Hedwich F. Kuipers

Common variants in P2RY11 are associated with narcolepsy

Growing evidence supports the hypothesis that narcolepsy with cataplexy is an autoimmune disease. We here report genome-wide association analyses for narcolepsy with replication and fine mapping across three ethnic groups (3,406 individuals of European ancestry, 2,414 Asians and 302 African Americans). We identify a SNP in the 3′ untranslated region of P2RY11, the purinergic receptor subtype P2Y11 gene, which is associated with narcolepsy (rs2305795, combined P = 6.1 × 10−10, odds ratio = 1.28, 95% CI 1.19–1.39, n = 5689). The disease-associated allele is correlated with reduced expression of P2RY11 in CD8+ T lymphocytes (72% reduced, P = 0.003) and natural killer (NK) cells (70% reduced, P = 0.031), but not in other peripheral blood mononuclear cell types. The low expression variant is also associated with reduced P2RY11-mediated resistance to ATP-induced cell death in T lymphocytes (P = 0.0007) and natural killer cells (P = 0.001). These results identify P2RY11 as an important regulator of immune-cell survival, with possible implications in narcolepsy and other autoimmune diseases.

Reversal of Paralysis and Reduced Inflammation from Peripheral Administration of β-Amyloid in TH1 and TH17 Versions of Experimental Autoimmune Encephalomyelitis

β-Amyloid reduces inflammation and improves symptoms in a mouse model of multiple sclerosis. When Being Bad Is Good β-Amyloid (Aβ) is one of the supervillains of the Alzheimer’s disease (AD) world. Aβ plaques are a hallmark of the disease and are thought to promote neurotoxicity and inflammation. But what happens when you take Aβ out of the context of AD and examine its role in another disease, such as multiple sclerosis (MS)? Grant et al. do just that in a mouse model of MS—experimental autoimmune encephalomyelitis (EAE)—and find that in some situations, Aβ might not be so bad after all. In four models of EAE that included both preventative and therapeutic ones, the authors found, contrary to their expectations, that treating with Aβ peptides decreased both inflammation and the severity of disease. Conversely, in EAE mice genetically altered to lack amyloid precursor protein, inflammation was more severe and MS-like symptoms were exacerbated. These anti-inflammatory effects of Aβ appear to depend on immune cells because treatment was also successful in models of EAE caused by transplantation of pathogenic T cells. Although more mechanistic and safety studies need to be performed before Aβ is ready for therapeutic prime time, these data highlight the molecule’s potential anti-inflammatory effects in selected contexts. Like some other presumed villains, Aβ might not be unequivocally bad, just misunderstood. β-Amyloid 42 (Aβ42) and β-amyloid 40 (Aβ40), major components of senile plaque deposits in Alzheimer’s disease, are considered neurotoxic and proinflammatory. In multiple sclerosis, Aβ42 is up-regulated in brain lesions and damaged axons. We found, unexpectedly, that treatment with either Aβ42 or Aβ40 peptides reduced motor paralysis and brain inflammation in four different models of experimental autoimmune encephalomyelitis (EAE) with attenuation of motor paralysis, reduction of inflammatory lesions in the central nervous system (CNS), and suppression of lymphocyte activation. Aβ42 and Aβ40 treatments were effective in reducing ongoing paralysis induced with adoptive transfer of either autoreactive T helper 1 (TH1) or TH17 cells. High-dimensional 14-parameter flow cytometry of peripheral immune cell populations after in vivo Aβ42 and Aβ40 treatment revealed substantial modulations in the percentage of lymphoid and myeloid subsets during EAE. Major proinflammatory cytokines and chemokines were reduced in the blood after Aβ peptide treatment. Protection conferred by Aβ treatment did not require its delivery to the brain: Adoptive transfer with lymphocytes from donors treated with Aβ42 attenuated EAE in wild-type recipient mice, and Aβ deposition in the brain was not detected in treated EAE mice by immunohistochemical analysis. In contrast to the improvement in EAE with Aβ treatment, EAE was worse in mice with genetic deletion of the amyloid precursor protein. Therefore, in the absence of Aβ, there is exacerbated clinical EAE disease progression. Because Aβ42 and Aβ40 ameliorate experimental autoimmune inflammation targeting the CNS, we might now consider its potential anti-inflammatory role in other neuropathological conditions.

Inhibition of hyaluronan synthesis restores immune tolerance during autoimmune insulitis.

