Title: Haematopoietic stem cell gene therapy with <scp>IL</scp> ‐1Ra rescues cognitive loss in mucopolysaccharidosis <scp>IIIA</scp>
Abstract: Article14 February 2020Open Access Source Data Haematopoietic stem cell gene therapy with IL-1Ra rescues cognitive loss in mucopolysaccharidosis IIIA Helen Parker Helen Parker Stem Cell and Neurotherapies, Division of Cell Matrix Biology & Regenerative Medicine, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK Search for more papers by this author Stuart M Ellison Stuart M Ellison Stem Cell and Neurotherapies, Division of Cell Matrix Biology & Regenerative Medicine, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK Search for more papers by this author Rebecca J Holley Rebecca J Holley Stem Cell and Neurotherapies, Division of Cell Matrix Biology & Regenerative Medicine, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK Search for more papers by this author Claire O'Leary Claire O'Leary Stem Cell and Neurotherapies, Division of Cell Matrix Biology & Regenerative Medicine, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK Search for more papers by this author Aiyin Liao Aiyin Liao Stem Cell and Neurotherapies, Division of Cell Matrix Biology & Regenerative Medicine, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK Search for more papers by this author Jalal Asadi Jalal Asadi Stem Cell and Neurotherapies, Division of Cell Matrix Biology & Regenerative Medicine, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK Search for more papers by this author Emily Glover Emily Glover Stem Cell and Neurotherapies, Division of Cell Matrix Biology & Regenerative Medicine, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK Search for more papers by this author Arunabha Ghosh Arunabha Ghosh Royal Manchester Children's Hospital, Manchester University Hospitals NHS Foundation Trust, Manchester, UK Search for more papers by this author Simon Jones Simon Jones Royal Manchester Children's Hospital, Manchester University Hospitals NHS Foundation Trust, Manchester, UK Search for more papers by this author Fiona L Wilkinson Fiona L Wilkinson Division of Biomedical Sciences, School of Healthcare Science, Manchester Metropolitan University, Manchester, UK The Centre for Bioscience, Manchester Metropolitan University, Manchester, UK Search for more papers by this author David Brough David Brough orcid.org/0000-0002-2250-2381 Division of Neuroscience & Experimental Psychology, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK Search for more papers by this author Emmanuel Pinteaux Emmanuel Pinteaux Division of Neuroscience & Experimental Psychology, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK Search for more papers by this author Hervé Boutin Hervé Boutin Division of Neuroscience & Experimental Psychology, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK Wolfson Molecular Imaging Centre, University of Manchester, Manchester, UK Search for more papers by this author Brian W Bigger Corresponding Author Brian W Bigger [email protected] orcid.org/0000-0002-9708-1112 Stem Cell and Neurotherapies, Division of Cell Matrix Biology & Regenerative Medicine, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK Search for more papers by this author Helen Parker Helen Parker Stem Cell and Neurotherapies, Division of Cell Matrix Biology & Regenerative Medicine, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK Search for more papers by this author Stuart M Ellison Stuart M Ellison Stem Cell and Neurotherapies, Division of Cell Matrix Biology & Regenerative Medicine, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK Search for more papers by this author Rebecca J Holley Rebecca J Holley Stem Cell and Neurotherapies, Division of Cell Matrix Biology & Regenerative Medicine, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK Search for more papers by this author Claire O'Leary Claire O'Leary Stem Cell and Neurotherapies, Division of Cell