Switch Of Macrophages Research Articles

Acute Iron Deprivation Reprograms Human Macrophage Metabolism and Reduces Inflammation In Vivo

SummaryIron is an essential metal that fine-tunes the innate immune response by regulating macrophage function, but an integrative view of transcriptional and metabolic responses to iron perturbation in macrophages is lacking. Here, we induced acute iron chelation in primary human macrophages and measured their transcriptional and metabolic responses. Acute iron deprivation causes an anti-proliferative Warburg transcriptome, characterized by an ATF4-dependent signature. Iron-deprived human macrophages show an inhibition of oxidative phosphorylation and a concomitant increase in glycolysis, a large increase in glucose-derived citrate pools associated with lipid droplet accumulation, and modest levels of itaconate production. LPS polarization increases the itaconate:succinate ratio and decreases pro-inflammatory cytokine production. In rats, acute iron deprivation reduces the severity of macrophage-dependent crescentic glomerulonephritis by limiting glomerular cell proliferation and inducing lipid accumulation in the renal cortex. These results suggest that acute iron deprivation has in vivo protective effects mediated by an anti-inflammatory immunometabolic switch in macrophages.

The cholesterol transporter ABCG1 modulates macrophage polarization in human monocyte-derived macrophages

Abstract The ATP-binding Cassette Transporter G1 (ABCG1) promotes cholesterol efflux from cells and regulates intracellular cholesterol homeostasis. Recent studies have shown that in mice in the absence of ABCG1, macrophages shift from a tumor-promoting M2 phenotype to a tumor-fighting M1 phenotype within the tumor and suppress bladder cancer growth in vivo. Two important aspects of this effect are still unknown: (1) whether ABCG1-would also impact polarization of human macrophages, and (2) the underlying molecular mechanism of this effect. Here, we show that in human monocyte-derived macrophages the inhibition of ABCG1 by siRNA significantly increased the expression of the M1 markers IDO1, CXCL10, CD64 and TNF after LPS/IFNγ (M1) stimulation. In contrast, after IL-4 (M2) stimulation the expression of the M2 markers MRC1, TGM2 and CD163 was significantly reduced. These data indicate that ABCG1-deficiency promotes the polarization of macrophage to an M1 phenotype in human macrophages. To address the molecular mechanism, we analysed cell signalling in bone marrow-derived macrophages from Abcg1−/− mice. These macrophages displayed reduced levels of Akt activation after stimulation with LPS/IFNγ or IL4. In addition, while Abcg1−/− macrophages stimulated with LPS/IFNγ produced more TNF compared to WT macrophages, after treatment with an Akt activator the TNF-production of Abcg1−/− and WT macrophages was comparable. These data suggest that the M1 bias of Abcg1−/− macrophages is mediated through the Akt signalling pathway. In summary, these findings deepen our mechanistic understanding of the M1/M2 switch in macrophages and suggest that ABCG1 could be a potential new target to modulate macrophage polarization for cancer immunotherapy.

Phenotypic switch of human and mouse macrophages and resultant effects on apoptosis resistance in hepatocytes

Acute-on-chronic liver failure (ACLF) carries a significant burden on critical care services and health care resources. However, the exact pathogenesis of ACLF remains to be elucidated, and novel treatments are desperately required. In our previous work, we utilized mice subjected to acute insult in the context of hepatic fibrosis to simulate the development of ACLF and documented the favorable hepatoprotection conferred by M2-like macrophages in vivo and in vitro. In the present study, we focused on the phenotypic switch of human and mouse macrophages and assessed the effects of this switch on apoptosis resistance in hepatocytes. For this purpose, human and mouse macrophages were isolated and polarized into M0, M(IFN-γ), M(IFN-γ→IL-4), M(IL-4) or M(IL-4→IFN-γ) subsets. Conditioned media (CM) from these subsets were applied to human and mouse hepatocytes followed by apoptosis induction. Cell apoptosis was evaluated by immunostaining for cleaved caspase-3. As a result, M(IFN-γ) or M(IL-4) macrophages switched their phenotype into M(IFN-γ→IL-4) or M(IL-4→IFN-γ) through reprogramming with IL-4 or IFN-γ, respectively. Importantly, hepatocytes pre-treated with M(IFN-γ→IL-4) CMs exhibited much weaker expression of cleaved caspase-3, compared to those pre-treated with M(IFN-γ) CM, and vice versa. Together, phenotypic switch of macrophages toward M(IL-4) phenotype confers hepatocytes enhanced resistance to apoptosis.

