Publications

The mycobiota are a critical part of the gut microbiome, but hostfungal interactions and specific functional contributions of commensal fungi to host fitness remain incompletely understood. Here, we report the identification of a new fungal commensal, Kazachstania heterogenica var. weizmannii, isolated from murine intestines. K. weizmannii exposure prevented Candida albicans colonization and significantly reduced the commensal C. albicans burden in colonized animals. Following immunosuppression of C. albicans colonized mice, competitive fungal commensalism thereby mitigated fatal candidiasis. Metagenome analysis revealed K. heterogenica or K. weizmannii presence among human commensals. Our results reveal competitive fungal commensalism within the intestinal microbiota, independent of bacteria and immune responses, that could bear potential therapeutic value for the management of C. albicansmediated diseases.

Langerhans cells (LCs) are distinct among phagocytes, functioning both as embryo-derived, tissue-resident macrophages in skin innervation and repair and as migrating professional antigen-presenting cells, a function classically assigned to dendritic cells (DCs). Here, we demonstrate that both intrinsic and extrinsic factors imprint this dual identity. Using ablation of embryo-derived LCs in the murine adult skin and tracking differentiation of incoming monocyte-derived replacements, we found intrinsic intraepidermal heterogeneity. We observed that ontogenically distinct monocytes give rise to LCs. Within the epidermis, Jagged-dependent activation of Notch signaling, likely within the hair follicle niche, provided an initial site of LC commitment before metabolic adaptation and survival of monocyte-derived LCs. In the human skin, embryo-derived LCs in newborns retained transcriptional evidence of their macrophage origin, but this was superseded by DC-like immune modules after postnatal expansion. Thus, adaptation to adult skin niches replicates conditioning of LC at birth, permitting repair of the embryo-derived LC network.

Global gene expression profiling has provided valuable insights into the specific contributions of different cell types to various physiological processes. Notably though, both bulk and single-cell transcriptomics require the prior retrieval of the cells from their tissue context to be analyzed. Isolation protocols for tissue macrophages are, however, notoriously inefficient and, moreover, prone to introduce considerable bias and artifacts. Here, we will discuss a valuable alternative, originally introduced by Amieux and colleagues. This so-called RiboTag approach allows, in combination with respective macrophage-specific Cre transgenic lines, to retrieve macrophage translatomes from crude tissue extracts. We will review our experience with this ingenious method, focusing on the study of brain macrophages, including microglia and border-associated cells. We will elaborate on the advantages of the RiboTag approach that render it a valuable complement to standard cell sorting-based profiling strategies, especially for the investigation of tissue macrophages.

Consecutive exposures to different pathogens are highly prevalent and often alter the host immune response. However, it remains unknown how a secondary bacterial infection affects an ongoing adaptive immune response elicited against primary invading pathogens. We demonstrated that recruitment of Sca-1+ monocytes into lymphoid organs during Salmonella Typhimurium (STm) infection disrupted pre-existing germinal center (GC) reactions. GC responses induced by influenza, plasmodium, or commensals deteriorated following STm infection. GC disruption was independent of the direct bacterial interactions with B cells and instead was induced through recruitment of CCR2-dependent Sca-1+ monocytes into the lymphoid organs. GC collapse was associated with impaired cellular respiration and was dependent on TNFα and IFNγ, the latter of which was essential for Sca-1+ monocyte differentiation. Monocyte recruitment and GC disruption also occurred during LPS-supplemented vaccination and Listeria monocytogenes infection. Thus, systemic activation of the innate immune response upon severe bacterial infection is induced at the expense of antibody-mediated immunity.

Microglial research has advanced considerably in recent decades yet has been constrained by a rolling series of dichotomies such as \u201cresting versus activated\u201d and \u201cM1 versus M2.\u201d This dualistic classification of good or bad microglia is inconsistent with the wide repertoire of microglial states and functions in development, plasticity, aging, and diseases that were elucidated in recent years. New designations continuously arising in an attempt to describe the different microglial states, notably defined using transcriptomics and proteomics, may easily lead to a misleading, although unintentional, coupling of categories and functions. To address these issues, we assembled a group of multidisciplinary experts to discuss our current understanding of microglial states as a dynamic concept and the importance of addressing microglial function. Here, we provide a conceptual framework and recommendations on the use of microglial nomenclature for researchers, reviewers, and editors, which will serve as the foundations for a future white paper.

Monocyte-derived macrophages (Mφs) are crucial regulators during muscularis inflammation. However, it is unclear which micro-environmental factors are responsible for monocyte recruitment and anti-inflammatory Mφ differentiation in this paradigm. Here, we investigate Mφ heterogeneity at different stages of muscularis inflammation and determine how environmental cues can attract and activate tissue-protective Mφs. Results showed that muscularis inflammation induced marked alterations in mononuclear phagocyte populations associated with a rapid infiltration of Ly6c⁺ monocytes that locally acquired unique transcriptional states. Trajectory inference analysis revealed two main pro-resolving Mφ subpopulations during the resolution of muscularis inflammation, i.e. Cd206⁺ MhcII^hi and Timp2⁺ MhcII^lo Mφs. Interestingly, we found that damage to the micro-environment upon muscularis inflammation resulted in EGC activation, which in turn stimulated monocyte infiltration and the consequent differentiation in anti-inflammatory CD206⁺ Mφs via CCL2 and CSF1, respectively. In addition, CSF1-CSF1R signaling was shown to be essential for the differentiation of monocytes into CD206⁺ Mφs and EGC proliferation during muscularis inflammation. Our study provides a comprehensive insight into pro-resolving Mφ differentiation and their regulators during muscularis inflammation. We deepened our understanding in the interaction between EGCs and Mφs, thereby highlighting pro-resolving Mφ differentiation as a potential novel therapeutic strategy for the treatment of intestinal inflammation.