Matrix Biology & Regenerative Medicine, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK Search for more papers by this author Aiyin Liao Aiyin Liao Stem Cell and Neurotherapies, Division of Cell Matrix Biology & Regenerative Medicine, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK Search for more papers by this author Jalal Asadi Jalal Asadi Stem Cell and Neurotherapies, Division of Cell Matrix Biology & Regenerative Medicine, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK Search for more papers by this author Emily Glover Emily Glover Stem Cell and Neurotherapies, Division of Cell Matrix Biology & Regenerative Medicine, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK Search for more papers by this author Arunabha Ghosh Arunabha Ghosh Royal Manchester Children's Hospital, Manchester University Hospitals NHS Foundation Trust, Manchester, UK Search for more papers by this author Simon Jones Simon Jones Royal Manchester Children's Hospital, Manchester University Hospitals NHS Foundation Trust, Manchester, UK Search for more papers by this author Fiona L Wilkinson Fiona L Wilkinson Division of Biomedical Sciences, School of Healthcare Science, Manchester Metropolitan University, Manchester, UK The Centre for Bioscience, Manchester Metropolitan University, Manchester, UK Search for more papers by this author David Brough David Brough orcid.org/0000-0002-2250-2381 Division of Neuroscience & Experimental Psychology, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK Search for more papers by this author Emmanuel Pinteaux Emmanuel Pinteaux Division of Neuroscience & Experimental Psychology, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK Search for more papers by this author Hervé Boutin Hervé Boutin Division of Neuroscience & Experimental Psychology, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK Wolfson Molecular Imaging Centre, University of Manchester, Manchester, UK Search for more papers by this author Brian W Bigger Corresponding Author Brian W Bigger [email protected] orcid.org/0000-0002-9708-1112 Stem Cell and Neurotherapies, Division of Cell Matrix Biology & Regenerative Medicine, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK Search for more papers by this author Author Information Helen Parker1, Stuart M Ellison1, Rebecca J Holley1, Claire O'Leary1, Aiyin Liao1, Jalal Asadi1, Emily Glover1, Arunabha Ghosh2, Simon Jones2, Fiona L Wilkinson3,4, David Brough5, Emmanuel Pinteaux5, Hervé Boutin5,6 and Brian W Bigger *,1 1Stem Cell and Neurotherapies, Division of Cell Matrix Biology & Regenerative Medicine, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK 2Royal Manchester Children's Hospital, Manchester University Hospitals NHS Foundation Trust, Manchester, UK 3Division of Biomedical Sciences, School of Healthcare Science, Manchester Metropolitan University, Manchester, UK 4The Centre for Bioscience, Manchester Metropolitan University, Manchester, UK 5Division of Neuroscience & Experimental Psychology, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK 6Wolfson Molecular Imaging Centre, University of Manchester, Manchester, UK *Corresponding author. Tel: +44 0161 306 0516; E-mail: [email protected] EMBO Mol Med (2020)12:e11185https://doi.org/10.15252/emmm.201911185 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mucopolysaccharidosis IIIA is a neuronopathic lysosomal storage disease, characterised by heparan sulphate and other substrates accumulating in the brain. Patients develop behavioural disturbances and cognitive decline, a possible consequence of neuroinflammation and abnormal substrate accumulation. Interleukin (IL)-1β and interleukin-1 receptor antagonist (IL-1Ra) expression were significantly increased in both murine models and human MPSIII patients. We identified pathogenic mechanisms of inflammasome activation, including that disease-specific 2-O-sulphated heparan sulphate was essential for priming an IL-1β response via the