Reparative Macrophages Research Articles

Abstract 102: MicroRNA-33 Inhibition Reprograms Monocyte/macrophage Dynamics in Atherosclerosis to Promote Plaque Regression

The inhibition of microRNA (miR)-33 promotes regression of atherosclerosis by increasing plasma HDL and reverse cholesterol transport levels, and reducing plaque inflammation. We sought to understand how anti-miR-33 treatment impacts monocyte and macrophage dynamics in the plaque to promote inflammation resolution. Ldlr -/- mice were fed a western diet for 14 weeks to establish plaques, after which mice were switched to chow diet to halt atherosclerosis progression and treated with anti-miR-33 or control anti-miR oligonucleotides for 4 weeks. As we reported previously, anti-miR-33 treatment increased plasma levels of HDL cholesterol by 30%, and concurrently reduced plaque size (-25%) and macrophage content (-30%), compared to control anti- miR treatment. Analysis of monocyte kinetic processes revealed that anti-miR-33 treatment normalized hypercholesterolemia associated monocytosis and reduced common myeloid progenitor (CMP) cells in the splenic reservoir. Interestingly, despite a 40% reduction in circulating monocytes, monocyte tracking assays revealed an increase in Ly6C hi monocyte recruitment into plaques of anti-miR-33 treated mice. These findings are consistent with our recent study showing that Ly6C hi monocytes are required for atherosclerosis regression and are a source of tissue reparative M2 macrophages. Indeed, we find that M2 macrophages, as well as atheroprotective regulatory T cells are enriched in plaques of anti-miR-33 treated mice. Finally, although the proliferation of macrophages in plaques did not change, we observed a 40% increase in the number of apoptotic cells in anti- miR33 treated mice, and an increase in macrophage efferocytosis (clearance of apoptotic cells) - markers that are consistent with resolution of inflammation. Collectively, our results provide insight into the mechanisms underlying anti-miR- 33’s atheroprotective actions, which now include reprogramming of monocyte/ macrophage dynamics to resolve inflammation in the plaque.

EGF-like repeats and discoidin I-like domain 3 (EDIL3) deficiency improves post-myocardial infarction healing via neutrophil extracellular traps (NETs) mediated pro-inflammatory macrophage polarization

Abstract After myocardial infarction (MI), injured cardiomyocytes recruit neutrophils, leading to extravasated monocytes polarized to inflammatory and reparative macrophages sequentially, which contribute to the healing and regenerative process. As an endogenous leukocyte-endothelial adhesion inhibitor, the role of EDIL3 in MI remains obscure. We found that serum EDIL3 level of MI patients was negatively associated with MI biomarkers, including CK-MB, cTnI and MGB. EDIL3−/− mice showed improved infarct healing with lower mortality, better cardiac function and decreased infarcted size compared with wild type (WT). Enhanced neutrophil infiltration resulted in the increase of Ly6Chi MHClo Mertk− macrophages, which we identified to be pro-inflammatory, protecting EDIL3−/− mice hearts from excessive fibrosis. A neutrophil infiltration antagonist not only eliminated the difference in cardiac function between WT and EDIL3−/− mice, but also reduced Ly6Chi MHClo Mertk− macrophage polarization. These data implied that improved cardiac healing was associated with increased infiltration of neutrophil and it mediated macrophage polarization. Notably, we confirmed that this polarization depended on NETs, which were more abundant in hearts and serum in EDIL3−/− mice than in WT. Indeed, self-DNA released from NETs primed the pro-inflammatory polarization of macrophages. The degradation of NETs by DNase I reduced this polarization and thus affected post-MI outcome, indicating the pivotal role of NETs in MI healing. Meanwhile, the pro-inflammatory macrophages induced apoptosis of neutrophils and restrained excessive inflammation. Our findings reveal a novel neutrophil–macrophage crosstalk essential to cardiac healing after MI.

Magnolol attenuates the cisplatin-induced muscle wasting via M2c macrophage activation

