A Custom Panel for Profiling Microglia Gene Expression.

Despite being immune cells of the central nervous system (CNS), microglia contribute to CNS development, maturation, and homeostasis, and microglia dysfunction has been implicated in several neurological disorders. Recent advancements in single-cell studies have uncovered unique microglia-specific gene expression. However, there is a need for a simple yet elegant multiplexed approach to quantifying microglia gene expression. To address this, we have designed a NanoString nCounter technology-based murine microglia-specific custom codeset comprising 178 genes. We analyzed RNA extracted from ex vivo adult mouse microglia, primary mouse microglia, the BV2 microglia cell line, and mouse bone marrow monocytes using our custom panel. Our findings reveal a pattern where homeostatic genes exhibit heightened expression in adult microglia, followed by primary cells, and are absent in BV2 cells, while reactive markers are elevated in primary microglia and BV2 cells. Analysis of publicly available data sets for the genes present in the panel revealed that the panel could reliably reflect the changes in microglia gene expression in response to various factors. These findings highlight that the microglia panel used offers a swift and cost-effective means to assess microglial cells and can be used to study them in varying contexts, ranging from normal homeostasis to disease models.

2,4-Dichlorophenoxyacetic Acid Induces Degeneration of mDA Neurons In Vitro.

Background: Parkinson's disease (PD) affects 1-2% of the population over the age of 60 and the majority of PD cases are sporadic, without any family history of the disease. Neuroinflammation driven by microglia has been shown to promote the progression of midbrain dopaminergic (mDA) neuron loss through the release of neurotoxic factors. Interestingly, the risk of developing PD is significantly higher in distinct occupations, such as farming and agriculture, and is linked to the use of pesticides and herbicides. Methods: The neurotoxic features of 2,4-Dichlorophenoxyacetic acid (2,4D) at concentrations of 10 µM and 1 mM were analyzed in two distinct E14 midbrain neuron culture systems and in primary microglia. Results: The application of 1 mM 2,4D resulted in mDA neuron loss in neuron-enriched cultures. Notably, 2,4D-induced neurotoxicity significantly increased in the presence of microglia in neuron-glia cultures, suggesting that microglia-mediated neurotoxicity could be one mechanism for progressive neuron loss in this in vitro setup. However, 2,4D alone was unable to trigger microglia reactivity. Conclusions: Taken together, we demonstrate that 2,4D is neurotoxic for mDA neurons and that the presence of glia cells enhances 2,4D-induced neuron death. These data support the role of 2,4D as a risk factor for the development and progression of PD and further suggest the involvement of microglia during 2,4D-induced mDA neuron loss.

Microglial CD74 Expression Is Regulated by TGFß Signaling.

Microglia play important roles during physiological and pathological situations in the CNS. Several reports have described the expression of Cd74 in disease-associated and aged microglia. Here, we demonstrated that TGFß1 controled the expression of Cd74 in microglia in vitro and in vivo. Using BV2 cells, primary microglia cultures as well as Cx3cr1CreERT2:R26-YFP:Tgfbr2fl/fl in combination with qPCR, flow cytometry, and immunohistochemistry, we were able to provide evidence that TGFß1 inhibited LPS-induced upregulation of Cd74 in microglia. Interestingly, TGFß1 alone was able to mediate downregulation of CD74 in vitro. Moreover, silencing of TGFß signaling in vivo resulted in marked upregulation of CD74, further underlining the importance of microglial TGFß signaling during regulation of microglia activation. Taken together, our data indicated that CD74 is a marker for activated microglia and further demonstrated that microglial TGFß signaling is important for regulation of Cd74 expression during microglia activation.

Characterization of the Leucocyte Immunoglobulin-like Receptor B4 (Lilrb4) Expression in Microglia.

As resident innate immune cells of the CNS, microglia play important essential roles during physiological and pathological situations. Recent reports have described the expression of Lilrb4 in disease-associated and aged microglia. Here, we characterized the expression of Lilrb4 in microglia in vitro and in vivo in comparison with bone marrow-derived monocytes and peritoneal macrophages in mice. Using BV2 cells, primary microglia cultures as well as ex vivo isolated microglia and myeloid cells in combination with qPCR and flow cytometry, we were able to provide a comprehensive characterization of Lilrb4 expression in distinct mouse myeloid cells. Whereas microglia in vivo display low expression of Lilrb4, primary microglia cultures present high levels of surface LILRB4. Among the analyzed peripheral myeloid cells, peritoneal macrophages showed the highest expression levels of Lilrb4. Moreover, LPS treatment and inhibition of microglial TGFß signaling resulted in significant increases of LILRB4 cell surface levels. Taken together, our data indicate that LILRB4 is a reliable surface marker for activated microglia and further demonstrate that microglial TGFß signaling is involved in the regulation of Lilrb4 expression during LPS-induced microglia activation.