Toll-like receptor 4 complex. However, mucopolysaccharidosis IIIA primary and secondary storage substrates, such as amyloid beta, were both required to activate the NLRP3 inflammasome and initiate IL-1β secretion. IL-1 blockade in mucopolysaccharidosis IIIA mice using IL-1 receptor type 1 knockout or haematopoietic stem cell gene therapy over-expressing IL-1Ra reduced gliosis and completely prevented behavioural phenotypes. In conclusion, we demonstrate that IL-1 drives neuroinflammation, behavioural abnormality and cognitive decline in mucopolysaccharidosis IIIA, highlighting haematopoietic stem cell gene therapy treatment with IL-1Ra as a potential neuronopathic lysosomal disease treatment. Synopsis This study reports that IL-1, stimulated by storage substrates, is a critical mediator in the mucopolysaccharidosis IIIA (MPSIIIA) inflammatory cascade. Haematopoietic stem cell gene therapy using IL-1Ra, the natural antagonist of IL-1, prevented cognitive and behavioural decline in mice. IL-1 blockade in MPSIIIA mice via lentiviral dependent haematopoietic stem cell overexpression of IL-1Ra corrected cognitive decline and reduced neuroinflammation. 2-O-sulphation of MPSIIIA heparan sulphate elicited production of TLR4 dependent IL-1β. MPSIIIA primary and secondary storage substrates activated the inflammasome and induced secretion of IL-1β. IL-1β and IL-1Ra are important immuno-biomarkers in MPSIIIA patients and mice. The paper explained Problem There is currently no cure for patients with the neuronopathic disease mucopolysaccharidosis IIIA. Anti-inflammatory therapies have shown some promise in alleviating clinical symptoms; however, long-term use of many anti-inflammatories and steroids is contraindicated. Results We demonstrate that interleukin-1 has a central role in the cognitive and behavioural phenotype seen in MPSIIIA. IL-1 signalling inhibition via knock out of IL-1 receptor 1 or IL-1 receptor blockade with its cognate inhibitor IL-1Ra, via stem cell gene therapy, alleviates neuroinflammation and cognitive decline in MPSIIIA mice. Impact IL-1 is a critical mediator of the MPSIIIA inflammatory cascade. Interleukin-1 inhibition is a potential anti-inflammatory therapy to treat cognitive decline and immunopathology in MPSIIIA. Introduction Many lysosomal storage diseases exhibit pronounced substrate deposition, neuroinflammation and neuronal degeneration (Platt et al, 2012; Bosch & Kielian, 2015). Several studies have suggested that correction of neuroinflammation could ameliorate abnormal behaviours in neuronopathic lysosomal diseases including Krabbe disease (Reddy et al, 2011), Sandhoff disease (Lee et al, 2007) and mucopolysaccharidoses (MPS) (Sergijenko et al, 2013; Holley et al, 2018). However, the series of events from initial enzyme deficiency and substrate accumulation, through to clinical manifestation of disease pathologies, remain obscure in all of these diseases, making the choice of anti-inflammatory target unclear. Additionally, the role of lysosomal substrates in precipitating these responses also remains uncertain. In MPSIIIA, it is known that a lack of lysosomal enzyme N-sulfoglucosamine sulfohydrolase (SGSH), which degrades the complex sugar heparan sulphate (HS), results in a progressive clinical disease, characterised by hyperactivity and other behavioural abnormalities, developmental regression and cognitive decline (Wraith, 2002; Heron et al, 2011; Valstar et al, 2011; Wijburg et al, 2013; Shapiro et al, 2016). To date, there are no clinically approved treatments; however, several potential therapies are currently under clinical evaluation, including adeno-associated virus-driven SGSH, recombinant SGSH and lentiviral-mediated haematopoietic stem cell gene therapy. In the MPSIIIA mouse model, the lack of SGSH results in intra- and extracellular accumulation of partially degraded and highly sulphated fragments of HS in all tissues, including the brain (Langford-Smith