Abstract Cancer chemotherapy induces malnutrition and rapid loss of muscle mass which directly restricts the daily activities leading to poor quality of life and increased mortality. Although the hormone-related therapies have been used to improve appetite and nutritional status, but current treatments are considered palliative. Thus, the maintenance of skeletal muscle mass without side effects is essential in sarcopenia. Magnolol from Magnolia officianlis has been reported to have several pharmacological effects including anti-cancer and anti-inflammatory effects, but the correlations of immune cell infiltrations and muscle protection in muscle atrophy are not well understood. In the present study, we investigated the effects of magnolol on muscle wasting and macrophage subtypes in cisplatin-induced sarcopenia mice model. We showed that magnolol significantly attenuated the body weight loss and muscle loss induced by cisplatin injection. The diameter of tiabialis anterior (TA) muscle was markedly increased by magnolol treatement compared with cisplatin group. Importantly, magnolol increased the percentages of total macrophages without affecting the percentages of T cells. Furthermore, magnolol reduced the CD206−CD163− M1 inflammatory macrophages whereas the CD206+CD163+ M2c tissue reparative macrophages were increased in CD45+CD11b+F4/80+ macrophages. We also showed that cisplatin increased the M1/M2 ratio and magnolol significantly lowered the M1/M2 ratio in TA muscle. Taken together, magnolol may be a promising chemoprotective agent for preventing muscle atrophy by upregulating M2c macrophages.

Insulin‐like growth factor 1 preserves cardiac function after myocardial infarction by affecting myeloid cells

Insulin‐like growth factor (IGF1) controls growth and metabolism of many cell types. In addition, IGF1 has been shown to provide cardioprotective effects after acute myocardial infarction (AMI). However, the cell type and mechanisms involved are not yet fully understood.Mice were exposed to 45 min LAD occlusion, followed by 4 weeks reperfusion. At the end of ischemia mice were injected with vehicle (Con) or IGF1 (40 ng/g ip), followed by continues treatment for 3 days via osmotic mini pumps (1 μg/g/d sc.). Cardiac function, determined by echocardiography at baseline and 1 and 4 weeks after ischemia, showed no differences at baseline in all mouse lines studied. IGF1 treatment improved cardiac function after 1 and 4 weeks in wild‐type mice ((EF, week 4: 49±4% (IGF1) vs 36±8% (Con)), but also in inducible cardiomyocyte‐specific IGF1‐receptor (IGF1R) KO mice (48±5% (IGF1) vs 35±5% (Con)). However, in myeloid cell specific IGF1R KO mice IGF1 was no longer protective (36±8% (IGF1) vs 38±8% (Con)), indicating that myeloid cells are responsible for the protective effect of IGF1 after MI. In addition, micro‐array analysis of the infarct area of the heart showed that mostly myeloid cell related pathways were altered in IGF treated mice 1 and 2 days after AMI.To look further into the mechanism involved, in vitro and subacute experiments were performed. FACS and qPCR analysis showed that treatment of monocyte derived macrophages with IGF1 induced macrophage polarization to macrophages with a reparative M2‐like phenotype, characterized by an increase in mannose receptor (CD206), arginase and resistin‐like α. Also, in vivo FACS analysis of the heart showed an increase in M2‐like (CD206+) macrophages in IGF1 treated mice 3 days after AMI (152 777±13510 (IGF1) vs 108 071±14023 (Con) cells per heart) without affecting total macrophage number. In these mice, no effects on the amount of lymphocytes or monocytes were observed in blood or heart. A trend towards an increased number of neutrophils in the heart was seen after IGF treatment (272 120±42478 (IGF1) vs 198 122±31741 (Con) cells per heart), without an effect on neutrophil amount in blood. In addition, one week after AMI, i.e. a timepoint where functional cardiac improvement was observed in echo analysis, histological analysis of scar size showed a significant reduction in scar size after IGF1 treatment (9.2±4% (IGF1) vs. 14.7±3.9% (Con) of left ventricle). This was accompanied by an increased vessel formation in the scar (654±132 (IGF1) vs 495±94 (Con) CD31+ cells/mm2) and borderzone (1676±72 (IGF1) vs 1467±110 (Con)) after IGF1 treatment.Interestingly, short term no protective effects of IGF1 were observed. IGF1 treatment did not reduce infarct size 2 hours after AMI (38±10% (IGF1) vs. 39±11% (Con) of left ventricle).Short term IGF1 treatment improves cardiac function in the subacute phase after AMI by affecting myeloid cells. This effect was independent of the IGF1R in cardiomyocytes. IGF1 treatment increases the amount of reparative M2 macrophages, and may thereby improve vasculature and reduce scar size.Support or Funding InformationFunded by SFB1116/A06This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neutrophils promote the development of reparative macrophages mediated by ROS to orchestrate liver repair

