Impaired Kupffer Cell Self-Renewal Alters the Liver Response to Lipid Overload during Non-alcoholic Steatohepatitis.

Kupffer cells (KCs) are liver-resident macrophages that self-renew by proliferation in the adult independently from monocytes. However, how they are maintained during non-alcoholic steatohepatitis (NASH) remains ill defined. We found that a fraction of KCs derived from Ly-6C+ monocytes during NASH, underlying impaired KC self-renewal. Monocyte-derived KCs (MoKCs) gradually seeded the KC pool as disease progressed in a response to embryo-derived KC (EmKC) death. Those MoKCs were partly immature and exhibited a pro-inflammatory status compared to EmKCs. Yet, they engrafted the KC pool for the long term as they remained following disease regression while acquiring mature EmKC markers. While KCs as a whole favored hepatic triglyceride storage during NASH, EmKCs promoted it more efficiently than MoKCs, and the latter exacerbated liver damage, highlighting functional differences among KCs with different origins. Overall, our data reveal that KC homeostasis is impaired during NASH, altering the liver response to lipids, as well as KC ontogeny.

Complement Factor H Inhibits CD47-Mediated Resolution of Inflammation.

Variants of the CFH gene, encoding complement factor H (CFH), show strong association with age-related macular degeneration (AMD), a major cause of blindness. Here, we used murine models of AMD to examine the contribution of CFH to disease etiology. Cfh deletion protected the mice from the pathogenic subretinal accumulation of mononuclear phagocytes (MP) that characterize AMD and showed accelerated resolution of inflammation. MP persistence arose secondary to binding of CFH to CD11b, which obstructed the homeostatic elimination of MPs from the subretinal space mediated by thrombospsondin-1 (TSP-1) activation of CD47. The AMD-associated CFH(H402) variant markedly increased this inhibitory effect on microglial cells, supporting a causal link to disease etiology. This mechanism is not restricted to the eye, as similar results were observed in a model of acute sterile peritonitis. Pharmacological activation of CD47 accelerated resolution of both subretinal and peritoneal inflammation, with implications for the treatment of chronic inflammatory disease.

DLK1/DIO3 locus upregulation by a ß-catenin-dependent enhancer drives cell proliferation and liver tumorigenesis.

The CTNNB1 gene, encoding ß-catenin, is frequently mutated in hepatocellular carcinoma (HCC, ∼30%) and in hepatoblastoma (HB, >80%), in which DLK1/DIO3 locus induction is correlated with CTNNB1 mutations. Here, we aim to decipher how sustained ß-catenin activation regulates DLK1/DIO3 locus expression and the role this locus plays in HB and HCC development in mouse models deleted for Apc (ApcΔhep) or Ctnnb1-exon 3 (ß-cateninΔExon3) and in human CTNNB1-mutated hepatic cancer cells. We identified an enhancer site bound by TCF-4/ß-catenin complexes in an open conformation upon sustained ß-catenin activation (DLK1-Wnt responsive element [WRE]) and increasing DLK1/DIO3 locus transcription in ß-catenin-mutated human HB and mouse models. DLK1-WRE editing by CRISPR-Cas9 approach impaired DLK1/DIO3 locus expression and slowed tumor growth in subcutaneous CTNNB1-mutated tumor cell grafts, ApcΔhep HB and ß-cateninΔExon3 HCC. Tumor growth inhibition resulted either from increased FADD expression and subsequent caspase-3 cleavage in the first case or from decreased expression of cell cycle actors regulated by FoxM1 in the others. Therefore, the DLK1/DIO3 locus is an essential determinant of FoxM1-dependent cell proliferation during ß-catenin-driven liver tumorigenesis. Targeting the DLK1-WRE enhancer to silence the DLK1/DIO3 locus might thus represent an interesting therapeutic strategy to restrict tumor growth in primary liver cancers with CTNNB1 mutations.

Phosphatidylserine enhances anti-inflammatory effects of reconstituted HDL in macrophages via distinct intracellular pathways.

