Changes In Macrophage Phenotype Research Articles

PPARγ activation following apoptotic cell instillation promotes resolution of lung inflammation and fibrosis via regulation of efferocytosis and proresolving cytokines.

Changes in macrophage phenotype have been implicated in apoptotic cell-mediated immune modulation via induction of peroxisome proliferator-activated receptor-γ (PPARγ). In this study, we characterized PPARγ induction by apoptotic cell instillation over the course of bleomycin-induced lung injury in C57BL/6 mice. Next, the role of PPARγ activation in resolving lung inflammation and fibrosis was investigated. Our data demonstrate that apoptotic cell instillation after bleomycin results in immediate and prolonged enhancement of PPARγ mRNA and protein in alveolar macrophages and lung. Moreover, PPARγ activity and expression of its target molecules, including CD36, macrophage mannose receptor, and arginase 1, were persistently enhanced following apoptotic cell instillation. Coadministration of the PPARγ antagonist, GW9662, reversed the enhanced efferocytosis, and the reduced proinflammatory cytokine expression, neutrophil recruitment, myeloperoxidase activity, hydroxyproline contents, and fibrosis markers, including type 1 collagen α2, fibronectin and α-smooth muscle actin (α-SMA), in the lung by apoptotic cell instillation. In addition, inhibition of PPARγ activity reversed the expression of transforming growth factor-β (TGF-β), interleukin (IL)-10, and hepatocyte growth factor (HGF). These findings indicate that one-time apoptotic cell instillation contributes to anti-inflammatory and antifibrotic responses via upregulation of PPARγ expression and subsequent activation, leading to regulation of efferocytosis and production of proresolving cytokines.

Epigenetic modifiers reduce inflammation and modulate macrophage phenotype during endotoxemia-induced acute lung injury.

Acute lung injury (ALI) during sepsis is characterized by bilateral alveolar infiltrates, lung edema and respiratory failure. Here, we examined the efficacy the DNA methyl transferase (DNMT) inhibitor 5-Aza 2-deoxycytidine (Aza), the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA), as well as the combination therapy of Aza and TSA (Aza+TSA) provides in the protection of ALI. In LPS-induced mouse ALI, post-treatment with a single dose of Aza+TSA showed substantial attenuation of adverse lung histopathological changes and inflammation. Importantly, these protective effects were due to substantial macrophage phenotypic changes observed in LPS-stimulated macrophages treated with Aza+TSA as compared with untreated LPS-induced macrophages or LPS-stimulated macrophages treated with either drug alone. Further, we observed significantly lower levels of pro-inflammatory molecules and higher levels of anti-inflammatory molecules in LPS-induced macrophages treated with Aza+TSA than in LPS-induced macrophages treated with either drug alone. The protection was ascribed to dual effects by an inhibition of MAPK-HuR-TNF and activation of STAT3-Bcl2 pathways. Combinatorial treatment with Aza+TSA reduces inflammation and promotes an anti-inflammatory M2 macrophage phenotype in ALI, and has a therapeutic potential for patients with sepsis.

Abstract 15724: Intermittent Hypoxia Induces Early Pro-Atherogenic Changes in Aortic Wall Macrophages in a Murine Model of Obstructive Sleep Apnea