Phagocytes, including neutrophils and macrophages, have been suggested to function in a cooperative way in the initial phase of inflammatory responses, but their interaction and integration in the resolution of inflammation and tissue repair remain unclear. Here we show that neutrophils have crucial functions in liver repair by promoting the phenotypic conversion of pro-inflammatory Ly6ChiCX3CR1lo monocytes/macrophages to pro-resolving Ly6CloCX3CR1hi macrophages. Intriguingly, reactive oxygen species (ROS), expressed predominantly by neutrophils, are important mediators that trigger this phenotypic conversion to promote liver repair. Moreover, this conversion is prevented by the depletion of neutrophils via anti-Ly6G antibody, genetic deficiency of granulocyte colony-stimulating factor, or genetic deficiency of NADPH oxidase 2 (Nox2). By contrast, adoptive transfer of WT rather than Nox2−/− neutrophils rescues the impaired phenotypic conversion of macrophages in neutrophil-depleted mice. Our findings thus identify an intricate cooperation between neutrophils and macrophages that orchestrate resolution of inflammation and tissue repair.

Characterization of M1 and M2 polarization phenotypes in peritoneal macrophages after treatment with graphene oxide nanosheets.

Macrophages play a key role in nanoparticle removal and are primarily responsible for their uptake and trafficking in vivo. Due to their functional plasticity, macrophages display a spectrum of phenotypes between two extremes indentified as pro-inflammatory M1 and reparative M2 macrophages, characterized by the expression of specific cell surface markers and the secretion of different cytokines. The influence of graphene oxide (GO) nanosheets functionalized with poly(ethylene glycol-amine) and labelled with fluorescein isothiocyanate (FITC-PEG-GO) on polarization of murine peritoneal macrophages towards M1 and M2 phenotypes was evaluated in basal and stimulated conditions by flow cytometry and confocal microscopy through the expression of different cell markers: CD80 and iNOS as M1 markers, and CD206 and CD163 as M2 markers. Although FITC-PEG-GO did not induce M1 or M2 macrophage polarization after 24 and 48 h in basal conditions, this nanomaterial decreased the percentage of M2 reparative macrophages. We have also compared control macrophages with macrophages that have or have not taken up FITC-PEG-GO after treatment with these nanosheets (GO+ and GO- cells, respectively). The CD80 expression diminished in GO+ macrophages after 48 h of GO treatment but the CD206 expression in GO+ population showed higher values than in both GO- population and control macrophages. In the presence of pro-inflammatory stimuli (LPS and IFN-γ), a significant decrease of CD80+ cells was observed after treatment with GO. This nanomaterial also induced significant decreases of CD206+ and CD163+ cells in the presence of reparative stimulus (IL-4). The CD80, iNOS and CD206 expression was lower in both GO- and GO+ cells than in control macrophages. However, higher CD163 expression was obtained in both GO- and GO+ cells in comparison with control macrophages. All these facts suggest that FITC-PEG-GO uptake did not induce the macrophage polarization towards the M1 pro-inflammatory phenotype, promoting the control of the M1/M2 balance with a slight shift towards M2 reparative phenotype involved in tissue repair, ensuring an appropriate immune response to these nanosheets.

Accelerate Healing of Severe Burn Wounds by Mouse Bone Marrow Mesenchymal Stem Cell-Seeded Biodegradable Hydrogel Scaffold Synthesized from Arginine-Based Poly(ester amide) and Chitosan.

Severe burns are some of the most challenging problems in clinics and still lack ideal modalities. Mesenchymal stem cells (MSCs) incorporated with biomaterial coverage of burn wounds may offer a viable solution. In this report, we seeded MSCs to a biodegradable hybrid hydrogel, namely ACgel, that was synthesized from unsaturated arginine-based poly(ester amide) (UArg-PEA) and chitosan derivative. MSC adhered to ACgels. ACgels maintained a high viability of MSCs in culture for 6 days. MSC seeded to ACgels presented well in third-degree burn wounds of mice at 8 days postburn (dpb) after the necrotic full-thickness skin of burn wounds was debrided and filled and covered by MSC-carrying ACgels. MSC-seeded ACgels promoted the closure, reepithelialization, granulation tissue formation, and vascularization of the burn wounds. ACgels alone can also promote vascularization but less effectively compared with MSC-seeded ACgels. The actions of MSC-seeded ACgels or ACgels alone involve the induction of reparative, anti-inflammatory interleukin-10, and M2-like macrophages, as well as the reduction of inflammatory cytokine TNFα and M1-like macrophages at the late inflammatory phase of burn wound healing, which provided the mechanistic insights associated with inflammation and macrophages in burn wounds. For the studied regimens of these treatments, no toxicity was identified to MSCs or mice. Our results indicate that MSC-seeded ACgels have potential use as a novel adjuvant therapy for severe burns to complement commonly used skin grafting and, thus, minimize the downsides of grafting.