Phosphatidylserine (PS) is a minor phospholipid constituent of high-density lipoprotein (HDL) that exhibits potent anti-inflammatory activity. It remains indeterminate whether PS incorporation can enhance anti-inflammatory effects of reconstituted HDL (rHDL). Human macrophages were treated with rHDL containing phosphatidylcholine alone (PC-rHDL) or PC and PS (PC/PS-rHDL). Interleukin (IL)-6 secretion and expression was more strongly inhibited by PC/PS-rHDL than PC-rHDL in both tumor necrosis factor (TNF)-α- and lipopolysaccharide (LPS)-stimulated macrophages. siRNA experiments revealed that the enhanced anti-inflammatory effects of PC/PS-rHDL required scavenger receptor class B type I (SR-BI). Furthermore, PC/PS-rHDL induced a greater increase in Akt1/2/3 phosphorylation than PC-rHDL. In addition, PC/PS but not PC-rHDL decreased the abundance of plasma membrane lipid rafts and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation. Finally, when these rHDL formulations were administered to dyslipidemic low-density lipoprotein (LDL)-receptor knockout mice fed a high-cholesterol diet, circulating IL-6 levels were significantly reduced only in PC/PS-rHDL-treated mice. In parallel, enhanced Akt1/2/3 phosphorylation by PC/PS-rHDL was observed in the mouse aortic tissue using immunohistochemistry. We concluded that the incorporation of PS into rHDLs enhanced their anti-inflammatory activity by modulating Akt1/2/3- and p38 MAPK-mediated signaling through SR-BI in stimulated macrophages. These data identify PS as a potent anti-inflammatory component capable of enhancing therapeutic potential of rHDL-based therapy.

Profound systemic alteration of the immune phenotype and an immunoglobulin switch in Erdheim-Chester disease in 78 patients from a single center.

Erdheim-Chester disease (ECD) is a rare, systemic, non-Langerhans cell histiocytosis neoplasm, which is characterized by the infiltration of CD63+ CD1a- histiocytes in multiple tissues. The BRAFV600E mutation is frequently present in individuals with ECD and has been detected in hematopoietic stem cells and immune cells from the myeloid and systemic compartments. Immune cells and pro-inflammatory cytokines are present in lesions, suggesting that ECD involves immune cell recruitment. Although a systemic cytokine T-helper-1-oriented signature has been reported in ECD, the immune cell network orchestrating the immune response in ECD has yet to be described. To address this issue, the phenotypes of circulating leukocytes were investigated in a large, single-center cohort of 78 patients with ECD and compared with those of a group of 21 control individuals. Major perturbations in the abundance of systemic immune cells were detected in patients with ECD, with decreases in circulating plasmacytoid, myeloid 1, and myeloid 2 dendritic cells, mostly in BRAFV600E carriers, in comparison with individuals in the control group. Similarly, marked decreases in blood Thelper, cytotoxic, and B-lymphocyte numbers were observed in patients with ECD, relative to the control group. Measurement of circulating immunoglobulin concentrations revealed an immunoglobulin G switch, from IgG1 to IgG4 subclasses, which are more frequently associated with the BRAF mutation. First-line therapies, including pegylated interferon-a and vemurafenib, were able to correct most of these alterations. This study reveals a profound disturbance in the systemic immune phenotype in patients with ECD, providing important new information, helping to understand the physiopathological mechanisms involved in this rare disease and improving the therapeutic management of patients.

Hypoalphalipoproteinemia and BRAFV600E Mutation Are Major Predictors of Aortic Infiltration in the Erdheim-Chester Disease.

Objective- Erdheim-Chester disease (ECD) is a rare non-Langerhans cell histiocytosis characterized by the infiltration of multiple tissues with lipid-laden histiocytes. Cardiovascular involvement is frequent in ECD and leads to a severe prognosis. The objective of this study was to determine whether an alteration of lipid metabolism participates in the lipid accumulation in histiocytes and the cardiovascular involvement in ECD. Approach and Results- An analysis of plasma lipid levels indicated that male ECD patients carrying the BRAFV600E (B-Raf proto-oncogene, serine/threonine kinase) mutation exhibited hypoalphalipoproteinemia, as demonstrated by low plasma HDL-C (high-density lipoprotein cholesterol) levels. Capacity of sera from male BRAFV600E ECD patients to mediate free cholesterol efflux from human macrophages was reduced compared with control individuals. Cardiovascular involvement was detected in 84% of the ECD patients, and we reported that the presence of the BRAFV600E mutation and hypoalphalipoproteinemia is an independent determinant of aortic infiltration in ECD. Phenotyping of blood CD14+ cells, the precursors of histiocytes, enabled the identification of a specific inflammatory signature associated with aortic infiltration which was partially affected by the HDL phenotype. Finally, the treatment with vemurafenib, an inhibitor of the BRAFV600E mutation, restored the defective sera cholesterol efflux capacity and reduced the aortic infiltration. Conclusions- Our findings indicate that hypoalphalipoproteinemia in male ECD patients carrying the BRAFV600E mutation favors the formation of lipid-laden histiocytes. In addition, we identified the BRAF status and the HDL phenotype as independent determinants of the aortic involvement in ECD with a potential role of HDL in modulating the infiltration of blood CD14+ cells into the aorta.