Transfusion-Induced Impairment of Macrophage Responses Is Partially Restored By the Iron Chelator Deferasirox

Iron overload is common in MDS, as a consequence of chronic red blood cell (RBC) transfusions and to a lesser extent, increased intestinal iron absorption to support erythropoiesis. Clinical evidence suggests that patients with transfusional iron overload show an enhanced susceptibility to infections, which represent one of the most common causes of morbidity and mortality in low risk MDS patients. Iron overload may increase the risk of infections by supporting bacterial growth and altering the immune response.We asked whether transfusions impair macrophage effector functions. Therefore we investigated the impact of transfusions on the phenotypic plasticity of monocytes and macrophages and their functional responses to infectious cues. We applied a mouse model of repeated transfusions of allogeneic RBCs in wild-type and NUP98-HOXD13 MDS mice. Monocytes as well as hepatic and splenic macrophages were analyzed in transfused and non-transfused mice, subjected or not to LPS challenge.Under steady-state condition, MDS mice displayed a mild but significant iron phenotype, hallmarked by elevated systemic and hepatic iron levels, due to inappropriately low hepcidin levels. Repeated transfusions increased serum iron levels and transferrin saturation and promoted iron deposition and oxidative damage in tissues.Transfusions caused macrophage iron loading, monocyte recruitment to the liver and spleen and massive splenic macrophage cell death. Macrophages from transfused mice developed an M2-like anti-inflammatory phenotype, hallmarked by elevated expression of M2 markers (CD206, Arg-1, Ym1) and reduced expression of M1 markers (MHCII, CD86). Accordingly, circulating pro-inflammatory cytokines (IL-6, TNFα, INFγ, IL-1β) were reduced, and anti-inflammatory cytokines (IL-10) increased. Consistently, after LPS challenge macrophages show a markedly reduced expression of M1 markers and inflammatory cytokines, and increased expression of M2 markers in transfused compared to non-transfused mice. Similar results were obtained both in wild-type and MDS mice. All together these observations indicate that transfusion-induced erythrophagocytosis dampens the inflammatory response to infectious cues.In an attempt to relieve macrophages from iron overload, transfused mice have been administered the iron chelator deferasirox during the transfusion period, up to 1 week after the last transfusion. Deferasirox significantly reduced iron levels in liver and spleen. Interestingly, chelation therapy partially restored the phenotype of macrophages by rescuing the expression of M1 markers, including MHCII and CD86, and M2 markers, such as CD206. Similar results have been obtained in the setting of transfusion with and without LPS stimulation.Our results show that transfusions lead to iron-overload mediated toxicity in macrophages blunting their inflammatory response to infectious stimuli by affecting cell plasticity. Our data support a role for transfusions in triggering a phenotypic switching of macrophages towards an anti-inflammatory phenotype, with reduced ability to counteract infections. By restoring cell polarization, iron chelation likely improves the functional response of macrophages to invading microorganisms. Combined with marked cytopenias, the weak pro-inflammatory activation of macrophages might explain the increased propensity of transfused MDS patients to develop infections. Transfusion practice might therefore increase the risk of infections not solely through increased iron availability to microorganisms, but also through the impairment of macrophage functions. Our results suggest that transfused MDS patients might benefit from chelation therapy by both restricting iron from pathogens and improving the innate immune response. DisclosuresVinchi:Novartis: Research Funding. Platzbecker:Celgene: Research Funding. Muckenthaler:Novartis: Research Funding.

Determining Macrophage Polarization upon Metabolic Perturbation.

Metabolic reprograming controlling macrophage activation and function is emerging as new regulatory circuit on shaping immune responses. Generally, lipopolysaccharides (LPS)-induced pro-inflammatory activated macrophages, known as M1 macrophages, display higher glycolysis. In contrast, interleukin-4 (IL-4)-skewed anti-inflammatory activated macrophages, known as M2 macrophages, mainly rely on oxidative phosphorylation for their bioenergetic demands. Emerging evidence reveals that these metabolic preferences further fine-tune macrophage polarization process, including signaling cascades and epigenetic reprogramming. Thus, specific nutrient microenvironments may affect inflammatory responses of macrophages by intervening these metabolic machineries. How to measure the metabolic switch of macrophages both in vitro and in vivo is an important issue for understanding immunometabolic regulations in macrophages. Here, we describe a basic protocol for examining how glutamine metabolism affects macrophage polarization by using the Extracellular Flux (XF(e)96) Analyzer (Seahorse Bioscience), which takes real-time measurements of oxidative phosphorylation and glycolysis. We also present a detailed procedure for detecting the expression of inflammatory genes in polarized macrophages under glutamine-replete or -deprived conditions.

