The deubiquitinase OTUD7B ameliorates central nervous system autoimmunity by inhibiting degradation of glial fibrillary acidic protein and astrocyte hyperinflammation

The deubiquitinase OTUD7B ameliorates central nervous system autoimmunity by inhibiting degradation of glial fibrillary acidic protein and astrocyte hyperinflammation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The deubiquitinase OTUD7B ameliorates central nervous system autoimmunity by inhibiting degradation of glial fibrillary acidic protein and astrocyte hyperinflammation Dirk Schlüter, Kunjan Harit, Wenjing Yi, Andreas Jeron, Jakob Schmidt, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5937561/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Oct, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Astrocytes are central to the pathogenesis of multiple sclerosis; however, their regulation by intrinsic post-translational ubiquitination and deubiquitination is unresolved. This study shows that the deubiquitinating enzyme OTUD7B in astrocytes confers protection against murine experimental autoimmune encephalomyelitis, a model of MS, by limiting neuroinflammation. RNA-sequencing of isolated astrocytes and spatial transcriptomics showed that in EAE OTUD7B downregulates the expression of chemokines in astrocytes of inflammatory lesions, which is associated with reduced recruitment of encephalitogenic CD4 + T cells. Furthermore, OTUD7B was essential for GFAP protein expression of astrocytes bordering inflammatory lesions. Mechanistically, OTUD7B (i) restricted TNF-induced chemokine production of astrocytes by sequential K63- and K48-deubiquitination of RIPK1 limiting NF-κB and MAPK activation and (ii) enabled GFAP protein expression by supporting GFAP mRNA expression and preventing its proteasomal degradation through K48-deubiquitination of GFAP. This dual action on TNF signaling and GFAP identifies astrocyte-intrinsic OTUD7B as a central inhibitor of astrocyte-mediated inflammation. Biological sciences/Immunology/Autoimmunity Biological sciences/Biochemistry/Proteins/Ubiquitylated proteins Figures Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Astrocytes are a highly abundant cell population present in all regions of the central nervous system (CNS) and play an important role in the maintenance of CNS homeostasis and health. Under physiological conditions, astrocytes provide physical and metabolic support for neurons and blood-brain barrier (BBB) functions 1–3 . Upon CNS damage, astrocytes are rapidly activated and undergo structural and functional changes, by a process known as reactive astrogliosis which critically regulates the pathogenesis, development and outcome of CNS disorders including autoimmune diseases, infections, neurodegenerative diseases and trauma 4 . Reactive astrogliosis is characterized by astrocyte hypertrophy and increased STAT3-mediated expression of the glia fibrillary acidic protein (GFAP) 5 . Depending on the underlying disease and the inflammatory milieu astrocytes can either support or suppress CNS inflammation, contribute to CNS damage but also to regeneration. This functional plasticity of astrocytes is based on the activation of different signaling pathways leading to the respective production and secretion of disease-modifying proteins. In multiple sclerosis (MS), a human inflammatory demyelinating disease and its murine model experimental autoimmune encephalitis (EAE), reactive astrocytes upregulate GFAP protein expression and are the major producers of chemokines 6,7 . These chemokines foster the recruitment of immune cells, primarily autoimmune CD4 + T cells to the site of inflammation. The CNS infiltrating CD4 + T cells orchestrate an attack on the myelin sheath resulting in demyelination and neurodegeneration 8,9 . The production of chemokines by astrocytes in MS and EAE is driven by several signaling pathways, including NF-κB, MAPK and JAK-STAT pathways 10–12 . Experimental studies have also shown that astrocyte-specific ablation of NF-κB signaling molecules attenuates EAE 11,12 . In addition to disease promoting functions, astrocytes can exert protective roles, in particular, at later stages of EAE and MS. These protective mechanisms include the local restriction and resolution of neuroinflammation and the promotion of remyelination and axonal repair 13–15 . In this regard, STAT3-mediated astrocyte survival, proliferation and up-regulation of GFAP expression ameliorates EAE by limiting the spread of encephalitogenic T cells and bordering of inflammatory lesions 16 . Depending on the subtype of MS and the EAE model, active and resolving inflammatory lesions exist in parallel throughout the CNS. In these different lesions astrocyte reactivity is diverse, shows