Deficiency of GATA3-Positive Macrophages Improves Cardiac Function Following Myocardial Infarction or Pressure Overload Hypertrophy

BackgroundMacrophages are highly plastic cells that play an important role in the pathogenesis of cardiovascular disease. ObjectivesThis study investigated the role of GATA3-positive macrophages in modulating cardiac function after myocardial infarction (MI) or in response to pressure overload hypertrophy. MethodsMyeloid-specific GATA3-deficient (mGATA3KO) mice were generated, MI or pressure overload was induced, and cardiac function was determined by echocardiography. GATA3-sufficient Cre mice were used as a control. Immunohistochemical staining, flow cytometry, MILLIPLEX Mouse Cytokine/Chemokine Assay, cultured macrophages, quantitative real-time polymerase chain reaction, and western blot were used to determine the role of GATA3 in macrophages. ResultsGATA3-positive macrophages rapidly accumulated in the infarcted region of the myocardium after acute MI. Deficiency of GATA3-positive macrophages led to a significant improvement of cardiac function in response to acute MI or pressure overload hypertrophy compared with the control mice. This improvement was associated with the presence of a large number of proinflammatory Ly6Chi monocytes/macrophages and fewer reparative Ly6Clo macrophages in the myocardium of mGATA3KO mice compared with control mice. Analysis of serum proteins from the 2 mouse genotypes revealed no major changes in the profile of serum growth factors and cytokines between the 2 mice genotypes before and after MI. GATA3 was found to be specifically and transiently induced by interleukin 4 in cultured macrophages through activity of the proximal promoter, whereas the distal promoter remained silent. In addition, the absence of GATA3 in macrophages markedly attenuated arginase-1 expression in cultured macrophages. ConclusionsWe demonstrated that the presence of GATA3-positive macrophages adversely affects remodeling of the myocardium in response to ischemia or pressure overload, whereas the absence of these macrophages led to a significant improvement in cardiac function. Targeting of signaling pathways that lead to the expression of GATA3 in macrophages may have favorable cardiac outcomes.

Graphene oxide nanosheets increase Candida albicans killing by pro-inflammatory and reparative peritoneal macrophages.

Graphene oxide (GO) is a new nanomaterial with different potential biomedical applications due to its excellent physicochemical properties and ease of surface functionalization. Macrophages play key roles in the control of fungal infections preventing invasive candidiasis by both limiting the growth of the opportunistic fungal pathogen Candida albicans and activating other immune effector cells. In order to know if macrophages maintain their immunocompetence against this microorganism after GO uptake, we have evaluated the interactions at the interface of GO nanosheets, macrophages and Candida albicans. Poly (ethylene glycol-amine)-derivatized GO nanosheets labelled with fluorescein isothiocyanate (FITC-PEG-GO), were efficiently taken up by peritoneal macrophages inducing a significant increase of C. albicans phagocytosis by both pro-inflammatory macrophages (M1/stimulated with LPS/IFN-γ) and reparative macrophages (M2/stimulated with IL-4). On the other hand, after FITC-PEG-GO treatment and C. albicans infection, the percentages of GO+ macrophages diminished when Candida uptake increased in every condition (macrophages with no stimuli, M1 and M2 macrophages), thus suggesting the exocytosis of this nanomaterial as a dynamic mechanism favoring fungal phagocytosis. For the first time, we have analyzed the effects of PEG-GO nanosheets on Candida albicans killing by unstimulated, M1 and M2 macrophages, evidencing that intracellular GO modulates the macrophage candidacidal activity in a multiplicity of infection (MOI) dependent manner. At MOI 1, the high intracellular GO levels increase the fungicidal activity of basal and stimulated macrophages. At MOI 5, as intracellular GO decreases, the previous pro-inflammatory or reparative stimulus predefines the killing ability of macrophages. In summary, GO treatment enhances classical M1 macrophage activation, important for pathogen eradication, and diminishes alternative activation of M2 macrophages, thus decreasing fungal persistence and avoiding chronic infectious diseases.

Azithromycin therapy reduces cardiac inflammation and mitigates adverse cardiac remodeling after myocardial infarction: Potential therapeutic targets in ischemic heart disease.

