Inflammasomes are multiprotein complexes that link pathogen recognition and cellular stress to the processing of the proinflammatory cytokine interleukin-1␤ (IL-1␤). Whereas inflammasome-mediated activation is heavily studied in hematopoietic macrophages and dendritic cells, much less is known about microglia, resident tissue macrophages of the brain that originate from a distinct progenitor. To directly compare inflammasome-mediated activation in different types of macrophages, we isolated primary microglia and hematopoietic macrophages from adult, healthy rhesus macaques. We analyzed the expression profile of NOD (nucleotide-binding oligomerization domain)-like receptors, adaptor proteins, and caspases and characterized inflammasome activation and regulation in detail. We here demonstrate that primary microglia can respond to the same innate stimuli as hematopoietic macrophages. However, microglial responses are more persistent due to lack of negative regulation on pro-IL-1␤ expression. In addition, we show that while caspase 1, 4, and 5 activation is pivotal for inflammasome-induced IL-1␤ secretion by hematopoietic macrophages, microglial secretion of IL-1␤ is only partially dependent on these inflammatory caspases. These results identify key cell type-specific differences that may aid the development of strategies to modulate innate immune responses in the brain.

Microglia activation is a prominent feature in many neuroinflammatory disorders. Unrestrained activation can generate a chronic inflammatory environment that might lead to neurodegeneration and autoimmunity. Extracellular adenosine modulates cellular activation through adenosine receptor (ADORA)-mediated signaling. There are four ADORA subtypes that can either increase (A2A and A2B receptors) or decrease (A1 and A3 receptors) intracellular cyclic AMP levels. The expression pattern of the subtypes thus orchestrates the cellular response to extracellular adenosine. We have investigated the expression of ADORA subtypes in unstimulated and TLR-activated primary rhesus monkey microglia. Activation induced an up-regulation of A2A and a down-regulation of A3 receptor (A3R) levels. The altered ADORA-expression pattern sensitized microglia to A2A receptor (A2AR)-mediated inhibition of subsequent TLR-induced cytokine responses. By using combinations of subtype-specific agonists and antagonists, we revealed that in unstimulated microglia, A2AR-mediated inhibitory signaling was effectively counteracted by A3R-mediated signaling. In activated microglia, the decrease in A3R-mediated signaling sensitized them to A2AR-mediated inhibitory signaling. We report a differential, activation state-specific expression of ADORA in microglia and uncover a role for A3R as dynamically regulated suppressors of A2AR-mediated inhibition of TLR-induced responses. This would suggest exploration of combinations of A2AR agonists and A3R antagonists to dampen microglial activation during chronic neuroinflammatory conditions.

Activated microglia are found in a variety of neuroinflammatory disorders where they have attributed roles as effector as well as antigen-presenting cells (APC). Critical determinants for the multifaceted role of microglia are the differentiation potential of microglia and their mode of activation. In this study, we have investigated the effects of M-CSF and GM-CSF-mediated differentiation of adult primate microglia on their cellular phenotype, antigen presentation, and phagocytic function as well as on Toll-like receptor (TLR)-mediated responses. We show that although cell morphology and expression levels of activation markers were markedly different, differentiation with either factor yielded microglia that phenotypically and functionally resemble macrophages. Both M-CSF and GM-CSF-differentiated microglia were responsive to TLR1/2, 2, 3, 4, 5, 6/2, and 8-mediated activation, but not to TLR7 or 9-mediated activation. Intriguingly, M-CSF-differentiated microglia expressed higher levels of TLR8-encoding mRNA and protein, and produced larger amounts of proinflammatory cytokines in response to TLR8-mediated activation as compared to GM-CSF-differentiated microglia. While differentiation of adult microglia by growth factors that can be produced endogenously in the central nervous system is thus unlikely to change their APC function, it can alter their innate responses to infectious stimuli such as ssRNA viruses. Resident primate microglia may thereby help shape rather than initiate adaptive immune responses.

Statins inhibit the endogenous intracellular mevalonate pathway and exposure to statins affects innate and adaptive immune responses. Different statins are currently under evaluation as (co)therapy in neuro-inflammatory diseases like multiple sclerosis. However, there are important discrepancies in the reported effects of statins on innate immune responses in different cell types. Studies to characterize such responses in clinically relevant primary cells are currently lacking. In this study, we investigated the effect of statins on Toll-like receptor (TLR)-induced responses of microglia, the resident macrophages of the central nervous system (CNS). Exposure of primary microglia from adult rhesus monkeys to different statins strongly amplified proinflammatory cytokine protein and mRNA levels in response to myeloid differentiation primary response gene 88-dependent TLR activation in particular. Rather than affecting nuclear facor-jB activation levels, statin exposure affected stress-activated protein/Jun-amino-terminal and p38 kinase signaling pathways. Mechanistic studies using specific pathway inhibitors and rescue experiments show that statininduced inhibition of cholesterol biosynthesis, rather than inhibition of isoprenylation, was mainly responsible for the amplified TLR responses. Additionally, microglia were more sensitive to statin-mediated effects than bone marrowderived macrophages of the same donor. This correlated to lower intrinsic microglial expression levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the enzyme targeted by statins. Amplification of TLR-induced responses in microglia by statin exposure might contribute to the generation of a more pro-inflammatory CNS microenvironment which can be of relevance for the pathogenesis of neuroinflammatory disorders. V

TLR-induced signaling potently activates cells of the innate immune system and is subject to regulation at different levels. Inflammatory conditions are associated with increased levels of extracellular adenosine, which can modulate TLR-induced production of cytokines through adenosine receptor–mediated signaling. There are four adenosine receptor subtypes that induce different signaling cascades. In this study, we demonstrate a pivotal contribution of adenosine A3 receptor (A3R)–mediated signaling to the TLR4-induced expression of IL-12 in different types of human myeloid APC. In dendritic cells, IL-12 and CCL2 responses as evoked by TLR2, 3, 4, 5, and 8, as well as IL-12 responses evoked by whole pathogens, were all reduced when A3R-mediated signaling was blocked. As a result, concomitant production of IFN-γ and IL-17 by T cells was significantly inhibited. We further show that selective inhibition of A3R-mediated signaling reduced TLR-induced phosphorylation of the transcription factor STAT1 at tyrosine 701. Next-generation sequencing revealed that A3R-mediated signaling controls the expression of metallothioneins, known inhibitors of STAT1 phosphorylation. Together our results reveal a novel regulatory layer of innate immune responses, with a central role for metallothioneins and autocrine/paracrine signaling via A3Rs.