Transcriptional network analysis of peripheral blood leukocyte subsets in multiple sclerosis identifies a pathogenic role for a cytotoxicity-associated gene network in myeloid cells.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system affecting predominantly adults. It is a complex disease associated with both environmental and genetic risk factors. Although over 230 risk single-nucleotide polymorphisms have been associated with MS, all are common human variants. The mechanisms by which they increase the risk of MS, however, remain elusive. We hypothesized that a complex genetic phenotype such as MS could be driven by coordinated expression of genes controlled by transcriptional regulatory networks. We, therefore, constructed a gene coexpression network from microarray expression analyses of five purified peripheral blood leukocyte subsets of 76 patients with relapsing remitting MS and 104 healthy controls. These analyses identified a major network (or module) of expressed genes associated with MS that play key roles in cell-mediated cytotoxicity which was downregulated in monocytes of patients with MS. Manipulation of the module gene expression was achieved in vitro through small interfering RNA gene knockdown of identified drivers. In a mouse model, network gene knockdown modulated the autoimmune inflammatory MS model disease-experimental autoimmune encephalomyelitis. This research implicates a cytotoxicity-associated gene network in myeloid cells in the pathogenesis of MS.

CSL324, a granulocyte colony-stimulating factor receptor antagonist, blocks neutrophil migration markers that are upregulated in hidradenitis suppurativa.

BACKGROUND: Neutrophils have been shown to contribute to the pathophysiology of hidradenitis suppurativa (HS), a chronic, painful and debilitating inflammatory skin disease, yet their exact role remains to be fully defined. Granulocyte colony-stimulating factor (G-CSF), a major regulator of neutrophil development and survival, can be blocked by the novel, fully human anti-G-CSF receptor (G-CSFR) monoclonal antibody CSL324. OBJECTIVES: We investigated the activation and migration of neutrophils in HS and the impact of blocking G-CSFR with CSL324. METHODS: Biopsy and peripheral blood samples were taken from participants of two studies: 2018.206, a noninterventional research study of systemic and dermal neutrophils and inflammatory markers in patients with neutrophilic skin diseases, and CSL324_1001 (ACTRN12616000846426), a single-dose ascending and repeated dose, randomized, double-blind, placebo-controlled study to assess the safety, pharmacokinetics and pharmacodynamics of CSL324 in healthy adult subjects. Ex vivo experiments were performed, including neutrophil enumeration and immunophenotyping, migration, receptor occupancy and transcriptome analysis. RESULTS: The number of cells positive for the neutrophil markers myeloperoxidase (MPO) and neutrophil elastase (NE) was significantly higher in HS lesions compared with biopsies from healthy donors (HDs) (P < 0.0001 and P = 0.0223, respectively). In peripheral blood samples, mean neutrophil counts were significantly higher in patients with HS than in HDs (2.98 vs. 1.60 × 109 L-1, respectively; P = 8.8 × 10-4). Neutrophil migration pathways in peripheral blood were increased in patients with HS and their neutrophils demonstrated an increased migration phenotype, with higher mean CXCR1 on the surface of neutrophils in patients with HS (24453.20 vs. 20798.47 for HD; P = 0.03). G-CSF was a key driver of the transcriptomic changes in the peripheral blood of patients with HS and was elevated in serum from patients with HS compared with HDs (mean 6.61 vs. 3.84 pg mL-1, respectively; P = 0.013). Administration of CSL324 inhibited G-CSF-induced transcriptional changes in HDs, similar to those observed in the HS cohort, as highlighted by expression changes in genes related to neutrophil migratory capacity. CONCLUSIONS: Data suggest that neutrophils contribute to HS pathophysiology and that neutrophils are increased in lesions due to an increase in G-CSF-driven migration. CSL324 counteracted G-CSF-induced transcriptomic changes and blocked neutrophil migration by reducing cell-surface levels of chemokine receptors.

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 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.

Common and Low Frequency Variants in MERTK Are Independently Associated with Multiple Sclerosis Susceptibility with Discordant Association Dependent upon HLA-DRB1*15:01 Status.

Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system. The risk of developing MS is strongly influenced by genetic predisposition, and over 100 loci have been established as associated with susceptibility. However, the biologically relevant variants underlying disease risk have not been defined for the vast majority of these loci, limiting the power of these genetic studies to define new avenues of research for the development of MS therapeutics. It is therefore crucial that candidate MS susceptibility loci are carefully investigated to identify the biological mechanism linking genetic polymorphism at a given gene to the increased chance of developing MS. MERTK has been established as an MS susceptibility gene and is part of a family of receptor tyrosine kinases known to be involved in the pathogenesis of demyelinating disease. In this study we have refined the association of MERTK with MS risk to independent signals from both common and low frequency variants. One of the associated variants was also found to be linked with increased expression of MERTK in monocytes and higher expression of MERTK was associated with either increased or decreased risk of developing MS, dependent upon HLA-DRB1*15:01 status. This discordant association potentially extended beyond MS susceptibility to alterations in disease course in established MS. This study provides clear evidence that distinct polymorphisms within MERTK are associated with MS susceptibility, one of which has the potential to alter MERTK transcription, which in turn can alter both susceptibility and disease course in MS patients.

A rare P2X7 variant Arg307Gln with absent pore formation function protects against neuroinflammation in multiple sclerosis.

Multiple sclerosis (MS) is a chronic relapsing-remitting inflammatory disease of the central nervous system characterized by oligodendrocyte damage, demyelination and neuronal death. Genetic association studies have shown a 2-fold or greater prevalence of the HLA-DRB1*1501 allele in the MS population compared with normal Caucasians. In discovery cohorts of Australasian patients with MS (total 2941 patients and 3008 controls), we examined the associations of 12 functional polymorphisms of P2X7, a microglial/macrophage receptor with proinflammatory effects when activated by extracellular adenosine triphosphate (ATP). In discovery cohorts, rs28360457, coding for Arg307Gln was associated with MS and combined analysis showed a 2-fold lower minor allele frequency compared with controls (1.11% for MS and 2.15% for controls, P = 0.0000071). Replication analysis of four independent European MS case-control cohorts (total 2140 cases and 2634 controls) confirmed this association [odds ratio (OR) = 0.69, P = 0.026]. A meta-analysis of all Australasian and European cohorts indicated that Arg307Gln confers a 1.8-fold protective effect on MS risk (OR = 0.57, P = 0.0000024). Fresh human monocytes heterozygous for Arg307Gln have >85% loss of 'pore' function of the P2X7 receptor measured by ATP-induced ethidium uptake. Analysis shows Arg307Gln always occurred with 270His suggesting a single 307Gln-270His haplotype that confers dominant negative effects on P2X7 function and protection against MS. Modeling based on the homologous zP2X4 receptor showed Arg307 is located in a region rich in basic residues located only 12 Å from the ligand binding site. Our data show the protective effect against MS of a rare genetic variant of P2RX7 with heterozygotes showing near absent proinflammatory 'pore' function.

The MS Risk Allele of CD40 Is Associated with Reduced Cell-Membrane Bound Expression in Antigen Presenting Cells: Implications for Gene Function.

Human genetic and animal studies have implicated the costimulatory molecule CD40 in the development of multiple sclerosis (MS). We investigated the cell specific gene and protein expression variation controlled by the CD40 genetic variant(s) associated with MS, i.e. the T-allele at rs1883832. Previously we had shown that the risk allele is expressed at a lower level in whole blood, especially in people with MS. Here, we have defined the immune cell subsets responsible for genotype and disease effects on CD40 expression at the mRNA and protein level. In cell subsets in which CD40 is most highly expressed, B lymphocytes and dendritic cells, the MS-associated risk variant is associated with reduced CD40 cell-surface protein expression. In monocytes and dendritic cells, the risk allele additionally reduces the ratio of expression of full-length versus truncated CD40 mRNA, the latter encoding secreted CD40. We additionally show that MS patients, regardless of genotype, express significantly lower levels of CD40 cell-surface protein compared to unaffected controls in B lymphocytes. Thus, both genotype-dependent and independent down-regulation of cell-surface CD40 is a feature of MS. Lower expression of a co-stimulator of T cell activation, CD40, is therefore associated with increased MS risk despite the same CD40 variant being associated with reduced risk of other inflammatory autoimmune diseases. Our results highlight the complexity and likely individuality of autoimmune pathogenesis, and could be consistent with antiviral and/or immunoregulatory functions of CD40 playing an important role in protection from MS.