et al, 2012; Wilkinson et al, 2012; Sergijenko et al, 2013). HS accumulation is accompanied by widespread inflammation, with substantial glial activation. This is accompanied by accumulation of secondary storage substrates including GM2 and GM3 gangliosides, cholesterol, amyloid beta, α-synuclein and hyper-phosphorylated tau (Ginsberg et al, 1999; McGlynn et al, 2004; Hamano et al, 2008; Ohmi et al, 2009; Wilkinson et al, 2012; Beard et al, 2017). MPSIIIA mice exhibit hyperactivity and memory impairment (Gliddon & Hopwood, 2004; Langford-Smith et al, 2011; Sergijenko et al, 2013), similar to observations seen in patients. Here, we sought to determine whether lysosomal substrates played a role in CNS inflammation and the cognitive behaviour observed in MPSIIIA. IL-1β is a key pro-inflammatory cytokine that is primarily produced in the periphery by immune cells but can also be synthesised by glia and neurons within the brain. The activation of TLR4 on immune cells by substrates, such as HS, leads to the expression of immature pro-IL-1β that is stored intracellularly (Takeuchi & Akira, 2010). However, a secondary inflammatory stimulus, which activates inflammasome complexes such as NLRP3, and the protease caspase-1, is required to drive the cleavage and release of mature, active IL-1β (Medzhitov, 2001; Takeda & Akira, 2004; Martinon et al, 2007; Lopez-Castejon & Brough, 2011). IL-1 signalling is mediated by the interleukin type 1 receptor (IL-1R1), and IL-1 binding to the IL-1R1 is blocked by the competitive IL-1 receptor antagonist (IL-1Ra), which inhibits all signal transduction (Weber et al, 2010). IL-1 signalling has been shown to play a key role in locomotor activity, explorative behaviour, anxiety and cognition (Kitazawa et al, 2011; Hein et al, 2012; Murray et al, 2013; Wohleb et al, 2014), and inhibition of IL-1 and inflammasome signalling has highlighted IL-1 as a key mediator in several neurological disorders, e.g. Alzheimer's disease, Parkinson's disease, stroke and multiple sclerosis (Halle et al, 2008; Jha et al, 2010; Heneka et al, 2013; Jesus & Goldbach-Mansky, 2014; Daniels et al, 2016; Sobowale et al, 2016; Dempsey et al, 2017). MPSIIIB is a clinically indistinguishable HS storage disease to MPSIIIA, where pathogenic HS appears to have a role in neuroinflammation (Ausseil et al, 2008). MPSIIIB HS, acting through Toll-like receptor 4 (TLR4), induces tumour necrosis factor (TNF)-α and interleukin (IL)-1β expression in primary microglial cultures (Ausseil et al, 2008). MPSIIIB mice crossed with either TLR4 knockout mice or MyD88 knockout mice (the adaptor protein downstream of TLR4) show initial improvements in brain microgliosis, but these inflammatory defects reappear at 3 months of age, coinciding with the accumulation of secondary storage substrates. This suggests that HS is not solely responsible for inflammation and that the inflammasome may not be completely activated by HS alone. Conversely, MPSVII mice, which develop a more somatic disease, and as such, less HS and more dermatan sulphate (DS) storage, show decreased levels of TNF-α and IL-1β, with commensurate improvements in somatic disease when crossed with TLR4 knockout mice (Simonaro et al, 2010). In terms of treating inflammatory manifestations of these diseases, broad spectrum anti-inflammatory therapies have shown some promise, with prednisolone showing correction of hyperactive behaviour in MPSIIIB mice despite only having reduced peripheral inflammation (DiRosario et al, 2009; Holley et al, 2018), and aspirin showing reduced brain cytokine profiles, including IL-1β in MPSIIIA (Arfi et al, 2011). Unravelling the inflammatory mechanisms responsible for cognitive decline and behavioural abnormalities in MPSIIIA and other lysosomal storage diseases is essential for the development of future therapies. Here, we addressed the role of lysosomal substrates in IL-1 pathways and whether IL-1 had a direct role in cognitive