Background: OSA is a highly prevalent condition throughout the lifespan, and has emerged as an independent risk factor for atherosclerosis. Macrophages play a key role in the pathogenesis of atherosclerosis through shifts in the continuum between the pro-inflammatory M1 and the athero-protective M2. We hypothesized that Intermittent Hypoxia (IH) during the sleep period, a model for OSA, will induce shifts towards a pro-atherogenic state in the spectrum of macrophages within the aortic vascular wall. Methods: 8-week old male C57BL/6J mice were exposed to IH for 12-hr/day during the light period or to room air (RA) for 6 weeks and were kept on a regular low-fat chow. Following exposure, full-length aortas were dissected and enzymatically digested. Single cell suspensions were prepared and cells were incubated with antibodies for macrophage markers previously implicated in atherogenesis and metabolic dysregulation - CD11b, F4/80, Ly6c, CD36, and CD64, and analyzed by FACS. Results: Macrophages were defined as CD11b+/F4/80+ cells. Significant increases in the percentage of macrophages emerged in IH-exposed mice (6.4%±0.3 vs. 8%±0.3, p=0.003, n=19-20/group). Furthermore, IH induced shifts in the macrophage population toward an M1 pro-inflammatory phenotype. Specifically, IH group showed significantly higher expression of Ly6c, (MFI:522±23 vs. 699±43, p=0.004, n=12/group) and increased the percentage of Ly6c+(hi) cells (9.2% vs. 11.7%, p=0.03). Conversely, tissue-resident marker CD64 expression in the aortic macrophages was down-regulated following IH exposures (MFI:3207±453 vs. 2967±483, p=0.03, n=12/group), while scavenger receptor CD36 expression was increased in the IH-exposed group (MFI:1694±56 vs.2129±209, p=0.005). Conclusion: Intermittent hypoxia during the sleep period leads to early expansion of the macrophage population in the aortic wall in a relatively resistant animal model for development of atherosclerosis, i.e., in the absence of a high-fat diet or of high atherogenic risk predisposing gene knockout. Furthermore IH induces changes in macrophage phenotypes, including a shift towards a pro-inflammatory phenotype, and recruitment of bone-marrow derived macrophages expressing indicators of metabolic activation.

Soluble factors from colonic epithelial cells contribute to gut homeostasis by modulating macrophage phenotype.

Intestinal macrophages originate from inflammatory blood monocytes which migrate to the intestine, where they differentiate into anti-inflammatory macrophages through a number of transitional stages. These macrophages typically remain hypo-responsive to commensal bacteria and food Ags in the intestine, yet also retain the ability to react to invading pathogens. In this study we examined the role of epithelial cells in inducing this intestinal macrophage phenotype. Using an in vitro system we showed that, in two-dimensional culture, epithelial cell-derived factors from a murine cell line, CMT-93, are sufficient to induce phenotypic changes in macrophages. Exposure of monocyte-derived macrophages, J774A.1, to soluble factors derived from epithelial cells, induced an altered phenotype similar to that of intestinal macrophages with decreased production of IL-12p40, IL-6 and IL-23 and expression of MHC ІІ and CD80 following TLR ligation. Furthermore, these conditioned macrophages showed enhanced phagocytic activity in parallel with low respiratory burst and NO production, similar to the response seen in intestinal macrophages. Our findings suggest a role for colonic epithelial cells in modulation of macrophage phenotype for maintenance of gut homeostasis. Further understanding of the cell interactions that maintain homeostasis in the gut could reveal novel therapeutic strategies to restore the balance in disease.

Changes in macrophage phenotype and induction of epithelial‐to‐mesenchymal transition genes following acute Achilles tenotomy and repair

Tendon injuries occur frequently in physically active individuals, but the clinical outcomes for these injuries can be poor. In many injured tissues the repair process is orchestrated by two types of cells, macrophages and fibroblasts. Macrophages, which have both pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes, can directly participate in tissue remodeling and direct the response of other cells through the secretion of cytokines and growth factors. In many organ systems, epithelial cells can trans-differentiate into fibroblasts, which can then regenerate damaged ECM. This process is triggered via activation of epithelial-to-mesenchymal transition (EMT) signaling programs. Most tendons are surrounded by sheets of epithelial cells, and these tissue layers could provide a source of fibroblasts to repair injured tendons. To gain greater insight into the biology of tendon repair, we performed a tenotomy and repair in Achilles tendons of adult rats and determined changes in macrophage phenotype, and ECM- and EMT-related genes over a 4-week time course. The results from this study suggest that changes in macrophage phenotype and activation of EMT-related programs likely contribute to the degradation and subsequent repair of injured tendon tissue.

