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.

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.

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.

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.

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.

Impact of X-irradiation on microglia.

Irradiation is widely used to treat brain tumors, and also to create bone marrow (BM) chimeras. BM chimeras are widely used to dissect functions and origin of microglia and blood-derived mononuclear cells under homeostatic or pathological conditions. This is facilitated by the fact that microglia survive irradiation and are thus regarded radio-resistant. In this study, we tested whether microglia are indeed radio-resistant and looked for potential mechanisms that might explain this phenomenon. We analyzed the radio-resistance of microglia independently of their physiological brain environment compared to other mononuclear cells from spleen and brain after X-irradiation with 7 Gy or 30 Gy. Furthermore, we investigated long-term effects of X-irradiation on microglia using organotypic hippocampal slice cultures (OHSCs). We found a significant higher survival rate of isolated microglia 4 hr after X-irradiation with 30 Gy accompanied by a decreased proliferation rate. Investigations of apoptosis-related genes revealed no regulation of a specific antiapoptotic pathway but ataxia telangiectasia mutated (ATM), a DNA-repair-related gene, was significantly upregulated in isolated microglia 4 hr after 30 Gy. Irradiation of OHSCs with 7 and 30 Gy revealed a highly and significantly decreased cell number, morphological changes and an increase in migration velocity of microglia. Furthermore, cell loss, increased soma size and process length of microglia was also found in BM chimeras irradiated with 9.5 Gy 5 weeks after irradiation. Here, we present new evidence implying that microglia are not a homogeneous population of radio-resistant cells and report on long-term alterations of microglia that survived irradiation.

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.

Microglial expression of CD83 governs cellular activation and restrains neuroinflammation in experimental autoimmune encephalomyelitis.

Microglial activation during neuroinflammation is crucial for coordinating the immune response against neuronal tissue, and the initial response of microglia determines the severity of neuro-inflammatory diseases. The CD83 molecule has been recently shown to modulate the activation status of dendritic cells and macrophages. Although the expression of CD83 is associated with early microglia activation in various disease settings, its functional relevance for microglial biology has been elusive. Here, we describe a thorough assessment of CD83 regulation in microglia and show that CD83 expression in murine microglia is not only associated with cellular activation but also with pro-resolving functions. Using single-cell RNA-sequencing, we reveal that conditional deletion of CD83 results in an over-activated state during neuroinflammation in the experimental autoimmune encephalomyelitis model. Subsequently, CD83-deficient microglia recruit more pathogenic immune cells to the central nervous system, deteriorating resolving mechanisms and exacerbating the disease. Thus, CD83 in murine microglia orchestrates cellular activation and, consequently, also the resolution of neuroinflammation.