a high plasticity, differs regionally and may also change over time 17–19 . Astrocyte reactivity is significantly determined by the differential activation of signaling pathways which regulate inflammatory reactions and glia scar formation. These signaling pathways are critically modulated by ubiquitination, a posttranslational modification mediated by the covalent linkage of ubiquitin, a 76-aa large protein, to substrates. Ubiquitination is catalyzed sequentially by an ubiquitin-activating enzyme (E1), an ubiquitin-conjugating enzyme (E2), and an ubiquitin ligase (E3). Ubiquitin molecules can be added to proteins in the form of monomers and polyubiquitin chains, in which ubiquitins are linked through the N-terminal methionine residue (M1) or one of the seven lysine residues K6, K11, K27, K33, K48, K63) 20 . The fate of ubiquitinated substrates is determined by the type of ubiquitin linkage. K48- and K11 chains lead to proteasomal degradation of substrates, whereas K63-linked ubiquitin chains modify protein function and can trigger signal transduction 21–24 . Ubiquitination is reversible and can be counteracted by deubiquitinating enzymes (DUBs). As fine-tuning modulators of cell signaling and activities, DUBs have emerged as important regulators of astrocytes and CNS autoimmunity mediating both protective and disease-promoting astrocyte functions 25 . Of note, the function of DUBs in CNS autoimmunity is also cell type-specific as illustrated by EAE promoting function of the DUB A20 (TNFAIP3) in T cells and its EAE inhibitory function in astrocytes 26,27 OTUD7B is a DUB belonging to the OTU subfamily and is expressed in all human and murine tissues. OTUD7B is expressed in all regions of the CNS, in particular in astrocytes and oligodendrocytes but not in neurons and microglia ( https://www.proteinatlas.org/ENSG00000264522-OTUD7B ). OTUD7B can hydrolyze K11- 28 , K48- 29 and K63- 30 linked ubiquitin chains from distinct substrates and regulates pro-inflammatory signaling by inhibiting TNF-mediated NF-κB activation through K63 deubiquitination of RIPK1 and TRAF6 30,31 . The regulation of TNF signaling by OTUD7B is cell type-specific since OTUD7B prevents TNF-mediated apoptosis in dendritic cells in vitro and in murine cerebral malaria by cleaving K48-ubiquitin chains from the E3 ubiquitin ligase TRAF2 29 . In T cells, OTUD7B inhibits T cell receptor (TCR) mediated activation by deubiquitination of ZAP70, a central molecule of proximal TCR signaling 32 . Consequently, OTUD7B-deficient mice are protected from myelin oligodendrocyte glycoprotein (MOG)-induced EAE due to impaired activation of encephalitogenic CD4 + T cells 32 further illustrating cell type- and disease-specific functions of OTUD7B in inflammatory diseases. To determine the unresolved astrocyte function of OTUD7B in CNS autoimmunity and its impact on astrocyte plasticity, we analyzed expression data of Otud7b in MS data sets and explored the in vivo astrocyte function of OTUD7B in murine EAE. We identified that OTUD7B is upregulated in inflammatory lesions of both MS patients and mice with EAE as compared to healthy CNS tissue. Murine studies revealed that astrocytic OTUD7B was not required for normal CNS development but alleviated EAE. Mechanistically OTUD7B inhibited proteasomal degradation of GFAP in reactive astrocytes by cleaving K48-polyubiquitin chains from GFAP and reducing GFAP mRNA production. In addition, OTUD7B suppressed TNF-induced chemokine production of astrocyte by sequential K63- and K48 -deubiquitination of RIPK1. The simultaneous upregulation of GFAP and inhibition of pro-inflammatroy TNF-signaling by OTUD7B resulted in reduced neuroinflammation, local containment of inflammatory lesions and reduced demyelination. Thus, deubiquitination of GFAP and RIPK1 by OTUD7B are key processes regulating astrocyte reactivity and inhibiting astrocyte-dependent damage in CNS autoimmunity. Results Upregulation of astrocytic Otud7b during CNS autoimmunity To investigate the role of OTUD7B in CNS autoimmunity, we first compared the expression of Otud7b in the astrocytes of patients with a fulminant, anti-MOG-mediated acute form of MS and healthy individuals. Analysis of public available microarray dataset (No. GSE32915) revealed that Otud7b mRNA expression was upregulated in the inflammatory lesions and normal appearing white matter of MS patients as compared to healthy individuals (Fig. 1 A). Next, we determined the expression of Otud7b in MOG-induced EAE, which is characterized by a peak of disease around day 15 post immunization (p.i.) followed by a gradual decline of clinical symptoms 33 . In EAE, Otud7b mRNA expression was upregulated and highest in spinal cords at day 15 p.i. and declined up to day 22 p.i. (Fig. 