Ly6Clo Macrophages Research Articles

Differential recruitment of monocyte-derived macrophages in control and stellate cell-depleted mice during recurrent carbon tetrachloride-induced acute liver injury.

Liver depleted of hepatic stellate cells (HSCs) is resistant to ischemia/reperfusion-, concanavalin A-, and acetaminophen-induced acute injury. Whether HSCs regulate carbon tetrachloride (CCl4 )-induced acute liver injury is not known. CCl4 treatment damages pericentral hepatocytes that express CCl4 -metabolizing Cyp2E1 and activates HSCs. We investigated whether HSC-depletion in mice transgenic for thymidine kinase under the glial fibrillary acidic protein promoter (GFAP-TK-Tg) confers resistance to injury and inflammation due to CCl4 rechallenge. GFAP-TK-Tg or wild type (WT) mice were administered 0.16 ml/kg CCl4 (3× at 3 days intervals), then 40 μg/g/day ganciclovir for 10 days. The treatment depletes ~70%-75% HSCs from GFAP-TK-Tg but not WT mice while the liver recovers from earlier CCl4 -induced injury. Mice were then administered CCl4 , and liver injury and inflammation were determined at 24 h. HSC-depleted and HSC-sufficient mice showed similar CCl4 -induced hepatocyte necrosis and oxidative stress. However, increase in F4/80+ macrophages, but not CD68+ cells, was greater in CCl4 rechallenged HSC-depleted compared to HSC-sufficient mice. Expression of tumor necrosis factor-α (TNF-α), CCL2, and CXCL1 increased similarly, whereas increase in interleukin-6 (IL6), IL1β, and IL10 expression was higher in CCl4 rechallenged HSC-depleted compared to HSC-sufficient mice. CCl4 rechallenge of HSC-sufficient mice rapidly activated HSCs causing significant fibrosis with increased expression of Col1a1, transforming growth factor β1 (TGFβ1), tissue inhibitors of metalloproteinases 1 (TIMP1); increase in TIPM1 was much lower and metalloproteinases 13 (MMP13) greater in CCl4 rechallenged HSC-depleted mice. Interestingly, hepatic recruitment of both profibrogenic (Ly6Chi ) and antifibrogenic restorative (Ly6Clo ) macrophages, and neutrophils was significantly greater in CCl4 rechallenged HSC-depleted mice. These data suggest that CCl4 directly damages hepatocytes but HSCs regulate inflammation. Rapid fibrogenesis in CCl4 rechallenged HSC-sufficient mice recovered from earlier injury indicates that even transiently activated HSCs that had reverted to the quiescent phenotype remain primed to become reactivated.

Occurrences and Functions of Ly6Chi and Ly6Clo Macrophages in Health and Disease.

Macrophages originating from the yolk sac or bone marrow play essential roles in tissue homeostasis and disease. Bone marrow-derived monocytes differentiate into Ly6Chi and Ly6Clo macrophages according to the differential expression of the surface marker protein Ly6C. Ly6Chi and Ly6Clo cells possess diverse functions and transcriptional profiles and can accelerate the disease process or support tissue repair and reconstruction. In this review, we discuss the basic biology of Ly6Chi and Ly6Clo macrophages, including their origin, differentiation, and phenotypic switching, and the diverse functions of Ly6Chi and Ly6Clo macrophages in homeostasis and disease, including in injury, chronic inflammation, wound repair, autoimmune disease, and cancer. Furthermore, we clarify the differences between Ly6Chi and Ly6Clo macrophages and their connections with traditional M1 and M2 macrophages. We also summarize the limitations and perspectives for Ly6Chi and Ly6Clo macrophages. Overall, continued efforts to understand these cells may provide therapeutic approaches for disease treatment.

C-C motif chemokine ligand 5 confines liver regeneration by down-regulating reparative macrophage-derived hepatocyte growth factor in a forkhead box O 3a-dependent manner.

