The therapeutic effect of GAS6 in remyelination is dependent upon Tyro3.

Multiple sclerosis is an autoimmune disease of the central nervous system (CNS) characterized by demyelination, axonal damage and, for the majority of people, a decline in neurological function in the long-term. Remyelination could assist in the protection of axons and their functional recovery, but such therapies are not, as yet, available. The TAM (Tyro3, Axl, and MERTK) receptor ligand GAS6 potentiates myelination in vitro and promotes recovery in pre-clinical models of MS. However, it has remained unclear which TAM receptor is responsible for transducing this effect and whether post-translational modification of GAS6 is required. In this study, we show that the promotion of myelination requires post-translational modification of the GLA domain of GAS6 via vitamin K-dependent γ-carboxylation. We also confirmed that the intracerebroventricular provision of GAS6 for 2 weeks to demyelinated wild-type (WT) mice challenged with cuprizone increased the density of myelinated axons in the corpus callosum by over 2-fold compared with vehicle control. Conversely, the provision of GAS6 to Tyro3 KO mice did not significantly improve the density of myelinated axons. The improvement in remyelination following the provision of GAS6 to WT mice was also accompanied by an increased density of CC1+ve mature oligodendrocytes compared with vehicle control, whereas this improvement was not observed in the absence of Tyro3. This effect occurs independent of any influence on microglial activation. This work therefore establishes that the remyelinative activity of GAS6 is dependent on Tyro3 and includes potentiation of oligodendrocyte numbers.

Mertk-expressing microglia influence oligodendrogenesis and myelin modelling in the CNS.

BACKGROUND: Microglia, an immune cell found exclusively within the CNS, initially develop from haematopoietic stem cell precursors in the yolk sac and colonise all regions of the CNS early in development. Microglia have been demonstrated to play an important role in the development of oligodendrocytes, the myelin producing cells in the CNS, as well as in myelination. Mertk is a receptor expressed on microglia that mediates immunoregulatory functions, including myelin efferocytosis. FINDINGS: Here we demonstrate an unexpected role for Mertk-expressing microglia in both oligodendrogenesis and myelination. The selective depletion of Mertk from microglia resulted in reduced oligodendrocyte production in early development and the generation of pathological myelin. During demyelination, mice deficient in microglial Mertk had thinner myelin and showed signs of impaired OPC differentiation. We established that Mertk signalling inhibition impairs oligodendrocyte repopulation in Xenopus tadpoles following demyelination. CONCLUSION: These data highlight the importance of microglia in myelination and are the first to identify Mertk as a regulator of oligodendrogenesis and myelin ultrastructure.

Mouse microglia express unique miRNA-mRNA networks to facilitate age-specific functions in the developing central nervous system.

Microglia regulate multiple processes in the central nervous system, exhibiting a considerable level of cellular plasticity which is facilitated by an equally dynamic transcriptional environment. While many gene networks that regulate microglial functions have been characterised, the influence of epigenetic regulators such as small non-coding microRNAs (miRNAs) is less well defined. We have sequenced the miRNAome and mRNAome of mouse microglia during brain development and adult homeostasis, identifying unique profiles of known and novel miRNAs. Microglia express both a consistently enriched miRNA signature as well as temporally distinctive subsets of miRNAs. We generated robust miRNA-mRNA networks related to fundamental developmental processes, in addition to networks associated with immune function and dysregulated disease states. There was no apparent influence of sex on miRNA expression. This study reveals a unique developmental trajectory of miRNA expression in microglia during critical stages of CNS development, establishing miRNAs as important modulators of microglial phenotype.

Cladribine Treatment for MS Preserves the Differentiative Capacity of Subsequently Generated Monocytes, Whereas Its Administration In Vitro Acutely Influences Monocyte Differentiation but Not Microglial Activation.