Roles for the CX3CL1/CX3CR1 and CCL2/CCR2 Chemokine Systems in Hypoxic Pulmonary Hypertension.

Monocytes/macrophages are major effectors of lung inflammation associated with various forms of pulmonary hypertension (PH). Interactions between the CCL2/CCR2 and CX3CL1/CX3CR1 chemokine systems that guide phagocyte infiltration are incompletely understood. Our objective was to explore the individual and combined actions of CCL2/CCR2 and CX3CL1/CX3CR1 in hypoxia-induced PH in mice; particularly their roles in monocyte trafficking, macrophage polarization, and pulmonary vascular remodeling. The development of hypoxia-induced PH was associated with marked increases in lung levels of CX3CR1, CCR2, and their respective ligands, CX3CL1 and CCL2. Flow cytometry revealed that both inflammatory Ly6Chi and resident Ly6Clo monocyte subsets exhibited sustained increases in blood and a transient peak in lung tissue, and that lung perivascular and alveolar macrophage counts showed sustained elevations. CX3CR1-/- mice were protected against hypoxic PH compared with wild-type mice, whereas CCL2-/- mice and double CX3CR1-/-/CCL2-/- mice exhibited similar PH severity, as did wild-type mice. The protective effects of CX3CR1 deficiency occurred concomitantly with increases in lung monocyte and macrophage counts and with a change from M2 to M1 macrophage polarization that markedly diminished the ability of conditioned media to induce pulmonary artery smooth muscle cell (PA-SMC) proliferation, which was partly dependent on CX3CL1 secretion. Results in mice given the CX3CR1 inhibitor F1 were similar to those in CX3CR1-/- mice. In conclusion, CX3CR1 deficiency protects against hypoxia-induced PH by modulating monocyte recruitment, macrophage polarization, and PA-SMC cell proliferation. Targeting CX3CR1 may hold promise for treating PH.

ECL1i, d(LGTFLKC), a novel, small peptide that specifically inhibits CCL2-dependent migration.

CC chemokine receptor type 2 (CCR2) is a key molecule in inflammatory diseases and is an obvious drug target for the treatment of inflammation. A number of nonpeptidic, competitive CCR2 antagonists have been developed, but none has yet been approved for clinical use. Our aim was to identify a short peptide that showed allosteric antagonism against human and mouse CCR2. On the basis of sequence analysis and 3-dimensional modeling, we identified an original 7-d-amino acid peptidic CCR2 inhibitor that we have called extracellular loop 1 inverso (ECL1i), d(LGTFLKC). In vitro, ECL1i selectively and potently inhibits CC chemokine ligand type 2 (CCL2)-triggered chemotaxis (IC50, 2 µM) but no other conventional CCL2-associated events. We used the classic competitive CCR2 antagonist, BMS22 {2-[(isopropylaminocarbonyl)amino]-N-[2-[[cis-2-[[4-(methylthio)benzoyl]amino]cyclohexyl]amino]-2-oxoethyl]-5-(trifluoromethyl)benzamide}, as positive control and inhibited CCL2-dependent chemotaxis with an IC50 of 18 nM. As negative control, we used a peptide with the same composition as ECL1i, but in a different sequence, d(FKLTLCG). In vivo, ECL1i (4 mg/kg) interfered with CCR2-positive cell recruitment and attenuated disease progression in experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. This study establishes ECL1i as the first allosteric inhibitor of CCR2 with functional selectivity. ECL1i is a promising new agent in therapeutic development, and it may, by its selective effect, increase our understanding of CCR2 signaling pathways and functions.-Auvynet, C., Baudesson de Chanville, C., Hermand, P., Dorgham, K., Piesse, C., Pouchy, C., Carlier, L., Poupel, L., Barthélémy, S., Felouzis, V., Lacombe, C., Sagan, S., Salomon, B., Deterre, P., Sennlaub, F., Combadière, C. ECL1i, d(LGTFLKC), a novel, small peptide that specifically inhibits CCL2-dependent migration.