Liver regeneration (LR) is vital for the recovery of liver function after hepatectomy. Limited regeneration capacity, together with insufficient remnant liver volume, is a risk factor for posthepatectomy liver failure (PHLF) resulting from small-for-size syndrome. Although inflammation plays an important role in controlling LR, the underlying mechanisms still remain obscure. We identified C-C motif chemokine ligand (CCL) 5 as an important negative regulator for LR. CCL5 levels were elevated after partial hepatectomy (PHx), both in healthy donors of living donor liver transplantation (LT) and PHx mouse models. Ccl5 knockout mice displayed improved survival after 90% PHx and enhanced LR 36 h after 70% PHx. However, primary hepatocytes from Ccl5-/- mice exposed to growth factors in vitro showed no proliferation advantage compared to those from wild-type (WT) mice. Flow cytometry analysis showed that proportions of Ly6Clo macrophages were significantly increased in Ccl5-/- mice after 70% PHx. RNA-sequencing analysis revealed that sorted macrophages (CD11b+ Ly6Clo&hi ) manifested enhanced expression of reparative genes in Ccl5-/- mice compared to WT mice. Mechanistically, CCL5 induced macrophages toward proinflammatory Ly6Chi phenotype, thereby inhibiting the production of hepatocyte growth factor (HGF) through the C-C motif chemokine receptor (CCR) 1- and CCR5-mediated forkhead box O (FoxO) 3a pathways. Finally, blockade of CCL5 greatly optimized survival and boosted LR in the mouse PHx model. Our findings suggest that inhibition of CCL5 is a promising strategy to improve regeneration restoration by enhancing HGF secretion from reparative macrophages through the FoxO3a pathway, which may potentially reduce the mortality of PHLF.

Mesenchymal stem cells attenuate liver fibrosis by targeting Ly6Chi/lo macrophages through activating the cytokine-paracrine and apoptotic pathways

Mesenchymal stem cell (MSC) therapy has become a promising treatment for liver fibrosis due to its predominant immunomodulatory performance in hepatic stellate cell inhibition and fibrosis resolution. However, the cellular and molecular mechanisms underlying these processes remain limited. In the present study, we provide insights into the functional role of bone marrow-derived MSCs (BM-MSCs) in alleviating liver fibrosis by targeting intrahepatic Ly6Chi and Ly6Clo macrophage subsets in a mouse model. Upon chronic injury, the Ly6Chi subset was significantly increased in the inflamed liver. Transplantation of BM-MSCs markedly promoted a phenotypic switch from pro-fibrotic Ly6Chi subset to restorative Ly6Clo subpopulation by secreting paracrine cytokines IL-4 and IL-10 from the BM-MSCs. The Ly6Chi/Ly6Clo subset switch significantly blocked the source of fibrogenic TGF-β, PDGF, TNF-α, and IL-1β cytokines from Ly6Chi macrophages. Unexpectedly, BM-MSCs experienced severe apoptosis and produced substantial apoptotic bodies in the fibrotic liver during the 72 h period of transplantation. Most apoptotic bodies were engulfed by Ly6Clo macrophages, and this engulfment robustly triggered MMP12 expression for fibrosis resolution through the PtdSer-MerTK-ERK signaling pathway. This paper is the first to show previously unrecognized dual regulatory functions of BM-MSCs in attenuating hepatic fibrosis by promoting Ly6Chi/Ly6Clo subset conversion and Ly6Clo macrophage restoration through secreting antifibrogenic-cytokines and activating the apoptotic pathway.

Basophils balance healing after myocardial infarction via IL-4/IL-13.

The inflammatory response after myocardial infarction (MI) is a precisely regulated process that greatly affects subsequent remodeling. Here, we show that basophil granulocytes infiltrated infarcted murine hearts, with a peak occurring between days 3 and 7. Antibody-mediated and genetic depletion of basophils deteriorated cardiac function and resulted in enhanced scar thinning after MI. Mechanistically, we found that basophil depletion was associated with a shift from reparative Ly6Clo macrophages toward increased numbers of inflammatory Ly6Chi monocytes in the infarcted myocardium. Restoration of basophils in basophil-deficient mice by adoptive transfer reversed this proinflammatory phenotype. Cellular alterations in the absence of basophils were accompanied by lower cardiac levels of IL-4 and IL-13, two major cytokines secreted by basophils. Mice with basophil-specific IL-4/IL-13 deficiency exhibited a similarly altered myeloid response with an increased fraction of Ly6Chi monocytes and aggravated cardiac function after MI. In contrast, IL-4 induction in basophils via administration of the glycoprotein IPSE/α-1 led to improved post-MI healing. These results in mice were corroborated by the finding that initially low counts of blood basophils in patients with acute MI were associated with a worse cardiac outcome after 1 year, characterized by a larger scar size. In conclusion, we show that basophils promoted tissue repair after MI by increasing cardiac IL-4 and IL-13 levels.