Leukocyte Differentiation by Histidine-Rich Glycoprotein/Stanniocalcin-2 Complex Regulates Murine Glioma Growth through Modulation of Antitumor Immunity

The plasma-protein histidine-rich glycoprotein (HRG) is implicated in phenotypic switching of tumor-associated macrophages, regulating cytokine production and phagocytotic activity, thereby promoting vessel normalization and antitumor immune responses. To assess the therapeutic effect of HRG gene delivery on CNS tumors, we used adenovirus-encoded HRG to treat mouse intracranial GL261 glioma. Delivery of Ad5-HRG to the tumor site resulted in a significant reduction in glioma growth, associated with increased vessel perfusion and increased CD45+ leukocyte and CD8+ T-cell accumulation in the tumor. Antibody-mediated neutralization of colony-stimulating factor-1 suppressed the effects of HRG on CD45+ and CD8+ infiltration. Using a novel protein interaction-decoding technology, TRICEPS-based ligand receptor capture (LRC), we identified Stanniocalcin-2 (STC2) as an interacting partner of HRG on the surface of inflammatory cells in vitro and colocalization of HRG and STC2 in gliomas. HRG reduced the suppressive effects of STC2 on monocyte CD14+ differentiation and STC2-regulated immune response pathways. In consequence, Ad5-HRG-treated gliomas displayed decreased numbers of IL35+ Treg cells, providing a mechanistic rationale for the reduction in GL261 growth in response to Ad5-HRG delivery. We conclude that HRG suppresses glioma growth by modulating tumor inflammation through monocyte infiltration and differentiation. Moreover, HRG acts to balance the regulatory effects of its partner, STC2, on inflammation and innate and/or acquired immunity. HRG gene delivery therefore offers a potential therapeutic strategy to control antitumor immunity. Mol Cancer Ther; 17(9); 1961-72. ©2018 AACR.

Nuclear Receptor Nur77 Limits the Macrophage Inflammatory Response through Transcriptional Reprogramming of Mitochondrial Metabolism

SummaryActivation of macrophages by inflammatory stimuli induces reprogramming of mitochondrial metabolism to support the production of pro-inflammatory cytokines and nitric oxide. Hallmarks of this metabolic rewiring are downregulation of α-ketoglutarate formation by isocitrate dehydrogenase (IDH) and accumulation of glutamine-derived succinate, which enhances the inflammatory response via the activity of succinate dehydrogenase (SDH). Here, we identify the nuclear receptor Nur77 (Nr4a1) as a key upstream transcriptional regulator of this pro-inflammatory metabolic switch in macrophages. Nur77-deficient macrophages fail to downregulate IDH expression and accumulate higher levels of succinate and other TCA cycle-derived metabolites in response to inflammatory stimulation in a glutamine-independent manner. Consequently, these macrophages produce more nitric oxide and pro-inflammatory cytokines in an SDH-dependent manner. In vivo, bone marrow Nur77 deficiency exacerbates atherosclerosis development and leads to increased circulating succinate levels. In summary, Nur77 induces an anti-inflammatory metabolic state in macrophages that protects against chronic inflammatory diseases such as atherosclerosis.

Targetted inhibition of CD74 attenuates adipose COX-2-MIF-mediated M1 macrophage polarization and retards obesity-related adipose tissue inflammation and insulin resistance.