1 B). Additionally, analysis of Otud7b mRNA expression of astrocytes isolated from the normal and EAE-diseased spinal cord revealed a prominent upregulation of Otud7b mRNA expression in astrocytes of mice with EAE (Fig. 1 C). For a detailed spatial analysis of the expression of astrocytic Otud7b in EAE, we performed spatial transcriptomics with single-molecule resolution on the Xenium platform. Upon immunization with MOG 35–55 peptide and a pertussis toxin boost, cell types were identified based on the transcriptome data using the pre-designed mouse brain panel with 247 genes in combination with a custom panel including additional 50 genes (Suppl. Table 1). We defined inflammatory lesions in the sections characterized by the accumulation of CD3 + CD4 + and CD3 + CD8 + T cells (Fig. 1 D). In addition, we defined a lesion rim in direct proximity to the lesions, with fewer T cells (200µm), and as a third region defined peri-lesion with no T cells (Fig. 1 D, black lines are defined lesion regions). We identified that Otud7b mRNA is upregulated about 1.5-fold in Sox2 + Sox9 + Aqp4 + astrocytes within the lesion core and lesion rim, but not in the peri-lesions as compared to non-immunized mice (Fig. 1 E). As expected, the number of identified cells per section increased almost two-fold upon immunization (Fig. 1 F), with a strong influx of CD68 + microglia and macrophages into the lesions (Fig. 1 F). We observed a strong induction of genes associated with inflammation including Isg15, the chemokines CXCL9, Ccl2, CXCL10 and the cytokines IL-6 and IL1β at d15 p.i. (Fig. 1 G), with the induction being particularly strong within the lesion core and a gradual decline in the lesion rim and peri-lesion. Taken together, astrocytes upregulate Otud7b expression in CNS autoimmunity with the highest expression in the T cell-enriched lesions and a gradual decline with increasing distance from the lesions core, suggesting that astrocytic OTUD7B is a regulator of neuroinflammation. Astrocytic OTUD7B-deficiency aggravates EAE To determine the astrocyte-specific function of OTUD7B in EAE, we crossed GFAP-cre mice with Otud7b fl/fl to generate GFAP-cre Otud7b fl/fl mice with deletion of Otud7b in astrocytes. The GFAP-cre strain used here expresses Cre late during embryonic development (day 14.5 34 ), has a high deletion efficacy in all spinal cord and brain astrocytes but only low deletion in neurons 10,11,16,34 , and allows analysis of astrocyte functions in inflammatory CNS disorders 10,11,16,17,35 . In vivo deletion of Otud7b in spinal cord astrocytes was validated by spatial transcriptomics showing a strong upregulation of Otud7b in astrocytes in EAE of Otud7b fl/fl mice, which was absent in GFAP-cre Otud7b fl/fl mice (Fig. 2 A). In Otud7b fl/fl mice, Otud7b was most strongly expressed in astrocytes, much weaker in neurons and marginally in pericytes, microglia, oligodendrocytes and endothelial cells (Fig. 2 A). Additional analysis of cultured astrocytes by Western blot (WB) also demonstrated efficient deletion of OTUD7B in astrocytes of GFAP-cre Otud7b fl/fl mice (Suppl. Figure 1A). GFAP-cre Otud7b fl/fl mice were born in a normal mendelian ratio and grew normally (data not shown). Histologically, spinal cords of GFAP-cre Otud7b fl/fl mice were normal with intact astrocytes and showed no signs of neurodegeneration and inflammation (Suppl. Figure 1B). Major leukocyte populations in the spinal cord, spleen, and lymph node were comparable between Otud7b fl/fl and GFAP-cre Otud7b fl/fl mice (Suppl. Figure 1C-F), consolidating that OTUD7B expression in astrocytes does not regulate inflammatory responses under homeostatic conditions. To explore the role of astrocytic OTUD7B in EAE, we immunized Otud7b fl/fl and GFAP-cre Otud7b fl/fl mice with MOG peptide. Although the two genotypes had similar disease onset at around day 12 p.i.,, GFAP-cre Otud7b fl/fl mice showed significantly increased clinical scores with higher maximal clinical scores and disease incidence (Fig. 2 B), and reduced body weight (Fig. 2 C). In accordance with the aggravated clinical symptoms, demyelination was more pronounced in GFAP-cre Otud7b fl/fl as compared to control mice at d15 p.i. (Fig. 2 D). In addition, inflammatory infiltrates were larger, more confluent and infiltrated deeper in the spinal cord tissue in GFAP-cre Otud7b fl/fl mice (Fig. 2 D, 2 E). In EAE, astrocyte morphology and reactivity was strongly altered. Adjacent to and bordering inflammatory lesions astrocytes of Otud7b fl/fl mice strongly expressed GFAP and showed prolonged GFAP + astrocytic processes (Fig. 2 F, 2 G). On the contrary, astrocytes of the GFAP-cre Otud7b fl/fl mice associated with inflammatory infiltrates only weakly expressed GFAP and, thus, did not form