During early embryogenesis, microglia arise from yolk sac progenitors that populate the developing central nervous system (CNS), but how the tissue-resident macrophages are maintained throughout the ...organism’s lifespan still remains unclear. Here, we describe a system that allows specific, conditional ablation of microglia in adult mice. We found that the microglial compartment was reconstituted within 1 week of depletion. Microglia repopulation relied on CNS-resident cells, independent from bone-marrow-derived precursors. During repopulation, microglia formed clusters of highly proliferative cells that migrated apart once steady state was achieved. Proliferating microglia expressed high amounts of the interleukin-1 receptor (IL-1R), and treatment with an IL-1R antagonist during the repopulation phase impaired microglia proliferation. Hence, microglia have the potential for efficient self-renewal without the contribution of peripheral myeloid cells, and IL-1R signaling participates in this restorative proliferation process.
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•Microglia repopulate within 5 days of depletion•Under defined host conditions, microglia can be replaced by BM cells•Without preconditioning, they renew themselves locally by massive proliferation•IL-1R1 on microglia is involved in self-renewal and maintenance
Microglia arise from yolk sac progenitors that populate the developing central nervous system (CNS), but how these cells are maintained in the adult remains unclear. Waisman and colleagues show via a genetic approach that after ablation, microglia repopulation is driven by local progenitors in response to IL-1R signaling.
The transfer of antigens from oligodendrocytes to immune cells has been implicated in the pathogenesis of autoimmune diseases. Here, we show that oligodendrocytes secrete small membrane vesicles ...called exosomes, which are specifically and efficiently taken up by microglia both in vitro and in vivo. Internalisation of exosomes occurs by a macropinocytotic mechanism without inducing a concomitant inflammatory response. After stimulation of microglia with interferon-γ, we observe an upregulation of MHC class II in a subpopulation of microglia. However, exosomes are preferentially internalised in microglia that do not seem to have antigen-presenting capacity. We propose that the constitutive macropinocytotic clearance of exosomes by a subset of microglia represents an important mechanism through which microglia participate in the degradation of oligodendroglial membrane in an immunologically 'silent' manner. By designating the capacity for macropinocytosis and antigen presentation to distinct cells, degradation and immune function might be assigned to different subtypes of microglia.
Microglia (tissue-resident macrophages) represent the main cell type of the innate immune system in the CNS; however, the mechanisms that control the activation of microglia are widely unknown. We ...systematically explored microglial activation and functional microglia–neuron interactions in organotypic hippocampal slice cultures, i.e., postnatal cortical tissue that lacks adaptive immunity. We applied electrophysiological recordings of local field potential and extracellular K⁺ concentration, immunohistochemistry, design-based stereology, morphometry, Sholl analysis, and biochemical analyses. We show that chronic activation with either bacterial lipopolysaccharide through Toll-like receptor 4 (TLR4) or leukocyte cytokine IFN-γ induces reactive phenotypes in microglia associated with morphological changes, population expansion, CD11b and CD68 up-regulation, and proinflammatory cytokine (IL-1β, TNF-α, IL-6) and nitric oxide (NO) release. Notably, these reactive phenotypes only moderately alter intrinsic neuronal excitability and gamma oscillations (30–100 Hz), which emerge from precise synaptic communication of glutamatergic pyramidal cells and fast-spiking, parvalbumin-positive GABAergic interneurons, in local hippocampal networks. Short-term synaptic plasticity and extracellular potassium homeostasis during neural excitation, also reflecting astrocyte function, are unaffected. In contrast, the coactivation of TLR4 and IFN-γ receptors results in neuronal dysfunction and death, caused mainly by enhanced microglial inducible nitric oxide synthase (iNOS) expression and NO release, because iNOS inhibition is neuroprotective. Thus, activation of TLR4 in microglia in situ requires concomitant IFN-γ receptor signaling from peripheral immune cells, such as T helper type 1 and natural killer cells, to unleash neurotoxicity and inflammation-induced neurodegeneration. Our findings provide crucial mechanistic insight into the complex process of microglia activation, with relevance to several neurologic and psychiatric disorders.