Inhibition of Microglial TGFß Signaling Increases Expression of Mrc1.

Microglia are constantly surveying their microenvironment and rapidly react to impairments by changing their morphology, migrating toward stimuli and adopting gene expression profiles characterizing their activated state. The increased expression of the M2-like marker Mannose receptor 1 (Mrc1), which is also referred to as CD206, in microglia has been reported after M2-like activation in vitro and in vivo. Mrc1 is a 175-kDa transmembrane pattern recognition receptor which binds a variety of carbohydrates and is involved in the pinocytosis and the phagocytosis of immune cells, including microglia, and thought to contribute to a neuroprotective microglial phenotype. Here we analyzed the effects of TGFß signaling on Mrc1 expression in microglia in vivo and in vitro. Using C57BL/6 wild type and Cx3cr1 CreERT2 :R26-YFP:Tgfbr2 fl/fl mice-derived microglia, we show that the silencing of TGFß signaling results in the upregulation of Mrc1, whereas recombinant TGFß1 induced the delayed downregulation of Mrc1. Furthermore, chromatin immunoprecipitation experiments provided evidence that Mrc1 is not a direct Smad2/Smad4 target gene in microglia. Altogether our data indicate that the changes in Mrc1 expression after the activation or the silencing of microglial TGFß signaling are likely to be mediated by modifications of the secondary intracellular signaling events influenced by TGFß signaling.

Silencing of TGFß signalling in microglia results in impaired homeostasis.

TGFß1 has been implicated in regulating functional aspects of several distinct immune cell populations including central nervous system (CNS) resident microglia. Activation and priming of microglia have been demonstrated to contribute to the progression of neurodegenerative diseases and, thus, underlie stringent control by endogenous regulatory factors including TGFß1. Here, we demonstrate that deletion of Tgfbr2 in adult postnatal microglia does neither result in impairment of the microglia-specific gene expression signatures, nor is microglial survival and maintenance affected. Tgfbr2-deficient microglia were characterised by distinct morphological changes and transcriptome analysis using RNAseq revealed that loss of TGFß signalling results in upregulation of microglia activation and priming markers. Moreover, protein arrays demonstrated increased secretion of CXCL10 and CCL2 accompanied by activation of immune cell signalling as evidenced by increased phosphorylation of TAK1. Together, these data underline the importance of microglial TGFß signalling to regulate microglia adaptive changes.

Aged Mouse Cortical Microglia Display an Activation Profile Suggesting Immunotolerogenic Functions.

Microglia are the resident immune cells of the central nervous system (CNS) and participate in physiological and pathological processes. Their unique developmental nature suggests age-dependent structural and functional impairments that might contribute to neurodegenerative diseases. In the present study, we addressed the age-dependent changes in cortical microglia gene expression patterns and the expression of M1- and M2-like activation markers. Iba1 immunohistochemistry, isolation of cortical microglia followed by fluorescence-activated cell sorting and RNA isolation to analyze transcriptional changes in aged cortical microglia was performed. We provide evidence that aging is associated with decreased numbers of cortical microglia and the establishment of a distinct microglia activation profile including upregulation of Ifi204, Lilrb4, Arhgap, Oas1a, Cd244 and Ildr2. Moreover, flow cytometry revealed that aged cortical microglia express increased levels of Cd206 and Cd36. The data presented in the current study indicate that aged mouse cortical microglia adopt a distinct activation profile, which suggests immunosuppressive and immuno-tolerogenic functions.

Postnatal maturation of microglia is associated with alternative activation and activated TGFß signaling.

Microglia are involved in a widespread set of physiological and pathological processes and further play important roles during neurodevelopmental events. Postnatal maturation of microglia has been associated with the establishment of microglia-specific gene expression patterns. The mechanisms governing microglia maturation are only partially understood but Tgfß1 has been suggested to be one important mediator. In the present study, we demonstrate that early postnatal microglia maturation is associated with alternative microglia activation, increased engulfment of apoptotic cells as well as activated microglial Tgfß signaling. Interestingly, microglial Tgfß signaling preceded the induction of the microglia-specific gene expression indicating the importance of Tgfß1 for postnatal microglia maturation. Moreover, we provide evidence that Tgfß1 is expressed by neurons in postnatal and adult brains defining neuron-microglia communication via Tgfß1 as an important event. Finally, we introduce the recently identified microglia marker Tmem119 as a direct Tgfß1-Smad2 target gene. Taken together, the data presented here further increase the understanding of Tgfß1-mediated effects in microglia and place emphasis on the importance of Tgfß1 for microglia maturation and maintenance.