Transcriptomic Analysis of Monocyte-Derived Non-Phagocytic Macrophages Favors a Role in Limiting Tissue Repair and Fibrosis.

Monocyte-derived macrophages are readily differentiating cells that adapt their gene expression profile to environmental cues and functional needs. During the resolution of inflammation, monocytes initially differentiate to reparative phagocytic macrophages and later to pro-resolving non-phagocytic macrophages that produce high levels of IFNβ to boost resolutive events. Here, we performed in-depth analysis of phagocytic and non-phagocytic myeloid cells to reveal their distinct features. Unexpectedly, our analysis revealed that the non-phagocytic compartment of resolution phase myeloid cells is composed of Ly6CmedF4/80− and Ly6ChiF4/80lo monocytic cells in addition to the previously described Ly6C−F4/80+ satiated macrophages. In addition, we found that both Ly6C+ monocytic cells differentiate to Ly6C−F4/80+macrophages, and their migration to the peritoneum is CCR2 dependent. Notably, satiated macrophages expressed high levels of IFNβ, whereas non-phagocytic monocytes of either phenotype did not. A transcriptomic comparison of phagocytic and non-phagocytic resolution phase F4/80+ macrophages showed that both subtypes express similar gene signatures that make them distinct from other myeloid cells. Moreover, we confirmed that these macrophages express closer transcriptomes to monocytes than to resident peritoneal macrophages (RPM) and resemble resolutive Ly6Clo macrophages and monocyte-derived macrophages more than their precursors, inflammatory Ly6Chi monocytes, recovered following liver injury and healing, and thioglycolate-induced peritonitis, respectively. A direct comparison of these subsets indicated that the non-phagocytic transcriptome is dominated by satiated macrophages and downregulate gene clusters associated with excessive tissue repair and fibrosis, ROS and NO synthesis, glycolysis, and blood vessel morphogenesis. On the other hand, non-phagocytic macrophages enhance the expression of genes associated with migration, oxidative phosphorylation, and mitochondrial fission as well as anti-viral responses when compared to phagocytic macrophages. Notably, conversion from phagocytic to satiated macrophages is associated with a reduction in the expression of extracellular matrix constituents that were demonstrated to be associated with idiopathic pulmonary fibrosis (IPF). Thus, macrophage satiation during the resolution of inflammation seems to bring about a transcriptomic transition that resists tissue fibrosis and oxidative damage while promoting the restoration of tissue homeostasis to complete the resolution of inflammation.

Controlling the Phenotype of Tumor-Infiltrating Macrophages via the PHD-HIF Axis Inhibits Tumor Growth in a Mouse Model.

SummaryThe tumor microenvironment (TME) polarizes tumor-infiltrating macrophages toward tumor support. Macrophage-abundant tumors are highly malignant and are the cause of poor prognosis and therapeutic resistance. In this study, we show that the prolyl hydroxylase (PHD) inhibitor FG-4592 (FG) inhibits tumor growth of macrophage-abundant tumors and prolongs mouse survival. FG not only normalizes tumor vessels and improves tumor oxygenation but also directly affects macrophages and activates phagocytosis through the PHD-hypoxia-inducible factor (HIF) axis. Remarkably, FG can promote phagocytic ability of the Ly6Clo subset of tumor-infiltrating macrophages, leading to tumor growth inhibition. Moreover, Ly6Cneg macrophages contributed to blood vessel normalization. Using a malignant tumor mouse model, we characterized macrophage function and subsets. Altogether, our findings suggest that the PHD inhibitor can promote the anti-tumor potential of macrophages to improve cancer therapy.