T cells undergo rigorous selection in the thymus to ensure self-tolerance and prevent autoimmunity, with this process requiring innocuous self-antigens (Ags) to be presented to thymocytes. Self-Ags are either expressed by thymic stroma cells or transported to the thymus from the periphery by migratory dendritic cells (DCs); meanwhile, small blood-borne peptides can access the thymic parenchyma by diffusing across the vascular lining. Here we describe an additional pathway of thymic Ag acquisition that enables circulating antigenic macromolecules to access both murine and human thymi. This pathway depends on a subset of thymus-resident DCs, distinct from both parenchymal and circulating migratory DCs, that are positioned in immediate proximity to thymic microvessels where they extend cellular processes across the endothelial barrier into the blood stream. Transendothelial positioning of DCs depends on DC-expressed CX3CR1 and its endothelial ligand, CX3CL1, and disrupting this chemokine pathway prevents thymic acquisition of circulating proteins and compromises negative selection of Ag-reactive thymocytes. Thus, transendothelial DCs represent a mechanism by which the thymus can actively acquire blood-borne Ags to induce and maintain central tolerance.

Microglia, the resident macrophages of the brain parenchyma, are key players in central nervous system (CNS) development, homeostasis, and disorders. Distinct brain pathologies seem associated with discrete microglia activation modules. How microglia regain quiescence following challenges remains less understood. Here, we explored the role of the interleukin-10 (IL-10) axis in restoring murine microglia homeostasis following a peripheral endotoxin challenge. Specifically, we show that lipopolysaccharide (LPS)-challenged mice harboring IL-10 receptor-deficient microglia displayed neuronal impairment and succumbed to fatal sickness. Addition of a microglial tumor necrosis factor (TNF) deficiency rescued these animals, suggesting a microglia-based circuit driving pathology. Single cell transcriptome analysis revealed various IL-10 producing immune cells in the CNS, including most prominently Ly49D+ NK cells and neutrophils, but not microglia. Collectively, we define kinetics of the microglia response to peripheral endotoxin challenge, including their activation and robust silencing, and highlight the critical role of non-microglial IL-10 in preventing deleterious microglia hyperactivation.

Microglia and central nervous system (CNS)-associated macrophages (CAMs), such as perivascular and meningeal macrophages, are implicated in virtually all diseases of the CNS. However, little is known about their cell-type-specific roles in the absence of suitable tools that would allow for functional discrimination between the ontogenetically closely related microglia and CAMs. To develop a new microglia gene targeting model, we first applied massively parallel single-cell analyses to compare microglia and CAM signatures during homeostasis and disease and identified hexosaminidase subunit beta (Hexb) as a stably expressed microglia core gene, whereas other microglia core genes were substantially downregulated during pathologies. Next, we generated HexbtdTomato mice to stably monitor microglia behavior in vivo. Finally, the Hexb locus was employed for tamoxifen-inducible Cre-mediated gene manipulation in microglia and for fate mapping of microglia but not CAMs. In sum, we provide valuable new genetic tools to specifically study microglia functions in the CNS.

Protective MHC class I-dependent immune responses require an overlap between repertoires of proteins directly presented on target cells and cross-presented by professional APC, specifically dendritic cells. How stable proteins that rely on defective ribosomal proteins for direct presentation are captured for cell-to-cell transfer remains enigmatic. In this study, we address this issue using a combination of in vitro (C57BL/6-derived mouse cell lines) and in vivo (C57BL/6 mouse strains) approaches involving stable and unstable versions of OVA model Ags displaying defective ribosomal protein-dependent and -independent Ag presentation, respectively. Apoptosis, but not necrosis, of donor cells was found associated with robust global protein aggregate formation and captured stable proteins permissive for cross-presentation. Potency of aggregates to serve as Ag source was directly demonstrated using polyglutamine-equipped model substrates. Collectively, our data implicate global protein aggregation in apoptotic cells as a mechanism that ensures the overlap between MHC class I epitopes presented directly or cross-presented by APC and demonstrate the unusual ability of dendritic cells to process stable protein aggregates.

Chronic lymphocytic leukemia (CLL) is a malignancy of mature B lymphocytes. The microenvironment of the CLL cells is a vital element in the regulation of the survival of these malignant cells. CLL cell longevity is dependent on external signals, originating from cells in their microenvironment including secreted and surface-bound factors. Dendritic cells (DCs) play an important part in tumor microenvironment, but their role in the CLL bone marrow (BM) niche has not been studied. We show here that CLL cells induce accumulation of bone marrow dendritic cells (BMDCs). Depletion of this population attenuates disease expansion. Our results show that the support of the microenvironment is partly dependent on CD84, a cell surface molecule belonging to the Signaling Lymphocyte Activating Molecule (SLAM) family of immunoreceptors. Our results suggest a novel therapeutic strategy whereby eliminating BMDCs or blocking the CD84 expressed on these cells may reduce the tumor load.

Recruited blood monocytes contribute to the establishment, perpetuation, and resolution of tissue inflammation. Specifically, in the inflamed intestine, monocyte ablation was shown to ameliorate colitis scores in preclinical animal models. However, the majority of intestinal macrophages that seed the healthy gut are also monocyte derived. Monocyte ablation aimed to curb inflammation would therefore likely interfere with intestinal homeostasis. In this study, we used a TLR2 trans-membrane peptide that blocks TLR2 dimerization that is critical for TLR2/1 and TLR2/6 heterodimer signaling to blunt inflammation in a murine colitis model. We show that although the TLR2 peptide treatment ameliorated colitis, it allowed recruited monocytes to give rise to macrophages that lack the detrimental proinflammatory gene signature and reduced potentially damaging neutrophil infiltrates. Finally, we demonstrate TLR blocking activity of the peptide on in vitro cultured human monocyte-derived macrophages. Collectively, we provide a significantly improved anti-inflammatory TLR2 peptide and critical insights in its mechanism of action toward future potential use in the clinic.

Microglia were first recognized as a distinct cell population in the CNS one century ago. For a long time, they were primarily considered to be phagocytes responsible for removing debris during CNS development and disease. More recently, advances in imaging and genetics and the advent of single-cell technology provided new insights into the much more complex and fascinatir; biology of microglia. The ontogeny of microglia was identified, and their functions in health and disease were better defined. Although many qL lions about microglia and their roles in human diseases remain unanswered, the prospect of targeting microglia for the treatment of neurological and psychiatric disorders is tantalizing.

