The extracellular heparan sulfatase SULF2 limits myeloid IFNβ signaling and Th17 responses in inflammatory arthritis

Heparan sulfate (HS) proteoglycans are important regulators of cellular responses to soluble mediators such as chemokines, cytokines and growth factors. We profiled changes in expression of genes encoding HS core proteins, biosynthesis enzymes and modifiers during macrophage polarisation, and found that the most highly regulated gene was Sulf2, an extracellular HS 6-O-sulfatase that was markedly downregulated in response to pro-inflammatory stimuli. We then generated Sulf2+/− bone marrow chimeric mice and examined inflammatory responses in antigen-induced arthritis, as a model of rheumatoid arthritis. Resolution of inflammation was impaired in myeloid Sulf2+/− chimeras, with elevated joint swelling and increased abundance of pro-arthritic Th17 cells in synovial tissue. Transcriptomic and in vitro analyses indicated that Sulf2 deficiency increased type I interferon signaling in bone marrow-derived macrophages, leading to elevated expression of the Th17-inducing cytokine IL6. This establishes that dynamic remodeling of HS by Sulf2 limits type I interferon signaling in macrophages, and so protects against Th17-driven pathology. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-024-05333-w.


Introduction
Inflammation is regulated by a network of cytokines, chemokines and growth factors that control the survival, proliferation, recruitment and signaling responses of innate and adaptive immune cells.Many of these extracellular mediators bind to heparan sulfate (HS) proteoglycans [1], and this interaction controls their localisation, stability, gradient formation and/or signaling [2][3][4].For example, binding to extracellular matrix HS is required for the in vivo chemotactic activity of CCL2, CCL4, CCL5 and CXCL4 [5,6], and regulates the in vivo half-life [7] and bioactivity of IFNγ [8].While less well-studied, cell surface HS has also been shown to modulate the responses of innate and adaptive immune cells to cytokines, chemokines and growth factors.For example, B cells require cell surface HS to proliferate and differentiate in response to A Proliferation-Inducing Ligand (APRIL) [9,10] and also to IL21 [11].
HS is a long, structurally diverse polysaccharide that can bind to > 400 protein ligands [1] with a range of affinities, and consequently a broad range of biological effects [12,13].Unlike the template-dependent synthesis of nucleic acids and proteins, synthesis of HS is a template-independent process, in which up to 20 enzymes act sequentially in the Golgi apparatus to add alternating uronic acid (either glucuronic acid or iduronic acid) and N-acetylglucosamine residues to the growing HS chain and sulfate it at various positions [13] i.e. glucuronic acid can be 2-O-sulfated (by HS2ST1), while N-acetylglucosamine can be N-sulfated (by NDST1-4), 6-O-sulfated (by HS6ST1-3) or 3-O-sulfated (by HS3ST1-6) during HS biosynthesis [13].After it has been synthesised and secreted from cells, HS can also be modified in the extracellular environment by heparanase, an enzyme that degrades the HS chains [13], or by the sulfatases SULF1 and SULF2, which specifically remove sulfate groups from the 6-carbon position of N-acetylglucosamine residues [13].Changes in expression of these biosynthetic and modifying enzymes lead to dynamic changes in HS structure and function during multiple physiological (development [14], wound healing and ageing [15][16][17]) and pathological (diabetes [18], fibrosis [19,20], Alzheimer's disease [21], osteoarthritis [22], cancer [20,23]) processes.
In myeloid cells, expression of HS sulfotransferases has been shown to change in response to cytokine-induced polarisation [24][25][26], leading to changes in HS abundance and sulfation [25,27].A key study by Gordts et al. [28] showed that myeloid-specific deletion of Ndst1 sensitised macrophages to type I interferon (IFN) signaling, and exacerbated inflammation in murine models of atherosclerosis and obesity.In vitro mechanistic studies indicated that Ndst1-dependent N-sulfation increased IFNβ binding to HS, potentially sequestering the cytokine from its cognate IFN receptors [28].Subsequent studies showed Ndst1 can also quench inflammatory responses in dendritic cells (DCs), with deletion of Ndst1 in DCs increasing antigen presentation and cytotoxic T cell responses in a lung carcinoma model [29].
Here, we systematically profiled changes in expression of the 12 HS core proteins and 25 HS biosynthesis and modifying enzymes in murine bone marrow-derived macrophages (BMDMs) in response to pro-and anti-inflammatory stimuli, and found that the 6-O-sulfatase Sulf2 was the most highly regulated gene.Using a bone marrow transplantation strategy, we generated chimeric mice with myeloid Sulf2-deficiency and examined inflammatory responses in vivo in an antigen-induced arthritis model.This showed that myeloid SULF2 limits inflammation, by suppressing type I interferon signaling and so reducing arthritogenic Th17 responses.