Ly6Chigh Monocytes Protect against Kidney Damage during Sepsis via a CX3CR1-Dependent Adhesion Mechanism.

Monocytes have a crucial role in both proinflammatory and anti-inflammatory phenomena occurring during sepsis. Monocyte recruitment and activation are orchestrated by the chemokine receptors CX3CR1 and CCR2 and their cognate ligands. However, little is known about the roles of these cells and chemokines during the acute phase of inflammation in sepsis. Using intravital microscopy in a murine model of polymicrobial sepsis, we showed that inflammatory Ly6C(high) monocytes infiltrated kidneys, exhibited altered motility, and adhered strongly to the renal vascular wall in a chemokine receptor CX3CR1-dependent manner. Adoptive transfer of Cx3cr1-proficient monocyte-enriched bone marrow cells into septic Cx3cr1-depleted mice prevented kidney damage and promoted mouse survival. Modulation of CX3CR1 activation in septic mice controlled monocyte adhesion, regulated proinflammatory and anti-inflammatory cytokine expression, and was associated with the extent of kidney lesions such that the number of lesions decreased when CX3CR1 activity increased. Consistent with these results, the pro-adhesive I249 CX3CR1 allele in humans was associated with a lower incidence of AKI in patients with sepsis. These data show that inflammatory monocytes have a protective effect during sepsis via a CX3CR1-dependent adhesion mechanism. This receptor might be a new therapeutic target for kidney injury during sepsis.

CCR5 as a treatment target in pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PH), whether idiopathic or related to underlying diseases such as HIV infection, results from complex vessel remodeling involving both pulmonary artery smooth muscle cell (PA-SMC) proliferation and inflammation. CCR5, a coreceptor for cellular HIV-1 entry expressed on macrophages and vascular cells, may be involved in the pathogenesis of PH. Maraviroc is a new CCR5 antagonist designed to block HIV entry. METHODS AND RESULTS: Marked CCR5 expression was found in lungs from patients with idiopathic PH, in mice with hypoxia-induced PH, and in Simian immunodeficiency virus-infected macaques, in which it was localized chiefly in the PA-SMCs. To assess the role for CCR5 in experimental PH, we used both gene disruption and pharmacological CCR5 inactivation in mice. Because maraviroc does not bind to murine CCR5, we used human-CCR5ki mice for pharmacological and immunohistochemical studies. Compared with wild-type mice, CCR5-/- mice or human-CCR5ki mice treated with maraviroc exhibited decreased PA-SMC proliferation and recruitment of perivascular and alveolar macrophages during hypoxia exposure. CCR5-/- mice reconstituted with wild-type bone marrow cells and wild-type mice reconstituted with CCR5-/- bone marrow cells were protected against PH, suggesting CCR5-mediated effects on PA-SMCs and macrophage involvement. The CCR5 ligands CCL5 and the HIV-1 gp120 protein increased intracellular calcium and induced growth of human and human-CCR5ki mouse PA-SMCs; maraviroc inhibited both effects. Maraviroc also reduced the growth-promoting effects of conditioned media from CCL5-activated macrophages derived from human-CCR5ki mice on PA-SMCs from wild-type mice. CONCLUSION: The CCL5-CCR5 pathway represents a new therapeutic target in PH associated with HIV or with other conditions.

CX3CR1 reduces Ly6Chigh-monocyte motility within and release from the bone marrow after chemotherapy in mice.

The chemokine receptor CCR2 controls the release of Ly6C(high) monocytes from the bone marrow and their recruitment to sites of inflammation. A second chemokine receptor, CX3CR1, is differentially expressed on monocyte subsets. We examined the role of CX3CR1 in monocyte trafficking during the recovery phase after cyclophosphamide (CP)-induced myeloablation and observed that, in the absence of CCR2, Ly6C(high) monocytes accumulated in the bone marrow and peripheral reconstitution was severely impaired compared with wild-type (WT) mice. In contrast, in the absence of CX3CR1, Ly6C(high) monocytes accumulated less rapidly in the marrow but recovered faster in the blood and were more recruited into the spleen, suggesting an opposite action between CCR2 and CX3CR1 in myelorestoration. During the recovery phase, marrow medullar monocytes displayed lower CX3CR1 expression and reduced their adherence to coated CX3CL1. Intravital imaging of the bone marrow showed that CP treatment impacts monocyte trafficking between the parenchyma and the vasculature. Medullar monocytes in CX3CR1(-/-) mice and mice treated with a specific antagonist of CX3CR1 displayed increased mean velocity and displacement and a reduced arrest coefficient compared with WT mice. This study indicates that CX3CR1 reduces the motility of Ly6C(high) monocytes in the bone marrow and thereby controls their release.