Cytokines maintain intestinal health, but precise intercellular communication networks remain poorly understood. Macrophages are immune sentinels of the intestinal tissue and are critical for gut homeostasis. Here, we show that in a murine inflammatory bowel disease (IBD) model based on macrophage-restricted interleukin-10 (IL-10) receptor deficiency (Cx3cr1^Cre:Il10ra^fl/fl mice), proinflammatory mutant gut macrophages cause severe spontaneous colitis resembling the condition observed in children carrying IL-10R mutations. We establish macrophage-derived IL-23 as the driving factor of this pathology. Specifically, we report that Cx3cr1^Cre:Il10ra^fl/fl:Il23a^fl/fl mice harboring macrophages deficient for both IL-10R and IL-23 are protected from colitis. By analyzing the epithelial response to proinflammatory macrophages, we provide evidence that T cells of colitic animals produce IL-22, which induces epithelial chemokine expression and detrimental neutrophil recruitment. Collectively, we define macrophage-specific contributions to the induction and pathogenesis of colitis, as manifested in mice harboring IL-10R deficiencies and human IBDs.

The involvement of macrophages in the pathogenesis of obesity has been recognized since 2003. Early studies mostly focused on the role of macrophages in adipose tissue (AT) and in obesity-associated chronic low-grade inflammation. Lately, AT macrophages were shown to undergo intrinsic metabolic changes that affect their immune function (i.e., immunometabolism), corresponding to their unique properties along the range of pro- versus anti-inflammatory activity. In parallel, recent studies in mice revealed critical neuronal-macrophage interactions, both in the CNS and in peripheral tissues, including in white and brown AT. These intercellular activities impinge on energy and metabolic homeostasis, partially by also engaging adipocytes in a neuronal-macrophage-adipocyte menage a trois. Finally, neuropeptides (NP), such as NPY and appetite-reducing NPFF, may prove as mediators in such intercellular network. In this concise review, we highlight some of these recent insights on adipose macrophage immunometabolism, as well as central and peripheral neuronal-macrophage interactions with emphasis on their impact on adipocyte biology and whole-body metabolism. We also discuss the expanding view on the role of the NP, NPY and NPFF, in obesity.

Background: Fractalkine (CX(3)CL1) and its receptor (CX(3)CR1) play an important role in regulating microglial function. We have previously shown that Cx(3)cr1 deficiency exacerbated tau pathology and led to cognitive impairment. However, it is still unclear if the chemokine domain of the ligand CX(3)CL1 is essential in regulating neuronal tau pathology.Methods: We used transgenic mice lacking endogenous Cx(3)cl1 (Cx(3)cl1(-/-)) and expressing only obligatory soluble form (with only chemokine domain) and lacking the mucin stalk of CX(3)CL1 (referred to as Cx(3)cl1(105 Delta) mice) to assess tau pathology and behavioral function in both lipopolysaccharide (LPS) and genetic (hTau) mouse models of tauopathy.Results: First, increased basal tau levels accompanied microglial activation in Cx(3)cl1(105 Delta) mice compared to control groups. Second, increased CD45(+) and F4/80(+) neuroinflammation and tau phosphorylation were observed in LPS, hTau/Cx(3)cl1(-/-), and hTau/Cx(3)cl1(105 Delta) mouse models of tau pathology, which correlated with impaired spatial learning. Finally, microglial cell surface expression of CX(3)CR1 was reduced in Cx(3)cl1(105 Delta) mice, suggesting enhanced fractalkine receptor internalization (mimicking Cx(3)cr1 deletion), which likely contributes to the elevated tau pathology.Conclusions: Collectively, our data suggest that overexpression of only chemokine domain of CX(3)CL1 does not protect against tau pathology.

Transcriptome profiling is widely used to infer functional states of specific cell types, as well as their responses to stimuli, to define contributions to physiology and pathophysiology. Focusing on microglia, the brain's macrophages, we report here a side-by-side comparison of classical cell-sorting-based transcriptome sequencing and the 'RiboTag' method, which avoids cell retrieval from tissue context and yields translatome sequencing information. Conventional whole-cell microglial transcriptomes were found to be significantly tainted by artifacts introduced by tissue dissociation, cargo contamination and transcripts sequestered from ribosomes. Conversely, our data highlight the added value of RiboTag profiling for assessing the lineage accuracy of Cre recombinase expression in transgenic mice. Collectively, this study indicates method-based biases, reveals observer effects and establishes RiboTag-based translatome profiling as a valuable complement to standard sorting-based profiling strategies.

Nitric oxide (NO) plays an established role in numerous physiological and pathological processes, but the specific cellular sources of NO in disease pathogenesis remain unclear, preventing the implementation of NO-related therapy. Argininosuccinate lyase (ASL) is the only enzyme able to produce arginine, the substrate for NO generation by nitric oxide synthase (NOS) isoforms. Here, we generated cell-specific conditional ASL knockout mice in combination with genetic and chemical colitis models. We demonstrate that NO derived from enterocytes alleviates colitis by decreasing macrophage infiltration and tissue damage, whereas immune cell-derived NO is associated with macrophage activation, resulting in increased severity of inflammation. We find that induction of endogenous NO production by enterocytes with supplements that upregulate ASL expression and complement its substrates results in improved epithelial integrity and alleviation of colitis and of inflammation-associated colon cancer.

Protective immune responses depend on the formation of immune synapses between T cells and antigen-presenting cells (APCs). The two main LFA-1 ligands, ICAM-1 and ICAM-2, are co-expressed on many cell types, including APCs and blood vessels. Although these molecules were suggested to be key players in immune synapses studied in vitro, their contribution to helper T cell priming in vivo is unclear. Here, we used transgenic mice and intravital imaging to examine the role of dendritic cell (DC) ICAM-1 and ICAM-2 in naive CD4 T cell priming and differentiation in skin-draining lymph nodes. Surprisingly, ICAM deficiency on endogenous CD40-stimulated lymph node DCs did not impair their ability to arrest and prime CD4 lymphocyte activation and differentiation into Th1 and Tfh effectors. Thus, functional T cell receptor (TCR)-specific helper T cell synapses with antigen-presenting DCs and subsequent proliferation and early differentiation into T effectors do not require LFA-1-mediated T cell adhesiveness to DC ICAMs.