Polymorphisms in the receptor tyrosine kinase MERTK gene are associated with multiple sclerosis susceptibility.

Multiple sclerosis (MS) is a debilitating, chronic demyelinating disease of the central nervous system affecting over 2 million people worldwide. The TAM family of receptor tyrosine kinases (TYRO3, AXL and MERTK) have been implicated as important players during demyelination in both animal models of MS and in the human disease. We therefore conducted an association study to identify single nucleotide polymorphisms (SNPs) within genes encoding the TAM receptors and their ligands associated with MS. Analysis of genotype data from a genome-wide association study which consisted of 1618 MS cases and 3413 healthy controls conducted by the Australia and New Zealand Multiple Sclerosis Genetics Consortium (ANZgene) revealed several SNPs within the MERTK gene (Chromosome 2q14.1, Accession Number NG_011607.1) that showed suggestive association with MS. We therefore interrogated 28 SNPs in MERTK in an independent replication cohort of 1140 MS cases and 1140 healthy controls. We found 12 SNPs that replicated, with 7 SNPs showing p-values of less than 10(-5) when the discovery and replication cohorts were combined. All 12 replicated SNPs were in strong linkage disequilibrium with each other. In combination, these data suggest the MERTK gene is a novel risk gene for MS susceptibility.

GM-CSF-induced autoimmune gastritis in interferon alpha receptor deficient mice.

Experimental autoimmune gastritis (EAG), a mouse model of human autoimmune gastritis, is characterised by gastric mononuclear cell infiltrates and parietal and zymogenic cell destruction. The gastritis is accompanied by circulating auto-antibodies to parietal cell-associated gastric H(+)/K(+) ATPase. As interferon alpha has been implicated in the regulation of immune responses, we asked whether EAG induced by the local transgenic expression of granulocyte-macrophage colony stimulating factor (GM-CSF) in the stomach (PC-GMCSF transgenic mice) would be affected by deficiency of its binding receptor. To address this, we crossed PC-GMCSF transgenic mice with mice deficient in interferon alpha (IFNalpha) receptor2 (IFNAR2). We found that EAG development in the PC-GMCSF transgenic mice was not affected by IFNAR2 deficiency. There was no difference in severity of gastric pathology, nor in autoantibody levels in the IFNAR2 deficient mice compared to wild-type, and heterozygous littermates. We conclude that the local transgenic expression of GM-CSF in the stomach overrides any possible modulatory effects of IFNAR2 on EAG development.

Transplantation of bone marrow genetically engineered to express proinsulin II protects against autoimmune insulitis in NOD mice.

BACKGROUND: Type 1 diabetes (T1D) is a T-cell-dependent autoimmune disease resulting from destructive inflammation (insulitis) of the insulin-producing pancreatic beta-cells. Transgenic expression of proinsulin II by a MHC class II promoter or transfer of bone marrow from these transgenic mice protects NOD mice from insulitis and diabetes. We assessed the feasibility of gene therapy in the NOD mouse as an approach to treat T1D by ex vivo genetic manipulation of normal hematopoietic stem cells (HSCs) with proinsulin II followed by transfer to recipient mice. METHODS: HSCs were isolated from 6-8-week-old NOD female mice and transduced in vitro with retrovirus encoding enhanced green fluorescent protein (EGFP) and either proinsulin II or control autoantigen. Additional control groups included mice transferred with non-manipulated bone marrow and mice which did not receive bone marrow transfer. EGFP-sorted or non-sorted HSCs were transferred into pre-conditioned 3-4-week-old female NOD mice and insulitis was assessed 8 weeks post-transfer. RESULTS: Chimerism was established in all major lymphoid tissues, ranging from 5-15% in non-sorted bone marrow transplants to 20-45% in EGFP-sorted bone marrow transplants. The incidence and degree of insulitis was significantly reduced in mice receiving proinsulin II bone marrow compared to controls. However, the incidence of sialitis in mice receiving proinsulin II bone marrow and control mice was not altered, indicating protection from insulitis was antigen specific. CONCLUSIONS: We show for the first time that ex vivo genetic manipulation of HSCs to express proinsulin II followed by transplantation to NOD mice can establish molecular chimerism and protect from destructive insulitis in an antigen-specific manner.