We recently reported that abundant deposits of the extracellular matrix polysaccharide hyaluronan (HA) are characteristic of autoimmune insulitis in patients with type 1 diabetes (T1D), but the relevance of these deposits to disease was unclear. Here, we have demonstrated that HA is critical for the pathogenesis of autoimmune diabetes. Using the DO11.10xRIPmOVA mouse model of T1D, we determined that HA deposits are temporally and anatomically associated with the development of insulitis. Moreover, treatment with an inhibitor of HA synthesis, 4-methylumbelliferone (4-MU), halted progression to diabetes even after the onset of insulitis. Similar effects were seen in the NOD mouse model, and in these mice, 1 week of treatment was sufficient to prevent subsequent diabetes. 4-MU reduced HA accumulation, constrained effector T cells to nondestructive insulitis, and increased numbers of intraislet FOXP3+ Tregs. Consistent with the observed effects of 4-MU treatment, Treg differentiation was inhibited by HA and anti-CD44 antibodies and rescued by 4-MU in an ERK1/2-dependent manner. These data may explain how peripheral immune tolerance is impaired in tissues under autoimmune attack, including islets in T1D. We propose that 4-MU, already an approved drug used to treat biliary spasm, could be repurposed to prevent, and possibly treat, T1D in at-risk individuals.

Hyaluronan synthesis is necessary for autoreactive T-cell trafficking, activation, and Th1 polarization

Significance 4-methylumbelliferone (4-MU) is an oral drug that inhibits synthesis of hyaluronan, an extracellular matrix polymer implicated in autoimmunity. In the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS), 4-MU drives the polarization of T cells away from a Th1 phenotype, associated with disease progression, and toward a FoxP3+ regulatory T-cell phenotype, associated with disease prevention. Moreover, 4-MU inhibits the reactive response of astrocytes, immunocompetent resident cells of the CNS, and prevents trafficking of activated T cells to the CNS. These data suggest that the extracellular matrix, and hyaluronan in particular, may be an active contributor to autoimmune pathogenesis in EAE and, furthermore, that 4-MU is a promising strategy for treatment of CNS autoimmunity. The extracellular matrix polysaccharide hyaluronan (HA) accumulates at sites of autoimmune inflammation, including white matter lesions in multiple sclerosis (MS), but its functional importance in pathogenesis is unclear. We have evaluated the impact of 4-methylumbelliferone (4-MU), an oral inhibitor of HA synthesis, on disease progression in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Treatment with 4-MU decreases the incidence of EAE, delays its onset, and reduces the severity of established disease. 4-MU inhibits the activation of autoreactive T cells and prevents their polarization toward a Th1 phenotype. Instead, 4-MU promotes polarization toward a Th2 phenotpye and induction of Foxp3+ regulatory T cells. Further, 4-MU hastens trafficking of T cells through secondary lymphoid organs, impairs the infiltration of T cells into the CNS parenchyma, and limits astrogliosis. Together, these data suggest that HA synthesis is necessary for disease progression in EAE and that treatment with 4-MU may be a potential therapeutic strategy in CNS autoimmunity. Considering that 4-MU is already a therapeutic, called hymecromone, that is approved to treat biliary spasm in humans, we propose that it could be repurposed to treat MS.

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.

The pharmacokinetics and dosing of oral 4-methylumbelliferone for inhibition of hyaluronan synthesis in mice.

Recently, there has been considerable interest in using 4‐methylumbelliferone (4‐MU) to inhibit hyaluronan (HA) synthesis in mouse models of cancer, autoimmunity and a variety of other inflammatory disorders where HA has been implicated in disease pathogenesis. In order to facilitate future studies in this area, we have examined the dosing, treatment route, treatment duration and metabolism of 4‐MU in both C57BL/6 and BALB/c mice. Mice fed chow containing 5% 4‐MU, a dose calculated to deliver 250 mg/mouse/day, initially lose substantial weight but typically resume normal weight gain after 1 week. It also takes up to a week to see a reduction in serum HA in these animals, indicating that at least a 1‐week loading period on the drug is required for most protocols. At steady state, more than 90% of the drug is present in plasma as the glucuronidated metabolite 4‐methylumbelliferyl glucuronide (4‐MUG), with the sulphated metabolite, 4‐methylumbelliferyl sulphate (4‐MUS) comprising most of the remainder. Chow containing 5% but not 0·65% 4‐MU was effective at preventing disease in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis, as well as in the DORmO mouse model of autoimmune diabetes. While oral 4‐MU was effective at preventing EAE, daily intraperitoneal injections of 4‐MU were not. Factors potentially affecting 4‐MU uptake and plasma concentrations in mice include its taste, short half‐life and low bioavailability. These studies provide a practical resource for implementing oral 4‐MU treatment protocols in mice.