Primary cell isolation and culture
All experiments were carried out in accordance with the United Kingdom Home Office Animals Scientific Procedures Act 1986 under relevant personal and project licenses.
Single cell suspensions were generated from joint synovium by digesting opened hindleg knee joints in liberase TL (Roche) and DNase I (Sigma) (30 min, 37 °C), and from lungs by digestion with collagenase IV (Sigma) and DNase I (Sigma) (40 min, 37 °C).Single cell suspensions were generated from spleen and lymph nodes by pressing tissues through a 70 μm cell strainer.All single cell suspensions, except those use for generating chimeras, were treated with red blood cell lysis buffer (2 min, room temperature) before further use.

Generation of Sulf2 +/− mice
Sulf2 +/− mice were generated from the Sulf2 tm1a(KOMP)Wtsi embryonic stem cell line (RRID: MMRRC_063144-UCD, obtained from the KOMP Repository at University of California, Davis), in which exon 4 of the Sulf2 gene is flanked by LoxP sites.Embryonic stem cells were injected into blastocysts and resulting chimeras bred to germline transmission of the Sulf2 tm1a allele, before crossing with Tg(Pgk1-cre) Lni mice [30], available from JAX, to facilitate recombination, and backcrossing to a C57BL/6 background to generate Sulf2 tm1b allele mice, with a reporter-tagged deletion allele, and missing critical exon 4 of the Sulf2 gene.The deletion of exon 4 is expected to generate a mis-sense mutation and to cause a premature stop codon in exon 5, which is anticipated to lead to nonsense-mediated decay of the mRNA and loss of protein expression.

RT-qPCR
RNA was reverse transcribed using a High Capacity Reverse Transcription cDNA kit (Applied Biosystems) after extraction from cultured cells (with RNeasy mini spin-columns, Qiagen), tissues (with TRIzol, Life Technologies), or joints [by homogenisation in a TissueLyser II using a Precellys hard tissue Lysing Kit (Bertin) followed by RNeasy mini spin-columns, Qiagen].TaqMan Low-Density Array (TLDA) cards (Applied Biosystems) were custom designed using TaqMan primer/probes (Applied Biosystems, Supplementary Table S1), with data acquired on a ViiA Real-Time PCR System (Applied Biosystems).Manual RT-qPCR was carried out using TaqMan primer/probes (Applied Biosystems) with data acquired on a ViiA Real-Time PCR System (Applied Biosystems), or with KiCqStart SYBR Green Primers with data acquired and melt curves examined on a QuantStudio 3 (ThermoFisher Scientific, Supplementary Table S2).Fold change in expression was calculated using the ΔΔCt method.

Generation of bone marrow chimeras and antigeninduced arthritis (AIA)
Recipient 8-week old wild-type (WT) C57BL/6 mice were irradiated (two doses of 5.5 Gy at a 4 h interval in a Gulmay X-ray irradiator), and injected 2 h later in the tail vein with bone marrow cells (5 million/mouse) from 8-week old WT or Sulf2 +/− donors.Seven weeks later, mice were subcutaneously injected with methylated bovine serum albumin (mBSA, 100 µg, Sigma) emulsified 1:1 with Complete Freund's adjuvant (BD Biosciences).Arthritis was induced 3 weeks later by intra-articular tibiofemoral injection of mBSA (100 µg in PBS, right knee) or PBS control (left