Matrix metalloproteinase-12 produced by Ly6Clow macrophages prolongs the survival after myocardial infarction by preventing neutrophil influx

BackgroundVarious immune cells are involved in different phases of cardiac repair after myocardial infarction (MI). Especially, Ly6Clow M2-like macrophages (Ly6Clo macrophages) are vital for cardiac repair after MI. However, the molecular mechanisms how Ly6Clo macrophages promote wound healing after MI are still largely unknown. Methods and resultsTranscriptome analysis of Ly6Clo macrophages and Ly6Chigh M1-like macrophages (Ly6Chi macrophages) harvested from the infarcted heart revealed that Ly6Clo macrophages highly expressed matrix metalloproteinase (MMP)-12 mRNA compared to Ly6Chi macrophages. MMP-12 expression was enhanced in the infarcted heart and preferentially observed in Ly6Clo macrophages. Importantly, the survival rate and cardiac function after MI were significantly impaired in MMP-12-deficient (mmp12−/−) mice compared with those in wild-type mice. In addition, the extent of myocardial fibrosis and the number of myofibroblasts in the infarct area were decreased in mmp12−/− mice. MMP-9 expression and neutrophils, which are the major cellular source of MMP-9, in the infarcted heart were increased in mmp12−/− mice. Moreover, mRNA expression of neutrophil-attracting chemokines including CXCL1, CXCL2, and CXCL5 was significantly higher in mmp12−/− mice. Consistently, treatment with anti-CXCR2 antibody significantly decreased neutrophil numbers and MMP-9 expression in the infarcted heart in mmp12−/− mice. Finally, the administration of recombinant MMP-12 into the infarcted heart decreased neutrophil numbers in the infarcted heart and promoted wound healing in both wild-type mice and mmp12−/− mice. ConclusionMMP-12 produced by Ly6Clo macrophages improves the survival after MI possibly through the promotion of wound healing by reducing neutrophil infiltration.

Dynamic changes to lipid mediators support transitions among macrophage subtypes during muscle regeneration.

Muscle damage elicits a sterile immune response that facilitates complete regeneration. Here, we utilized mass spectrometry-based lipidomics to map the mediator lipidome during the transition from inflammation to resolution and regeneration in skeletal muscle injury. We observed the temporal regulation of glycerophospholipids and the production of pro-inflammatory (e.g., leukotrienes, prostaglandins) and specialized pro-resolving (e.g., resolvins, lipoxins) lipid mediators, which were modulated by ibuprofen. These time-dependent profiles were recapitulated in sorted neutrophils and Ly6Chi and Ly6Clo muscle-infiltrating macrophages, with a distinct pro-resolving signature observed in Ly6Clo macrophages. RNA-seq of macrophages stimulated with resolvin D2 (RvD2) showed similarities to transcriptional changes found during the temporal Ly6Chi to Ly6Clo macrophage transition. In vivo, RvD2 increased Ly6Clo macrophages and functional improvement of the regenerating muscle. These results reveal dynamic lipid mediator signatures of innate immune cells and provide a proof-of-concept for their exploitable effector roles in muscle regeneration.