decline associated with MPSIIIA. We found that both the MPSIIIA murine model and patients with MPSIII had elevated levels of secreted IL-1β, suggesting inflammasome activation. We found that glial activation in vitro is primed by pathogenic 2-O-sulphated MPSIIIA glycosaminoglycans (GAGs) through TLR4 signalling. Secondary storage substrates drove IL-1β secretion through an NLRP3 inflammasome-dependent mechanism, but only when pre-primed with MPSIIIA GAG. We attenuated IL-1 signalling in MPSIIIA mice using either lentiviral-mediated haematopoietic stem cell gene therapy to over-express IL-1Ra or through the generation of IL-1R1-deficient MPSIIIA mice. Both approaches prevented working memory deficits and hyperactivity, and reduced brain glial activation, without any reduction in neuronal lysosomal storage. These data suggest that IL-1 is an important mediator in the MPSIIIA inflammatory cascade, and precipitates at least some of the abnormal behaviours observed. We highlight haematopoietic stem cell gene therapy using IL-1Ra as a potential anti-inflammatory therapy to treat cognitive decline in MPSIIIA and other neuronopathic lysosomal storage diseases. Results Interleukin-1-dependent inflammation is observed in MPSIIIA An array of pathological and immunological markers was quantified in 9-month-old MPSIIIA mouse brain to establish an observational study of events that may be associated with IL-1-mediated neuroinflammation. Significant brain astrocyte (GFAP) and microglial (ILB4) reactivity were observed within cortical layers II-VI (Fig 1A) in MPSIIIA mice compared to wild-type (WT) mice, as previously described (Wilkinson et al, 2012). Quantitative PCR within whole brain extracts from WT and MPSIIIA mice revealed significant up-regulation of pro-inflammatory cytokines TNF-α (Tnfa; P < 0.001), IL-1β (Il1b; P < 0.001), IL-1α (Il1a; P < 0.05), IL-6 (Il6; P < 0.001) and IL-1Ra (Il1rn; P < 0.001) (Fig 1B). Figure 1. Interleukin-1 and related cytokines are elevated in MPSIIIA mice and patients A. Representative sections of WT and MPSIIIA brains at 9 months of age stained for astrocytes (GFAP) or microglia (isolectin B4, ILB4). Images correspond to high power views covering cortical layers II-V. Scale bar, 100 μm (n = 4 mice per group). B. Quantitative PCR for various inflammatory cytokines in whole brains from 9-month-old WT and MPSIIIA mice (n = 6 mice per group). Data are expressed as mean ± STDEV, and data for each cytokine were tested by unpaired t-test; *P < 0.05, **P < 0.01, ***P < 0.001. Symbols above bars are versus WT. Exact P-values are indicated in Appendix Table S1. C, D. Quantification of IL-1β and IL-1Ra levels in plasma from human healthy control and MPSIII patients (n = 6 control participants and n = 21 MPSIII patients). Data are expressed as mean ± STDEV and were tested by unpaired t-test. (C) IL-1β, control versus MPSIII *P = 0.0407; (D) IL-1Ra, control versus MPSIII ***P = 0.0001. E, F. Quantification of IL-1β and IL-1Ra levels in cerebrospinal fluid (CSF) from control and MPSIII patients (n = 3 control participants and n = 21 MPSIII patients). Data are expressed as mean ± STDEV and were tested by unpaired t-test. (F) IL-1Ra, control versus MPSIII *P = 0.0167. Download figure Download PowerPoint We also found an analogous change in protein levels of inflammatory mediators in plasma and cerebrospinal fluid (CSF) from MPSIII patients. We found the inflammatory markers IL-1β and IL-1Ra to be significantly elevated in MPSIIIA, IIIB and IIIC patients when compared to healthy participants; this increase was observed in both plasma (Fig 1C and D) and cerebrospinal fluid for IL-1Ra (Fig 1E and F). There were no significant differences between subtypes. MPSIIIA GAG primes an intracellular IL-1β inflammatory response Heparan sulphate is known to control inflammatory responses, including acting as a co-receptor for cytokines/chemokines, modulation of leucocyte–endothelium interactions and