Inflammasome activation
Macrophages were stimulated with 100 ng/ml LPS (100 ng/ ml, 6 h) with nigericin (5 µM) added for the last 2 h of culture.Cell death was assessed using the CytoTox 96 Cytotoxicity assay (Promega) and IL1β secretion measured by ELISA (Invitrogen).knees).Knee swelling was measured daily for 7 days after intra-articular injection using a calliper, and pain assessed by measuring weight bearing on mBSA-injected compared to PBS-injected knees using a dynamic weight-bearing 2.0 chamber (Bioseb).Mice were sacrificed after 7 days, and immune infiltration and total histological score, composed of the sub-synovium, synovium and bone marrow density scores, calculated from blinded scoring [31].Immunohistochemical analysis of pSTAT1 (Y701) (D4A7, Cell Signaling, followed by goat anti-rabbit Alexa 488, ThermoFisher Scientific) and CD206 (C068C2, BioLegend, followed by goat anti-rat Alexa 568, ThermoFisher Scientific) abundance was done following antigen retrieval in Tris-EDTA.Fluorescent signal (mean fluorescence per cell cluster x area) was quantified for at least 700 cell clusters per genotype using a BioTek Cytation 7 imaging reader and Gen5 v 3.14 software.To analyse T cell subsets, single cell suspensions from knee joints and lymph nodes were stimulated for 4 h with PMA (20 ng/ml) and ionomycin (1 mg/ml) in the presence of protein transport inhibitors, before CD3 + , CD4 + and CD8 + subsets were analysed by flow cytometry.

Bulk RNASeq
RNA was isolated (RNeasy Micro Kits, Qiagen) from single cell suspensions prepared from knee joints of WT and Sulf2-deficient bone marrow chimera mice sacrificed 7 days after induction of arthritis.Oxford Genomics Centre prepared bulk RNA libraries using an NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina (New England Biolabs) and sequenced the samples at a depth of 25 million reads per sample on a NovaSeq6000 (Illumina).PolyA transcripts were analysed with the Nextflow RNA-Seq pipeline [32], using STAR for alignment with the GRCm38 reference murine genome.Deconvolution of cell types was performed with MuSIC [33], utilising single cell data from Park et al. [34].DeSeq2 was used for differential gene expression analysis, with correction for multiple comparisons carried out using the Benjamini and Hochberg method.Functional enrichment analysis was performed using gProfiler2 (v0.2.0) utilising the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database [35] for terms.The gSCS (Set Counts and Sizes) correction method was used to determine significantly enriched pathways with significance p < 0.05.Differentially regulated genes were manually compared with the Interferome database (interferome.org,[36]), and Homer was used to analyse promoter regions (8 bp motifs, -400 to -100 base pairs upstream of transcriptional start sites) of differentially expressed genes [37].
that they expressed detectable levels of 22 of these genes (8 of the possible 12 core proteins and 14 of the possible 25 modifying enzymes).Polarisation of the cells in vitro with IFNγ + LPS or IL4 + IL13 significantly altered expression of 60% of the expressed genes (Fig. 1A), with 3 distinct groups on a PCA analysis (Fig. 1B).
The most strongly regulated gene was Sulf2, a sulfatase that removes 6-O sulfate groups from N-acetylglucosamine residues of HS.Expression of this gene was markedly down-regulated in response to IFNγ + LPS treatment (25fold compared to unpolarised BMDMs, Fig. 1A).

Sulf2-deficiency altered macrophage polarisation in vitro
We observed perinatal lethality in Sulf2 −/− mice, as has been reported by others [38,39], so we utilised Sulf2 +/− BMDMs to investigate the impact of partial Sulf2 deficiency in macrophages.Given that unpolarised BMDMs expressed high levels of Sulf2, we investigated the effect of Sulf2-deficiency on polarisation of these cells (Fig. 2A).IL4 + IL13-treated Sulf2 +/− BMDMs expressed significantly lower levels of Arg1, Cd206 and Fizz1, suggesting that Sulf2-deficiency may reduce polarisation to an anti-inflammatory phenotype.IFNγ + LPS-treated Sulf2 +/− BMDMs expressed significantly higher levels of the prototypic pro-inflammatory marker Tnf (i.e.TNFα) and reduced levels of Socs3, a suppressor of cytokine signaling (Fig. 2A), indicating a potentially more inflammatory phenotype, although these cells also expressed lower levels of Nos2.
Potential mechanisms leading to these changes in polarisation were investigated.There was no evidence for a change in IL6 release in response to IFNγ, LPS, poly(I: C), FSL-1 or Pam3CSK4, and a small effect size increase in IL6 release in response to IFNγ + LPS (Fig. S1A), indicating TLR signaling was not markedly impacted by Sulf2-deficiency.

Cytotoxicity assay
A CytoTox 96 Cytotoxicity assay (Promega) was performed according to manufacturer's instructions.This assay detects release of lactate dehydrogenase from damaged cells.