Changes in macrophage phenotype and induction of epithelial‐to‐mesenchymal transition genes following acute Achilles tenotomy and repair (1166.3)

Tendon injuries occur frequently in physically active individuals, but the clinical outcomes for these injuries can be poor. In many injured tissues the repair process is orchestrated by two types of cells, macrophages and fibroblasts. Macrophages, which have both proinflammatory (M1) and antiinflammatory (M2) phenotypes, can directly participate in tissue remodeling and direct the response of other cells through the secretion of cytokines and growth factors. Fibroblasts have a well‐described role in synthesizing and remodeling extracellular matrix (ECM) after tissue injury. In many organ systems, epithelial cells can transdifferentiate into fibroblasts, which can then regenerate damaged ECM. This process is triggered via activation of epithelial‐to‐mesenchymal transition (EMT) signaling programs. Most tendons are surrounded by sheets of epithelial cells, and these tissue layers could provide a source of fibroblasts to repair injured tendons. To gain greater insight into the biology of tendon regeneration, we performed a tenotomy and repair in Achilles tendons of adult rats and determined changes in macrophage phenotype, and ECM‐ and EMT‐related genes over a four week time course. The results from this study suggest that changes in macrophage phenotype and activation of canonical EMT programs likely contribute to the degradation and subsequent repair of injured tendon tissue.Grant Funding Source: NIH grants R01‐AR063649, T32‐GM008616 and T32‐GM008322

Peripheral administration of morphine attenuates postincisional pain by regulating macrophage polarization through COX-2-dependent pathway.

BackgroundMacrophage infiltration to inflammatory sites promotes wound repair and may be involved in pain hypersensitivity after surgical incision. We recently reported that the development of hyperalgesia during chronic inflammation is regulated by macrophage polarity, often referred to as proinflammatory (M1) or anti-inflammatory (M2) macrophages. Although opioids such as morphine are known to alter the inflammatory milieu of incisional wounds through interactions with immunocytes, the macrophage-mediated effects of morphine on the development of postincisional pain have not been well investigated. In this study, we examined how morphine alters pain hypersensitivity through phenotypic shifts in local macrophages during the course of incision-induced inflammation.ResultsLocal administration of morphine in the early phase, but not in the late phase alleviated mechanical hyperalgesia, and this effect was reversed by clodronate-induced peripheral depletion of local macrophages. At the morphine-injected incisional sites, the number of pro-inflammatory F4/80+iNOS+M1 macrophages was decreased during the course of pain development whereas increased infiltration of wound healing F4/80+CD206+M2 macrophages was observed during the early phase. Morphine increased the gene expression of endogenous opioid, proenkephalin, and decreased the pronociceptive cytokine, interleukin-1β. Heme oxygenase (HO)-1 promotes the differentiation of macrophages to the M2 phenotype. An inhibitor of HO-1, tin protoporphyrin reversed morphine-induced analgesic effects and the changes in macrophage phenotype. However, local expression levels of HO-1 were not altered by morphine. Conversely, cyclooxygenase (COX)-2, primarily produced from peripheral macrophages in acute inflammation states, was up-regulated in the early phase at morphine-injected sites. In addition, the analgesic effects and a phenotype switching of infiltrated macrophages by morphine was reversed by local administration of a COX inhibitor, indomethacin.ConclusionsLocal administration of morphine alleviated the development of postincisional pain, possibly by altering macrophage polarity at the incisional sites. A morphine-induced shift in macrophage phenotype may be mediated by a COX-2-dependent mechanism. Therefore, μ-opioid receptor signaling in macrophages may be a potential therapeutic target during the early phase of postincisional pain development.