Cladribine (2-chlorodeoxyadenosine, 2CdA) is one of the most effective disease-modifying drugs for multiple sclerosis (MS). Cladribine is a synthetic purine nucleoside analog that induces cell death of lymphocytes and oral cladribine treatment leads to a long-lasting disease stabilization, potentially attributable to immune reconstitution. In addition to its effects on lymphocytes, cladribine has been shown to have immunomodulatory effects on innate immune cells, including dendritic cells and monocytes, which could also contribute to its therapeutic efficacy. However, whether cladribine can modulate human macrophage/microglial activation or monocyte differentiation is currently unknown. The aim of this study was to determine the immunomodulatory effects of cladribine upon monocytes, monocyte-derived macrophages (MDMs) and microglia. We analyzed the phenotype and differentiation of monocytes from MS patients receiving their first course of oral cladribine both before and three weeks after the start of treatment. Flow cytometric analysis of monocytes from MS patients undergoing cladribine treatment revealed that the number and composition of CD14/CD16 monocyte subsets remained unchanged after treatment. Furthermore, after differentiation with M-CSF, such MDMs from treated MS patients showed no difference in gene expression of the inflammatory markers compared to baseline. We further investigated the direct effects of cladribine in vitro using human adult primary MDMs and microglia. GM-CSF-derived MDMs were more sensitive to cell death than M-CSF-derived MDMs. In addition, MDMs treated with cladribine showed increased expression of costimulatory molecules CD80 and CD40, as well as expression of anti-inflammatory, pro-trophic genes IL10 and MERTK, depending on the differentiation condition. Cladribine treatment in vitro did not modulate the expression of activation markers in human microglia. Our study shows that cladribine treatment in vitro affects the differentiation of monocytes into macrophages by modulating the expression of activation markers, which might occur similarly in tissue after their infiltration in the CNS during MS.

White matter tract conductivity is resistant to wide variations in paranodal structure and myelin thickness accompanying the loss of Tyro3: an experimental and simulated analysis.

Myelination within the central nervous system (CNS) is crucial for the conduction of action potentials by neurons. Variation in compact myelin morphology and the structure of the paranode are hypothesised to have significant impact on the speed of action potentials. There are, however, limited experimental data investigating the impact of changes in myelin structure upon conductivity in the central nervous system. We have used a genetic model in which myelin thickness is reduced to investigate the effect of myelin alterations upon action potential velocity. A detailed examination of the myelin ultrastructure of mice in which the receptor tyrosine kinase Tyro3 has been deleted showed that, in addition to thinner myelin, these mice have significantly disrupted paranodes. Despite these alterations to myelin and paranodal structure, we did not identify a reduction in conductivity in either the corpus callosum or the optic nerve. Exploration of these results using a mathematical model of neuronal conductivity predicts that the absence of Tyro3 would lead to reduced conductivity in single fibres, but would not affect the compound action potential of multiple myelinated neurons as seen in neuronal tracts. Our data highlight the importance of experimental assessment of conductivity and suggests that simple assessment of structural changes to myelin is a poor predictor of neural functional outcomes.

Development of [18F]MIPS15692, a radiotracer with in vitro proof-of-concept for the imaging of MER tyrosine kinase (MERTK) in neuroinflammatory disease.

MER tyrosine kinase (MERTK) upregulation is associated with M2 polarization of microglia, which plays a vital role in neuroregeneration following damage induced by neuroinflammatory diseases such as multiple sclerosis (MS). Therefore, a radiotracer specific for MERTK could be of great utility in the clinical management of MS, for the detection and differentiation of neuroregenerative and neurodegenerative processes. This study aimed to develop an [18F] ligand with high affinity and selectivity for MERTK as a potential positron emission tomography (PET) radiotracer. MIPS15691 and MIPS15692 were synthesized and kinase assays were utilized to determine potency and selectivity for MERTK. Both compounds were shown to be potent against MERTK, with respective IC50 values of 4.6 nM and 4.0 nM, and were also MERTK-selective. Plasma and brain pharmacokinetics were measured in mice and led to selection of MIPS15692 over MIPS15691. X-ray crystallography was used to visualize how MIPS15692 is recognized by the enzyme. [18F]MIPS15692 was synthesized using an automated iPHASE FlexLab module, with a molar activity (Am) of 49 ± 26 GBq/µmol. The radiochemical purity of [18F]MIPS15692 was >99% and the decay-corrected radiochemical yields (RCYs) were determined as 2.45 ± 0.85%. Brain MERTK protein density was measured by a saturation binding assay in the brain slices of a cuprizone mouse model of MS. High levels of specific binding of [18F]MIPS15692 to MERTK were found, especially in the corpus callosum/hippocampus (CC/HC). The in vivo PET imaging study of [18F]MIPS15692 suggested that its neuroPK is sub-optimal for clinical use. Current efforts are underway to optimize the neuroPK of our next generation PET radiotracers for maximal in vivo utility.

The TAM receptor TYRO3 is a critical regulator of myelin thickness in the central nervous system.