Adipose tissue (AT) inflammation is crucial to the development of obesity-associated insulin resistance. Our aim was to investigate the contribution of cyclooxygenase-2 (COX-2)/macrophage migration inhibitory factor (MIF)-mediated cross-talk between hypertrophic adipocytes and macrophages to the etiology of AT inflammation and the involvement of CD74 using human SGBS adipocytes, THP-1 macrophages and mice fed a high-fat (HF) diet. The MIF and CD74 mRNA levels in the adipocytes and stromal vascular cells (SVCs) of white fat were highly correlated with body weight (BW), homeostatic model assessment for insulin resistance (HOMA-IR), and adipose macrophage marker expression levels, especially those in SVCs. COX-2 inhibition suppressed the elevation of MIF production in HF white adipocytes as well as palmitate and hypoxic-treated SGBS adipocytes. Treatment of adipocytes transfected with shCOX-2 and siMIF or subjected to MIF depletion in the medium reversed the pro-inflammatory responses in co-incubated THP-1 cells. Inhibition of NF-κB activation reversed the COX2-dependent MIF secretion from treated adipocytes. The targetted inhibition of macrophage CD74 prevented M1 macrophage polarization in the above co-culture model.The COX-2-dependent increases in CD74 gene expression and MIF release in M1-polarized macrophages facilitated the expression of COX-2 and MIF in co-cultured SGBS adipocytes. CD74 shRNA intravenous injection suppressed HF-induced AT M1 macrophage polarization and inflammation as well as insulin resistance in mice. The present study suggested thatCOX-2-mediated MIF secretion through NF-κB activation from hypertrophic and hypoxic adipocytes as well as M1 macrophages might substantially contribute to the phenotypic switch of AT macrophages through CD74 in obesity. Inhibition of CD74 could attenuate AT inflammation and insulin resistance in the development of HF diet-induced obesity.

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.

Targeted intracerebral delivery of the anti-inflammatory cytokine IL13 promotes alternative activation of both microglia and macrophages after stroke

BackgroundSubtle adjustment of the activation status of CNS resident microglia and peripheral macrophages, to promote their neuroprotective and neuroregenerative functions, may facilitate research towards curing neurodegenerative disorders. In the present study, we investigated whether targeted intracerebral delivery of the anti-inflammatory cytokine interleukin (IL)13, by means of transplanting IL13-expressing mesenchymal stem cells (IL13-MSCs), can promote a phenotypic switch in both microglia and macrophages during the pro-inflammatory phase in a mouse model of ischemic stroke.MethodsWe used the CX3CR1eGFP/+ CCR2RFP/+ transgenic mouse model to separately recognize brain-resident microglia from infiltrated macrophages. Quantitative immunohistochemical analyses were applied to characterize polarization phenotypes of both cell types.ResultsDistinct behaviors of both cell populations were noted dependent on the anatomical site of the lesion. Immunohistochemistry revealed that mice grafted with IL13-MSCs, in contrast to non-grafted and MSC-grafted control mice, were able to drive recruited microglia and macrophages into an alternative activation state, as visualized by a significant increase of Arg-1 and a noticeable decrease of MHC-II expression at day 14 after ischemic stroke. Interestingly, both Arg-1 and MHC-II were expressed more abundantly in macrophages than in microglia, further confirming the distinct behavior of both cell populations.ConclusionsThe current data highlight the importance of controlled and localized delivery of the anti-inflammatory cytokine IL13 for modulation of both microglia and macrophage responses after ischemic stroke, thereby providing pre-clinical rationale for the application of L13-MSCs in future investigations of neurodegenerative disorders.

Abstract 586: Oxidized LDL Induce a Metabolic Switch Through CD36 in Macrophages

Under atherogenic conditions, low-density lipoproteins are converted to oxidized forms (oxLDL) by reactive oxygen species. They are then recognized by scavenger receptors such as CD36 in macrophages and stimulate pro-inflammatory functions of those innate immune cells. Although oxLDL are believed to contribute to chronic inflammation during atherosclerosis development, how the inflammatory status is sustained is not well understood. In this study, we explored how oxLDL affects the metabolic status of macrophages, which is increasingly being recognized as a key regulator of macrophage phenotypes. Exposure of wild type macrophages to oxLDL, but not native LDL, suppressed mitochondria oxidative phosphorylation (OXPHOS) as measured by oxygen consumption rate (OCAR), and increased glycolysis as measured by extracellular acidification rate (ECAR), in a dose-dependent manner. At 50μg/ml oxLDL for 24h there was a 79% reduction in OXPHOS and 60% elevation in glycolysis. Exposure to 50μg/ml oxLDL increased glucose uptake by 1.4 fold. Inhibition of glycolysis with 2-deoxy-glucose blocked oxLDL-induced HIF-1α expression as well as cytokine production, linking glycolytic metabolism to the pro-inflammatory response to oxLDL. These metabolic changes were not seen in cd36 null macrophages. In conclusion, oxLDL induce a metabolic switch in macrophages from mitochondrial OXPHOS to glycolysis through CD36-mediated signaling. This metabolic switch is important for inflammatory activation of macrophages and may be an underlying mechanism for sustained inflammatory status of those cells in the context of atherosclerosis.