a GFAP + border surrounding the inflammatory infiltrate (Fig. 2 E, 2 G, 2 H). In both control and GFAP-cre Otud7b fl/fl mice, equal numbers of SOX2 + SOX9 + astrocytes were present indicating that the reduction of GFAP expression was not caused by a loss of OTUD7B-deficient astrocytes (Fig. 2 G, 2 H). Interestingly, a comparison of GFAP mRNA levels by spatial transcriptomics showed upregulation particularly in the lesion core and the lesion rim, which was higher in Otud7b fl/fl than in GFAP-cre Otud7b fl/fl mice. (Fig. 2 I) indicating that the diminished transcription of GFAP may contribute to the reduced GFAP protein in GFAP-cre Otud7b fl/fl mice (Fig. 2 F-H). OTUD7B suppressed chemokine production, and recruitment of encephalitogenic CD4 T cells to the CNS Reactive astrocytes contribute to the development of EAE by producing pro-inflammatory mediators, including chemokines, which induce the recruitment of encephalitogenic autoimmune CD4 + T cells into the CNS 36,37 . Since GFAP-cre Otud7b fl/fl mice exhibited increased CNS inflammation, we next determined the impact of OTUD7B on the transcriptome of astrocytes isolated from the spinal cords of non-immunized and MOG-immunized (d15 p.i.) Otud7b fl/fl and GFAP-cre Otud7b fl/fl mice. We detected 1944 genes differentially regulated between OTUD7B-deficient and -sufficient astrocytes (Fig. 3 A). Of these 1994 genes, 689 genes were differentially regulated under homeostatic conditions, 1090 genes were regulated at d15 p.i. and 165 genes were regulated under both conditions (Fig. 3 A). Clustering of the genes using the K-means clustering algorithm resulted in 10 clusters. Ontology and KEGG pathway enrichment analysis showed that the pathways related to chemokine and cytokine signaling (Fig. 3 B) were upregulated in cluster 6 and 2 of OTUD7B-deficient astrocytes upon EAE. In addition, OTUD7B-deficient astrocytes of cluster 6 and 2 had increased expression of genes regulating transendothelial migration of leukocytes, pro-inflammatory cytokine and chemokine signaling, cell adhesion and angiogenesis (Fig. 3 C, 3 D). A detailed analysis of chemokine genes showed that upon induction of EAE, expression of CCL and CXCL chemokines was upregulated in both genotypes but expression of CCL2, 3, 4, 5, 6, 7, 8, 11, 19 and CXCL 1, 2, 9, 10, 12, 13, 16 were higher in OTUD7B-deficient astrocytes. This suggests that the increased disease pathology in GFAP-cre Otud7b fl/fl mice was due to increased chemokine and cytokine mediated infiltration of leukocytes into the spinal cord, (Fig. 3 E). Only Ccl22, Cxcl16, and Cxcl12, which are stronger expressed by microglia and brain macrophages as compared to astrocytes ( https://www.proteinatlas.org ) were increased upregulated in OTUD7B-comptetent astrocytes (Fig. 3 E). Spatial transcriptome analysis of spinal cord tissue provided further information on the anatomic distribution of chemokines and cytokines. Chemokine mRNA expression was higher in OTUD7B-deficient astrocytes in the lesion cores and in the rims surrounding the lesion in relation to peri-lesion areas where the chemokine levels were comparable (Fig. 3 F). Among the cytokines studied, only IL-6 and IL-1β but not TNF, TGF-β1 were highly expressed in OTUD7B-deficient astrocytes in EAE. In contrast to astrocytes, chemokine and cytokine expression of microglia was equal between the two genotypes. Consistent with the astrocyte RNA-sequencing data, RT-PCR-based quantification of chemokine mRNA in spinal cords of mice with EAE revealed that GFAP-cre Otud7b fl/fl mice had significantly higher levels of CXCL1, CXCL10, CXCL11, Ccl2, and Ccl20 mRNA at day 15 p.i., which are major attractants for lymphoid and monocytic cells (Fig. 3 G). Increased recruitment of encephalitogenic CD4 T cells in GFAP-cre Otud7b mice To determine whether the increased chemokine production of OTUD7B-deficient astrocytes resulted in an enhanced recruitment of leukocytes to the CNS in MOG-immunized mice, we performed a flow cytometry analysis of leukocytes isolated from the spinal cords. In contrast to non-immunized mice (Suppl. Figure 1E), relative and absolute numbers of CD4 + but not of CD8 + T cells were significantly increased in Otud7b fl/fl and GFAP-cre Otud7b fl/fl mice with EAE (Fig. 4 A). Additionally, numbers of CD11c + dendritic cells, Ly6C high CD11b + inflammatory monocytes and F4/80 + CD11b + macrophages/microglia but not of CD19 + B cells were higher in GFAP-cre OTUD7b fl/fl mice (Fig. 4 B). Among CD4 + T cell subsets, IFN-producing Th1 cells, IL-17-producing Th17 cells, and GM-CSF-producing CD4 + T cells are the major effectors in EAE and each of them can induce EAE independently 38–40 . We identified that the absolute but not relative numbers of infiltrating GM-CSF + , IFN-γ + , and IL17 + CD4 cells were significantly increased in the spinal cord of GFAP-cre Otud7b fl/fl mice with EAE (Fig. 4 C, D). This indicates that astrocyte-specific OTUD7B limits the recruitment of encephalitogenic CD4 + T cell subsets to the CNS but does not influence the differentiation and composition of the recruited CD4 + T cell subsets during EAE. In accordance with the increased recruitment of encephalitogenic CD4 + T cells, inflammatory monocytes and macrophages into the CNS of GFAP-cre Otud7b fl/fl mice with EAE, mRNA production of IFN-γ,TNF, IL-17, GM-CSF and NOS2, which all contribute to demyelination in EAE 41–45 , was also increased, Collectively, OTUD7B expression in astrocytes suppressed astrocyte chemokine production, recruitment of encephalitogenic leukocytes, demyelination and disease symptoms in EAE illustrating the important neuroprotective function of astrocytic OTUD7B. OTUD7B suppressed early TNF-induced pro-inflammatory signaling in astrocytes The increased astrocyte activation and chemokine production of GFAP-cre Otud7b fl/fl mice under inflammatory but not under homeostatic conditions indicates that pro-inflammatory cytokines induced OTUD7B-dependent immunoregulatory astrocyte function. To identify whether astrocyte chemokine and cytokine production induced by the encephalitogenic cytokines TNF, IFN-γ and IL-17, respectively, is regulated by OTUD7B, we cultured OTUD7B-deficient and -competent astrocytes, stimulated them with the cytokines and determined chemokine and cytokine production by RT-PCR. Upon stimulation with TNF, mRNA expression of CXCL1, CXCL11, CCL20, IL-6, CCL2 and NOS2 were significantly increased in OTUD7B-deficient astrocytes (Fig. 5 A). In contrast, OTUD7B did not regulate IFN-γ- and IL-17-induced chemokine and cytokine production (Fig. 5 A) indicating that TNF signaling is the major pathway regulated by OTUDB in astrocytes. Thus, both increased TNF mRNA expression in EAE (Fig. 4 E) and TNF-induced astrocytic cytokine and chemokine production are under control of astrocytic OTUD7B. To directly assess the effect of OTUD7B on TNF, IFN-γ and IL-17 signaling, we stimulated cultivated astrocytes with these cytokines and analyzed activation of the respective signaling pathways by WB. Upon stimulation with TNF, activation of both the NF-κB pathway, indicated by increased phosphorylation of p65 and degradation of IκBα, and the phosphorylation of ERK, p38 and JNK in the MAPK pathways were stronger in OTUD7B-deficient astrocytes (Fig. 5 B). Of note, these differences were detectable as early as 10 min post TNF stimulation. In contrast, stimulation with IFN-γ did not result in increased STAT1 phosphorylation (Fig. 5 C) and activation with IL-17 had no effect on phosphorylation of IκBα, ERK, p38 and JNK (Fig. 5 D). Thus, OTUD7B is an inhibitor of TNF but not IFN-γ and IL-17 signaling in astrocytes. OTUD7B regulates TNF signaling by sequential K63- and K48- deubiquitination of RIPK1 Since chemokine production of OTUD7B-deficient astrocytes was highest in the lesion core (Fig. 3 E) and OTUD7B-regulated TNF-dependent chemokine production, we next analyzed the spatial distribution of TNF mRNA in Otud7b fl/fl and GFAP-cre Otud7b fl/fl mice with EAE. In accordance with the chemokine data, expression of TNF was highest in the lesion core with a gradual decline in the lesion rim and the peri-lesional tissue (Fig. 6 A). These data show a gradient of TNF production and also indicate that astrocytes will be exposed to TNF for several days based on the persistence of the lesions. TNF signaling is dynamically regulated by K63 and K48 poly-ubiquitination of RIPK1. Rapidly after TNF stimulation, RIPK1 is K63 poly-ubiquitinated by TRAF2 and cIAP1 leading to RIPK1-dependent activation of NF-κB and MAPKs 29,46 . The activation of NF-κB induces expression of A20 (TNFAIP3), which cleaves K63 chains from RIPK1 and can also induce K48-dependent proteasomal degradation of RIPK1 47 , which also leads to degradation of TRAF2 and termination of NF-κB and MAPK signaling 48,49 . Thus, we determined the impact of OTUD7B on RIPK1, TRAF2 and cIAP1 over a period of 5 days to reflect that under in vivo conditions astrocytes are exposed to TNF for several day. WB analysis showed that RIPK1, TRAF2 and cIAP1 protein levels remained unchanged in both genotypes but that phosphorylation of p65, p38 and JNK was increased in OTUD7B-deficient astrocytes at days 1 and 2 of TNF stimulation (Fig. 6 B). From day 3 to 5, RIPK1 (Fig. 6 B, Suppl. Figure 2B) and from day 4 to 5 TRAF2 gradually declined in OTUD7B-deficient but not OTUD7B-competent astrocytes, whereas cIAP1 remained unchanged (Fig. 6 B). The decline of RIPK1 protein in OTUD7B-deficient astrocytes was preceded by a strong increase