Objective Postoperative ileus (POI), the most frequent complication after intestinal surgery, depends on dendritic cells (DCs) and macrophages. Here, we have investigated the mechanism that activates these cells and the contribution of the intestinal microbiota for POI induction.Design POI was induced by manipulating the intestine of mice, which selectively lack DCs, monocytes or macrophages. The disease severity in the small and large intestine was analysed by determining the distribution of orally applied fluorescein isothiocyanate-dextran and by measuring the excretion time of a retrogradely inserted glass ball. The impact of the microbiota on intestinal peristalsis was evaluated after oral antibiotic treatment.Results We found that Cd11c-Cre(+) Irf4(flox/flox) mice lack CD103(+) CD11b(+) DCs, a DC subset unique to the intestine whose function is poorly understood. Their absence in the intestinal muscularis reduced pathogenic inducible nitric oxide synthase (iNOS) production by monocytes and macrophages and ameliorated POI. Pathogenic iNOS was produced in the jejunum by resident Ly6C(-) macrophages and infiltrating chemokine receptor 2-dependent Ly6C(+) monocytes, but in the colon only by the latter demonstrating differential tolerance mechanisms along the intestinal tract. Consistently, depletion of both cell subsets reduced small intestinal POI, whereas the depletion of Ly6C(+) monocytes alone was sufficient to prevent large intestinal POI. The differential role of monocytes and macrophages in small and large intestinal POI suggested a potential role of the intestinal microbiota. Indeed, antibiotic treatment reduced iNOS levels and ameliorated POI.Conclusions Our findings reveal that CD103(+) CD11b(+) DCs and the intestinal microbiome are a prerequisite for the activation of intestinal monocytes and macrophages and for dysregulating intestinal motility in POI.

Vaccination is a promising strategy to trigger and boost immune responses against cancer or infectious disease. We have designed, synthesized and characterized aliphatic-polyester (poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP) to investigate how the nature of protein association (adsorbed versus entrapped) and polymer/surfactant concentrations impact on the generation and modulation of antigen-specific immune responses. The ability of the NP formulations to target dendritic cells (DC), be internalized and activate the T cells was characterized and optimized in vitro and in vivo using markers of DC activation and co-stimulatory molecules. Ovalbumin (OVA) was used as a model antigen in combination with the engraftment of CD4⁺ and CD8⁺ T cells, carrying a transgenic OVA-responding T cell receptor (TCR), to trace and characterize the activation of antigen-specific CD4⁺ and CD8⁺ lymph node T cells upon NP vaccination. Accordingly, the phenotype and frequency of immune cell stimulation induced by the NP loaded with OVA, isolated or in combination with synthetic unmethylated cytosine-phosphate-guanine (CpG) oligodeoxynucleotide (ODN) motifs, were characterized. DC-NP interactions increased with incubation time, presenting internalization values between 50 and 60% and 3040%, in vitro and in vivo, respectively. Interestingly, animal immunization with antigen-adsorbed NP up-regulated major histocompatibility complex (MHC) class II (MHCII), while NP entrapping the antigen up-regulated MHCI, suggesting a more efficient cross-presentation. On the other hand, rather surprisingly, the surfactant used in the NP formulation had a major impact on the activation of antigen presenting cells (APC). In fact, DC collected from lymph nodes of animals immunized with NP prepared using poly(vinil alcohol) (PVA), as a surfactant, expressed significantly higher levels of CD86, MHCI and MHCII. In addition, those NP prepared with PVA and co-entrapping OVA and the toll-like receptor (TLR) ligand CpG, induced the most profound antigen-specific T cell response, by both CD4⁺ and CD8⁺ T cells, in vivo. Overall, our data reveal the impact of NP composition and surface properties on the type and extension of induced immune responses. Deeper understanding on the NP-immune cell crosstalk can guide the rational development of nano-immunotherapeutic systems with improved and specific therapeutic efficacy and avoiding off-target effects.

Microglia seed the embryonic neuro-epithelium, expand and actively sculpt neuronal circuits in the developing central nervous system, but eventually adopt relative quiescence and ramified morphology in the adult. Here, we probed the impact of post-transcriptional control by microRNAs (miRNAs) on microglial performance during development and adulthood by generating mice lacking microglial Dicer expression at these distinct stages. Conditional Dicer ablation in adult microglia revealed that miRNAs were required to limit microglial responses to challenge. After peripheral endotoxin exposure, Dicer-deficient microglia expressed more pro-inflammatory cytokines than wild-type microglia and thereby compromised hippocampal neuronal functions. In contrast, prenatal Dicer ablation resulted in spontaneous microglia activation and revealed a role for Dicer in DNA repair and preservation of genome integrity. Accordingly, Dicer deficiency rendered otherwise radio-resistant microglia sensitive to gamma irradiation. Collectively, the differential impact of the Dicer ablation on microglia of the developing and adult brain highlights the changes these cells undergo with time. Microglia of developing and adult brain differ in activation state and function. Here, Varol and colleagues ablated microglial Dicer expression at distinct times. Adult microglia tolerated the perturbation but became hyper-responsive to challenge compromising hippocampus functions. Dicer and microRNA absence during development caused spontaneous microglia activation and impaired genome integrity.

Monocyte-derived macrophages (MoMF) play a pivotal role in the resolution of acetaminophen-induced liver injury (AILI). Timely termination of neutrophil activity and their clearance are essential for liver regeneration following injury. Here, we show that infiltrating Ly6C^hi monocytes, their macrophage descendants, and neutrophils spatially and temporally overlap in the centrilobular necrotic areas during the necroinflammatory and resolution phases of AILI. At the necroinflammatory phase, inducible ablation of circulating Ly6C^hi monocytes resulted in reduced numbers and fractions of reactive oxygen species (ROS)-producing neutrophils. In alignment with this, neutrophils sorted from monocyte-deficient livers exhibited reduced expression of NADPH oxidase 2. Moreover, human CD14⁺ monocytes stimulated with lipopolysaccharide or hepatocyte apoptotic bodies directly induced ROS production by cocultured neutrophils. RNA-seq-based transcriptome profiling of neutrophils from Ly6C^hi monocyte-deficient versus normal livers revealed 449 genes that were differentially expressed with at least twofold change (p ≤ 0.05). In the absence of Ly6C^hi monocytes, neutrophils displayed gene expression alterations associated with decreased innate immune activity and increased cell survival. At the early resolution phase, Ly6C^hi monocytes differentiated into ephemeral Ly6C^lo MoMF and their absence resulted in significant accumulation of late apoptotic neutrophils. Further gene expression analysis revealed the induced expression of a specific repertoire of bridging molecules and receptors involved with apoptotic cell clearance during the transition from Ly6C^hi monocytes to MoMF. Collectively, our findings establish a phase-dependent task division between liver-infiltrating Ly6C^hi monocytes and their MoMF descendants with the former regulating innate immune functions and cell survival of neutrophils and the later neutrophil clearance.