Pharmacological inhibition of the chemokine receptor, CX3CR1, reduces atherosclerosis in mice.

OBJECTIVE: Alterations of the chemokine receptor CX3CR1 gene were associated with a reduced risk of myocardial infarction in human and limited atherosclerosis in mice. In this study, we addressed whether CX3CR1 antagonists are potential therapeutic tools to limit acute and chronic inflammatory processes in atherosclerosis. APPROACH AND RESULTS: Treatment with F1, an amino terminus-modified CX3CR1 ligand endowed with CX3CR1 antagonist activity, reduced the extent of atherosclerotic lesions in both Apoe(-/-) and Ldlr(-/-) proatherogenic mouse models. Macrophage accumulation in the aortic sinus was reduced in F1-treated Apoe(-/-) mice but the macrophage density of the lesions was similar in F1-treated and control mice. Both in vitro and in vivo F1 treatment reduced CX3CR1-dependent inflammatory monocyte adhesion, potentially limiting their recruitment. In addition, F1-treated Apoe(-/-) mice displayed reduced numbers of blood inflammatory monocytes, whereas resident monocyte numbers remained unchanged. Both in vitro and in vivo F1 treatment reduced CX3CR1-dependent inflammatory monocyte survival. Finally, F1 treatment of Apoe(-/-) mice with advanced atherosclerosis led to smaller lesions than untreated mice but without reverting to the initial phenotype. CONCLUSIONS: The CX3CR1 antagonist F1 is a potent inhibitor of the progression of atherosclerotic lesions by means of its selective impact on inflammatory monocyte functions. Controlling monocyte trafficking and survival may be an alternative or complementary therapy to lipid-lowering drugs classically used in the treatment of atherosclerosis.

Chemokine receptor CCR1 disruption limits renal damage in a murine model of hemolytic uremic syndrome.

Shiga toxin (Stx)-producing Escherichia coli is the main etiological agent that causes hemolytic uremic syndrome (HUS), a microangiopathic disease characterized by hemolytic anemia, thrombocytopenia, and acute renal failure. Although direct cytotoxic effects on endothelial cells by Stx are the primary pathogenic event, there is evidence that indicates the inflammatory response mediated by polymorphonuclear neutrophils and monocytes as the key event during HUS development. Because the chemokine receptor CCR1 participates in the pathogenesis of several renal diseases by orchestrating myeloid cell kidney infiltration, we specifically addressed the contribution of CCR1 in a murine model of HUS. We showed that Stx type 2-treated CCR1(-/-) mice have an increased survival rate associated with less functional and histological renal damage compared with control mice. Stx type 2-triggered neutrophilia and monocytosis and polymorphonuclear neutrophil and monocyte renal infiltration were significantly reduced and delayed in CCR1(-/-) mice compared with control mice. In addition, the increase of the inflammatory cytokines (tumor necrosis factor-α and IL-6) in plasma was delayed in CCR1(-/-) mice compared with control mice. These data demonstrate that CCR1 participates in cell recruitment to the kidney and amplification of the inflammatory response that contributes to HUS development. Blockade of CCR1 could be important to the design of future therapies to restrain the inflammatory response involved in the development of HUS.

The chemokine decoy receptor D6 prevents excessive inflammation and adverse ventricular remodeling after myocardial infarction.