IntroductionAcute myocardial infarction (MI) is a primary cause of worldwide morbidity and mortality. Macrophages are fundamental components of post-MI inflammation. Pro-inflammatory macrophages can lead to adverse cardiac remodeling and heart failure while anti-inflammatory/reparative macrophages enhance tissue healing. Shifting the balance between pro-inflammatory and reparative macrophages post-MI is a novel therapeutic strategy. Azithromycin (AZM), a commonly used macrolide antibiotic, polarizes macrophages towards the anti-inflammatory phenotype, as shown in animal and human studies. We hypothesized that AZM modulates post-MI inflammation and improves cardiac recovery.Methods and resultsMale WT mice (C57BL/6, 6–8 weeks old) were treated with either oral AZM (160 mg/kg/day) or vehicle (control) starting 3 days prior to MI and continued to day 7 post-MI. We observed a significant reduction in mortality with AZM therapy. AZM-treated mice showed a significant decrease in pro-inflammatory (CD45+/Ly6G-/F4-80+/CD86+) and increase in anti-inflammatory (CD45+/Ly6G-/F4-80+/CD206+) macrophages, decreasing the pro-inflammatory/anti-inflammatory macrophage ratio in the heart and peripheral blood as assessed by flow cytometry and immunohistochemistry. Macrophage changes were associated with a significant decline in pro- and increase in anti-inflammatory cytokines. Mechanistic studies confirmed the ability of AZM to shift macrophage response towards an anti-inflammatory state under hypoxia/reperfusion stress. Additionally, AZM treatment was associated with a distinct decrease in neutrophil count due to apoptosis, a known signal for shifting macrophages towards the anti-inflammatory phenotype. Finally, AZM treatment improved cardiac recovery, scar size, and angiogenesis.ConclusionAzithromycin plays a cardioprotective role in the early phase post-MI through attenuating inflammation and enhancing cardiac recovery. Post-MI treatment and human translational studies are warranted to examine the therapeutic applications of AZM.

Macrophage CD31 Signaling in Dissecting Aortic Aneurysm

BackgroundThe authors recently found that a CD31 agonist peptide reaches macrophages in injured aortas and exerts beneficial effects on apolipoprotein E-knockout (Apo E−/−) mice subjected to angiotensin (Ang) II infusion, a model of experimental acute aortic dissection and intramural hematoma (ADIM). ObjectivesThe purpose of this study was to evaluate the therapeutic potential of a drug-suitable agonist peptide in experimental ADIM. MethodsP8RI, a retro-inverso sequence of the best candidate identified by functional in vitro screening of a peptide library, passed an absorption, distribution, metabolism, excretion and toxicology analysis. Apo E−/− mice (male, 28-week-old) implanted with Ang II-releasing pumps received P8RI (2.5 mg/kg/day) or vehicle from day 14 (n = 10/group). Leukocytes were analyzed by flow cytometry. Healing features of human and mouse dissected aortic segments were assessed by histology and immunofluorescence. The effect of CD31 on macrophages was evaluated using cells from CD31−/− mice and P8RI, in vitro. ResultsHuman and experimental ADIM were characterized by the infiltration of proinflammatory macrophages. The absence of CD31 enhanced the proinflammatory polarization of macrophages, whereas the CD31 agonist P8RI favored reparative macrophages both in vitro and in vivo. The administration of P8RI after the occurrence of ADIM prevented aneurysmal transformation by promoting the resolution of intramural hematoma and the production of collagen in dissected aortas in vivo, associated with enrichment of M2 macrophages at the site of injury. ConclusionsCD31 signaling promotes the switching of proinflammatory macrophages to the reparative phenotype and favors the healing of experimental dissected aortas. Treatment with a drug-suitable CD31 agonist may facilitate the clinical management of ADIM.

P38 MAPK and the compromised regenerative response of the infarcted adult heart

Mononucleated cardiomyocytes promoted the hyperplasic growth of the embryonic mammalian heart and the proliferative phenotype was retained by neonatal cardiomyocytes for a short duration after birth. Despite the cell cycle re-entry of pre-existing cardiomyocytes, regeneration of the damaged neonatal heart required the coordinated effort of reparative embryonic macrophages. During postnatal development, pre-existing cardiomyocytes underwent karyokinesis in the absence of cytokinesis and the ability to proliferate was lost. However, a paucity of mononucleated cardiomyocytes persisted in the adult heart and an ischemic insult facilitated re-entry into the cell cycle. The latter paradigm provided a semblance of hope that a cardiac regenerative response was possible. However, the selective expansion of pro-inflammatory monocyte-derived macrophages following ischemic injury to the adult heart may have further impeded an already compromised regenerative response. The serine/threonine kinase p38 MAPK was identified as a seminal target of pro-inflammatory cytokines and a smaller infarct was reported after inhibition. The smaller infarct was attributed in part to cardiac regeneration as p38 MAPK suppressed the cell cycle re-entry of neonatal/adult cardiomyocytes in response to peptide growth factors. p38 MAPK-mediated inhibition of cell cycle re-entry may be related in part to the attenuation of nestin expression by pre-existing cardiomyocytes as the intermediate filament protein was directly implicated in the cell proliferation of normal and tumorigenic cells. Thus, targeting the inflammatory response via p38 MAPK inhibition may represent a rational approach to unmask the proliferative potential of pre-existing mononucleated cardiomyocytes and initiate a partial regenerative response of the infarcted adult mammalian heart.