CD24 regulates Ly6Chi macrophages and BDL-induced liver fibrosis

Objective To investigate the regulatory effects of CD24 on Ly6Chi macrophages in liver and its influences on bile duct ligation (BDL)-induced hepatic fibrosis in mice. Methods Liver fibrosis was induced in wild-type (WT) and CD24-/- mice by surgical ligation of the biliary duct. Levels of alanine amino transferase (ALT) in serum were detected and liver sections were stained with haematoxylin and eosin (H&E) staining to assess the severity of liver injury. Sirius Red staining was used to observe the degree of liver fibrosis. Real-time PCR was performed for detecting the expression of hepatic fibrosis-related markers and TGF-β1 at mRNA level. The percentage of macrophages and the number of TGF-β1-producing macrophages were measured by flow cytometry. Results BDL-induced liver fibrosis was exacerbated in CD24-/- mice than in WT mice as demonstrated by more serious hyperplasia in bile duct, more inflammatory infiltration at the portal area and higher levels of ALT in serum. Results of Sirius red staining also showed that the liver fibrosis was more severe in CD24-/- mice than in WT mice. Moreover, α-SMA and collagen typeⅠ alpha 1 (Col1a1) were significantly upregulated in CD24-/- mice. Flow cytometry analysis revealed that CD24 was highly expressed by hepatic macrophages in BDL-induced WT mice, and the percentages of hepatic macrophages were significantly elevated in CD24-/- mice compared with those in WT mice. Further analysis revealed that the percentages of Ly6Chi hepatic macrophages in CD24-/- mice were higher than those in WT mice, but there was no significant difference in the percentages of Ly6Clo macrophages. The expression of TGF-β1 at mRNA level was increased in CD24-/- mice as compared with that in WT mice after BDL. Moreover, intracellular staining showed that Ly6Chi hepatic macrophages in CD24-/- mice secreted more TGF-β1 than the macrophages in WT mice. Conclusions CD24 might attenuate the BDL-induced liver fibrosis in mice via regulating the percentage of hepatic Ly6Chi macrophages and the secretion of TGF-β1. Key words: CD24; Bile duct ligation (BDL); Liver fibrosis; Ly6Chi hepatic macrophage; TGF-β1

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.

Abstract 4744: Disruption of Clever-1 in macrophages activates the innate immune response and mediates tumor rejection

Abstract Macrophages are considered the main obstacles for successful cancer treatment since they can dampen anti-tumor immune responses and promote tumor progression and resistance formation. Macrophages are highly eligible candidates for targeted therapies since these cells are abundantly present in various tumors, they are very plastic and can be converted into pro-inflammatory macrophages supporting T cell activation and tumor rejection. Clever-1 (also known as Stabilin-1) is a multifunctional molecule conferring scavenging ability on a subset of alternatively activated macrophages. The aim of this study is to uncover previously unknown functions of Clever-1 in the regulation of macrophage-mediated anti-tumor immune responses and develop anti-Clever-1 therapy into clinical use for those suffering from refractory, untreatable solid tumors. By using in vivo tumor models and sophisticated immunological assays, our data show that mice carrying a macrophage-specific genetic deletion of Clever-1 (Lyz2-Cre/Stab1fl/fl) reject implanted Lewis lung carcinoma and EL4 lymphoma tumors. Macrophage Clever-1 deficiency induced proliferation of Ly6Chi monocytes in the tumor microenvironment but impaired their maturation and polarization into tumorigenic Ly6Clo macrophages. Tumors in Lyz2-Cre/Stab1fl/fl mice consisted almost completely of proliferating CD8 T cells with an exhausted PD-1+Lag3+ phenotype two weeks after implantation. Functionally, Clever-1 depleted macrophages showed enhanced pro-inflammatory responses following TLR activation with a shift in IL-12/IL-10 secretion levels. This was due to enhanced activation of mTORC1 and NFκB signaling, which resulted in increased secretion of TNFα in steady state and after LPS induction. In conclusion, our data show an important function of Clever-1 in controlling macrophage mediated local immune responses. Moreover, our data supports Clever-1 targeting as a novel approach to increase host defenses against immune compromised tumors and promotes anti-Clever-1 immunotherapy into clinical trials where it may have benefits in comparison with currently available immune activating drugs. Citation Format: Miro Viitala, Reetta Virtakoivu, Sina Tadayon, Sirpa Jalkanen, Maija Hollmén. Disruption of Clever-1 in macrophages activates the innate immune response and mediates tumor rejection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4744.

Infiltrating macrophages are broadly activated at the early stage to support acute skeletal muscle injury repair

Acute skeletal muscle injury repair requires an adequate inflammatory response predominated by macrophage infiltration. We studied the activation of infiltrating macrophages by analyzing the expression of M1/M2 signature genes. Most of the intramuscular macrophages were Ly6Chi at day 1 after BaCl2 injection, while many were Ly6Clo at day 3. Ly6Chi macrophages at day 1 expressed a high level of both M1 and M2 genes, and the Ly6Chi and Ly6Clo macrophages at day 3 expressed a similar level of many M1/M2 genes. Infiltrating macrophages are broadly activated rather than polarized at the early stage to support acute skeletal muscle injury repair.