Statistical analysis
Generation of heatmaps and principal component analysis (PCA) analysis was performed in R (version 3.5.2) and R studio (version 1.1.453).For PCA analysis, the prcomp function was used, without any maximum number of ranks and without clustering.The samples were then plotted on the obtained and rotated principal components, together with ellipsoids indicating 95% confidence around the centroids of the data groups.All other data were analysed using GraphPad Prism (version 8.4.1).Data were analysed for normality using the D'Agostino-Pearson test, and for statistical significance using relevant tests as detailed in the figure legends.Data are presented as mean ± SEM or median ± IQR where appropriate.

Macrophage expression of HS core proteins and modifying enzymes was regulated by polarisation in vitro
We profiled the expression of genes encoding the 37 HS core proteins and modifying enzymes in BMDMs, and found increased expression of the efferocytosis receptor Tyro3, but not Mertk, Axl or Cd36 (Fig. 2D).Expression of integrin αvβ3 (Itgb3) and the efferocytosis bridging molecules Gas6 and Pros1 was similarly unaffected.
There was no evidence that in vitro uptake of OVA antigen and cell surface expression of costimulatory molecules (MHC-I, CD80, CD86) was altered in Sulf2-deficient BMDMs and BMDCs, but expression of MHC II was reduced with a small effect size in Sulf2 +/− BMDCs (Fig. S2).Similarly, there was no evidence that Sulf2-deficiency had an effect on in vitro presentation of OVA to CD4 + T cells by BMDCs, with unaltered CD4 + T cell proliferation and expression of CD25 (Fig. S3).
Similarly, there was no significant difference in pyroptotic cell death and IL1β release in response to LPS + nigerycin was unaffected (Fig. S1B), indicating unaltered inflammasome activation in Sulf2-deficient BMDMs.

Sulf2-deficiency increased phagocytosis in vitro
To further assess the phenotype of Sulf2-deficient BMDMs, we profiled their phagocytic and antigen-presenting abilities.
Mean phagocytosis of polystyrene beads was increased by 25% (Fig. 2B, P < 0.05) and phagocytosis of apoptotic cells was increased by 43% (Fig. 2C, P < 0.05) in Sulf2deficient cells.This was accompanied by significantly  A-C).Bone marrow-derived macrophages were polarised in vitro with IFNγ + LPS or IL4 + IL13 (18 h, n = 7-9 mice per group).mRNA expression of the 37 HS core proteins and modifying enzymes was quantified by RT-qPCR using customdesigned TaqMan low-density array cards, and expressed relative to the geometric mean of the housekeeping genes 18s, Gapdh, and Rplp0, and to the average expression in IFNγ + LPS-polarised cells.(A).Fold change in expression of the 22 detectably-expressed HS core proteins and modifying enzymes (median ± IQR, analysed by Kruskall-Wallis test followed by multiplicity correction using the two-stage step-up method of Benjamini, Krieger and Yekutieli.*, P < 0.05; **, P < 0.01, shown in orange for IFNγ + LPS and in green for and IL4 + IL13).(B).PCA analysis of differentially expressed genes, with ellipses indicating the 95% confidence interval.(C).Heatmap showing changes in expression of the 22 detectably-expressed HS genes.For PCA analysis, the prcomp function was used, without any maximum number of ranks and without clustering.The samples were then plotted on the obtained and rotated principal components, together with ellipsoids indicating 95% confidence around the centroids of the data groups day 7 (Fig. 3B-C).Paradoxically, Sulf2 +/− chimeric animals exhibited less pain at day 7 (Fig. 3C).

Myeloid Sulf2 deficiency increased Th17/Treg ratio in knees
The increased joint swelling in Sulf2 +/− chimeric mice was not explained by increased immune cell infiltration, as flow cytometry showed there was no significant difference in the abundance or frequency of myeloid or adaptive immune cell types in the knees of WT and Sulf2 +/− chimeric mice 2 or 7 days after initiation of AIA (Fig. S6).
While the overall number of CD4 + T cells was not altered (Fig. S6), closer examination of this compartment revealed a significant increase in Th17 abundance in the knees of Sulf2 +/− chimeric mice on day 7 of AIA (Fig. 4A-B), leading to a 2-fold increase in the Th17/Treg ratio (Fig. 4C-D).This shift in the Th17/Treg ratio was not observed in the inguinal lymph nodes (Fig. S7), indicating it was a local rather than a systemic effect.