initiation of immune responses. To understand whether GAG may be responsible for IL-1-dependent inflammation in MPSIIIA, we delivered either MPSIIIA GAG, WT GAG (amounts administered based on quantity of HS detected; Fig EV1), bovine kidney HS, heparin, PBS or lipopolysaccharides (LPS), a known pro-inflammatory response initiator (Cunningham et al, 2005) into 4-month-old C57BL/6J mice by intravenous injection. One hour, 2 and 6 h after treatment, the expression of Il1b in the brain was measured by qPCR (Fig 2A). Mice treated with MPSIIIA GAG showed significant increases in Il1b gene expression 2 and 6 h post-treatment (P < 0.001), when compared to saline treatment (Fig 2B). Il1b gene expression in MPSIIIA GAG-treated mice significantly increased between 1 and 2 h, and declined by 6 h (Fig 2B). No response was achieved with WT GAG, control bovine kidney HS or heparin treatment whilst the positive control of LPS elicited significant responses at 1 h, declining steadily with time. Thus, MPSIIIA GAG specifically initiates an acute IL-1β inflammatory response in vivo. Click here to expand this figure. Figure EV1. Heparan sulphate analysis A. Total relative amounts of HS. Amounts are expressed as μg HS per mg of liver protein and calculated from AMAC fluorescent reads compared to known amounts of HS standards (n = 3 GAG samples). Data are expressed as mean ± STDEV, and data for each cytokine were tested by unpaired t-test; WT versus MPSIIIA ***P < 0.0001. B. Compositional disaccharide analysis for HS from WT and MPSIIIA (n = 3 GAG samples). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; **P < 0.01, ***P < 0.001. Symbols above bars are versus WT. Exact P-values are indicated in Appendix Table S7. NAc, N-acetylated glucosamine; NS, N-sulphated glucosamine; 2S, 2-O-sulphate group; 6S, 6-O-sulphate group. Download figure Download PowerPoint Figure 2. MPSIIIA 2-O-sulphated HS induces intracellular IL-1β expression in vivo and in vitro A. Mice were systemically challenged via intraperitoneal (I.P.) injection with PBS, bovine kidney HS (2.5 mg/kg), heparin (20 U/kg), WT GAG (2.5 mg/kg), MPSIIIA GAG (2.5 mg/kg) or LPS (250 μg/kg). Animals were sacrificed 1, 2 and 6 h after I.P. challenge. B. The expression of Il1b was measured in whole brain using quantitative PCR 1, 2 and 6 h after I.P. challenge (n = 6 mice per group). Data are expressed as mean ± STDEV and were tested by two-way ANOVA with Bonferroni's post-test; **P < 0.01, ***P < 0.001. Symbols above bars are versus PBS at the relevant time-point. Exact P-values are indicated in Appendix Table S2. C. In vitro modelling. WT or MPSIIIA GAG (0.8 μg/ml HS) was applied to WT-mixed glial cultures for 24 h before harvesting the media or cell extract and measuring levels of IL-1β. D. Quantification of intracellular production of IL-1β (n = 3 independent experiments each with 3 intra experimental replicates). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; ***P < 0.001. Symbols above bars are versus PBS. Exact P-values are indicated in Appendix Table S2. E. Quantification of IL-1β secreted into the media (n = 3 independent experiments each with three inter-experimental replicates). A horizontal dotted line represents the limit of detection of the ELISA. Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; **P < 0.01, ***P < 0.001. Symbols above bars are versus PBS. Exact P-values are indicated in Appendix Table S2. F. WT-mixed glial cultures were co-treated with MPSIIIA GAG (4 μg/ml) and/or the TLR4 inhibitor CLI-095 (1 μg/ml) for 24 h. The intracellular production of IL-1β was measured (n = 3 independent experiments each with 3 intra experimental replicates). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; ***P < 0.001. Symbols above bars are versus MPSIIIA GAG + vehicle. Exact P-values are indicated in Appendix Table S2. G. Multi-heparinase (HepM) and/or chondroitinase ABC (cABC) digests were performed on MPSIIIA GAG, and the resulting oligosaccharides applied to a WT-mixed glial culture for 24 h alongside heparin, bovine HS (bHS), porcine DS (pDS) and WT GAG. The intracellular production of IL-1β was measured (n = 3 independent experiments each with three intra experimental replicates). A horizontal dotted line represents the limit of detection of the ELISA. Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; *P < 0.05, ***P < 0.001. Symbols above bars are versus MPSIIIA GAG alone. Exact P-values are indicated in Appendix Table S2. H. MPSIIIA GAG was 2-O-desulphated (hashed bars) utilising 120 mM NaOH, and compositional HS disaccharide analysis performed via RP-HPLC against untreated MPSIIIA GAG (black bars) to validate complete 2-O-desulphation. Percentage contribution of each modification was determined (n = 3 GAG samples). Data are expressed as mean ± STDEV and were tested by two-way ANOVA with Bonferroni's post-test; 2S MPSIIIA GAG versus 2S 2-ODS MPSIIIA GAG ***P < 0.0001. NS, N-sulphated glucosamine; 2S, 2-O-sulphate group; 6S, 6-O-sulphate group. I. MPSIIIA GAG was 2-O-desulphated and the resulting oligosaccharides applied to a WT-mixed glial culture for 24 h. The intracellular production of IL-1β was measured (n = 3 independent experiments each with three intra experimental replicates). A horizontal dotted line represents the limit of detection of the ELISA. Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; MPSIIIA GAG versus 2ODS MPSIIIA GAG **P < 0.0001. 2ODS, 2-O-desulphated. Download figure Download PowerPoint IL-1β is initially produced in a pro-form, requiring inflammasome-mediated caspase 1 cleavage for activation and secretion. To explore whether GAGs alone stimulated the full IL-1β secretion response, equivalent amounts of MPSIIIA and WT GAGs were used to stimulate WT-mixed glial cultures in vitro (Fig 2C). Stimulation with MPSIIIA GAG significantly increased intracellular IL-1β production (P < 0.001) (Fig 2D), although no significant secretion of IL-1β into culture media was detected (Fig 2E) (below limit of detection) suggesting that GAGs alone prime IL-1β production. MPSIII pathogenic GAG has previously been shown to act via a TLR4-dependent response in vitro (Ausseil et al, 2008). When MPSIIIA GAG or LPS (positive control) was applied to WT-mixed glial cultures together with CLI-095 (a TLR4 intracellular domain inhibitor), complete abrogation of the inflammatory response was achieved, with significantly decreased intracellular production of IL-1β (P < 0.001), suggesting that GAGs do not act through any other innate immune receptor pathway (Fig 2F). In order to determine whether the composition of MPSIIIA GAG was instrumental in eliciting the inflammatory response, MPSIIIA GAGs were digested with heparinases I, II and III (HepM) and/or chondroitinase ABC (cABC) to selectively remove HS and/or CS/DS, respectively and boiled to denature any co-purified GAG-binding proteins or lipids. Upon de-polymerisation of HS to constituent disaccharides, intracellular production of IL-1β was significantly reduced (P < 0.001; Fig 2G), to below the limit of detection. Degradation of CS/DS alone significantly reduced the intracellular production of IL-1β (P < 0.001; Fig 2G). Digestion of MPSIIIA GAG with both HepM and cABC resulted in a cumulative reduction in the production of IL-1β when compared to untreated MPSIIIA GAGs (P < 0.001) (Fig 2G). This suggests that the inflammatory response induced by MPSIIIA GAG can be largely attributed to HS but also partly to CS/DS. 2-O-sulphation of HS plays a role in protein-binding interactions and subsequent signalling; e.g. HS oligosaccharides containing 2-O-sulphated IdoA demonstrate a high-binding affinity for FGF2, IL-8, MCP-1 and other co-factors (Gallagher & Turnbull, 1992; Lortat-Jacob et al, 1995; Frevert et al, 2003; Simon Davis & Parish, 2013). To determine whether the 2-O-sulphated regions in MPSIIIA HS were involved in immune signalling, MPSIIIA GAG was selectively