of A20, which was much weaker in OTUD7B-competent astrocytes (Fig. 6 B). In OTUD7B-deficient astrocytes the decline of RIPK1 and TRAF2 protein was paralleled by strong reduction of p65, p38 and JNK phosphorylation, which was not detectable in OTUD7B-expressing astrocytes (Fig. 6 B). Since RIPK1 activity and stability are regulated by its K63 and K48 poly-ubiquitination, respectively, we analyzed next whether the DUB OTUD7B regulates the RIPK1 ubiquitination status. Immunoprecipitation of RIPK1 at the various time points up to day 5 of TNF stimulation, showed that the activating K63-ubiqutination of RIPK1 was higher in OTUD7B-deficient astrocytes at days 1 and 2 but reduced at days 3 to 5 as compared to OTUD7B-expressing astrocytes (Fig. 6 C). On the opposite, K48 poly-ubiquitination of RIPK1 strongly increased from days 3 to 5 in OTUD7B-deficient but to a much lesser extent in OTUD7B-competent astrocytes (Fig. 6 C). In the RIPK1 complexes, higher amounts of A20 and TRAF2 were present in OTUD7B-deficient as compared to OTUD7B-expressing astrocytes (Fig. 6 C). To validate that OTUD7B counteracts A20-mediated proteasomal degradation of RIPK1, we silenced A20 by siRNA in TNF-stimulated astrocytes. Inhibition of A20 restored RIPK1 protein in OTUD7B-deficient astrocytes to the same level as in OTUD7B-competent astrocytes (Fig. 6 D). In parallel to the increased RIPK1 protein, TRAF2 increased in A20 siRNA-treated OTUD7B-deficient astrocytes (Fig. 6 D), which is explained by the stabilizing role of RIPK1 for TRAF2 49 . In addition, proteasome inhibition by MG132 prevented degradation of RIPK1 in OTUD7B-deficient astrocytes (Fig. 6 E), which is in line with both the K48-dependent proteasomal degradation of RIPK1 in OTUD7B-deficient astrocytes and the prevention of proteasomal degradation by K48-deubiquitination of RIPK1 by OTUD7B in OTUD7B-competent astrocytes (Fig. 6 C). Since spatial transcriptomics revealed an increase of TNF mRNA from the healthy tissue to the core of EAE lesions (Fig. 6 A), we stimulated cultivated astrocytes with increasing TNF concentrations for 5 days (Fig. 6 E). These studies revealed that TNF-stimulation reduced RIPK1 proteins in OTUD7B-deficient but not in OTUD7B-competent astrocytes in dose-dependent manner (Fig. 6 E). In parallel TRAF2 levels declined, whereas cIAP1 protein levels were not affected by OTUD7B-deficiency. In addition, the decrease of downstream p65, p38 and JNK phosphorylation in OTUD7B-deficient astrocytes was higher upon stimulation with increased amount of TNF (Fig. 6 E). In both OTUD7B-competent and -deficient astrocytes, A20 was induced by TNF-stimulation and A20 protein levels were independent of the TNF concentration. Importantly and in agreement with the analysis of the kinetic of A20 expression (Fig. 6 B), A20 levels were strongly increased in OTUD7B-deficient astrocytes, and, thus, were not affected by the reduced NF-κB activation. K48 poly-ubiquitination of RIPK1 increased in OTUD7B-deficient astrocytes stimulated with higher concentrations of TNF and this was paralleled by a TNF-dependent increase of A20/RIPK1 complex formation (Fig. 6 F, Suppl. Figure 2B). Collectively, these data identify that OTUD7B regulates both K63- and K48-ubiquitination of RIPK1 in a time- and TNF dose-dependent manner. OTUD7B mediates GFAP-stability by its K48 deubiquitination Increased GFAP protein expression is a hallmark of reactive astrocytes but the mechanism regulating GFAP protein levels, i.e. the impact of GFAP mRNA transcription and, in particular, GFAP protein stability and turnover are largely unresolved. Histological analysis of GFAP showed that OTUD7B was required for GFAP protein expression in EAE (Fig. 2 F-H). Moreover, the reduced GFAP mRNA of GFAP-Otud7b fl/fl mice with EAE (Fig. 2 I) indicates that the reduced GFAP transcription may contribute to the reduction of GFAP protein in GFAP-cre OTUD7b fl/fl mice. Since IL-6 activates STAT3 and both are important for the induction of GFAP mRNA, we analyzed first whether IL-6 mRNA production was reduced in GFAP-cre OTUD7b fl/fl mice with EAE. On the contrary, spatial transcriptomics showed an increase in the levels of IL-6 mRNA in GFAP-cre Otud7b fl/fl mice (Fig. 7 A), therefore, we next analyzed whether OTUD7B might regulate IL-6 signaling, in particular STAT3 activation. In TNF-stimulated OTUD7B-deficient astrocytes, the phosphorylation of STAT3 was increased in the first two days and subsequently strongly reduced as compared to OTUD7B-competent astrocytes (Fig. 7 B). GFAP mRNA (Fig. 7 C) was regulated in the same kinetic indicating that OTUD7B-dependent STAT3 phosphorylation is an important factor regulating GFAP mRNA expression. Of note, total STAT3 protein levels were OTUD7B-independent (Fig. 