Myeloid Sulf2 deficiency increased joint damage and inflammation in vivo
Sulf2 is highly expressed by myeloid cells (Fig. S4), but it is also expressed by multiple other cell types [40].To study the function of myeloid Sulf2, we thus generated chimeric mice by irradiating WT animals and reconstituting their myeloid population with BMDMs isolated from either WT or Sulf2 +/− donors.The effect of Sulf2-deficiency on myeloid function was then assessed using the antigeninduced arthritis (AIA) model, in which immunisation and boosting of mice with mBSA leads to myeloid and lymphoid cell-dependent joint inflammation and damage in mice [31].
WT and Sulf2 +/− chimeric mice developed similar acute inflammation in the first 4 days after intra-articular injection with mBSA, with no significant difference in initial knee swelling (Fig. 3A) or histological joint damage (Fig. S5).However, the resolution of inflammation was significantly impaired in Sulf2 +/− chimeric mice, with knee swelling significantly elevated at days 5-7.This was accompanied by increased joint damage in the Sulf2 +/− chimeric mice, with a significantly increased total histological score, synovial and subsynovial thickening and bone marrow density at or IL4 + IL13, with significant changes in expression of at least one gene from each family of HS modifying enzymes.The most highly regulated gene was the extracellular HS 6-O-sulfatase Sulf2, which was downregulated 25-fold by IFNγ + LPS.This extends previous work by Martinez et al. [25], who demonstrated significant changes in expression of HS sulfotransferases during macrophage polarisation, but did not examine HS polymerases (Ext1, Ext2, Extl1, Extl2, Extl3), epimerase (Glce), degrading enzymes (Hpse, Hpse2) or sulfatases (Sulf1, Sulf2).
The strong regulation of Sulf2 expression is striking because SULF1 and SULF2 are the only enzymes that are able to modify HS sulfation in the extracellular environment [13].N-, 2-O-, 3-O-and 6-O-sulfation are all added to HS during its synthesis in the Golgi apparatus, but once HS is secreted into the extracellular environment, its structure can only be modified by heparanase (encoded by Hspe), which cleaves HS into smaller fragments, or by the sulfatases (encoded by Sulf1 and Sulf2), which remove sulfate groups specifically from the 6-carbon position of N-acetylglucosamine residues [40].SULFs thus carry out the only 'editing' of HS that occurs in the extracellular environment, giving these enzymes the unique ability to rapidly fine-tune HS affinity for ligands and so dynamically modulate downstream signaling pathways [43].It should be noted that no change in overall levels of 6-O-sulfation were observed in some studies of Sulf2-null animals [39], suggesting SULF1 and SULF2 may act co-operatively [39], or that SULF2 may may target micro-regions of HS with functional importance.

Sulf2-deficient macrophages have an elevated inflammatory phenotype
The marked reduction in Sulf2 expression in response to treatment with IFNγ + LPS suggests that SULF2 may have an anti-inflammatory or regulatory role in macrophages.We found that Sulf2 +/− macrophages exhibited a generally more inflammatory phenotype in vitro, with higher expression of Tnf, and lower expression of Socs3, Arg1, Cd206 and Fizz in response to polarising cytokines.This is in agreement with Zhang et al. [44], who showed that loss of SULF2 expression in bladder cancer cells promoted polarisation of cocultured THP-1 cells towards an inflammatory phenotype.Further in vitro investigations showed that no evidence that macrophage Sulf2-deficiency altered TLR signaling, inflammasome activation, or antigen presentation, so we sought to investigate the role of myeloid Sulf2 on inflammation in vivo.
Sulf2 is widely expressed [40], so we adopted a bone marrow transfer approach, to generate chimeric mice with heterozygous deficiency of Sulf2 in the myeloid lineage.We observed perinatal lethality of Sulf2 −/− animals, as has been