OBJECTIVE: Leukocyte infiltration in ischemic areas is a hallmark of myocardial infarction, and overwhelming infiltration of innate immune cells has been shown to promote adverse remodeling and cardiac rupture. Recruitment of inflammatory cells in the ischemic heart depends highly on the family of CC-chemokines and their receptors. Here, we hypothesized that the chemokine decoy receptor D6, which specifically binds and scavenges inflammatory CC-chemokines, might limit inflammation and adverse cardiac remodeling after infarction. METHODS AND RESULTS: D6 was expressed in human and murine infarcted myocardium. In a murine model of myocardial infarction, D6 deficiency led to increased chemokine (C-C motif) ligand 2 and chemokine (C-C motif) ligand 3 levels in the ischemic heart. D6-deficient (D6(-/-)) infarcts displayed increased infiltration of pathogenic neutrophils and Ly6Chi monocytes, associated with strong matrix metalloproteinase-9 and matrix metalloproteinase-2 activities in the ischemic heart. D6(-/-) mice were cardiac rupture prone after myocardial infarction, and functional analysis revealed that D6(-/-) hearts had features of adverse remodeling with left ventricle dilation and reduced ejection fraction. Bone marrow chimera experiments showed that leukocyte-borne D6 had no role in this setting, and that leukocyte-specific chemokine (C-C motif) receptor 2 deficiency rescued the adverse phenotype observed in D6(-/-) mice. CONCLUSIONS: We show for the first time that the chemokine decoy receptor D6 limits CC-chemokine-dependent pathogenic inflammation and is required for adequate cardiac remodeling after myocardial infarction.

Targeted delivery of LXR-agonists to atherosclerotic lesions mediated by polydiacetylene micelles.

We report the development of compact and stabilized micelles incorporating a synthetic LXR agonist prodrug for the passive targeting of atherosclerotic lesions and therapeutic intervention. In vivo studies showed that the nanohybrid micelles exhibited favorable pharmacokinetics/biodistribution and were able to upregulate, to some extent, LXR target genes with no alteration of lipid metabolism.

Integrated omics approach for the identification of HDL structure-function relationships in PCSK9-related familial hypercholesterolemia.

BACKGROUND: The role of proprotein convertase subtilisin/kexin type 9 (PCSK9) in dyslipidemia may go beyond its immediate effects on low-density lipoprotein receptor (LDL-R) activity. OBJECTIVE: This study aimed to assess PCSK9-derived alterations of high-density lipoprotein (HDL) physiology, which bear a potential to contribute to cardiovascular risk profile. METHODS: HDL was isolated from 33 patients with familial autosomal dominant hypercholesterolemia (FH), including those carrying PCSK9 gain-of-function (GOF) genetic variants (FH-PCSK9, n = 11), together with two groups of dyslipidemic patients employed as controls and carrying genetic variants in the LDL-R not treated (ntFH-LDLR, n = 11) and treated (tFH-LDLR, n = 11) with statins, and 11 normolipidemic controls. Biological evaluations paralleled by proteomic, lipidomic and glycomic analyses were applied to characterize functional and compositional properties of HDL. RESULTS: Multiple deficiencies in the HDL function were identified in the FH-PCSK9 group relative to dyslipidemic FH-LDLR patients and normolipidemic controls, which involved reduced antioxidative, antiapoptotic, anti-thrombotic and anti-inflammatory activities. By contrast, cellular cholesterol efflux capacity of HDL was unchanged. In addition, multiple alterations of the proteomic, lipidomic and glycomic composition of HDL were found in the FH-PCSK9 group. Remarkably, HDLs from FH-PCSK9 patients were systematically enriched in several lysophospholipids as well as in A2G2S2 (GP13) glycan and apolipoprotein A-IV. Based on network analysis of functional and compositional data, a novel mosaic structure-function model of HDL biology involving FH was developed. CONCLUSION: Several metrics of anti-atherogenic HDL functionality are altered in FH-PCSK9 patients paralleled by distinct compositional alterations. These data provide a first-ever overview of the impact of GOF PCSK9 genetic variants on structure-function relationships in HDL.

Network analysis of large-scale ImmGen and Tabula Muris datasets highlights metabolic diversity of tissue mononuclear phagocytes.

The diversity of mononuclear phagocyte (MNP) subpopulations across tissues is one of the key physiological characteristics of the immune system. Here, we focus on understanding the metabolic variability of MNPs through metabolic network analysis applied to three large-scale transcriptional datasets: we introduce (1) an ImmGen MNP open-source dataset of 337 samples across 26 tissues; (2) a myeloid subset of ImmGen Phase I dataset (202 MNP samples); and (3) a myeloid mouse single-cell RNA sequencing (scRNA-seq) dataset (51,364 cells) assembled based on Tabula Muris Senis. To analyze such large-scale datasets, we develop a network-based computational approach, genes and metabolites (GAM) clustering, for unbiased identification of the key metabolic subnetworks based on transcriptional profiles. We define 9 metabolic subnetworks that encapsulate the metabolic differences within MNP from 38 different tissues. Obtained modules reveal that cholesterol synthesis appears particularly active within the migratory dendritic cells, while glutathione synthesis is essential for cysteinyl leukotriene production by peritoneal and lung macrophages.