Identification of a distinct glucocorticosteroid-insensitive pulmonary macrophage phenotype in patients with chronic obstructive pulmonary disease

In patients with chronic obstructive pulmonary disease (COPD), pulmonary macrophages increase in number, release increased levels of inflammatory mediators, and respond poorly to glucocorticosteroids. Whether this is due to a change in macrophage phenotype or localized activation is unknown. We sought to investigate whether macrophages from patients with COPD are a distinct phenotype. Macrophage populations were isolated from human lung tissue from nonsmokers, smokers, and patients with COPD by using Percoll density gradients. Five macrophage populations were isolated on the basis of density (1.011-1.023, 1.023-1.036, 1.036-1.048, 1.048-1.061, and 1.061-1.073 g/mL), and cell-surface expression of CD14, CD16, CD163, CD40, and CD206 was assessed by using flow cytometry. Release of active matrix metalloproteinase 9, TNF-α, CXCL8, and IL-10 was measured by using ELISA. The 2 least dense fractions were more than 90% apoptotic/necrotic, with the remaining fractions greater than 70% viable. Macrophages from nonsmokers and smokers were CD163(+), CD206(+), CD14(+), and CD40(-), whereas macrophages from patients with COPD were less defined, showing significantly lower expression of all receptors. There were no differences in receptor expression associated with density. Macrophages from patients with COPD of a density of 1.036 to 1.048 g/mL released higher levels of active matrix metalloproteinase 9 compared with cells from nonsmokers, with no difference between the remaining fractions. This population of macrophages from patients with COPD was less responsive to budesonide compared with those from nonsmokers and smokers when stimulated with LPS. Glucocorticosteroid insensitivity was selective for proinflammatory cytokines because budesonide inhibition of LPS-stimulated IL-10 release was similar for all macrophages. This study identifies a specific macrophage phenotype in the lungs of patients with COPD who are glucocorticosteroid insensitive with a density of 1.036 to 1.048 g/mL but do not correspond to the current concept of macrophage phenotypes.

Macrophage Phenotype in the Mammary Gland Fluctuates over the Course of the Estrous Cycle and Is Regulated by Ovarian Steroid Hormones1

The mammary gland undergoes development and regression over the course of the ovarian cycle under the regulation of ovarian hormones. Macrophages are implicated as local mediators of this tissue remodeling and may also affect immune surveillance and tumor incidence. To investigate cycle-related changes in macrophage phenotype, mammary gland cells from naturally cycling Cfms-Gfp mice recovered at estrus, metestrus, diestrus, and proestrus were analyzed by flow cytometry. Macrophage expression of MHCII was highest in the proestrus phase, with a 1.6-fold increase compared to the metestrus phase. Similarly, macrophage expression of CD204 was 1.9-fold higher at proestrus compared to estrus. Conversely, macrophage expression of NKG2D was increased at metestrus and diestrus by 7-fold and 5-fold, respectively, compared to estrus. To investigate hormonal regulation of macrophage phenotype, an ovariectomy and hormone replacement model was utilized. Ovariectomized mice were stimulated with exogenous estradiol and progesterone to induce early alveolar development, then given progesterone receptor antagonist RU486 to elicit alveolar bud regression. Progesterone and estradiol in combination reduced macrophage expression of MHCII and CD204 by 5-fold and 3-fold, respectively, and increased macrophage expression of NKG2D by 4-fold. Administration of RU486, following estradiol and progesterone, reversed the macrophage phenotype. These results reveal an essential requirement for ovarian hormones in regulating macrophage phenotype in the mammary gland and indicate that progesterone is particularly critical for controlling macrophage antigen presentation and immune surveillance capacity.