Locus ceruleus (LC)-supplied norepinephrine (NE) suppresses neuroinflammation in the brain. To elucidate the effect of LC degeneration and subsequent NE deficiency on Alzheimer's disease pathology, ...we evaluated NE effects on microglial key functions. NE stimulation of mouse microglia suppressed Aβ-induced cytokine and chemokine production and increased microglial migration and phagocytosis of Aβ. Induced degeneration of the locus ceruleus increased expression of inflammatory mediators in APP-transgenic mice and resulted in elevated Aβ deposition. In vivo laser microscopy confirmed a reduced recruitment of microglia to Aβ plaque sites and impaired microglial Aβ phagocytosis in NE-depleted APP-transgenic mice. Supplying the mice the norepinephrine precursor L-threo-DOPS restored microglial functions in NE-depleted mice. This indicates that decrease of NE in locus ceruleus projection areas facilitates the inflammatory reaction of microglial cells in AD and impairs microglial migration and phagocytosis, thereby contributing to reduced Aβ clearance. Consequently, therapies targeting microglial phagocytosis should be tested under NE depletion.
The proinflammatory cytokine interleukin 1 (IL-1) is crucially involved in the pathogenesis of multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Herein, we ...studied the role of IL-1 signaling in blood–brain barrier (BBB) endothelial cells (ECs), astrocytes and microglia for EAE development, using mice with the conditional deletion of its signaling receptor IL-1R1. We found that IL-1 signaling in microglia and astrocytes is redundant for the development of EAE, whereas the IL-1R1 deletion in BBB-ECs markedly ameliorated disease severity. IL-1 signaling in BBB-ECs upregulated the expression of the adhesion molecules Vcam-1, Icam-1 and the chemokine receptor Darc, all of which have been previously shown to promote CNS-specific inflammation. In contrast, IL-1R1 signaling suppressed the expression of the stress-responsive heme catabolizing enzyme heme oxygenase-1 (HO-1) in BBB-ECs, promoting disease progression via a mechanism associated with deregulated expression of the IL-1-responsive genes
Vcam1
,
Icam1
and
Ackr1
(Darc). Mechanistically, our data emphasize a functional crosstalk of BBB-EC IL-1 signaling and HO-1, controlling the transcription of downstream proinflammatory genes promoting the pathogenesis of autoimmune neuroinflammation.
Foxp3+ regulatory T (Treg) cells restrict immune pathology in inflamed tissues; however, an inflammatory environment presents a threat to Treg cell identity and function. Here, we establish a ...transcriptional signature of central nervous system (CNS) Treg cells that accumulate during experimental autoimmune encephalitis (EAE) and identify a pathway that maintains Treg cell function and identity during severe inflammation. This pathway is dependent on the transcriptional regulator Blimp1, which prevents downregulation of Foxp3 expression and “toxic” gain-of-function of Treg cells in the inflamed CNS. Blimp1 negatively regulates IL-6- and STAT3-dependent Dnmt3a expression and function restraining methylation of Treg cell-specific conserved non-coding sequence 2 (CNS2) in the Foxp3 locus. Consequently, CNS2 is heavily methylated when Blimp1 is ablated, leading to a loss of Foxp3 expression and severe disease. These findings identify a Blimp1-dependent pathway that preserves Treg cell stability in inflamed non-lymphoid tissues.
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•Most Foxp3+ Treg cells in the inflamed CNS express Blimp1•Blimp1 inhibits Dnmt3a and prevents methylation of the Foxp3 locus•IL-6 contributes to methylation of the Foxp3 locus in a Dnmt3a-dependent manner•Blimp1 counteracts the IL-6-driven destabilization of Treg cells
An inflammatory environment threatens the stability of Foxp3+ Treg cells. Garg et al. show that by expressing the transcriptional regulator Blimp1, Treg cells resist the IL-6-driven loss of Foxp3 in inflamed tissues. Blimp1 prevents the methylation and reduced expression of Foxp3 through inhibition of the methyltransferase Dnmt3a.