Gene therapy strategies towards immune tolerance to treat the autoimmune diseases.

Autoimmune diseases such as type 1 diabetes and multiple sclerosis pose a significant health burden on our society. As a whole, autoimmune diseases affect approximately 6% of the population and are the third largest disease burden after heart disease and cancer. Such pathologic manifestations arise by way of damaging reactions of B-cell derived antibodies and/or T-cells to self-antigens and are triggered by genetic and environmental factors. Currently there is no known cure, with treatment restricted to toxic, long-term immunosuppressive regimes, replacement therapy and in intractable cases, transplantation of autologous or allogeneic haematopoietic stem cells. In experimental models of autoimmunity, gene therapeutic approaches have demonstrated promise in treating the autoimmune diseases. These include delivery of anti-inflammatory cytokines and exploitation of regulatory T cells. However, none of these approaches provide lasting, long-term benefit. We hypothesise that therapeutically transduced haematopoietic stem cells followed by transplantation is an alternative strategy to establish permanent immune tolerance that can not only prevent autoimmunity but also cure these diseases. Our approach is focused on directing autoimmune disease-specific autoantigen expression in the thymus by genetic manipulation of haematopoietic stem cells to establish molecular chimeras. Our hypothesis originates from experimental studies with a mouse model of experimental autoimmune gastritis (EAG) and more recently with the non-obese diabetic (NOD) mouse model for type 1 diabetes (T1D).

Haematopoietic stem cell gene therapy to treat autoimmune disease.

Autoimmune diseases affect approximately 6% of the population and are characterised by a pathogenic immune response that targets self-antigens. Well known diseases of this nature include type 1 diabetes, systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis. Treatment is often restricted to replacement therapy or immunosuppressive regimes and to date there are no cures. The strategy of utilising autologous or allogeneic haematopoietic stem cell transplantation to treat autoimmunity and induce immunological tolerance has been trailed with various levels of success. A major issue is disease relapse as the autoimmune response is reinitiated. Cells of the immune system originate from bone marrow and have a central role in the induction of immunological tolerance. The ability to isolate and genetically manipulate bone marrow haematopoietic stem cells therefore makes these cells a suitable vehicle for driving ectopic expression of defined autoantigens and induction of immunological tolerance.

Reversing the autoimmune condition: experience with experimental autoimmune gastritis.

Autoimmune diseases remain a significant health problem in our society, despite the best efforts to understand and treat these conditions. Current clinical treatments are aimed at alleviating the consequences of these diseases, with limited prospects for cure. Our studies with the experimental model of autoimmune gastritis have led us to explore potential curative strategies that can reverse the autoimmune condition. Using mouse models, we have shown that expression of the known gastric autoantigen in the thymus results in immunological tolerance and resistance to the induction of autoimmune gastritis. Also, induced tolerance in donor mice can be transferred to syngeneic recipient mice by bone marrow cells. Strategies based on these observations could lead to reversal of established disease. Transfer of ensuing knowledge to the cure of serious human autoimmune diseases is our ultimate goal.

Chemokine receptor CCR5 is not required for development of experimental autoimmune gastritis.

Experimental autoimmune gastritis (EAG) is a model of human autoimmune gastritis, the underlying cause of pernicious anaemia. It is characterised by gastric mononuclear cell infiltrates, destruction of parietal and zymogenic cells, and autoantibodies to parietal cell-associated H(+)/K(+) ATPase. Here, we have investigated the role of CCR5 in the development of EAG. We found that the development of EAG was not prevented in CCR5-deficient mice. Using reverse-transcriptase analysis of stomachs from normal and gastritic mice we found no difference in expression of CCR5 and its chemokine ligands MIP-1alpha, MIP-1beta, and RANTES. We also found that the CCR5 antagonist met-RANTES failed to prevent the development of EAG induced by neonatal thymectomy. These observations suggest that the CC chemokine receptor CCR5 is not essential for development of EAG.