Adipocytes Modulate the Phenotype of Human Macrophages through Secreted Lipids

Previous studies have shown accumulation and an enhanced proinflammatory profile of macrophages in adipose tissue of obese mice, indicating the presence of an interaction between adipocytes and macrophages in this tissue. However, the consequences of this interaction in humans are yet incompletely understood. In this study, we explored the modulating effects of adipocytes on the phenotype of macrophages in humans and studied the possible molecular pathways involved. Adipocyte-conditioned media (ACM) treatment of macrophages for 48 h strongly reduced the LPS-induced IL-12p40 secretion by macrophages, whereas the production of TNF-α and other cytokines remained largely unaffected. This effect was independent of the source of adipocytes. Interestingly, the level of inhibition correlated directly with body mass index (BMI) of the adipocyte donor. Because adipocytes release many different cytokines, adipokines, and lipids, we have separated the protein and lipid fractions of ACM, to obtain insight into the molecular nature of the soluble mediators underlying the observed effect. These experiments revealed that the inhibitory effect resided predominantly in the lipid fraction. Further studies revealed that PGE2 and linoleic and oleic acid were potent inhibitors of IL-12p40 secretion. Interestingly, concentrations of these ACM-derived lipids increased with increase in BMI of the adipocyte donor, suggesting that they could mediate the BMI-dependent effects of ACM. To our knowledge, these results provide first evidence that obesity-related changes in adipose tissue macrophage phenotype could be mediated by adipocyte-derived lipids in humans. Intriguingly, these changes appear to be different from those in murine obesity.

IL-17–Mediated M1/M2 Macrophage Alteration Contributes to Pathogenesis of Bisphosphonate-Related Osteonecrosis of the Jaws

Osteonecrosis of the jaw (ONJ) is emerging as one of the important complications in cancer patients treated with antiresorptive agents. This study explored the potential role of interleukin (IL)-17-mediated M1/M2 macrophage alterations in the pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ). The expression of IL-17 and M1 and M2 macrophage markers at the local mucosal site of human BRONJ lesions was examined by immunofluorescence studies. BRONJ-like disease was induced in C57BL/6 mice and multiple myeloma-burdened mice by intravenous injection of zoledronate to evaluate the correlation of elevated IL-17 levels with changes in M1 and M2 macrophage phenotypes and the therapeutic effects of blocking IL-17 on pathogenesis of BRONJ-like disease. Increased T-helper (TH)17 cells and IL-17 cytokine correlate with an increase in M1/M2 macrophages ratio at the local mucosal site of both murine and human BRONJ lesion. Convincingly, in mice burdened with multiple myeloma, a combination of elevated suprabasal level and drug-induced IL-17 activity augmented the incidence of BRONJ; both systemic increase of IL-17 and disease severity could be reversed by adoptive transfer of ex vivo expanded M2 macrophages. Targeting IL-17 via specific neutralizing antibodies or a small inhibitory molecule, laquinimod, significantly decreased M1/M2 ratio and concomitantly suppressed BRONJ-like condition in mice. Mechanistically, IL-17 enhanced IFN-γ-induced M1 polarization through augmenting STAT-1 phosphorylation while suppressing IL-4-mediated M2 conversion via inhibiting STAT-6 activation. These findings have established a compelling linkage between activated IL-17-mediated polarization of M1 macrophages and the development of BRONJ-like conditions in both human disease and murine models.

Changes in macrophage phenotype as the immune response evolves

Mononuclear phagocytic cells (MPCs), including macrophages and dendritic cells (DCs), are widely distributed throughout our organs where they perform important homeostatic, surveillance and regenerative tasks. In response to infection or injury, the composition and number of MPCs change remarkably, in part due to the recruitment of inflammatory monocytes from bone marrow. In infection or injury, macrophages and DCs perform important innate and adaptive immune roles from the initial insult through repair and regeneration of the tissue and resolution of inflammation. Evidence from mouse models of disease has shown increasing complexity and subtlety to the mononuclear phagocytic system, which will be reviewed here. New studies show that in addition to monocytes, the resident populations of mononuclear phagocytes expand in disease states and play distinct but important roles in the immune response. Finally, new insights into these functionally diverse cells are now translating into therapeutics to treat human disease.

AB0085 Macrophages are modulated by factors secreted by adipocytes

BackgroundObesity has been associated with development and progression of osteoarthritis. Although the biological mechanisms underlying this association are unknown, several studies have indicated that adipose tissue secretes a large variety...