The intestinal lamina propria (LP) is a leukocyte-rich cornerstone of the immune system owing to its vital role in immune surveillance and barrier defense against external pathogens. Here, we present ...a protocol for isolating and analyzing immune cell subsets from the mouse intestinal LP for further downstream applications. Starting from tissue collection and cleaning, epithelium removal, and enzymatic digestion to collection of single cells, we explain each step in detail to maximize the yield of immune cells from the intestinal LP.
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•Protocol for the isolation of murine leukocytes from the intestinal lamina propria•Comparative analysis of the isolation efficiency of three widely used digestion enzymes•Assessment of intestinal immune cells using minimal 8-color flow cytometry panels•Advice for choosing the digestion enzyme based on the cell of interest to maximize yield
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
The intestinal lamina propria (LP) is a leukocyte-rich cornerstone of the immune system owing to its vital role in immune surveillance and barrier defense against external pathogens. Here, we present a protocol for isolating and analyzing immune cell subsets from the mouse intestinal LP for further downstream applications. Starting from tissue collection and cleaning, epithelium removal, and enzymatic digestion to collection of single cells, we explain each step in detail to maximize the yield of immune cells from the intestinal LP.
Chronic hepatitis leads to liver fibrosis and cirrhosis. Cirrhosis is a major cause of worldwide morbidity and mortality. Macrophages play a key role in fibrosis progression and reversal. However, ...the signals that determine fibrogenic vs fibrolytic macrophage function remain ill defined. We studied the role of interleukin-4 receptor α (IL-4Rα), a potential central switch of macrophage polarization, in liver fibrosis progression and reversal. We demonstrate that inflammatory monocyte infiltration and liver fibrogenesis were suppressed in general IL-4Rα−/− as well as in macrophage-specific IL-4Rα−/− (IL-4RαΔLysM) mice. However, with deletion of IL-4RαΔLysM spontaneous fibrosis reversal was retarded. Results were replicated by pharmacological intervention using IL-4Rα-specific antisense oligonucleotides. Retarded resolution was linked to the loss of M2-type resident macrophages, which secreted MMP-12 through IL-4 and IL-13-mediated phospho-STAT6 activation. We conclude that IL-4Rα signaling regulates macrophage functional polarization in a context-dependent manner. Pharmacological targeting of macrophage polarization therefore requires disease stage-specific treatment strategies.
Alternative (M2-type) macrophage activation through IL-4Rα promotes liver inflammation and fibrosis progression but speeds up fibrosis reversal. This demonstrates context dependent, opposing roles of M2-type macrophages. During reversal IL-4Rα induces fibrolytic MMPs, especially MMP-12, through STAT6. Liver-specific antisense oligonucleotides efficiently block IL-4Rα expression and attenuate fibrosis progression.
•IL-4Rα is considered a central switch for alternative macrophage polarization.•IL-4Rα on macrophages is shown to differentially regulate liver fibrosis progression vs reversal.•Therapeutic IL-4Rα antisense oligonucleotides replicate these findings.•MMP-12 induced via macrophage IL-4Rα is a key promoter of fibrosis reversal.
Toll-like receptors (TLRs) are crucial pattern recognition receptors in innate immunity that are expressed in microglia, the resident macrophages of the brain. TLR2, -4, and -9 are important in the ...responses against Streptococcus pneumoniae, the most common agent causing bacterial meningitis beyond the neonatal period. Murine microglial cultures were stimulated with agonists for TLR1/2 (Pam₃CSK₄), TLR4 (lipopolysaccharide), and TLR9 (CpG oligodeoxynucleotide) for 24 h and then exposed to either the encapsulated D39 (serotype 2) or the nonencapsulated R6 strain of S. pneumoniae. After stimulation, the levels of interleukin-6 and CCL5 (RANTES regulated upon activation normal T-cell expressed and secreted) were increased, confirming microglial activation. The TLR1/2, -4, and -9 agonist-stimulated microglia ingested significantly more bacteria than unstimulated cells (P < 0.05). The presence of cytochalasin D, an inhibitor of actin polymerizaton, blocked >90% of phagocytosis. Along with an increased phagocytic activity, the intracellular bacterial killing was also increased in TLR-stimulated cells compared to unstimulated cells. Together, our data suggest that microglial stimulation by these TLRs may increase the resistance of the brain against pneumococcal infections.
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