Abstract 190: Mir-33 Inhibition Alters Monocyte/macrophage Kinetic Processes to Promote Atherosclerotic Plaque Regression

Previous studies have identified microRNA-33 (miR-33) as a key regulator of metabolic gene pathways involved in cholesterol and fatty acid homeostasis. Inhibition of miR-33 promotes the regression of atherosclerosis, in part, by enhancing plasma HDL and reverse cholesterol transport levels. However, it has become clear that miR-33 inhibition can also reduce plaque inflammation, independent of its effects on cholesterol homeostasis, yet the mechanisms of these actions are still not well understood. To investigate how miR-33 inhibition alters monocyte/macrophage dynamics during atherosclerosis regression, we fed Ldlr -/- mice a western diet for 14 weeks to establish plaques, after which mice were switched to chow diet and treated with anti-miR-33 or control anti-miR oligonucleotides for 4 weeks. As we reported previously, anti-miR-33 treatment increased plasma levels of HDL cholesterol by 30%, and concurrently reduced plaque size, macrophage content and necrotic area, compared to control anti-miR treatment. Analysis of the monocyte/macrophage kinetic processes contributing to macrophage burden in plaques revealed that anti-miR-33 treatment normalized the monocytosis associated with hypercholesterolemia. Mice treated with anti-miR33 had 40% fewer circulating monocytes than control anti-miR treated mice, and this was associated with a reduction of splenic common myeloid progenitor (CMP) cells. Surprisingly, monocyte tracking assays also revealed an increase in Ly6C hi monocyte recruitment into plaques of anti-miR-33 treated mice. Although somewhat counterintuitive, these data are consistent with recent findings that Ly6C hi monocytes are required for atherosclerosis regression and are a source of tissue reparative M2 macrophages. Indeed, we find that M2 macrophages, as well as atheroprotective regulatory T cells are enriched in plaques of anti-miR-33 treated mice. Collectively, our results provide insight into the mechanisms underlying anti-miR-33’s atheroprotective actions, which include both anti-inflammatory and cholesterol homeostatic effects.

It’s the Metabolism That Makes Macrophages Detectable in the Magnetic Resonance Scanner

Modulation of the immune response becomes more and more attractive for therapeutic interventions of cardiovascular diseases, aiming to improve healing and prevent remodeling after myocardial infarction or in myocarditis.1,2 However, there are also conflicting and sometimes disappointing results.3,4 Although a huge body of knowledge concerning immunologic pathways and networks is available, translation to the human setup and clinical progress are rather slow. One reason for this discrepancy might be that most of the latter is—roughly speaking—based on in vitro studies, which, for example, led to the concept of distinct classes of proinflammatory and reparative macrophages. However, there is increasing evidence that immune cells as macrophages behave differently in tissue. They are plastic and their polarization into different states strongly depends on the environment, which is determined by the affected organ and type of pathology.5 Article, see p 1084 Hence, it is of paramount importance that immunologic processes and targets should be assessed in their natural surroundings—at best in vivo, which demands the development of appropriate imaging techniques. Such techniques should provide new insights into basic processes and might guide therapeutic interventions in humans. In their article, Lewis et al6 apply a cutting-edge technology of nuclear magnetic resonance imaging which makes macrophage metabolism visible in vivo, namely 13C …

Immune responsive resolvin D1 programs myocardial infarction-induced cardiorenal syndrome in heart failure.