Adaptive thermogenesis is the process of heat generation in response to cold stimulation. It is under the control of the sympathetic nervous system, whose chief effector is the catecholamine norepinephrine (NE). NE enhances thermogenesis through beta 3-adrenergic receptors to activate brown adipose tissue and by 'browning' white adipose tissue. Recent studies have reported that alternative activation of macrophages in response to interleukin (IL)-4 stimulation induces the expression of tyrosine hydroxylase (TH), a key enzyme in the catecholamine synthesis pathway, and that this activation provides an alternative source of locally produced catecholamines during the thermogenic process. Here we report that the deletion of Th in hematopoietic cells of adult mice neither alters energy expenditure upon cold exposure nor reduces browning in inguinal adipose tissue. Bone marrow-derived macrophages did not release NE in response to stimulation with IL-4, and conditioned media from IL-4-stimulated macrophages failed to induce expression of thermogenic genes, such as uncoupling protein 1 (Ucp1), in adipocytes cultured with the conditioned media. Furthermore, chronic treatment with IL-4 failed to increase energy expenditure in wild-type, Ucp1(-/-) and interleukin-4 receptor-alpha double-negative (Il4ra(-/-)) mice. In agreement with these findings, adipose-tissue-resident macrophages did not express TH. Thus, we conclude that alternatively activated macrophages do not synthesize relevant amounts of catecholamines, and hence, are not likely to have a direct role in adipocyte metabolism or adaptive thermogenesis.

Hematopoietic-specific microRNA-142 is a critical regulator of various blood cell lineages, but its role in erythrocytes is unexplored. Herein, we characterize the impact of microRNA-142 on erythrocyte physiology and molecular cell biology, using a mouse loss-of-function allele. We report that microRNA-142 is required for maintaining the typical erythrocyte biconcave shape and structural resilience, for the normal metabolism of reactive oxygen species, and for overall lifespan. microRNA-142 further controls ACTIN filament homeostasis and membrane skeleton organization. The analyses presented reveal previously unappreciated functions of microRNA-142 and contribute to an emerging view of small RNAs as key players in erythropoiesis. Finally, the work herein demonstrates how a housekeeping network of cytoskeletal regulators can be reshaped by a single micro-RNA denominator in a cell type specific manner.

Hematopoietic-specific miR-142 is a critical regulator of various blood cell lineages including CD4+ dendritic cells1 and platelet biogenesis in megakaryocytes.2 Furthermore, we recently reported that miR-142 is required in order to maintain the biconcave shape of erythrocytes, their structural resilience and lifespan, through a mechanism that involves actin filament homeostasis.3 Here, we used a mouse loss of function allele to characterize a new axis, where miR-142 functions upstream of Rac1 in regulating erythropoiesis.

Recent data suggest that ramified microglia fulfil various tasks in the brain. However, to investigate this unique cell type cultured primary microglia are only a poor model. We here describe a method to deplete and repopulate organotypic hippocampal slice cultures (OHSC) with ramified microglia isolated from adult mouse brain creating microglia-replenished OHSC (Mrep-OHSC). Replenished microglia integrate into the tissue and ramify to a degree indistinguishable from their counterparts in the mouse brain. Moreover, wild-type slices replenished with microglia from TNF alpha-deficient animals provide similar results as OHSC prepared from microglia-specific TNF alpha-knockout mice (CX3CR1(cre)/TNF alpha(fl/fl)). Furthermore, this study demonstrates that replenished microglia in OHSC maintain original functions and properties acquired in vivo. Microglia from ERCC1(Delta/ko) mice, a mouse model of accelerated aging, maintain enhanced Mac2 expression and their activated phenotype after replenishment to wild-type OHSC tissue. Thus, the present study demonstrates that Mrep-OHSC are a unique tool to construct chimeric brain slices allowing studying the function of different phenotypes of in vivo like microglia in a tissue culture setting.

Perivascular, subdural meningeal and choroid plexus macrophages are non-parenchymal macrophages that mediate immune responses at brain boundaries. Although the origin of parenchymal microglia has recently been elucidated, much less is known about the precursors, the underlying transcriptional program and the dynamics of the other macrophages in the central nervous system (CNS). It was assumed that they have a high turnover from blood-borne monocytes. However, using parabiosis and fate-mapping approaches in mice, we found that CNS macrophages arose from hematopoietic precursors during embryonic development and established stable populations, with the notable exception of choroid plexus macrophages, which had dual origins and a shorter life span. The generation of CNS macrophages relied on the transcription factor PU.1, whereas the MYB, BATF3 and NR4A1 transcription factors were not required.

Background The developmental origin of the c-kit expressing progenitor cell pool in the adult heart has remained elusive. Recently, it has been discovered that the injured heart is enriched with c-kit⁺ cells, which also express the hematopoietic marker CD45. Methods and results In this study, we characterize the phenotype and transcriptome of the c-kit +/CD45 +/CD11b +/Flk-1 +/Sca-1 ± (B-type) cell population, originating from the left atrial appendage. These cells are defined as cardiac macrophage progenitors. We also demonstrate that the CD45 + progenitor cell population activates heart development, neural crest and pluripotency-associated pathways in vitro, in conjunction with CD45 down-regulation, and acquire a c-kit +/CD45 -/CD11b -/Flk-1 -/Sca-1 + (A-type) phenotype through cell fusion and asymmetric division. This putative spontaneous reprogramming evolves into a highly proliferative, partially myogenic phenotype (C-type). Conclusions Our data suggests that A-type cells and cardiac macrophage precursor cells (B-type) have a common lineage origin, possibly resolving some current conundrums in the field of cardiac regeneration.