Deficiency of TDAG51 Protects Against Atherosclerosis by Modulating Apoptosis, Cholesterol Efflux, and Peroxiredoxin‐1 Expression

BackgroundApoptosis caused by endoplasmic reticulum (ER) stress contributes to atherothrombosis, the underlying cause of cardiovascular disease (CVD). T‐cell death‐associated gene 51 (TDAG51), a member of the pleckstrin homology‐like domain gene family, is induced by ER stress, causes apoptosis when overexpressed, and is present in lesion‐resident macrophages and endothelial cells.Methods and ResultsTo study the role of TDAG51 in atherosclerosis, male mice deficient in TDAG51 and apolipoprotein E (TDAG51−/−/ApoE−/−) were generated and showed reduced atherosclerotic lesion growth (56±5% reduction at 40 weeks, relative to ApoE−/− controls, P<0.005) and necrosis (41±4% versus 63±8% lesion area in TDAG51−/−/ApoE−/− and ApoE−/−, respectively; P<0.05) without changes in plasma levels of lipids, glucose, and inflammatory cytokines. TDAG51 deficiency caused several phenotypic changes in macrophages and endothelial cells that increase cytoprotection against oxidative and ER stress, enhance PPARγ‐dependent reverse cholesterol transport, and upregulate peroxiredoxin‐1 (Prdx‐1), an antioxidant enzyme with antiatherogenic properties (1.8±0.1‐fold increase in Prdx‐1 protein expression, relative to control macrophages; P<0.005). Two independent case–control studies found that a genetic variant in the human TDAG51 gene region (rs2367446) is associated with CVD (OR, 1.15; 95% CI, 1.07 to 1.24; P=0.0003).ConclusionsThese findings provide evidence that TDAG51 affects specific cellular pathways known to reduce atherogenesis, suggesting that modulation of TDAG51 expression or its activity may have therapeutic benefit for the treatment of CVD.

Dietary modification dampens liver inflammation and fibrosis in obesity-related fatty liver disease

Alms1 mutant (foz/foz) mice develop hyperphagic obesity, diabetes, metabolic syndrome, and fatty liver (steatosis). High-fat (HF) feeding converts pathology from bland steatosis to nonalcoholic steatohepatitis (NASH) with fibrosis, which leads to cirrhosis in humans. We sought to establish how dietary composition contributes to NASH pathogenesis. foz/foz mice were fed HF diet or chow 24 weeks, or switched HF to chow after 12 weeks. Serum ALT, NAFLD activity score (NAS), fibrosis severity, neutrophil, macrophage and apoptosis immunohistochemistry, uncoupling protein (UCP)2, ATP, NF-κB activation/expression of chemokines/adhesion molecules/fibrogenic pathways were determined. HF intake upregulated liver fatty acid and cholesterol transporter, CD36. Dietary switch expanded adipose tissue and decreased hepatomegaly by lowering triglyceride, cholesterol ester, free cholesterol and diacylglyceride content of liver. There was no change in lipogenesis or fatty acid oxidation pathways; instead, CD36 was suppressed. These diet-induced changes in hepatic lipids improved NAS, reduced neutrophil infiltration, normalized UCP2 and increased ATP; this facilitated apoptosis with a change in macrophage phenotype favoring M2 cells. Dietary switch also abrogated NF-κB activation and chemokine/adhesion molecule expression, and arrested fibrosis by dampening stellate cell activation. Reversion to a physiological dietary composition after HF feeding in foz/foz mice alters body weight distribution but not obesity. This attenuates NASH severity and fibrotic progression by suppressing NF-κB activation and reducing neutrophil and macrophage activation. However, adipose inflammation persists and is associated with continuing apoptosis in the residual fatty liver disease. Taken together, these findings indicate that other measures, such as weight reduction, may be required to fully reverse obesity-related NASH.