7 B) demonstrating that OTUD7B did not regulate STAT3 stability. To determine whether GFAP protein stability might be additionally regulated by OTUD7B, we stimulated astrocytes with TNF, inhibited IL-6, blocked new protein synthesis by CHX and prevented proteasomal degradation of proteins by MG132 treatment (Fig. 7 D, E). We limited these experiments until day three post TNF stimulation, since from that time point onwards the impaired p38 and JNK activity of OTUD7B-deficient astrocytes might impact on GFAP mRNA expression. These experiments revealed that (i) TNF-stimulation increased GFAP protein in OTUD7B-competent but not in OTUD7B-deficient astrocytes, (ii) additional inhibition of IL-6 reduced GFAP protein in both genotypes, (iii) additional inhibition of protein synthesis by CHX further reduced GFAP and that (iv) inhibition of proteasomes by MG132 restored GFAP levels in OTUD7B-deficient astrocytes to the same level as in OTUD7B-competent astrocytes (Fig. 7 D). This uncovers that OTUD7B stabilizes GFAP by preventing its proteasomal degradation. In good agreement, K48 poly-ubiquitination of GFAP was strongly increased in TNF-stimulated OTUD7B-deficient astrocytes (Fig. 7 E). Collectively, OTUD7B supported GFAP protein levels/abundance by two independent but synergistic mechanisms: STAT3-mediated GFAP mRNA expression based on sustained RIPK1, p38 and JNK activation leading to continued STAT3 phosphorylation and by K48-deubiquitination of GFAP critical for stabilization and increased GFAP proteins in reactive astrocytes (Fig. 7 F). Discussion Astrocytes are an important regulator of CNS inflammation, which can both limit and augment neuroinflammation 10,11,27,50–52 . However, the astrocyte intrinsic molecular mechanisms determining the pro- and anti-inflammatory function of astrocytes in CNS autoimmunity are incompletely understood. Analysis of public available transcriptome data of MS tissue ( 53 , Fig. 1 A) and spatial transcriptome analysis of mice with EAE (Fig. 2 G) identified that OTUD7B is expressed in astrocytes under homeostatic conditions and upregulated in astrocytes associated with the inflammatory lesions. Data presented here identify that the upregulation of OTUD7B is a protective astrocyte-intrinsic mechanism ameliorating CNS autoimmunity by dynamic modulation of pro-inflammatory TNF signaling through RIPK1 deubiquitination and by upregulation of GFAP protein levels. A hallmark of reactive astrocytes in most CNS pathologies is an increased expression of GFAP protein. Mice with OTUD7B deletion in astrocytes had normal GFAP expression under homeostatic conditions but greatly reduced or even absent GFAP protein proximal to inflammatory lesions in EAE. Mechanistically, prolonged stimulation with TNF induced direct interaction of OTUD7B with GFAP and prevented K48-dependent proteasomal degradation of GFAP. This shows for the first time that GFAP protein abundance is under direct control of the ubiquitin system. Thus, in addition to cleavage of GFAP by caspase-3 54 , the deubiquitinating function of OTUD7B plays a non-redundant role for GFAP stability in reactive astrocytes. In addition, sustained GFAP mRNA expression was dependent on OTUD7B in vivo and in vitro . STAT3 is the major transcription factor inducing GFAP mRNA expression and can be activated by TNF and IL-6, respectively. Deletion of STAT3 or the cognate receptors for IL-6 family cytokines leads to strongly diminished GFAP protein expression and impaired containment of inflammatory lesions by reactive astrogliosis 5,33,55–57 . Since IL-6 mRNA levels did not differ in inflammatory lesions of OTUD7b fl/fl and GFAP-cre OTUD7b fl/fl mice but p38 and JNK were less activated due to impaired RIPK1 stability in OTUD7B-deficient astrocytes, OTUD7B might regulate GFAP mRNA expression indirectly. Key factors for the phosphorylation of STAT3 are the MAPK kinases p38 and JNK 58–63 , which can be activated by RIPK1 upon TNF stimulation. Our analysis of the impact of OTUD7B on the activation of p38 and JNK had revealed that OTUD7B regulated RIPK1 activity and downstream p38 and JNK activation (Fig. 6 B) in the identical kinetic as STAT3 phosphorylation (Fig. 7 B). Thus, OTUD7B might regulate GFAP mRNA expression indirectly via the RIPK1-p38/JNK-STAT3 axis. Since OTUD7B-deficiency did not regulate STAT3 protein stability (Fig. 7 B), a direct K48-deubiquitinating activity of OTUD7B on STAT3 leading to reduced GFAP mRNA production can be ruled out. In addition, a K63-deubiquitination of STAT3 by OTUD7B would impair STAT3 activity and, thus, cannot underly the increased in vivo and in vitro GFAP mRNA expression of OTUD7B-competent astrocytes. In EAE and other inflammatory diseases of the CNS, reactive astrocytes can form borders