HS biosynthesis is significantly regulated during macrophage polarization
Both the immune system and HS proteoglycans are complex information networks, in which dynamic interactions between multiple nodes determine biological outcomes.Here, we comprehensively profiled changes in expression of HS biosynthetic machinery during macrophage polarisation, and found that almost two-thirds of the expressed genes were significantly regulated in response to IFNγ + LPS and/ that N-sulfation of HS could promote IFNβ binding to cell surface HS proteoglycans in preference to its cognate receptors, while 6-O-sulfation generates a different structural motif that may promote formation and/or stabilisation of IFNβ/receptor complexes.This model predicts that homeostatic expression of SULF2 would keep 6-O-sulfation of cell surface HS at low levels, leading to accumulation of IFNβ on N-sulfated HS proteoglycans and keeping IFNβ signaling in check, while in inflammatory environments (or Sulf2-deficiency, which mimics and/or exacerbates this), reduced SULF2 would lead to increased HS 6-O-sulfation and elevated IFNβ signaling.This suggests that IFNβ signaling would be further elevated in macrophages deficient for both Sulf2 and Ndst1.
Our findings suggest it is worth investigating whether Sulf2 could also be protective in other autoimmune conditions where IFNβ signaling promotes Th17-driven inflammation e.g.psoriasis, systemic lupus erythematosus, Sjögren's syndrome, and neuromyelitis optica [57].However, IFNβ signaling can also suppress auto-immune responses, such as in relapsing remitting multiple sclerosis, where IFNβ is used therapeutically to slow disease progression [57], and Sulf2 may have deleterious effects in such contexts.Indeed, Saraswat et al. [58] showed that Sulf2 expression is elevated in multiple sclerosis lesions, and propose that it contributes to an inhibitory microenvironment that limits remyelination.HS sulfation in numerous tissues and cells types is known to change with ageing [15][16][17], inflammation [59], infection [60] and metabolic state [18,61], suggesting a novel mechanism by which co-morbidities could modulate physiological, pathological and therapeutic type I interferon signaling.Establishing what regulates Sulf2 expression and HS structure more broadly is thus likely to be of importance for multiple auto-immune conditions.

Further considerations
Looking beyond type I interferon signaling, Sulf2-deficiency is also likely to impact the biological activity of multiple other HS-binding proteins, with experimental identification of an affected pathway dependent on the model analysed and its molecular drivers.For example, Sulf2-deficient mice have worse outcomes in models of osteoarthritis [62], myocardial infarction [63], and bleomycin-induced lung injury [64], which has been attributed to impaired bone morphogenetic protein [62] and vascular endothelial growth factor [63] signaling and elevated fibroblast growth factor 2 [62] and TGFb [64] signaling.
Both cell-associated [65][66][67] and secreted [40,63,66] forms of SULF2 have been described, suggesting the enzyme can act in both an autocrine and paracrine manner.Recently, El Masri et al. [68] found that SULF2 localisation and activity can be modulated by post-translational modification, with attachment of a chondroitin or dermatan sulfate glycosaminoglycan chain reported for some other Sulf2 −/− lines [38,39], but not others [45,46].Such strain-dependent effects have also been reported for mice lacking other HS biosynthetic enzymes, such as Hs3st1 [47] and Hs6st1 [48], suggesting HS biosynthesis is strongly influenced by modifier genes.
Sulf2 +/− bone marrow chimeric mice showed significantly increased joint swelling and histological damage in the resolution phase of the macrophage- [49,50]-and CD4 + T cell-dependent [51] antigen-induced arthritis model.This was accompanied by an increased abundance of Th17 cells in joints of Sulf2 +/− chimeras, consistent with the known ability of this subset to exacerbate inflammation in this [52] and other [53] models of arthritis.

