Classical monocyte ontogeny dictates their functions and fates as tissue macrophages.

Classical monocytes (CMs) are ephemeral myeloid immune cells that circulate in the blood. Emerging evidence suggests that CMs can have distinct ontogeny and originate from either granulocyte-monocyte- or monocyte-dendritic-cell progenitors (GMPs or MDPs). Here, we report surface markers that allowed segregation of murine GMP- and MDP-derived CMs, i.e., GMP-Mo and MDP-Mo, as well as their functional characterization, including fate definition following adoptive cell transfer. GMP-Mo and MDP-Mo yielded an equal increase in homeostatic CM progeny, such as blood-resident non-classical monocytes and gut macrophages; however, these cells differentially seeded various other selected tissues, including the dura mater and lung. Specifically, GMP-Mo and MDP-Mo differentiated into distinct interstitial lung macrophages, linking CM dichotomy to previously reported pulmonary macrophage heterogeneity. Collectively, we provide evidence for the existence of two functionally distinct CM subsets in the mouse that differentially contribute to peripheral tissue macrophage populations in homeostasis and following challenge.

Competitive fungal commensalism mitigates candidiasis pathology.

The mycobiota are a critical part of the gut microbiome, but host-fungal 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. albicans-mediated diseases.

Translatome Profiling of Tissue-Resident Macrophages Using the RiboTag Approach.

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.

Tackling Tissue Macrophage Heterogeneity by SplitCre Transgenesis.

Macrophages represent a broad spectrum of distinct, but closely related tissue-resident immune cells. This presents a major challenge for the study of functional aspects of these cells using classical Cre recombinase-mediated conditional mutagenesis in mice, since single promoter-driven Cre transgenic models often display limited specificity toward their intended target. The advent of CRISPR/Cas9 technology has now provided a time- and cost-effective method to explore the full potential of binary transgenic, intersectional genetics. Specifically, the use of two promoters driving inactive Cre fragments that, when co-expressed, dimerize and only then gain recombinase activity allows the characterization and manipulation of genetically defined tissue macrophage subpopulations. Here, we will elaborate on the use of this protocol to capitalize on these recent technological advances in mouse genetics and discuss their strengths and pitfalls to improve the study of tissue macrophage subpopulations in physiology and pathophysiology.

Cognate microglia-T cell interactions shape the functional regulatory T cell pool in experimental autoimmune encephalomyelitis pathology.

Microglia, the parenchymal brain macrophages of the central nervous system, have emerged as critical players in brain development and homeostasis. The immune functions of these cells, however, remain less well defined. We investigated contributions of microglia in a relapsing-remitting multiple sclerosis paradigm, experimental autoimmune encephalitis in C57BL/6 x SJL F1 mice. Fate mapping-assisted translatome profiling during the relapsing-remitting disease course revealed the potential of microglia to interact with T cells through antigen presentation, costimulation and coinhibition. Abundant microglia-T cell aggregates, as observed by histology and flow cytometry, supported the idea of functional interactions of microglia and T cells during remission, with a bias towards regulatory T cells. Finally, microglia-restricted interferon-γ receptor and major histocompatibility complex mutagenesis significantly affected the functionality of the regulatory T cell compartment in the diseased central nervous system and remission. Collectively, our data establish critical non-redundant cognate and cytokine-mediated interactions of microglia with CD4+ T cells during autoimmune neuroinflammation.

A Binary Cre Transgenic Approach Dissects Microglia and CNS Border-Associated Macrophages.

The developmental and molecular heterogeneity of tissue macrophages is unravelling, as are their diverse contributions to physiology and pathophysiology. Moreover, also given tissues harbor macrophages in discrete anatomic locations. Functional contributions of specific cell populations can in mice be dissected using Cre recombinase-mediated mutagenesis. However, single promoter-based Cre models show limited specificity for cell types. Focusing on macrophages in the brain, we establish here a binary transgenic system involving complementation-competent NCre and CCre fragments whose expression is driven by distinct promoters: Sall1ncre: Cx3cr1ccre mice specifically target parenchymal microglia and compound transgenic Lyve1ncre: Cx3cr1ccre animals target vasculature-associated macrophages, in the brain, as well as other tissues. We imaged the respective cell populations and retrieved their specific translatomes using the RiboTag in order to define them and analyze their differential responses to a challenge. Collectively, we establish the value of binary transgenesis to dissect tissue macrophage compartments and their functions.