Sensors of Succinate: Neural Stem Cell Grafts Fight Neuroinflammation.

In this issue of Cell Stem Cell, Peruzzotti-Jametti etal. (2018) demonstrate how neural stem cells, transplanted in a mouse model of multiple sclerosis, respond to extracellular succinate and modulate neuroinflammation by releasing anti-inflammatory prostaglandin E2 and scavenging succinate. This mechanism reduces CNS damage and ameliorates motor impairment.

Macrophage phenotype switch by sequential action of immunomodulatory cytokines from hydrogel layers on titania nanotubes.

Inflammatory response occurring between tissues and implants after implantation has attracted increasing attention because it can cause local tissue necrosis and even implant failure. Macrophages play a key role in all stages of inflammation. Pro-inflammatory (M1) and anti-inflammatory (M2) macrophages comprise two main phenotypes and the switch from M1 to M2 at specific time points is important for wound healing and tissue regeneration. Therefore, we hypothesized that biomaterial systems capable of facilitating macrophage phenotype switching should attenuate inflammation and enhance healing. To this end, a system of double hydrogel layers on titania nanotubes (TNT) was prepared as reservoir to modulate the release of interleukin-4 (IL-4) and interferon-γ (IFN-γ). In this system, IL-4, an anti-inflammatory cytokine, was loaded in TNT and IFN-γ, a pro-inflammatory cytokine, was located between two hydrogel layers of chitosan/β-glycerophosphate disodium and carboxymethyl chitosan/genipin. IFN-γ released rapidly in 3 days, whereas IL-4 exhibited a sustained release profile. In culture with mesenchymal stem cells and macrophages, this system displayed good cytocompatibility and significantly promoted cell proliferation. Macrophage phenotype switch was determined by ELISA, FACS and PCR. The results manifested that IFN-γ released from the system stimulated switching of macrophages to M1 in 3 days, whereas sustained release of IL-4 polarized macrophages to M2 after 4 days. This system can modulate macrophage phenotype switching from M1 to M2 by sequential action of the two cytokines, and might be used to research immune response between tissues and implants. The present study also provided a novel strategy for designing functional biomaterials.

High-mobility group box 1 protein orchestrates responses to tissue damage via inflammation, innate and adaptive immunity, and tissue repair.

A single protein, HMGB1, directs the triggering of inflammation, innate and adaptive immune responses, and tissue healing after damage. HMGB1 is the best characterized damage-associated molecular pattern (DAMP), proteins that are normally inside the cell but are released after cell death, and allow the immune system to distinguish between antigens that are dangerous or not. Notably, cells undergoing severe stress actively secrete HMGB1 via a dedicated secretion pathway: HMGB1 is relocated from the nucleus to the cytoplasm and then to secretory lysosomes or directly to the extracellular space. Extracellular HMGB1 (either released or secreted) triggers inflammation and adaptive immunological responses by switching among multiple oxidation states, which direct the mutually exclusive choices of different binding partners and receptors. Immune cells are first recruited to the damaged tissue and then activated; thereafter, HMGB1 supports tissue repair and healing, by coordinating the switch of macrophages to a tissue-healing phenotype, activation and proliferation of stem cells, and neoangiogenesis. Inevitably, HMGB1 also orchestrates the support of stressed but illegitimate tissues: tumors. Concomitantly, HMGB1 enhances the immunogenicity of mutated proteins in the tumor (neoantigens), promoting anti-tumor responses and immunological memory. Tweaking the activities of HMGB1 in inflammation, immune responses and tissue repair could bring large rewards in the therapy of multiple medical conditions, including cancer.

Anti-inflammatory and wound healing activities of calophyllolide isolated from Calophyllum inophyllum Linn.