The Cardiac Microenvironment Instructs Divergent Monocyte Fates and Functions in Myocarditis

Two types of monocytes, Ly6Chi and Ly6Clo, infiltrate the heart in murine experimental autoimmune myocarditis (EAM). We discovered a previously unappreciated role for cardiac fibroblasts in facilitating both Ly6Chi and Ly6Clo monocyte-to-macrophage differentiation, allowing these macrophages to perform divergent functions in myocarditis progression. During the acute phase of EAM, IL-17A is highly abundant. It signals through cardiac fibroblasts to attenuate efferocytosis of Ly6Chi monocyte-derived macrophages and simultaneously prevents Ly6Clo monocyte-to-macrophage differentiation. We demonstrated an inverse correlation of cardiac IL-17A and efferocytic receptor expression in human heart failure. During the resolution of inflammation, Ly6Clo monocyte-to-macrophage differentiation is restored as IL-17A signaling through cardiac fibroblast subsides. Ly6Clo monocyte-derived macrophages exhibit robust MHC class II-associated antigen presentation but are incapable of engulfing debris. We propose that MHCII+ Ly6Clo monocyte-derived macrophages may prevent fibrosis and heart failure.

Innate Immune Mechanisms in Myocardial Infarction - An Update

Abstract Acute myocardial infarction (AMI) is a disease associated with high morbidity and mortality. Currently there are no available treatments specifically targeting the post-ischemic myocardial processes that lead to heart failure and recurrent coronary events. The innate immune system plays a central role in the two consecutive phases that follow an acute ischemic event: the inflammatory phase and the reparatory phase. The inflamatory phase involves a massive infiltration of neutrophils and inflammatory Ly6Chi monocytes into the injured myocardium. The reparatory phase is orchestrated by reparatory Ly6Clo macrophages that clear necrotic and apoptotic cells through efferocytosis, secrete anti-inflammatory mediators and stimulate fibrosis and repair. Important recent studies provided proof that Ly6Chi monocytes that enter the myocardium in the inflammatory phase upregulate the orphan nuclear receptor Nr4a1 and switch phenotype to Ly6CloNr4a1hi reparatory macrophages. Additionally, neutrophils have been shown to promote cardiac recovery by upregulating expression of the efferocytosis receptor MerTK on reparatory macrophages. A finely tuned balance between the inflammatory and the reparatory phases is thus essential for limiting myocardial damage and promoting efficient recovery. Treatment strategies targeting only the inflammatory phase have so far failed to improve prognosis in AMI patients. A detailed understanding of the interplay between the two phases of the innate immune response is paramount for designing efficient therapies able to improve post- AMI prognosis. In the current review, we summarize the state-of-the-art of the field and discuss previous therapeutic attempts and currently ongoing clinical trials targeting innate immune mechanisms in AMI patients.

A heart-brain-kidney network controls adaptation to cardiac stress through tissue macrophage activation.

Heart failure is a complex clinical syndrome characterized by insufficient cardiac function. In addition to abnormalities intrinsic to the heart, dysfunction of other organs and dysregulation of systemic factors greatly affect the development and consequences of heart failure. Here we show that the heart and kidneys function cooperatively in generating an adaptive response to cardiac pressure overload. In mice subjected to pressure overload in the heart, sympathetic nerve activation led to activation of renal collecting-duct (CD) epithelial cells. Cell-cell interactions among activated CD cells, tissue macrophages and endothelial cells within the kidney led to secretion of the cytokine CSF2, which in turn stimulated cardiac-resident Ly6Clo macrophages, which are essential for the myocardial adaptive response to pressure overload. The renal response to cardiac pressure overload was disrupted by renal sympathetic denervation, adrenergic β2-receptor blockade or CD-cell-specific deficiency of the transcription factor KLF5. Moreover, we identified amphiregulin as an essential cardioprotective mediator produced by cardiac Ly6Clo macrophages. Our results demonstrate a dynamic interplay between the heart, brain and kidneys that is necessary for adaptation to cardiac stress, and they highlight the homeostatic functions of tissue macrophages and the sympathetic nervous system.

