Statins amplify TLR-induced responses in microglia via inhibition of cholesterol biosynthesis.

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 pro-inflammatory cytokine protein and mRNA levels in response to myeloid differentiation primary response gene 88-dependent TLR activation in particular. Rather than affecting nuclear facor-κB 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 statin-induced 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 marrow-derived 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.

Differential expression of adenosine A3 receptors controls adenosine A2A receptor-mediated inhibition of TLR responses in microglia.

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 (A(2A) and A(2B) receptors) or decrease (A(1) and A(3) 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 A(2A) and a down-regulation of A(3) receptor (A(3)R) levels. The altered ADORA-expression pattern sensitized microglia to A(2A) receptor (A(2A)R)-mediated inhibition of subsequent TLR-induced cytokine responses. By using combinations of subtype-specific agonists and antagonists, we revealed that in unstimulated microglia, A(2A)R-mediated inhibitory signaling was effectively counteracted by A(3)R-mediated signaling. In activated microglia, the decrease in A(3)R-mediated signaling sensitized them to A(2A)R-mediated inhibitory signaling. We report a differential, activation state-specific expression of ADORA in microglia and uncover a role for A(3)R as dynamically regulated suppressors of A(2A)R-mediated inhibition of TLR-induced responses. This would suggest exploration of combinations of A(2A)R agonists and A(3)R antagonists to dampen microglial activation during chronic neuroinflammatory conditions.

Exposure of Microglia to Interleukin-4 Represses NF-κB-Dependent Transcription of Toll-Like Receptor-Induced Cytokines.

Interleukin (IL)-4 is a cytokine that affects both adaptive and innate immune responses. In the central nervous system, microglia express IL-4 receptors and it has been described that IL-4-exposed microglia acquire anti-inflammatory properties. We here demonstrate that IL-4 exposure induces changes in the cell surface protein expression profile of primary rhesus macaque microglia and enhances their potential to induce proliferation of T cells with a regulatory signature. Moreover, we show that Toll like receptor (TLR)-induced cytokine production is broadly impaired in IL-4-exposed microglia at the transcriptional level. IL-4 type 2 receptor-mediated signaling is shown to be crucial for the inhibition of microglial innate immune responses. TLR-induced nuclear translocalization of NF-κB appeared intact, and we found no evidence for epigenetic modulation of target genes. By contrast, nuclear extracts from IL-4-exposed microglia contained significantly less NF-κB capable of binding to its DNA consensus site. Further identification of the molecular mechanisms that underlie the inhibition of TLR-induced responses in IL-4-exposed microglia may aid the design of strategies that aim to modulate innate immune responses in the brain, for example in gliomas.

Induction of progressive demyelinating autoimmune encephalomyelitis in common marmoset monkeys using MOG34-56 peptide in incomplete freund adjuvant.

Experimental autoimmune encephalomyelitis in the neotropical primate common marmoset (Callithrix jacchus) is a relevant autoimmune animal model of multiple sclerosis. T cells specific for peptide 34 to 56 of myelin/oligodendrocyte glycoprotein (MOG34-56) have a central pathogenic role in this model. The aim of this study was to assess the requirement for innate immune stimulation for activation of this core pathogenic autoimmune mechanism. Marmoset monkeys were sensitized against synthetic MOG34-56 peptide alone or in combination with the nonencephalitogenic peptide MOG74-96 formulated in incomplete Freund adjuvant, which lacks microbial components. Experimental autoimmune encephalomyelitis development was recorded by monitoring neurological signs, brain magnetic resonance imaging, and longitudinal profiling of cellular and humoral immune parameters. All monkeys developed autoimmune inflammatory/demyelinating central nervous system disease characterized by massive brain and spinal cord demyelinating white matter lesions with activated macrophages and CD3+ T cells. Immune profiling ex vivo demonstrated the activation of mainly CD3+CD4+/8+CD56+ T cells against MOG34-56. Upon ex vivo stimulation, these T cells produced more interleukin 17A compared with TH1 cytokines (e.g. interferon-gamma) and displayed peptide-specific cytolytic activity. These results indicate that the full spectrum of marmoset experimental autoimmune encephalomyelitis can be induced by sensitization against a single MOG peptide in incomplete Freund adjuvant lacking microbial compounds for innate immune activation and by eliciting antigen-specific T-cell cytolytic activity.

Oligodendrocyte-specific protein is encephalitogenic in rhesus macaques and induces specific demyelination of the optic nerve.

Oligodendrocyte-specific protein (OSP) is a candidate autoantigen in the development of multiple sclerosis (MS). We evaluated the potential of OSP to induce EAE in rhesus monkeys, an out bred animal model for MS that is immunologically close to humans. Since OSP is a four-membrane spanning protein with highly hydrophobic regions, we synthesized recombinant proteins encompassing only the hydrophilic regions of human OSP (soluble (s)hOSP). Immunization with shOSP proteins induced clinical signs and histological features of optic neuritis in four out of ten rhesus monkeys. The development of clinical disease was associated with the presence of a strong cellular proliferative response to the immunizing shOSP protein. Analysis of the cellular responses in combination with neuropathological observations also indicates an important role for neutrophils in the disease process. Interestingly, all immunized monkeys developed antibody responses to OSP peptide 103-123, a B cell epitope previously identified in MS patients. These responses did not correlate with the development of clinical disease, but may have relevance as a biomarker for immunoreactivity towards OSP in myelin disorders. Our data demonstrate that in rhesus monkeys immune responses directed at OSP are encephalitogenic, leading to inflammatory responses throughout the central nervous system and to selective demyelination of the optic nerve.