Multiple sclerosis (MS) is an autoimmune, demyelinating disease of the central nervous system (CNS). Major deficits arise in MS patients due to an inability to repair damaged myelin sheaths following CNS insult, resulting in prolonged axonal exposure and neurodegeneration. The TAM receptors (Tyro3, Axl, and Mertk) have been implicated in MS susceptibility, demyelination and remyelination. Previously, we have shown that Tyro3 regulates developmental myelination and myelin thickness within the optic nerve and rostral region of the corpus callosum (CC) of adult mice. In this study we have verified and extended our previous findings via a comprehensive analysis of axonal ensheathment and myelin thickness in the CC of unchallenged mice, following demyelination and during myelin repair. We show that the loss of the Tyro3 receptor correlates with significantly thinner myelin sheaths in both unchallenged mice and during remyelination, particularly in larger caliber axons. The hypomyelinated phenotype observed in the absence of Tyro3 occurs independently of any influence upon oligodendrocyte precursor cell (OPC) maturation, or density of oligodendrocytes (OLs) or microglia. Rather, the primary effect of Tyro3 is upon the radial expansion of myelin. The loss of Tyro3 leads to a reduction in the number of myelin lamellae on axons, and is therefore most likely a key component of the regulatory mechanism by which oligodendrocytes match myelin production to axonal diameter.

The TAM receptor Tyro3 regulates myelination in the central nervous system.

Myelin is an essential component of the mammalian nervous system, facilitating rapid conduction of electrical impulses by axons, as well as providing trophic support to neurons. Within the central nervous system, the oligodendrocyte is the specialized neural cell responsible for producing myelin by a process that is thought to be regulated by both activity dependent and independent mechanisms but in incompletely understood ways. We have previously identified that the protein Gas6, a ligand for a family of tyrosine kinase receptors known as the TAM (Tyro3, Axl, and Mertk) receptors, directly increases oligodendrocyte induced myelination in vitro. Gas6 can bind to and activate all three TAM receptors, but the high level of expression of Tyro3 on oligodendrocytes makes this receptor the principal candidate for transducing the pro-myelinating effect of Gas6. In this study, we establish that in the absence of Tyro3, the pro-myelinating effect of Gas6 is lost, that developmental myelination is delayed and that the myelin produced is thinner than normal. We show that this effect is specific to the myelination process and not due to changes in the proliferation or differentiation of oligodendrocyte precursor cells. We have further demonstrated that the reduction in myelination is due to the loss of Tyro3 on oligodendrocytes, and this effect may be mediated by activation of Erk1. Collectively, our findings indicate the critical importance of Tyro3 in potentiating central nervous system myelination. GLIA 2017 GLIA 2017;65:581-591.

Raised soluble P-selectin moderately accelerates atherosclerotic plaque progression.

Soluble P-selectin (sP-selectin), a biomarker of inflammatory related pathologies including cardiovascular and peripheral vascular diseases, also has pro-atherosclerotic effects including the ability to increase leukocyte recruitment and modulate thrombotic responses in vivo. The current study explores its role in progressing atherosclerotic plaque disease. Apoe-/- mice placed on a high fat diet (HFD) were given daily injections of recombinant dimeric murine P-selectin (22.5 µg/kg/day) for 8 or 16 weeks. Saline or sE-selectin injections were used as negative controls. In order to assess the role of sP-selectin on atherothrombosis an experimental plaque remodelling murine model, with sm22α-hDTR Apoe-/- mice on a HFD in conjunction with delivery of diphtheria toxin to induce targeted vascular smooth muscle apoptosis, was used. These mice were similarly given daily injections of sP-selectin for 8 or 16 weeks. While plaque mass and aortic lipid content did not change with sP-selectin treatment in Apoe-/- or SM22α-hDTR Apoe-/- mice on HFD, increased plasma MCP-1 and a higher plaque CD45 content in Apoe-/- HFD mice was observed. As well, a significant shift towards a more unstable plaque phenotype in the SM22α-hDTR Apoe-/- HFD mice, with increased macrophage accumulation and lower collagen content, leading to a lower plaque stability index, was observed. These results demonstrate that chronically raised sP-selectin favours progression of an unstable atherosclerotic plaque phenotype.

Ensuring animal welfare while meeting scientific aims using a murine pneumonia model of septic shock.

With animal models, death as an intentional end point is ethically unacceptable. However, in the study of septic shock, death is still considered the only relevant end point. We defined eight humane end points into four stages of severity (from healthy to moribund) and used to design a clinically relevant scoring tool, termed "the mouse clinical assessment score for sepsis" (M-CASS). The M-CASS was used to enable a consistent approach to the assessment of disease severity. This allowed an ethical and objective assessment of disease after which euthanasia was performed, instead of worsening suffering. The M-CASS displayed a high internal consistency (Cronbach α = 0.97) with a high level of agreement and an intraclass correlation coefficient equal to 0.91. The plasma levels of cytokines and markers of oxidative stress were all associated with the M-CASS score (Kruskal-Wallis test, P < 0.05). The M-CASS allows tracking of disease progression and animal welfare requirements.