Changes in Macrophage Phenotype after Infection of Pigs with Haemophilus parasuis Strains with Different Levels of Virulence

Haemophilus parasuis is a colonizer of healthy piglets and the etiological agent of Glässer's disease. Differences in virulence among strains of H. parasuis have been widely observed. In order to explore the host-pathogen interaction, snatch-farrowed colostrum-deprived piglets were intranasally infected with 4 strains of H. parasuis: reference virulent strain Nagasaki, reference nonvirulent strain SW114, field strain IT29205 (from a systemic lesion and virulent in a previous challenge), and field strain F9 (from the nasal cavity of a healthy piglet). At different times after infection, two animals of each group were euthanized and alveolar macrophages were analyzed for the expression of CD163, CD172a, SLA I (swine histocompatibility leukocyte antigen I), SLA II, sialoadhesin (or CD169), and CD14. At 1 day postinfection (dpi), virulent strains induced reduced expression of CD163, SLA II, and CD172a on the surfaces of the macrophages, while nonvirulent strains induced increased expression of CD163, both compared to noninfected controls. At 2 dpi, the pattern switched into a strong expression of CD172a, CD163, and sialoadhesin by the virulent strains, which was followed by a steep increase in interleukin 8 (IL-8) and soluble CD163 in serum at 3 to 4 dpi. The early increase in surface expression of CD163 induced by nonvirulent strains went along with higher levels of IL-8 in serum than those induced by virulent strains in the first 2 days of infection. Alpha interferon (IFN-α) induction was observed only in animals infected with nonvirulent strains. Overall, these results are compatible with a delay in macrophage activation by virulent strains, which may be critical for disease production.

Targeting the EP1 receptor reduces Fas ligand expression and increases the antitumor immune response in an in vivo model of colon cancer

Despite studies demonstrating that inhibition of cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2 ) has significant chemotherapeutic benefits in vitro and in vivo, inhibition of COX enzymes is associated with serious gastrointestinal and cardiovascular side effects, limiting the clinical utility of these drugs. PGE2 signals through four different receptors (EP1-EP4) and targeting individual receptor(s) may avoid these side effects, while retaining significant anticancer benefits. Here, we show that targeted inhibition of the EP1 receptor in the tumor cells and the tumor microenvironment resulted in the significant inhibition of tumor growth in vivo. Both dietary administration and direct injection of the EP1 receptor-specific antagonist, ONO-8713, effectively reduced the growth of established CT26 tumors in BALB/c mice, with suppression of the EP1 receptor in the tumor cells alone less effective in reducing tumor growth. This antitumor effect was associated with reduced Fas ligand expression and attenuated tumor-induced immune suppression. In particular, tumor infiltration by CD4(+) CD25(+) Foxp3(+) regulatory T cells was decreased, whereas the cytotoxic activity of isolated splenocytes against CT26 cells was increased. F4/80(+) macrophage infiltration was also decreased; however, there was no change in macrophage phenotype. These findings suggest that the EP1 receptor represents a potential target for the treatment of colon cancer.

Abstract 19264: Regression of Atherosclerosis is Characterized by Molecular Re-programming of the Transcriptome: Insights into the Identification of New Therapeutic Targets Against Cardiovascular Disease

Atherosclerosis is the leading underlying cause of strokes and myocardial infarctions worldwide. We reported a mouse model of atherosclerosis regression that involves transplanting an atherosclerotic aortic arch into a normolipidemic mouse. The CD68+ cell population (predominantly macrophages) in the regressing plaques exhibited migratory behavior. To identify pathways and factors responsible for the beneficial changes in macrophage content and phenotype in the regressing plaques, we performed microarray analysis on RNA purified from laser captured CD68+ plaque cells isolated from atherosclerosis progression and regression environments. This yielded two major findings. First, genes associated with the contractile apparatus (such as titin, actinin and myopalladin) that are responsible for cellular movement were differentially up-regulated under regression conditions (by 3-4 fold) with members of the cadherin family (serves in adhesion functions) being significantly down-regulated by 2-3 fold. Both changes are consistent with the macrophage migratory behavior we observed in the regression environment. Secondly, the most highly up-regulated gene under regression conditions was arginase I (~8 fold), a classical marker of the M2 alternatively activated macrophage. Further examination revealed that regression was characterized by macrophages displaying other M2 markers, such as CD163, C-lectin receptor, mannose receptor, and Fizz-1, consistent with a less inflammatory state in the regression environment. In addition, we applied recently introduced local causal pathway discovery methods to the microarray data that revealed that vinculin and ApoCII, known factors in cell adhesion and lipid metabolism, respectively, may be important regulators in the pathophysiology of atherosclerosis regression. Ultimately, the insights gained from the regression model and the different modes of global analyses should lead to new therapeutic targets against cardiovascular disease.