Due to the high-cost and limitations of current wound healing treatments, the search for alternative approaches or drugs, particularly from medicinal plants, is of key importance. In this study, we report anti-inflammatory and wound healing activities of the major calophyllolide (CP) compound isolated from Calophyllum inophyllum Linn. The results showed that CP had no effect on HaCaT cell viability over a range of concentrations. CP reduced fibrosis formation and effectively promoted wound closure in mouse model without causing body weight loss. The underlying molecular mechanisms of wound repair by CP was investigated. CP markedly reduced MPO activity, and increased M2 macrophage skewing, as shown by up-regulation of M2-related gene expression, which is beneficial to the wound healing process. CP treatment prevented a prolonged inflammatory process by down-regulation of the pro-inflammatory cytokines—IL-1β, IL-6, TNF-α, but up-regulation of the anti-inflammatory cytokine, IL-10. This study is the first to indicate a plausible role for CP in accelerating the process of wound healing through anti-inflammatory activity mechanisms, namely, by regulation of inflammatory cytokines, reduction in MPO, and switching of macrophages to an M2 phenotype. These findings may enable the utilization of CP as a potent therapeutic for cutaneous wound healing.

In situ macrophage phenotypic transition is affected by altered cellular composition prior to acute sterile muscle injury.

The in situ phenotypic switch of macrophages is delayed in acute injury following irradiation. The combination of bone marrow transplantation and local muscle radiation protection allows for the identification of a myeloid cell contribution to tissue repair. PET-MRI allows monitoring of myeloid cell invasion and metabolism. Altered cellular composition prior to acute sterile injury affects the in situ phenotypic transition of invading myeloid cells to repair macrophages. There is reciprocal intercellular communication between local muscle cell compartments, such as PAX7 positive cells, and recruited macrophages during skeletal muscle regeneration. Skeletal muscle regeneration is a complex interplay between various cell types including invading macrophages. Their recruitment to damaged tissues upon acute sterile injuries is necessary for clearance of necrotic debris and for coordination of tissue regeneration. This highly dynamic process is characterized by an in situ transition of infiltrating monocytes from an inflammatory (Ly6Chigh ) to a repair (Ly6Clow ) macrophage phenotype. The importance of the macrophage phenotypic shift and the cross-talk of the local muscle tissue with the infiltrating macrophages during tissue regeneration upon injury are not fully understood and their study lacks adequate methodology. Here, using an acute sterile skeletal muscle injury model combined with irradiation, bone marrow transplantation and in vivo imaging, we show that preserved muscle integrity and cell composition prior to the injury is necessary for the repair macrophage phenotypic transition and subsequently for proper and complete tissue regeneration. Importantly, by using a model of in vivo ablation of PAX7 positive cells, we show that this radiosensitive skeletal muscle progenitor pool contributes to macrophage phenotypic transition following acute sterile muscle injury. In addition, local muscle tissue radioprotection by lead shielding during irradiation preserves normal macrophage transition dynamics and subsequently muscle tissue regeneration. Taken together, our data suggest the existence of a more extensive and reciprocal cross-talk between muscle tissue compartments, including satellite cells, and infiltrating myeloid cells upon tissue damage. These interactions shape the macrophage in situ phenotypic shift, which is indispensable for normal muscle tissue repair dynamics.

REG3\u03b2 modifies cell tumor function by impairing extracellular vesicle uptake

Extracellular vesicles (EVs), including exosomes and microvesicles, are nano-sized membrane vesicles containing proteins and nucleic acids, which act as intercellular messengers. They play an important role in a variety of physiological processes, as well as in pathological situations such as inflammation or cancer. Here, we show that in the case of pancreatic ductal adenocarcinoma (PDAC), the healthy pancreatic tissue surrounding the tumor releases REG3β, a lectin that binds to the glycoproteins present in the surface of EVs, thus interfering with their uptake and internalization by target cells. In vitro, the disruption of the signaling mediated by EVs due to the presence of REG3β, prevents the EV-induced phenotypic switch in macrophages, inhibits the increased cell migration of cancer cells and reverses a number of metabolomic changes promoted by EVs. In vivo, the uptake of REG3β+ EVs by tumor cells is significantly impaired. Furthermore, it results in an increase of circulating REG3β+ EVs in blood of pancreatic cancer patients. Our findings highlight the effect of a lectin released by the healthy pancreatic tissue surrounding the tumor in modulating the EV-mediated interactions between different cell types in PDAC.