Anti-inflammatory functions of apolipoprotein A-I and high-density lipoprotein are preserved in trimeric apolipoprotein A-I.

Raising high-density lipoprotein (HDL) levels is proposed as an attractive target to treat cardiovascular disease. However, a number of clinical studies examining the effect of HDL-raising therapies have been prematurely halted due to futility. Therefore there is a need for alternative therapies. Infusion of reconstituted HDL (rHDL) particles is still considered as a viable approach to increasing HDL levels. In this study we have profiled the anti-inflammatory effects of a trimeric-HDL particle. We show that trimeric apoA-I and rHDL particles promote cholesterol efflux to a similar rate as native apoA-I particles in both ABCA1-dependent and -independent pathways. Trimeric particles inhibited ICAM-1 and VCAM-1 expression and the ability of the endothelium to capture monocytes under shear flow. Monocyte activation, CD11b-dependent adhesion, and monocyte recruitment under shear flow conditions were perturbed by the trimeric particles. Our data suggest that trimeric rHDL particles can be constructed without any loss of function, preserving the anti-inflammatory effects of HDL that are key to its in vivo actions.

Interleukin 10 antioxidant effect decreases leukocytes/endothelial interaction induced by tumor necrosis factor α.

Little is known about the endothelial mechanisms involved in the anti-inflammatory effects of interleukin 10 (IL-10). The goal of this study was to evaluate the effects of IL-10 on endothelial oxidative stress and endothelial inflammation induced by tumor necrosis factor α (TNF-α). Production of reactive oxygen species (ROS) in perfused human umbilical vein endothelial cells (HUVECs) was studied by fluorescent microscopy using dichlorodihydrofluorescein diacetate. Tumor necrosis factor α (1 ng/mL) was added to the perfusion medium in the absence and presence of IL-10 (1 ng/mL). The role of phosphatidylinositol 3-kinase (PI3-kinase) was assessed using wortmannin and LY 2940002 (inhibitors of PI3-kinase). Specific inhibition of p110 α and p110 γ/δ PI3-kinase subunits was studied using A66 and TG100-115. As well, levels of ceramide and intercellular adhesion molecule 1 (ICAM-1) expression were measured. Finally, the effect of IL-10 on TNF-α-induced leukocyte/endothelium interaction was examined using an ex vivo perfused vessel model. Interleukin 10 significantly reduced dichlorodihydrofluorescein diacetate fluorescence induced by TNF-α in HUVECs (12.5% ± 3.2% vs. 111.7% ± 21.6% at 60 min). Pretreatment by LY2940002 or wortmannin restored ROS production induced by TNF-α in the presence of IL-10. In HUVECs treated by TNF-α + IL-10, inhibition of p110 α PI3-kinase subunit significantly increased ROS production, whereas p110 γ/δ inhibition did not have a significant effect. Pretreatment with IL-10 significantly decreased TNF-α-induced increased levels of ceramide (TNF-α vs. TNF-α + IL-10: 6,278 ± 1,013 vs. 1,440 ± 130 pmol/mg prot), as well as ICAM-1 expression and leukocyte adhesion (TNF-α vs. TNF-α + IL-10: 26.8 ± 2.6 vs. 6.7 ± 0.4 adherent leukocytes/field at 15 min). Interleukin 10 decreases the level of inflammation induced by TNF-α in endothelial cells by reducing the TNF-α-induced ROS production, ICAM-1 expression, and leukocyte adhesion to the endothelium. The antioxidant effect of IL-10 is mediated through PI3-kinase and is paralleled by a decrease in ceramide synthesis induced by TNF-α.

High molecular weight gingipains from Porphyromonas gingivalis induce cytokine responses from human macrophage-like cells via a nonproteolytic mechanism.