Homo and heterozygote cx₃cr1 mutant mice, which harbor a green fluorescent protein (EGFP) in their cx₃cr1 loci, represent a widely used animal model to study microglia and peripheral myeloid cells. Here we report that microglia in the dentate gyrus (DG) of cx₃cr1^-/- mice displayed elevated microglial sirtuin 1 (SIRT1) expression levels and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) p65 activation, despite unaltered morphology when compared to cx₃cr1+/- or cx₃cr1+/+ controls. This phenotype was restricted to the DG and accompanied by reduced adult neurogenesis in cx₃cr1^-/- mice. Remarkably, adult neurogenesis was not affected by the lack of the CX3CR1-ligand, fractalkine (CX₃CL1). Mechanistically, pharmacological activation of SIRT1 improved adult neurogenesis in the DG together with an enhanced performance of cx₃cr1^-/- mice in a hippocampusdependent learning and memory task. The reverse condition was induced when SIRT1 was inhibited in cx₃cr1^-/- mice, causing reduced adult neurogenesis and lowered hippocampal cognitive abilities. In conclusion, our data indicate that deletion of CX3CR1 from microglia under resting conditions modifies brain areas with elevated cellular turnover independent of CX₃CL1.

Multiple sclerosis is the most frequent chronic inflammatory disease of the CNS. The entry and survival of pathogenic T cells in the CNS are crucial for the initiation and persistence of autoimmune neuroinflammation. In this respect, contradictory evidence exists on the role of the most potent type of antigen-presenting cells, dendritic cells. Applying intravital two-photon microscopy, we demonstrate the gatekeeper function of CNS professional antigen-presenting CD11c⁺ cells, which preferentially interact with Th17 cells. IL-17 expression correlates with expression of GM-CSF by T cells and with accumulation of CNS CD11c⁺ cells. These CD11c⁺ cells are organized in perivascular clusters, targeted by T cells, and strongly express the inflammatory chemokines Ccl5, Cxcl9, and Cxcl10. Our findings demonstrate a fundamental role of CNS CD11c⁺ cells in the attraction of pathogenic T cells into and their survival within the CNS.

Macrophages can be of dual origin. Most tissue-resident macrophage compartments are generated before birth and subsequently maintain themselves independently from each other locally in healthy tissue. Under inflammatory conditions, these cells can however be complemented by macrophages derived from acute monocyte infiltrates. Due to the lack of suitable experimental systems, differential functional contributions of central nervous system (CNS)-resident microglia and monocyte-derived macrophages (MoMF) to CNS inflammation, such as experimental autoimmune encephalomyelitis (EAE), the mouse model of multiple sclerosis (MS), remain poorly understood. Here, we will review recent progress in this field that suggest distinct roles of microglia and MoMF in disease induction and progression, capitalizing on novel transgenic mouse models. The latter finding could have major implications for the rationale development of therapeutic approaches to the management of brain inflammation and MS therapy.

Emerging evidence suggests that immunological mechanisms underlie metabolic control of adipose tissue. Here, we have shown the regulatory impact of a rare subpopulation of dendritic cells, rich in perforin-containing granules (perf-DCs). Using bone marrow transplantation to generate animals selectively lacking perf-DCs, we found that these chimeras progressively gained weight and exhibited features of metabolic syndrome. This phenotype was associated with an altered repertoire of T cells residing in adipose tissue and could be completely prevented by T cell depletion in vivo. A similar impact of perf-DCs on inflammatory T cells was also found in a well-defined model of multiple sclerosis, experimental autoimmune encephlalomyelitis (EAE). Thus, perf-DCs probably represent a regulatory cell subpopulation critical for protection from metabolic syndrome and autoimmunity.

Localization of memory CD8⁺ T cells to lymphoid or peripheral tissues is believed to correlate with proliferative capacity or effector function. Here we demonstrate that the fractalkine-receptor/CX 3 CR1 distinguishes memory CD8⁺ T cells with cytotoxic effector function from those with proliferative capacity, independent of tissue-homing properties. CX₃ CR1-based transcriptome and proteome-profiling defines a core signature of memory CD8⁺ T cells with effector function. We find CD62L hi CX³ CR1⁺ memory T cells that reside within lymph nodes. This population shows distinct migration patterns and positioning in proximity to pathogen entry sites. Virus-specific CX₃CR1⁺ memory CD8⁺ T cells are scarce during chronic infection in humans and mice but increase when infection is controlled spontaneously or by therapeutic intervention. This CX₃ CR1-based functional classification will help to resolve the principles of protective CD8⁺ T-cell memory.

Mutations in MECP2, encoding the epigenetic regulator methyl-CpG-binding protein 2, are the predominant cause of Rett syndrome, a disease characterized by both neurological symptoms and systemic abnormalities. Microglial dysfunction is thought to contribute to disease pathogenesis, and here we found microglia become activated and subsequently lost with disease progression in Mecp2-null mice. Mecp2 was found to be expressed in peripheral macrophage and monocyte populations, several of which also became depleted in Mecp2-null mice. RNA-seq revealed increased expression of glucocorticoid- and hypoxia-induced transcripts in Mecp2-deficient microglia and peritoneal macrophages. Furthermore, Mecp2 was found to regulate inflammatory gene transcription in response to TNF stimulation. Postnatal re-expression of Mecp2 using Cx3cr1(creER) increased the lifespan of otherwise Mecp2-null mice. These data suggest that Mecp2 regulates microglia and macrophage responsiveness to environmental stimuli to promote homeostasis. Dysfunction of tissue-resident macrophages might contribute to the systemic pathologies observed in Rett syndrome.

Macrophages are myeloid immune cells that are strategically positioned throughout the body tissues, where they ingest and degrade dead cells, debris, and foreign material and orchestrate inflammatory processes. Here we review two major recent paradigm shifts in our understanding of tissue macrophage biology. The first is the realization that most tissue-resident macrophages are established prenatally and maintained through adulthood by longevity and self-renewal. Their generation and maintenance are thus independent from ongoing hematopoiesis, although the cells can be complemented by adult monocyte-derived macrophages. Second, aside from being immune sentinels, tissue macrophages form integral components of their host tissue. This entails their specialization in response to local environmental cues to contribute to the development and specific function of their tissue of residence. Factors that govern tissue macrophage specialization are emerging. Moreover, tissue specialization is reflected in discrete gene expression profiles of macrophages, as well as epigenetic signatures reporting actual and potential enhancer usage.