Role of exogenous interleukin 4 in the dynamics of acute liver injury

BackgroundMortality in acute liver failure is very high, and the only reliable treatment is transplantation. Interleukin 4 (IL4) has pleiotropic effects on a network of cells directing repair, regeneration, and fibrosis. Pretreatment with subcutaneous IL4 complexed with anti-IL4 antibody (IL4c) promotes repair of carbon tetrachloride-mediated murine liver injury by stimulating hepatocyte proliferation. We hypothesised that IL4c could be administered therapeutically and act by a variety of mechanisms beyond engagement of hepatocytes. MethodsC57Bl/6 mice were given carbon tetrachloride intraperitoneally to induce acute liver injury. Preinjury and postinjury dosing regimens of IL4c were compared. The role of IL4 receptor α-chain (IL4Rα) signalling in bone-marrow-derived cells was investigated using tissue-protected chimeras generated with wild type or IL4Rα−/– donor bone marrow. Liver was analysed by immunohistochemistry and fluorescence-activated cell sorting (FACS). FindingsWhen compared with vehicle, administration of IL4c after injury produced a significant reduction in mean alanine aminotransferase (242 U/L [61·9] vs 131 [30·8], p<0·0001) and necrotic cell area (11·6% [7·04] vs 0·36 [0·6], p<0·0001). Although necrosis was reduced, the necrotic area in IL4c-treated liver was not replaced with hepatocytes but with a cellular infiltrate characterised by high F4/80 expression. FACs analysis showed a reduced proportion and number of Ly6chi MHCIIlo monocytes and a concomitant increase in the number of Ly6clo MHCIIhi F4/80hi macrophages in injured livers treated with IL4c. This phenotypic change was dependent on IL4c signalling to Ly6chi MHCIIlo monocytes, since these cells exhibited developmental arrest when derived from IL4Rα-deficient bone marrow in chimeric mice. InterpretationWe demonstrate that IL4c can be administered therapeutically after carbon tetrachloride injury to improve indices of hepatic injury. Furthermore, IL4c treatment mediates a change in the monocyte–macrophage compartment which is consistent with a switch from a proinflammatory (Ly6chi) to a proreparative (Ly6clo) macrophage phenotype. We postulate that these Ly6clo macrophages mediate clearance of necrotic debris and facilitate hepatic regeneration. FundingWellcome Trust.

Editor's Highlight: CCR2 Regulates Inflammatory Cell Accumulation in the Lung and Tissue Injury following Ozone Exposure.

Ozone-induced lung injury is associated with an accumulation of activated macrophages in the lung. Chemokine receptor CCR2 mediates the migration of inflammatory monocytes/macrophages to sites of tissue injury. It is also required for monocyte egress from the bone marrow. In the present studies, we analyzed the role of CCR2 in inflammatory cell trafficking to the lung in response to ozone. Treatment of mice with ozone (0.8 ppm, 3 h) resulted in increases in proinflammatory CCR2+ macrophages in the lung at 24 h, as well as proinflammatory CD11b + Ly6CHi and iNOS+ macrophages at 24 and 48 h. Mannose receptor+ anti-inflammatory macrophages were also observed in the lung 24 and 48 h post-ozone. Loss of CCR2 was associated with reduced numbers of proinflammatory macrophages in the lung and decreased expression of the proinflammatory cytokines, IL-1β and TNFα. Decreases in anti-inflammatory CD11b + Ly6CLo macrophages were also observed in lungs of CCR2-/- mice treated with ozone, whereas mannose receptor+ macrophage accumulation was delayed; conversely, CX3CL1 and CX3CR1 were upregulated. Changes in lung macrophage subpopulations and inflammatory gene expression in CCR2-/- mice were correlated with reduced ozone toxicity and oxidative stress, as measured by decreases in bronchoalveolar lavage protein content and reduced lung expression of heme-oxygenase-1, 4-hydroxynonenal and cytochrome b5. These data demonstrate that CCR2 plays a role in both pro- and anti-inflammatory macrophage accumulation in the lung following ozone exposure. The fact that ozone-induced lung injury and oxidative stress are reduced in CCR2-/- mice suggests more prominent effects on proinflammatory macrophages.