Resolvins are innate, immune responsive, bioactive mediators generated after myocardial infarction (MI) to resolve inflammation. The MI-induced bidirectional interaction between progressive left ventricle (LV) remodeling and kidney dysfunction is known to advance cardiorenal syndrome (CRS). Whether resolvins limit MI-induced cardiorenal inflammation is unclear. Thus, to define the role of exogenous resolvin D (RvD)-1 in post-MI CRS, we subjected 8- to 12-wk-old male C57BL/6 mice to coronary artery ligation. RvD1 was injected 3 h after MI. MI mice with no treatment served as MI controls (d 1 and 5). Mice with no surgery served as naive controls. In the injected mice, RvD1 promoted neutrophil (CD11b+/Ly6G+) egress from the infarcted LV, compared with the MI control group at d 5, indicative of neutrophil clearance and thereby resolved inflammation. Further, RvD1-injected mice showed higher reparative macrophages (F4/80+/Ly6Clow/CD206+) in the infarcted LV than did MI control mice at d 5 after MI. RvD1 suppressed the miRNA storm at d 1 and limited the MI-induced edematous milieu in a remote area of the LV compared with the MI control at d 5 after MI. Also, RvD1 preserved the nephrin expression that was diffuse in the glomerular membrane at d 5 and 28 in MI controls, indicating renal injury. RvD1 attenuated MI-induced renal inflammation, decreasing neutrophil gelatinase-associated lipocalin and proinflammatory cytokines and chemokines in the kidney compared with the MI control. In summary, RvD1 clears MI-induced inflammation by increasing resolving leukocytes and facilitates renoprotective mechanisms to limit CRS in acute and chronic heart failure.-Halade, G. V., Kain, V., Serhan, C. N. Immune responsive resolvin D1 programs myocardial infarction-induced cardiorenal syndrome in heart failure.

Translocator Protein as an Imaging Marker of Macrophage and Stromal Activation in Rheumatoid Arthritis Pannus.

PET radioligands targeted to translocator protein (TSPO) offer a highly sensitive and specific means of imaging joint inflammation in rheumatoid arthritis (RA). Through high expression of TSPO on activated macrophages, TSPO PET has been widely reported in several studies of RA as a means of imaging synovial macrophages in vivo. However, this premise does not take into account the ubiquitous expression of TSPO. This study aimed to investigate TSPO expression in major cellular constituents of RA pannus-monocytes, macrophages, fibroblastlike synoviocytes (FLS cells), and CD4-positive (CD4+) T lymphocytes (T cells)-to more accurately interpret TSPO PET signal from RA synovium. Methods: Three RA patients and 3 healthy volunteers underwent PET of both knees using the TSPO radioligand 11C-PBR28. Through 3H-PBR28 autoradiography and immunostaining of synovial tissue in 6 RA patients and 6 healthy volunteers, cellular expression of TSPO in synovial tissue was evaluated. TSPO messenger RNA expression and 3H-PBR28 radioligand binding was assessed using in vitro monocytes, macrophages, FLS cells, and CD4+ T cells. Results:11C-PBR28 PET signal was significantly higher in RA joints than in healthy joints (average SUV, 0.82 ± 0.12 vs. 0.03 ± 0.004; P < 0.01). Further, 3H-PBR28-specific binding in synovial tissue was approximately 10-fold higher in RA patients than in healthy controls. Immunofluorescence revealed TSPO expression on macrophages, FLS cells, and CD4+ T cells. The in vitro study demonstrated the highest TSPO messenger RNA expression and 3H-PBR28-specific binding in activated FLS cells, nonactivated M0 macrophages, and activated M2 reparative macrophages, with the least TSPO expression being in activated and nonactivated CD4+ T cells. Conclusion: To our knowledge, this study was the first evaluation of cellular TSPO expression in synovium, with the highest TSPO expression and PBR28 binding being found on activated synovial FLS cells and M2 macrophages. TSPO-targeted PET may therefore have a unique sensitivity in detecting FLS cells and macrophage-predominant inflammation in RA, with potential utility for assessing treatment response in trials using novel FLS-cell-targeted therapies.

A CD103+ Conventional Dendritic Cell Surveillance System Prevents Development of Overt Heart Failure during Subclinical Viral Myocarditis

Innate and adaptive immune cells modulate heart failure pathogenesis during viral myocarditis, yet their identities and functions remain poorly defined. We utilized a combination of genetic fate mapping, parabiotic, transcriptional, and functional analyses and demonstrated that the heart contained two major conventional dendritic cell (cDC) subsets, CD103+ and CD11b+, which differentially relied on local proliferation and precursor recruitment to maintain their tissue residency. Following viral infection of the myocardium, cDCs accumulated in the heart coincident with monocyte infiltration and loss of resident reparative embryonic-derived cardiac macrophages. cDC depletion abrogated antigen-specific CD8+ Tcell proliferative expansion, transforming subclinical cardiac injury to overt heart failure. These effects were mediated by CD103+ cDCs, which are dependent on the transcription factor BATF3 for their development. Collectively, our findings identified resident cardiac cDC subsets, defined their origins, and revealed an essential role for CD103+ cDCs in antigen-specific Tcell responses during subclinical viral myocarditis.