IL-10 Triggers Changes in Macrophage Phenotype That Promote Muscle Growth and Regeneration

We examined the function of IL-10 in regulating changes in macrophage phenotype during muscle growth and regeneration following injury. Our findings showed that the Th1 cytokine response in inflamed muscle is characterized by high levels of expression of CD68, CCL-2, TNF-α, and IL-6 at 1 d postinjury. During transition to the Th2 cytokine response, expression of those transcripts declined, whereas CD163, IL-10, IL-10R1, and arginase-1 increased. Ablation of IL-10 amplified the Th1 response at 1 d postinjury, causing increases in IL-6 and CCL2, while preventing a subsequent increase in CD163 and arginase-1. Reductions in muscle fiber damage that normally occurred between 1 and 4 d postinjury did not occur in IL-10 mutants. In addition, muscle regeneration and growth were greatly slowed by loss of IL-10. Furthermore, myogenin expression increased in IL-10 mutant muscle at 1 d postinjury, suggesting that the mutation amplified the transition from the proliferative to the early differentiation stages of myogenesis. In vitro assays showed that stimulation of muscle cells with IL-10 had no effect on cell proliferation or expression of MyoD or myogenin. However, coculturing muscle cells with macrophages activated with IL-10 to the M2 phenotype increased myoblast proliferation without affecting MyoD or myogenin expression, showing that M2 macrophages promote the early, proliferative stage of myogenesis. Collectively, these data show that IL-10 plays a central role in regulating the switch of muscle macrophages from a M1 to M2 phenotype in injured muscle in vivo, and this transition is necessary for normal growth and regeneration of muscle.

Macrophage markers and C3d in the central &amp; peripheral choroid of young, aged and amd eyes

AbstractPurpose To investigate the distribution and expression of macrophage phenotypes and the complement activation protein, C3d, in human choroid/retina in young, aged and AMD eyes.Methods Paraffin sections of central and peripheral choroid/retina from young (&lt;40 years, n=6), aged (&gt;70 years, n=4) and AMD (&gt;70 years, n=8) human post‐mortem eyes were co‐immunolabelled with antibodies to leukocyte and macrophage markers (CD68, CD163, Iba1 &amp; CD45) and complement activation component, C3d. Localisation and distribution of antibodies was assessed using confocal microscopy. Counts of macrophage sub‐populations were compared in central and peripheral choroid for each group.Results Heterogeneous populations of macrophages were found in the choroid/retina of all groups. Iba1+ and CD45+ retinal microglia and macrophages, and choroidal macrophages were observed in all eyes. In AMD eyes, Iba1+ and CD45+ cells were seen in the subretinal space amongst photoreceptors, as well as in inner retina. Compared to young eyes, aged and AMD specimens showed increased numbers of CD163+ macrophages (M2, proangiogenic) in choroid, and in central vs peripheral retina. Fewer CD68+ cells were seen in all specimens, regardless of age or location. C3d was expressed in young, aged and AMD eyes, localised to Bruch’s membrane and choriocapillaris, with more extensive immunostaining in aged and AMD eyes. C3d was also seen in drusen in aged and AMD eyes, and more peripherally, compared to young eyes.Conclusion Age‐related changes in macrophage phenotypes and in complement pathway activation at the choroidal/retinal interface may play a role in development and progression of AMD. Supported by Sydney Foundation for Medical Research.