±0.1%, p&amp;lt;0.05), decreased TNF-α (130 vs 39 pg/ml, p&amp;lt;0.05), and anti-CII in the serum. Our study indicated that M1 MΦ differentiation and function is orchestrated by fucosylation, which is a novel therapeutic target in RA.

Dysfunction of Inflammation-Resolving Pathways Is Associated with Exaggerated Postoperative Cognitive Decline in a Rat Model of the Metabolic Syndrome

The cholinergic antiinflammatory pathway (CAP), which terminates in the spleen, attenuates postoperative cognitive decline (PCD) in rodents. Surgical patients with metabolic syndrome exhibit exaggerated and persistent PCD that is reproduced in postoperative rats selectively bred for easy fatigability and that contain all features of metabolic syndrome (low-capacity runners [LCRs]). We compared the CAP and lipoxin A(4) (LXA(4)), another inflammation-resolving pathway in LCR, with its counterpart high-capacity runner (HCR) rats. Isoflurane-anesthetized LCR and HCR rats either underwent aseptic trauma involving tibial fracture (surgery) or not (sham). At postoperative d 3 (POD3), compared with HCR, LCR rats exhibited significantly exaggerated PCD (trace fear conditioning freezing time 43% versus 57%). Separate cohorts were killed at POD3 to collect plasma for LXA4 and to isolate splenic mononuclear cells (MNCs) to analyze CAP signaling, regulatory T cells (Tregs) and M2 macrophages (M2 Mφ). Under lipopolysaccharide (LPS) stimulation, tumor necrosis factor (TNF)-α produced by splenic MNCs was 117% higher in LCR sham and 52% higher in LCR surgery compared with HCR sham and surgery rats; LPS-stimulated TNF-α production could not be inhibited by an α7 nicotinic acetylcholine receptor agonist, whereas inhibition by the β(2) adrenergic agonist, salmeterol, was significantly less (-35%) than that obtained in HCR rats. Compared to HCR, sham and surgery LCR rats had reduced β(2) adrenergic receptor-expressing T lymphocytes (59%, 44%), Tregs (47%, 54%) and M2 Mφ (45%, 39%); surgical LCR rats' hippocampal M2 Mφ was 66% reduced, and plasma LXA4 was decreased by 120%. Rats with the metabolic syndrome have ineffective inflammation-resolving mechanisms that represent plausible reasons for the exaggerated and persistent PCD.

Human Cytomegalovirus Infection of M1 and M2 Macrophages Triggers Inflammation and Autologous T-Cell Proliferation

Macrophages (MΦ) are first targets during human cytomegalovirus (HCMV) infection and are thought to be crucial for viral persistence and dissemination. However, since MΦ are also a first line of defense and key modulators of the immune response, these cells are at the crossroad between protection and viral pathogenesis. To date, the MΦ-specific contribution to the immune response against HCMV is still poorly understood. In view of the opposite roles of M1 and M2 MΦ during initiation and resolution of the immune response, we characterized the effects of HCMV infection on classically activated M1 MΦ and alternatively activated M2 MΦ. Although HCMV susceptibility was higher in M2 MΦ, HCMV established a productive and persistent infection in both types of MΦ. Upon HCMV encounter, both types of MΦ acquired similar features of classical activation and secreted high levels of proinflammatory cytokines and chemokines. As a functional consequence, conditioned media obtained from HCMV-infected M1 and M2 MΦ potently activated freshly isolated monocytes. Finally, compared to HCMV-infected monocyte-derived dendritic cells, infected M1 and M2 MΦ were more efficient in stimulating proliferation of autologous T cells from HCMV-seropositive donors at early times (24 h) postinfection, while the MΦ immunostimulatory properties were reduced, but not abrogated, at later times (72 h postinfection). In summary, our findings indicate that MΦ preserve proper antigen presentation capacity upon HCMV infection while enhancing inflammation, thus suggesting that MΦ play a role in the maintenance of the large HCMV-specific T-cell repertoire in seropositive individuals.