Periodontal disease is an oral inflammatory disease affecting the supporting structures of teeth. Porphyromonas gingivalis, a major pathogenic agent for the disease, expresses a number of virulence factors, including cysteine proteases called the gingipains. The arginine- and lysine-specific gingipains, HRgpA and Kgp, respectively, are expressed as high molecular weight forms containing both catalytic and adhesin subunits. We examined the expression pattern of cytokines and their receptors in differentiated macrophages following exposure to active and inactive forms of the gingipains, using a cDNA array, quantitative PCR and ELISA analysis. Amongst other pro-inflammatory cytokines, results from the cDNA array suggested that interleukin-1beta, granulocyte-macrophage colony stimulatory factor and interferon-gamma were upregulated after exposure of the macrophages to the gingipains. Quantitative PCR analysis substantiated these observations and indicated that active or inactive forms of the high molecular weight gingipains were able to upregulate expression of transcripts for these cytokines. The strongly enhanced production of interleukin-1beta and granulocyte-macrophage colony stimulatory factor by differentiated macrophages in response to active or inactive forms of the high molecular weight gingipains was confirmed at the protein level by ELISA analysis. The results indicate that the adhesin subunits of the gingipains mediate strong upregulation of the expression of pro-inflammatory cytokines in macrophages.

Leucine-rich repeats 2-4 (Leu60-Glu128) of platelet glycoprotein Ibalpha regulate shear-dependent cell adhesion to von Willebrand factor.

Glycoprotein (GP) Ib-IX-V binds von Willebrand factor (VWF), initiating thrombosis at high shear stress. The VWF-A1 domain binds the N-terminal domain of GPIbalpha (His1-Glu282); this region contains seven leucine-rich repeats (LRR) plus N- and C-terminal flanking sequences and an anionic sequence containing three sulfated tyrosines. Our previous analysis of canine/human and human/canine chimeras of GPIbalpha expressed on Chinese hamster ovary (CHO) cells demonstrated that LRR2-4 (Leu60-Glu128) were crucial for GPIbalpha-dependent adhesion to VWF. Paradoxically, co-crystal structures of the GPIbalpha N-terminal domain and GPIbalpha-binding VWF-A1 under static conditions revealed that the LRR2-4 sequence made minimal contact with VWF-A1. To resolve the specific functional role of LRR2-4, we compared wild-type human GPIbalpha with human GPIbalpha containing a homology domain swap of canine for human sequence within Leu60-Glu128 and a reverse swap (canine GPIbalpha with human Leu60-Glu128) for the ability to support adhesion to VWF under flow. Binding of conformation-specific anti-GPIbalpha antibodies and VWF binding in the presence of botrocetin (which does not discriminate between species) confirmed equivalent expression of wild-type and mutant receptors in a functional form competent to bind ligand. Compared with CHO cells expressing wild-type GPIbalpha, cells expressing GPIbalpha, where human Leu60-Glu128 sequence was replaced by canine sequence, supported adhesion to VWF at low shear rates but became increasingly ineffective as shear increased from 50 to 2000 s(-1). Together, these data demonstrate that LRR2-4, encompassing a pronounced negative charge patch on human GPIbalpha, is essential for GPIbalpha.VWF-dependent adhesion as hydrodynamic shear increases.

Functional analysis of the C-terminal flanking sequence of platelet glycoprotein Ib alpha using canine-human chimeras.

Platelet glycoprotein Ib-IX-V (GPIb-IX-V) mediates adhesion to von Willebrand factor (vWF) in (patho)physiological thrombus formation. vWF binds the N-terminal 282 residues of GPIb alpha, consisting of an N-terminal flank (His1-Ile35), 7 leucine-rich repeats (Leu36-Ala200), a C-terminal flank (Phe201-Gly268), and a sulfated tyrosine sequence (Asp269-Glu282). By expressing canine-human chimeras of GPIb alpha on Chinese hamster ovary cells, binding sites for functional anti-GPIb alpha antibodies to individual domains were previously mapped, and it was shown that leucine-rich repeats 2 to 4 were required for optimal vWF recognition under static or flow conditions. Using novel canine-human chimeras dissecting the C-terminal flank, it is now demonstrated that (1) Phe201-Glu225 contains the epitope for AP1, an anti-GPIb alpha monoclonal antibody that inhibits both ristocetin- and botrocetin-dependent vWF binding; (2) VM16d, an antibody that preferentially inhibits botrocetin-dependent vWF binding, recognizes the sequence Val226-Gly268, surrounding Cys248, which forms a disulfide-bond with Cys209; (3) vWF binding to chimeric GPIb alpha is comparable to wild-type in 2 chimeras in which the sixth leucine-rich repeat was of the same species as the first disulfide loop (Phe201-Cys248) of the C-terminal flank, suggesting an interaction between these domains may be important for optimal vWF binding; and (4) replacing the C-terminal flank second disulfide loop (Asp249-Gly268) in human GPIb alpha with the corresponding canine sequence enhanced vWF binding under static and flow conditions, providing the first evidence for a gain-of-function phenotype associated with the second loop of the C-terminal flank.