The spleen as an extramedullary source of inflammatory cells responding to acetaminophen-induced liver injury

Macrophages have been shown to play a role in acetaminophen (APAP)-induced hepatotoxicity, contributing to both pro- and anti-inflammatory processes. In these studies, we analyzed the role of the spleen as an extramedullary source of hepatic macrophages. APAP administration (300mg/kg, i.p.) to control mice resulted in an increase in CD11b+ infiltrating Ly6G+ granulocytic and Ly6G− monocytic cells in the spleen and the liver. The majority of the Ly6G+ cells were also positive for the monocyte/macrophage activation marker, Ly6C, suggesting a myeloid derived suppressor cell (MDSC) phenotype. By comparison, Ly6G− cells consisted of 3 subpopulations expressing high, intermediate, and low levels of Ly6C. Splenectomy was associated with increases in mature (F4/80+) and immature (F4/80−) pro-inflammatory Ly6Chi macrophages and mature anti-inflammatory (Ly6Clo) macrophages in the liver after APAP; increases in MDSCs were also noted in the livers of splenectomized (SPX) mice after APAP. This was associated with increases in APAP-induced expression of chemokine receptors regulating pro-inflammatory (CCR2) and anti-inflammatory (CX3CR1) macrophage trafficking. In contrast, APAP-induced increases in pro-inflammatory galectin-3+ macrophages were blunted in livers of SPX mice relative to control mice, along with hepatic expression of TNF-α, as well as the anti-inflammatory macrophage markers, FIZZ-1 and YM-1. These data demonstrate that multiple subpopulations of pro- and anti-inflammatory cells respond to APAP-induced injury, and that these cells originate from distinct hematopoietic reservoirs.

Bone Marrow and Spleen as Sources of Inflammatory Macrophages Responding to Ozone‐Induced Lung Injury

Ozone is an urban air pollutant known to damage the lung. Inflammatory macrophages (MP) have been shown to contribute to ozone toxicity; however, the origin of these cells has not been established. Herein, we analyzed the bone marrow and spleen as potential sources of inflammatory lung MP. Inflammatory monocyte (MO) trafficking from the bone marrow is mediated by CCR2/CCL2, while migration of splenic MOs is controlled by angiotensin-1 receptor (AT1R). Exposure of mice to ozone (0.8 ppm, 3 h) resulted in an increase in CCR2 and AT1R expression in the lung which peaked at 24 h. This was associated with increases in mature F4/80+ proinflammatory CD11b+Ly6Chi and antiinflammatory CD11b+Ly6CLo MP in the lung. Loss of CCR2 resulted in a reduction in mature CD11b+Ly6Chi and iNOS+ proinflammatory MP responding to ozone. In contrast, CD11b+Ly6CLo antiinflammatory MP were increased in lungs of CCR2-/- mice; this was correlated with reduced ozone toxicity, measured by bronchoalveolar lavage protein content and 4-HNE staining of the lung. Splenectomized (SPX) mice were used to evaluate the spleen as an extramedullary source of inflammatory MOs. Ozone-induced increases in CD11b+Ly6Chi proinflammatory MP were significantly reduced in lungs of SPX mice, when compared to control mice at all post exposure times, with no effect on iNOS+ MP; conversely antiinflammatory CD11b+Ly6CLo MP were increased in the lung, most prominently at 72 h; this was associated with increased CCR2 expression and deceased toxicity. These data demonstrate that proinflammatory lung MPs are derived from both spleen and bone marrow reservoirs. Moreover, in the absence of the spleen, signaling to the bone marrow to generate inflammatory MOs increases. NIH ES004738, AR055073, ES007148 and ES005022.