Phosphorylation of αB-crystallin supports reactive astrogliosis in demyelination

Significance αB-crystallin (CRYAB) is a protein involved in the protection of cells from stress and cell death, and is very highly produced in brain lesions in multiple sclerosis (MS). Astrocytes are the main non-neuronal cell type in the central nervous system. Although they are involved in many processes in health and injury, their precise role in MS is poorly understood. Using a mouse model of MS, as well as brain tissue from MS patients, we found that CRYAB enables astrocytes to become activated and that these activated astrocytes play a pathogenic role in MS, worsening injury. Furthermore, we found that a specific stress-induced modification of CRYAB supports its role in astrocyte activation and affects the intracellular signaling pathways it regulates. The small heat shock protein αB-crystallin (CRYAB) has been implicated in multiple sclerosis (MS) pathogenesis. Earlier studies have indicated that CRYAB inhibits inflammation and attenuates clinical disease when administered in the experimental autoimmune encephalomyelitis model of MS. In this study, we evaluated the role of CRYAB in primary demyelinating events. Using the cuprizone model of demyelination, a noninflammatory model that allows the analysis of glial responses in MS, we show that endogenous CRYAB expression is associated with increased severity of demyelination. Moreover, we demonstrate a strong correlation between the expression of CRYAB and the extent of reactive astrogliosis in demyelinating areas and in in vitro assays. In addition, we reveal that CRYAB is differentially phosphorylated in astrocytes in active demyelinating MS lesions, as well as in cuprizone-induced lesions, and that this phosphorylation is required for the reactive astrocyte response associated with demyelination. Furthermore, taking a proteomics approach to identify proteins that are bound by the phosphorylated forms of CRYAB in primary cultured astrocytes, we show that there is clear differential binding of protein targets due to the specific phosphorylation of CRYAB. Subsequent Ingenuity Pathway Analysis of these targets reveals implications for intracellular pathways and biological processes that could be affected by these modifications. Together, these findings demonstrate that astrocytes play a pivotal role in demyelination, making them a potential target for therapeutic intervention, and that phosphorylation of CRYAB is a key factor supporting the pathogenic response of astrocytes to oligodendrocyte injury.

Piet Mondrian's trees and the evolution in understanding multiple sclerosis, Charcot Prize Lecture 2011

Four questions were posed about multiple sclerosis (MS) at the 2011 Charcot Lecture, Oct. 22, 2011. 1. The Male/Female Disparity: Why are women developing MS so much more frequently than men? 2. Neuronal and Glial Protection: Are there guardian molecules that protect the nervous system in MS? 3. Predictive Medicine: With all the approved drugs, how can we rationally decide which one to use? 4. The Precise Scalpel vs. the Big Hammer for Therapy: Is antigen-specific therapy for demyelinating disease possible? To emphasize how our views on the pathogenesis and treatment of MS are evolving, and given the location of the talk in Amsterdam, Piet Mondrian’s progressive interpretations of trees serve as a heuristic.

Hyaluronan in immune dysregulation and autoimmune diseases

The tissue microenvironment contributes to local immunity and to the pathogenesis of autoimmune diseases - a diverse set of conditions characterized by sterile inflammation, immunity against self-antigens, and destruction of tissues. However, the specific factors within the tissue microenvironment that contribute to local immune dysregulation in autoimmunity are poorly understood. One particular tissue component implicated in multiple autoimmune diseases is hyaluronan (HA), an extracellular matrix (ECM) polymer. HA is abundant in settings of chronic inflammation and contributes to lymphocyte activation, polarization, and migration. Here, we first describe what is known about the size, amount, and distribution of HA at sites of autoimmunity and in associated lymphoid structures in type 1 diabetes, multiple sclerosis, and rheumatoid arthritis. Next, we examine the recent literature on HA and its impact on adaptive immunity, particularly in regards to the biology of lymphocytes and Foxp3+ regulatory T-cells (Treg), a T-cell subset that maintains immune tolerance in healthy individuals. We propose that HA accumulation at sites of chronic inflammation creates a permissive environment for autoimmunity, characterized by CD44-mediated inhibition of Treg expansion. Finally, we address potential tools and strategies for targeting HA and its receptor CD44 in chronic inflammation and autoimmunity.

The role of hyaluronan and 4-methylumbelliferone treatment in multiple sclerosis

cells with a naive phenotype decreased from baseline to Month 1 (36.7% to 2.3%), whereas the percentage of CD4 memory cells increased (62.5% to 97.4%). The percentages of both cell types then gradually returned toward baseline levels, but did not reach baseline levels by Month 12. The proportion of CD4 T cells with a regulatory phenotype increased from baseline to Month 1 (3.8% to 12.5%), and remained elevated at Month 12. A similar pattern was observed for CD8 T-cell subsets. The proportion of B cells with a mature naive phenotype decreased from 56.4% to 5.4% from baseline to Month 1, whereas the immature cell fraction increased from 4.7% to 36.8%; relative proportions then approached baseline levels by Month 6. Conclusions:Alemtuzumab-induced lymphocytedepletionwasselective andresulted inanincreasedproportionofmemoryandregulatoryTcells. A distinctive pattern of lymphocyte repopulation was observed within weeks. These effects may explain alemtuzumabs sustained benefit despite infrequent (yearly) administration. Study Supported by: Genzyme, a Sanofi Company, and Bayer Healthcare Pharmaceuticals.