Increased type I interferon signaling in Sulf2deficient macrophages
To understand how Sulf2-deficiency in macrophages could promote Th17 differentiation, we conducted bulk RNA sequencing of cells isolated from AIA joints during the resolution phase of joint inflammation.This showed that differentially expressed genes were related to viral infection responses, suggesting altered type I interferon signaling, with 56% of the differentially expressed genes being known type I interferon-responsive genes.In vitro analyses confirmed elevated responses to IFNβ in Sulf2 +/− BMDMs, with increased STAT1 phosphorylation and increased induction of Ccl5, Ccl7, Tlr3 and Il6 expression.STAT1 phosphorylation was also increased in vivo.Increased IL6 expression would lead to the observed increase in Th17 abundance and Th7/Threg ratio [54].This proposed mechanism is consistent with a previous study in lung carcinoma cells, where siRNA or epigenetic silencing of SULF2 was found to activate expression of interferon-inducible genes [55].
Considering the molecular mechanism by which SULF2 could reduce type I interferon signaling, Gordts et al. [28] previously showed that IFNβ signaling in macrophages is modulated by cell surface HS.IFNβ binds strongly to HS (K i of 1.4 nM) [28,56], while IFNα4, interferon-alpha/beta receptor alpha chain (IFNAR1) and interferon-alpha/beta receptor beta chain (IFNAR2) do not [28], and as with all HS ligands, IFNβ/HS affinity is likely to be modulated by the overall level and pattern of HS sulfation.N-sulfation of HS was shown to reduce IFNβ signaling and to be protective in models of atherosclerosis and obesity [28].Our data here suggest that 6-O-sulfation of HS has an opposite effect, promoting IFNβ signaling and leading to increased Il6 expression, which promotes the generation of Th17 cells and exacerbates inflammatory arthritis.
Further experiments are required to test the molecular mechanism by which HS alters IFNβ signaling, and we suggest the following model to stimulate such investigations.We propose Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/.
to the enzyme's HD domain increasing cell surface association and reducing 6-O-sulfatase activity.Degradation of the glycosaminoglycan chain by hyaluronidase reduced cell surface binding and increased SULF2 activity [68].Conditions that increase hyaluronidase expression could thus switch SULF2 from autocrine to enhanced paracrine activity.Our in vivo data are likely to reflect autocrine activity of SULF2, because Sulf2-deficiency in myeloid cells was not compensated for by expression in other cells of the joint, such as synovial fibroblasts, which express abundant Sulf2 in RA [69].Intriguingly, Siegel et al. [69] found that SULF2 expressed by rheumatoid synovial fibroblasts promotes inflammation, by increasing transcriptomic and phenotypic responses of these cells to TNF.This suggests that synovial fibroblast and myeloid SULF2 have opposing effects on joint inflammation, with synovial SULF2 potentially contributing to initiation of inflammation, and myeloid SULF2 promoting resolution.Further investigation is required to define the kinetics of SULF2 expression and activity in these cell types.

Fig. 1
Fig. 1 Expression of HS core proteins and modifying enzymes was downregulated by IFNγ + LPS.(A-C).Bone marrow-derived macrophages were polarised in vitro with IFNγ + LPS or IL4 + IL13 (18 h, n = 7-9 mice per group).mRNA expression of the 37 HS core proteins and modifying enzymes was quantified by RT-qPCR using customdesigned TaqMan low-density array cards, and expressed relative to the geometric mean of the housekeeping genes 18s, Gapdh, and Rplp0, and to the average expression in IFNγ + LPS-polarised cells.(A).Fold change in expression of the 22 detectably-expressed HS core proteins and modifying enzymes (median ± IQR, analysed by Kruskall-Wallis

Fig. 2 Fig. 4 Fig. 3
Fig. 2 Macrophage polarisation and phagocytosis was modulated by Sulf2.BMDMs from WT and Sulf2 +/-mice were stimulated with IFNγ + LPS or IL4 + IL13 (18 h).(A).Fold change in gene expression was quantified relative to Rplp0 (n = 3, mean ± SEM, analysed by two-way ANOVA with Bonferroni's correction for multiple comparisons.***, P < 0.001).(B-C).Cells were incubated with fluorescent polystyrene beads at 50 MOI (B) or apoptotic cells at 1 MOI, and the percentage of percentage of fluorescence positive cells determined by flow cytometry (n = 3, mean ± SEM, analysed by two-way Student's t-tests.*, P < 0.05).(D).Fold change in gene expression was quantified relative to Rplp0 and to WT cells (n = mean ± SEM, analysed by multiple unpaired t-tests, followed by multiplicity correction using the two-stage step-up method of Benjamini, Krieger and Yekutieli, **, q < 0.01)

Fig. 5
Fig. 5 Type I interferon signaling was increased in in Sulf2-deficient macrophages.(A-B).Single cell suspensions were isolated from knees of WT and Sulf2 +/− bone marrow chimeric mice (n = 3) 7 days after initiation of arthritis and bulk RNA sequencing performed.(A).The volcano plot shows differentially expressed genes (fold change in Sulf2 +/− compared to WT) identified by DeSeq2, with significantly altered type I interferon-responsive genes[36] labelled in red, other significantly altered genes in black, and non-significantly altered genes in grey.(B).The top 15 KEGG identifiers enriched in differentially expressed genes are shown, indicating their P-value and gene ratio (fraction of the differentially-expressed genes in each KEGG identifier).(C).BMDMs from WT and Sulf2 +/-mice were stimulated with IFNβ (50 ng/ml,