Microglia are highly specialized tissue macrophages of the brain with dedicated functions in neuronal development, homeostasis and recovery from pathology Despite their unique localization in the central nervous system (CNS), microglia are ontogenetically and functionally related to their peripheral counterparts of the mononuclear phagocytic system in the body, namely tissue macrophages and circulating myeloid cells. Recent developments provided new insights into the myeloid system in the body with microglia emerging as intriguing unique archetypes. Similar to other tissue macrophages, microglia develop early during embryogenesis from immature yolk sac progenitors. But in contrast to most of their tissue relatives microglia persist throughout the entire life of the organism without any significant input from circulating blood cells due to their longevity and their capacity of self-renewal. Notably, microglia share some features with short-lived blood monocytes to limit CNS tissue damage in pathologies, but only bone marrow-derived cells display the ability to become permanently integrated in the parenchyma. This emphasizes the therapeutic potential of bone marrow-derived microglia-like cells. Further understanding of both fate and function of microglia during CNS pathologies and considering their uniqueness among other tissue macrophages will be pivotal for potential manipulation of immune cell function in the CNS, thereby reducing disease burden. Here, we discuss new aspects of myeloid cell biology in general with special emphasis on the brain-resident macrophages and microglia.

Chromatin modifications are crucial for development, yet little is known about their dynamics during differentiation. Hematopoiesis provides a well-defined model to study chromatin state dynamics; however, technical limitations impede profiling of homogeneous differentiation intermediates. We developed a high-sensitivity indexing-first chromatin immunoprecipitation approach to profile the dynamics of four chromatin modifications across 16 stages of hematopoietic differentiation. We identify 48,415 enhancer regions and characterize their dynamics.We find that lineage commitment involves de novo establishment of 17,035 lineage-specific enhancers. These enhancer repertoire expansions foreshadow transcriptional programs in differentiated cells. Combining our enhancer catalog with gene expression profiles, we elucidate the transcription factor network controlling chromatin dynamics and lineage specification in hematopoiesis. Together, our results provide a comprehensive model of chromatin dynamics during development.

Interleukin-10 (IL-10) is a pleiotropic anti-inflammatory cytokine produced and sensed by most hematopoietic cells. Genome-wide association studies and experimental animal models point at a central role of the IL-10 axis in inflammatory bowel diseases. Here we investigated the importance of intestinal macrophage production of IL-10 and their IL-10 exposure, as well as the existence of an IL-10-based autocrine regulatory loop in the gut. Specifically, we generated mice harboring IL-10 or IL-10 receptor (IL-10Rα) mutations in intestinal lamina propria-resident chemokine receptor CX₃CR1-expressing macrophages. We found macrophage-derived IL-10 dispensable for gut homeostasis and maintenance of colonic T regulatory cells. In contrast, loss of IL-10 receptor expression impaired the critical conditioning of these monocyte-derived macrophages and resulted in spontaneous development of severe colitis. Collectively, our results highlight IL-10 as a critical homeostatic macrophage-conditioning agent in the colon and define intestinal CX₃CR1^hi macrophages as a decisive factor that determines gut health or inflammation.

In multicellular organisms, biological function emerges when heterogeneous cell types form complex organs. Nevertheless, dissection of tissues into mixtures of cellular subpopulations is currently challenging. We introduce an automated massively parallel single-cell RNA sequencing (RNA-seq) approach for analyzing in vivo transcriptional states in thousands of single cells. Combined with unsupervised classification algorithms, this facilitates ab initio cell-type characterization of splenic tissues. Modeling single-cell transcriptional states in dendritic cells and additional hematopoietic cell types uncovers rich cell-type heterogeneity and gene-modules activity in steady state and after pathogen activation. Cellular diversity is thereby approached through inference of variable and dynamic pathway activity rather than a fixed preprogrammed cell-type hierarchy. These data demonstrate single-cell RNA-seq as an effective tool for comprehensive cellular decomposition of complex tissues.

Classical dendritic cells (cDC) are specialized antigen-presenting cells mediating immunity and tolerance. cDC cell-lineage decisions are largely controlled by transcriptional factor regulatory cascades. Using an in vivo cell-specific targeting of Runx3 at various stages of DC lineage development we show that Runx3 is required for cell-identity, homeostasis and function of splenic Esam^hi DC. Ablation of Runx3 in DC progenitors led to a substantial decrease in splenic CD4⁺/CD11b⁺ DC. Combined chromatin immunoprecipitation sequencing and gene expression analysis of purified DC-subsets revealed that Runx3 is a key gene expression regulator that facilitates specification and homeostasis of CD11b⁺Esam^hi DC. Mechanistically, loss of Runx3 alters Esam^hi DC gene expression to a signature characteristic of WT Esam^low DC. This transcriptional reprogramming caused a cellular change that diminished phagocytosis and hampered Runx3^-/- Esam^hi DC capacity to prime CD4⁺ T cells, attesting to the significant role of Runx3 in specifying Esam^hi DC identity and function.

Monocyte-derived macrophages are essential for recovery after spinal cord injury, but their homing mechanism is poorly understood. Here, we show that although of common origin, the homing of proinflammatory (M1) and the " alternatively activated" anti-inflammatory (M2) macrophages to traumatized spinal cord (SC) was distinctly regulated, neither being through breached blood-brain barrier. The M1 macrophages (Ly6c^hiCX3CR1^lo) derived from monocytes homed in a CCL2 chemokine-dependent manner through the adjacent SC leptomeninges. The resolving M2 macrophages (Ly6c^loCX3CR1^hi) derived from monocytes trafficked through a remote blood-cerebrospinal-fluid (CSF) barrier, the brain-ventricular choroid plexus (CP), via VCAM-1-VLA-4 adhesion molecules and epithelial CD73 enzyme for extravasation and epithelial transmigration. Blockage of these determinants, or mechanical CSF flow obstruction, inhibited M2 macrophage recruitment and impaired motor-function recovery. The CP, along with the CSF and the central canal, provided an anti-inflammatory supporting milieu, potentially priming the trafficking monocytes. Overall, our finding demonstrates that the route of monocyte entry to central nervous system provides an instructional environment to shape their function.