Interleukin-10 Prevents Pathological Microglia Hyperactivation following Peripheral Endotoxin Challenge.

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.

Defining murine monocyte differentiation into colonic and ileal macrophages.

Monocytes are circulating short-lived macrophage precursors that are recruited on demand from the blood to sites of inflammation and challenge. In steady state, classical monocytes give rise to vasculature-resident cells that patrol the luminal side of the endothelium. In addition, classical monocytes feed macrophage compartments of selected organs, including barrier tissues, such as the skin and intestine, as well as the heart. Monocyte differentiation under conditions of inflammation has been studied in considerable detail. In contrast, monocyte differentiation under non-inflammatory conditions remains less well understood. Here we took advantage of a combination of cell ablation and precursor engraftment to investigate the generation of gut macrophages from monocytes. Collectively, we identify factors associated with the gradual adaptation of monocytes to tissue residency. Moreover, comparison of monocyte differentiation into the colon and ileum-resident macrophages revealed the graduated acquisition of gut segment-specific gene expression signatures.

Comparative analysis of CreER transgenic mice for the study of brain macrophages: A case study.

Conditional mutagenesis and fate mapping have contributed considerably to our understanding of physiology and pathology. Specifically, Cre recombinase-based approaches allow the definition of cell type-specific contributions to disease development and of inter-cellular communication circuits in respective animal models. Here we compared Cx3 cr1CreER and Sall1CreER transgenic mice and their use to decipher the brain macrophage compartment as a showcase to discuss recent technological advances. Specifically, we highlight the need to define the accuracy of Cre recombinase expression, as well as strengths and pitfalls of these particular systems that should be taken into consideration when applying these models.

Obesity and dysregulated central and peripheral macrophage-neuron cross-talk.

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 ménage à 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.

Re-evaluating microglia expression profiles using RiboTag and cell isolation strategies.

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.

Dicer Deficiency Differentially Impacts Microglia of the Developing and Adult Brain.

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.

Brown-adipose-tissue macrophages control tissue innervation and homeostatic energy expenditure.

Tissue macrophages provide immunological defense and contribute to the establishment and maintenance of tissue homeostasis. Here we used constitutive and inducible mutagenesis to delete the nuclear transcription regulator Mecp2 in macrophages. Mice that lacked the gene encoding Mecp2, which is associated with Rett syndrome, in macrophages did not show signs of neurodevelopmental disorder but displayed spontaneous obesity, which was linked to impaired function of brown adipose tissue (BAT). Specifically, mutagenesis of a BAT-resident Cx3Cr1+ macrophage subpopulation compromised homeostatic thermogenesis but not acute, cold-induced thermogenesis. Mechanistically, malfunction of BAT in pre-obese mice with mutant macrophages was associated with diminished sympathetic innervation and local titers of norepinephrine, which resulted in lower expression of thermogenic factors by adipocytes. Mutant macrophages overexpressed the signaling receptor and ligand PlexinA4, which might contribute to the phenotype by repulsion of sympathetic axons expressing the transmembrane semaphorin Sema6A. Collectively, we report a previously unappreciated homeostatic role for macrophages in the control of tissue innervation. Disruption of this circuit in BAT resulted in metabolic imbalance.

MicroRNA-142 controls thymocyte proliferation.

T-cell development is a spatially and temporally regulated process, orchestrated by well-defined contributions of transcription factors and cytokines. Here, we identify the noncoding RNA miR-142 as an additional regulatory layer within murine thymocyte development and proliferation. MiR-142 deficiency impairs the expression of cell cycle-promoting genes in mature mouse thymocytes and early progenitors, accompanied with increased levels of cyclin-dependent kinase inhibitor 1B (Cdkn1b, also known as p27Kip1 ). By using CRISPR/Cas9 technology to delete the miR-142-3p recognition element in the 3'UTR of cdkn1b, we confirm that this gene is a novel target of miR-142-3p in vivo. Increased Cdkn1b protein expression alone however was insufficient to cause proliferation defects in thymocytes, indicating the existence of additional critical miR-142 targets. Collectively, we establish a key role for miR-142 in the control of early and mature thymocyte proliferation, demonstrating the multifaceted role of a single miRNA on several target genes.