Melatonin protects mice against stress-induced inflammation through enhancement of M2 macrophage polarization

Stress is known to cause neuropsychiatric diseases, and it has a detrimental impact on the function of the immune system. Melatonin (MLT), a pineal gland hormone, exhibits potent anti-inflammatory properties and it plays a fundamental role in neuroimmunomodulation. In the present study, we investigated the molecular mechanisms of MLT in stress-induced inflammation, focusing on macrophage polarization. MLT (50 and 100mg/kg) or a vehicle control (5% ethanol in saline) was intraperitoneally administered to mice once a day for 5days. After the last treatment, mice were subjected to restraint stress (RS) for 2h. MLT markedly decreased serum levels of corticosterone after RS. RS significantly increased serum interleukin (IL)-1β and IL-6 levels, and it decreased serum IL-10 levels. MLT administration attenuated these changes. After RS, MLT markedly decreased lipid peroxidation and increased hepatic glutathione content. In purified Kupffer cells (KCs) and peritoneal macrophages from mice exposed to RS, the expression levels of M1 marker genes (NOS2a and CD40) increased, while the expression levels of an M2 marker gene (Arg1) decreased. MLT attenuated this increase in expression of M1 marker genes and decrease in the expression levels of the M2 marker gene. Furthermore, in KCs and peritoneal macrophages exposed to RS, MLT decreased the number of F4/80+CD86+ cells and increased the number of F4/80+MRC1+ cells. In splenocytes exposed to RS, MLT inhibited the increase in mRNA expression of NOS2a. MLT downregulated expression of phosphorylated signal transducer and activator of transcription (STAT) 1 and upregulated STAT3 protein expression. Our findings suggest that MLT reduces stress-induced inflammatory responses by inducing an M1 to M2 phenotype switch in macrophages via activation of STAT3 signaling.

Anti-inflammatory effect of IL-10 mediated by metabolic reprogramming of macrophages.

Interleukin 10 (IL-10) is an anti-inflammatory cytokine that plays a critical role in the control of immune responses. However, its mechanisms of action remain poorly understood. Here, we show that IL-10 opposes the switch to the metabolic program induced by inflammatory stimuli in macrophages. Specifically, we show that IL-10 inhibits lipopolysaccharide-induced glucose uptake and glycolysis and promotes oxidative phosphorylation. Furthermore, IL-10 suppresses mammalian target of rapamycin (mTOR) activity through the induction of an mTOR inhibitor, DDIT4. Consequently, IL-10 promotes mitophagy that eliminates dysfunctional mitochondria characterized by low membrane potential and a high level of reactive oxygen species. In the absence of IL-10 signaling, macrophages accumulate damaged mitochondria in a mouse model of colitis and inflammatory bowel disease patients, and this results in dysregulated activation of the NLRP3 inflammasome and production of IL-1β.

3’UTR AU-Rich Elements (AREs) and the RNA-Binding Protein Tristetraprolin (TTP) Are Not Required for the LPS-Mediated Destabilization of Phospholipase-Cβ-2 mRNA in Murine Macrophages

We have shown previously that bacterial lipopolysaccharide (LPS)-mediated suppression of phospholipase-Cβ-2 (PLCβ-2) expression is involved in M1 (inflammatory) to M2-like (wound healing) phenotypic switching of macrophages triggered by adenosine. This suppression is mediated post-transcriptionally by destabilization of PLCβ-2 mRNA (messenger ribonucleic acid). To investigate the mechanism of this LPS-mediated destabilization, we examined the roles of RNA-binding agents including microRNAs and RNA-binding proteins that are involved in regulating stability of mRNAs encoding growth factors, inflammatory mediators, and proto-oncogenes. Adenylate and uridylate (AU)-rich elements (AREs) in 3'UTRs are specific recognition sites for RNA-binding proteins including tristetraprolin (TTP), HuR, and AUF1 and for microRNAs that are involved in regulating mRNA stability. In this study, we investigated the role of TTP and AREs in regulating PLCβ-2 mRNA stability. The 3'UTR of the PLCβ-2 gene was inserted into the pLightswitch luciferase reporter plasmid and transfected into RAW264.7 cells. LPS suppressed luciferase expression from this reporter. Luciferase expression from mutant 3'UTR constructs lacking AREs was similarly downregulated, suggesting that these regions are not required for LPS-mediated suppression of PLCβ-2. TTP was rapidly upregulated in both primary murine macrophages and RAW264.7 cells in response to LPS. Suppression of PLCβ-2 by LPS was examined using macrophages from mice lacking TTP (TTP-/-). LPS suppressed PLCβ-2 expression to the same extent in wild type (WT) and TTP-/- macrophages. Also, the rate of decay of PLCβ-2 mRNA in LPS-treated macrophages following transcriptional blockade was similar in WT and TTP-/- macrophages, clearly indicating that TTP is not involved in LPS-mediated destabilization of PLCβ-2 mRNA in macrophages.