The peritoneum: healing, immunity, and diseases.

The peritoneum defines a confined microenvironment, which is stable under normal conditions, but is exposed to the damaging effect of infections, surgical injuries, and other neoplastic and non-neoplastic events. Its response to damage includes the recruitment, proliferation, and activation of a variety of haematopoietic and stromal cells. In physiological conditions, effective responses to injuries are organized; inflammatory triggers are eliminated; inflammation quickly abates; and the normal tissue architecture is restored. However, if inflammatory triggers are not cleared, fibrosis or scarring occurs and impaired tissue function ultimately leads to organ failure. Autoimmune serositis is characterized by the persistence of self-antigens and a relapsing clinical pattern. Peritoneal carcinomatosis and endometriosis are characterized by the persistence of cancer cells or ectopic endometrial cells in the peritoneal cavity. Some of the molecular signals orchestrating the recruitment of inflammatory cells in the peritoneum have been identified in the last few years. Alternative activation of peritoneal macrophages was shown to guide angiogenesis and fibrosis, and could represent a novel target for molecular intervention. This review summarizes current knowledge of the alterations to the immune response in the peritoneal environment, highlighting the ambiguous role played by persistently activated reparative macrophages in the pathogenesis of common human diseases. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Obesity-induced vascular inflammation involves elevated arginase activity.

Obesity-induced vascular dysfunction involves pathological remodeling of the visceral adipose tissue (VAT) and increased inflammation. Our previous studies showed that arginase 1 (A1) in endothelial cells (ECs) is critically involved in obesity-induced vascular dysfunction. We tested the hypothesis that EC-A1 activity also drives obesity-related VAT remodeling and inflammation. Our studies utilized wild-type and EC-A1 knockout (KO) mice made obese by high-fat/high-sucrose (HFHS) diet. HFHS diet induced increases in body weight, fasting blood glucose, and VAT expansion. This was accompanied by increased arginase activity and A1 expression in vascular ECs and increased expression of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), interleukin-10 (IL-10), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) mRNA and protein in both VAT and ECs. HFHS also markedly increased circulating inflammatory monocytes and VAT infiltration by inflammatory macrophages, while reducing reparative macrophages. Additionally, adipocyte size and fibrosis increased and capillary density decreased in VAT. These effects of HFHS, except for weight gain and hyperglycemia, were prevented or reduced in mice lacking EC-A1 or treated with the arginase inhibitor 2-(S)-amino-6-boronohexanoic acid (ABH). In mouse aortic ECs, exposure to high glucose (25 mM) and Na palmitate (200 μM) reduced nitric oxide production and increased A1, TNF-α, VCAM-1, ICAM-1, and MCP-1 mRNA, and monocyte adhesion. Knockout of EC-A1 or ABH prevented these effects. HFHS diet-induced VAT inflammation is mediated by EC-A1 expression/activity. Limiting arginase activity is a possible therapeutic means of controlling obesity-induced vascular and VAT inflammation.

IL-4 as a Repurposed Biological Drug for Myocardial Infarction through Augmentation of Reparative Cardiac Macrophages: Proof-of-Concept Data in Mice

Recent research has shown that reparative (alternatively activated or M2) macrophages play a role in repair of damaged tissues, including the infarcted hearts. Administration of IL-4 is known to augment M2 macrophages. This translational study thus aimed to investigate whether IL-4 administration is useful for the treatment of myocardial infarction. Long-acting IL-4 complex (IL-4c; recombinant IL-4 mixed with anti-IL-4 monoclonal antibody as a stabilizer) was administered after coronary artery ligation in mice. It was observed that IL-4c administration increased accumulation of CD206+F4/80+ M2-like macrophages predominantly in the injured myocardium, compared to the control. Sorted cardiac M2-like macrophages highly expressed wide-ranging tissue repair-related genes. Indeed, IL-4c administration enhanced cardiac function in association with reduced infarct size and enhanced tissue repair (strengthened connective tissue formation, improved microvascular formation and attenuated cardiomyocyte hypertrophy). Experiments using Trib1−/− mice that had a depleted ability to develop M2 macrophages and other in-vitro studies supported that these IL-4-mediated effects were induced via M2-like macrophages. On the other hand, when administered at Day 28 post-MI, the effects of IL-4c were diminished, suggesting a time-frame for IL-4 treatment to be effective. These data represent proof-of-concept of efficacy of IL-4 treatment for acute myocardial infarction, encouraging its further development.