Prolonged Elimination of Negative Feedback Control Mechanisms Along the Insulin Signaling Pathway Impairs ß-Cell Function In Vivo.

Cellular stress and proinflammatory cytokines induce phosphorylation of insulin receptor substrate (IRS) proteins at Ser sites that inhibit insulin and IGF-I signaling. We therefore examined the effects of mutation of five "inhibitory" Ser phosphorylation sites on IRS2 function in transgenic mice that overexpress, selectively in pancreatic ß-cells, either wild-type (WT) or a mutated IRS2 protein (IRS25A). Islets size, number, and mRNA levels of catalase and superoxide dismutase were increased, whereas those of nitric oxide synthase were decreased, in 7- to 10-week-old IRS25A-ß mice compared with IRS2WT-ß mice. However, glucose homeostasis and insulin secretion in IRS25A-ß mice were impaired when compared with IRS2WT-ß mice or to nontransgenic mice. This was associated with reduced mRNA levels of Glut2 and islet ß-cell transcription factors such as Nkx6.1 and MafA Similarly, components mediating the unfolded protein response were decreased in islets of IRS25A-ß mice in accordance with their decreased insulin secretion. The beneficial effects of IRS25A on ß-cell proliferation and ß-cell transcription factors were evident only in 5- to 8-day-old mice. These findings suggest that elimination of inhibitory Ser phosphorylation sites of IRS2 exerts short-term beneficial effects in vivo; however, their sustained elimination leads to impaired ß-cell function.

TM7SF3, a novel p53-regulated homeostatic factor, attenuates cellular stress and the subsequent induction of the unfolded protein response.

Earlier reported small interfering RNA (siRNA) high-throughput screens, identified seven-transmembrane superfamily member 3 (TM7SF3) as a novel inhibitor of pancreatic ß-cell death. Here we show that TM7SF3 maintains protein homeostasis and promotes cell survival through attenuation of ER stress. Overexpression of TM7SF3 inhibits caspase 3/7 activation. In contrast, siRNA-mediated silencing of TM7SF3 accelerates ER stress and activation of the unfolded protein response (UPR). This involves inhibitory phosphorylation of eukaryotic translation initiation factor 2α activity and increased expression of activating transcription factor-3 (ATF3), ATF4 and C/EBP homologous protein, followed by induction of apoptosis. This process is observed both in human pancreatic islets and in a number of cell lines. Some of the effects of TM7SF3 silencing are evident both under basal conditions, in otherwise untreated cells, as well as under different stress conditions induced by thapsigargin, tunicamycin or a mixture of pro-inflammatory cytokines (tumor necrosis factor alpha, interleukin-1 beta and interferon gamma). Notably, TM7SF3 is a downstream target of p53: activation of p53 by Nutlin increases TM7SF3 expression in a time-dependent manner, although silencing of p53 abrogates this effect. Furthermore, p53 is found in physical association with the TM7SF3 promoter. Interestingly, silencing of TM7SF3 promotes p53 activity, suggesting the existence of a negative-feedback loop, whereby p53 promotes expression of TM7SF3 that acts to restrict p53 activity. Our findings implicate TM7SF3 as a novel p53-regulated pro-survival homeostatic factor that attenuates the development of cellular stress and the subsequent induction of the UPR.

Nedd4 family interacting protein 1 (Ndfip1) promotes death of pancreatic beta cells.

High-throughput siRNA screening was employed to identify novel genes that regulate cytokine-induced death of pancreatic ß-cells. One of the 'hits' was Nedd4 family interacting protein 1 (Ndfip1), an adaptor and activator of Nedd4-family ubiquitin ligases. Silencing of Ndfip1 inhibited cytokine-induced apoptosis of mouse and human pancreatic islets and promoted glucose-stimulated insulin secretion. These effects were associated with an increase in the cellular content of JunB, a potent inhibitor of ER stress and apoptosis. Silencing of Ndfip1 also increased the expression of ATF4, IRE-1α, and the spliced form of XBP that govern the unfolded protein response (UPR) and relieve cytokine-induced ER stress, while overexpression of Ndfip1 exerted opposite effects. These findings implicate Ndfip1 in the degradation of JunB; inhibition of the UPR and insulin secretion; and promotion of cytokine-induced death of pancreatic ß-cells.