The mononuclear phagocyte system comprises cells as diverse as monocytes, macrophages, and dendritic cells (DCs), which collectively play key roles in innate immune responses and the triggering of adaptive immunity. Recent studies have highlighted the role of growth and transcription factors in defining developmental pathways and lineage relations within this cellular compartment. However, contributions of miRNAs to the development of mononuclear phagocytes remain largely unknown. In the present study, we report a comprehensive map of miRNA expression profiles for distinct myeloid populations, including BM-resident progenitors, monocytes, and mature splenic DCs. Each of the analyzed cell populations displayed a distinctive miRNA profile, suggesting a role for miRNAs in defining myeloid cell identities. Focusing on DC development, we found miR-142 to be highly expressed in classic FLT3-L-dependent CD4⁺ DCs, whereas reduced expression was observed in closely related CD8α ⁺ or CD4^-CD8α^- DCs. Moreover, mice deficient for miR-142 displayed an impairment of CD4⁺ DC homeostasis both in vitro and in vivo. Furthermore, loss of miR-142-dependent CD4 ⁺ DCs was accompanied by a severe and specific defect in the priming of CD4⁺ T cells. The results of our study establish a novel role for miRNAs in myeloid cell specification and define miR-142 as a pivotal genetic component in the maintenance of CD4⁺ DCs.

The bone marrow (BM) hosts memory lymphocytes and supports secondary immune responses against blood-borne antigens, but it is unsettled whether primary responses occur there and which cells present the antigen. We used 2-photon microscopy in the BM of live mice to study these questions. Naïve CD8+ T cells crawled rapidly at steady state but arrested immediately upon sensing antigenic peptides. Following infusion of soluble protein, various cell types were imaged ingesting the antigen, while antigen-specific T cells decelerated, clustered, upregulated CD69, and were observed dividing in situ to yield effector cells. Unlike in the spleen, T-cell responses persisted when BM-resident dendritic cells (DCs) were ablated but failed when all phagocytic cells were depleted. Potential antigen-presenting cells included monocytes and macrophages but not B cells. Collectively, our results suggest that the BM supports crosspresentation of blood-borne antigens similar to the spleen; uniquely, alongside DCs, other myeloid cells participate in crosspresentation.

Ly6C^hi monocytes seed the healthy intestinal lamina propria to give rise to resident CX₃CR1⁺ macrophages that contribute to the maintenance of gut homeostasis. Here we report on two alternative monocyte fates in the inflamed colon. We showed that CCR2 expression is essential to the recruitment of Ly6C^hi monocytes to the inflamed gut to become the dominant mononuclear cell type in the lamina propria during settings of acute colitis. In the inflammatory microenvironment, monocytes upregulated TLR2 and NOD2, rendering them responsive to bacterial products to become proinflammatory effector cells. Ablation of Ly6C^hi monocytes ameliorated acute gut inflammation. With time, monocytes differentiated into migratory antigen-presenting cells capable of priming naive T cells, thus acquiring hallmarks reminiscent of dendritic cells. Collectively, our results highlight cellular dynamics in the inflamed colon and the plasticity of Ly6C^hi monocytes, marking them as potential targets for inflammatory bowel disease (IBD) therapy.

Mature dendritic cells (DCs) are established as unrivaled antigen-presenting cells (APCs) in the initiation of immune responses, whereas steady-state DCs induce peripheral T cell tolerance. Using various genetic approaches, we depleted CD11c⁺ DCs in mice and induced autoimmune CNS inflammation. Unexpectedly, mice lacking DCs developed aggravated disease compared to control mice. Furthermore, when we engineered DCs to present a CNS-associated autoantigen in an induced manner, we found robust tolerance that prevented disease, which coincided with an upregulation of the PD-1 receptor on antigen-specific T cells. Additionally, we showed that PD-1 was necessary for DC-mediated induction of regulatory T cells. Our results show that a reduction of DCs interferes with tolerance, resulting in a stronger inflammatory response, and that other APC populations could compensate for the loss of immunogenic APC function in DC-depleted mice.

BID, a BH3-only BCL2 family member, functions in apoptosis as well as the DNA-damage response. Our previous data demonstrated that BID is an ATM effector acting to induce cell-cycle arrest and inhibition of apoptosis following DNA damage. Here we show that ATM-mediated BID phosphorylation plays an unexpected role in maintaining the quiescence of haematopoietic stem cells (HSCs). Loss of BID phosphorylation leads to escape from quiescence of HSCs, resulting in exhaustion of the HSC pool and a marked reduction of HSC repopulating potential in vivo. We also demonstrate that BID phosphorylation plays a role in protecting HSCs from irradiation, and that regulating both quiescence and survival of HSCs depends on BID's ability to regulate oxidative stress. Moreover, loss of BID phosphorylation, ATM knockout or exposing mice to irradiation leads to an increase in mitochondrial BID, which correlates with an increase in mitochondrial oxidative stress. These results show that the ATM-BID pathway serves as a critical checkpoint for coupling HSC homeostasis and the DNA-damage stress response to enable long-term regenerative capacity.

Although most genes are expressed biallelically, a number of key genomic sites-including immune and olfactory receptor regions-are controlled monoallelically in a stochastic manner, with some cells expressing the maternal allele and others the paternal allele in the target tissue(1,2). Very little is known about how this phenomenon is regulated and programmed during development. Here, using mouse immunoglobulin-kappa (Ig kappa) as a model system, we demonstrate that although individual haematopoietic stem cells are characterized by allelic plasticity, early lymphoid lineage cells become committed to the choice of a single allele, and this decision is then stably maintained in a clonal manner that predetermines monoallelic rearrangement in B cells. This is accompanied at the molecular level by underlying allelic changes in asynchronous replication timing patterns at the kappa locus. These experiments may serve to define a new concept of stem cell plasticity.

Dendritic cells (DCs) in tissues and lymphoid organs comprise distinct functional subsets that differentiate in situ from circulating progenitors. Tissue-specific signals that regulate DC subset differentiation are poorly understood. We report that DC-specific deletion of the Notch2 receptor caused a reduction of DC populations in the spleen. Within the splenic CD11b ⁺ DC subset, Notch signaling blockade ablated a distinct population marked by high expression of the adhesion molecule Esam. The Notch-dependent Esam ^hi DC subset required lymphotoxin beta receptor signaling, proliferated in situ, and facilitated CD4 ⁺ T cell priming. The Notch-independent Esam ^lo DCs expressed monocyte-related genes and showed superior cytokine responses. In addition, Notch2 deletion led to the loss of CD11b ⁺CD103 ⁺ DCs in the intestinal lamina propria and to a corresponding decrease of IL-17-producing CD4 ⁺ T cells in the intestine. Thus, Notch2 is a common differentiation signal for T cell-priming CD11b ⁺ DC subsets in the spleen and intestine.