Targeted invalidation of SR-B1 in macrophages reduces macrophage apoptosis and accelerates atherosclerosis.

AIMS: SR-B1 is a cholesterol transporter that exerts anti-atherogenic properties in liver and peripheral tissues in mice. Bone marrow (BM) transfer studies suggested an atheroprotective role in cells of haematopoietic origin. Here, we addressed the specific contribution of SR-B1 in the monocyte/macrophage. METHODS AND RESULTS: We generated mice deficient for SR-B1 in monocytes/macrophages (Lysm-Cre × SR-B1f/f) and transplanted their BM into Ldlr-/- mice. Fed a cholesterol-rich diet, these mice displayed accelerated aortic atherosclerosis characterized by larger macrophage-rich areas and decreased macrophage apoptosis compared with SR-B1f/f transplanted controls. These findings were reproduced in BM transfer studies using another atherogenic mouse recipient (SR-B1 KOliver × Cholesteryl Ester Transfer Protein). Haematopoietic reconstitution with SR-B1-/- BM conducted in parallel generated similar results to those obtained with Lysm-Cre × SR-B1f/f BM; thus suggesting that among haematopoietic-derived cells, SR-B1 exerts its atheroprotective role primarily in monocytes/macrophages. Consistent with our in vivo data, free cholesterol (FC)-induced apoptosis of macrophages was diminished in the absence of SR-B1. This effect could not be attributed to differential cellular cholesterol loading. However, we observed that expression of apoptosis inhibitor of macrophage (AIM) was induced in SR-B1-deficient macrophages, and notably upon FC-loading. Furthermore, we demonstrated that macrophages were protected from FC-induced apoptosis by AIM. Finally, AIM protein was found more present within the macrophage-rich area of the atherosclerotic lesions of SR-B1-deficient macrophages than controls. CONCLUSION: Our findings suggest that macrophage SR-B1 plays a role in plaque growth by controlling macrophage apoptosis in an AIM-dependent manner.

Immune surveillance of the lung by migrating tissue monocytes.

Monocytes are phagocytic effector cells in the blood and precursors of resident and inflammatory tissue macrophages. The aim of the current study was to analyse and compare their contribution to innate immune surveillance of the lung in the steady state with macrophage and dendritic cells (DC). ECFP and EGFP transgenic reporters based upon Csf1r and Cx3cr1 distinguish monocytes from resident mononuclear phagocytes. We used these transgenes to study the migratory properties of monocytes and macrophages by functional imaging on explanted lungs. Migratory monocytes were found to be either patrolling within large vessels of the lung or locating at the interface between lung capillaries and alveoli. This spatial organisation gives to monocytes the property to capture fluorescent particles derived from both vascular and airway routes. We conclude that monocytes participate in steady-state surveillance of the lung, in a way that is complementary to resident macrophages and DC, without differentiating into macrophages.

CX3CR1 deficiency promotes muscle repair and regeneration by enhancing macrophage ApoE production.

Muscle injury triggers inflammation in which infiltrating mononuclear phagocytes are crucial for tissue regeneration. The interaction of the CCL2/CCR2 and CX3CL1/CX3CR1 chemokine axis that guides phagocyte infiltration is incompletely understood. Here, we show that CX3CR1 deficiency promotes muscle repair and rescues Ccl2(-/-) mice from impaired muscle regeneration as a result of altered macrophage function, not infiltration. Transcriptomic analysis of muscle mononuclear phagocytes reveals that Apolipoprotein E (ApoE) is upregulated in mice with efficient regeneration. ApoE treatment enhances phagocytosis by mononuclear phagocytes in vitro, and restores phagocytic activity and muscle regeneration in Ccl2(-/-) mice. Because CX3CR1 deficiency may compensate for defective CCL2-dependant monocyte recruitment by modulating ApoE-dependent macrophage phagocytic activity, targeting CX3CR1 expressed by macrophages might be a powerful therapeutic approach to improve muscle regeneration.