around inflammatory lesions, which contributes to the local restriction of the neuroinflammation and CNS damage 64–66 . Functionally important, studies in GFAP-deficient mice revealed that deletion of GFAP leads to a more widespread inflammation and more severe disease in EAE and bacterial and parasitic CNS infections 64,67–69 . This contributed to the concept that bordering of inflammatory lesions by reactive astrocytes with increased GFAP expression contributes to the local containment of inflammation (reviewed by Sofroniew 9 ). Of note, a low level of K48 ubiquitination and GFAP degradation were also detected in OTUD7B-competent astrocytes (Fig. 7 E). This may be an important mechanism to prevent pathological GFAP accumulation in reactive astrocytes. In this regard, excessive accumulation of GFAP in astrocytes induced by gain-of-function mutations in the GFAP gene and by impaired proteasomal GFAP degradation is toxic and underlies human Alexander disease and its corresponding mouse models 70 . Perspectival, it would be interesting to explore OTUD7B in this astrocytopathy and to determine whether GFAP degradation induced by OTUD7B inhibition might have a therapeutic effect. In addition, it remains to be determined which E3 ligases mediate K48 ubiquitination of GFAP. Collectively, these data imply that the regulation of GFAP abundance by OTUD7B-dependent deubiquitination is an important general mechanism potentially regulating the function of reactive astrocytes independent of the underlying disease. In addition to reduced GFAP protein, the dominant phenotype of OTUD7B-deficient astrocytes was increased chemokine production in the core and rim of inflammatory EAE lesions, which was associated with an increased recruitment of encephalitogenic T cells, more widespread inflammation and demyelination. Chemokine production of astrocytes occurs also in other CNS disorders and is regarded as a key function of astrocytes leading to the recruitment of leukocytes to the CNS and neuroinflammation 71,72 . Detailed analysis of the molecular functions of OTUD7B identified that OTUD7B rapidly interacted with RIPK1 upon TNF exposure. RIPK1 is a central signaling molecule regulating the activation of pro-inflammatory NF-κB and MAPK signaling as well as cell death pathways 73,74 . The ubiquitination status of RIPK1 is critical to induce or inhibit these individual cellular pathways 29,46 . OTUD7B limited NF-κB and MAPK activation by reducing RIPK1 K63-ubiquitination, which is important for NF-κB - and MAPK-dependent cytokine and chemokine production of TNF-stimulated astrocytes. The reduced chemo- and cytokine expression of OTUD7B-competent astrocytes was evident in vitro, in the bulk RNAseq analysis of ex vivo isolated astrocytes and spatial transcriptomic analysis of mice with EAE. In TNF activated cells, NF-κB activation leads to the expression of A20 which subsequently interacts with and inhibits RIPK1. In this negative feedback loop, A20 inhibits sustained RIPK1 activation by RIPK1 K63-deubiqutination and by inducing K48-dependent proteasomal degradation of RIPK1. Here, we identified that the A20-mediated dynamic change of RIPK1 ubiquitination, resulted in a shift of the targeted ubiquitin chains of OTUD7B from K63 to K48 of RIPK1, and that OTUD7B interacted with A20 and diminished K48 polyubiquitination of RIPK1. In the absence of OTUD7B, A20 induced K48 ubiquitination of RIPK1 induced its proteasomal degradation. Functionally important and in agreement with the present study deletion of A20 in astrocytes results in an augmented and sustained NF-κB and also STAT1 activation leading to aggravation of EAE 27 . Of note, the dominant in vivo phenotype of OTUD7B-deficient astrocytes was increased activation and chemokine production at day 15 p.i., i.e. when clinical symptoms of EAE already existed for several days. Thus, the OTUD7B-regulated in vitro shift from TNF-induced increased to reduced astrocyte activation was not detectable in vivo. In this regard, it should be stressed that (i) other NF-κB and MAPK activating pathways, in particular IL-17 signaling, were not regulated by OTUD7B (Fig. 5 D), and (ii) IL-17 was increased expressed in the CNS of GFAP-cre Otud7b fl/fl mice (Fig. 4 E). In addition, disturbance of GFAP protein expression induces endoplasmic reticulum stress, increased activation of MAPK kinases and neuroinflammation as observed in murine models of Alexanders diseases 70 . Thus, the in vitro diminished RIPK1 signaling upon prolonged TNF exposure might be in vivo compensated by other pro-inflammatory signaling pathways contributing to the sustained increased chemokine production by astrocytes. TNF can induce RIPK1-dependent cell death, if K63 ubiquitination of RIPK1 is impaired. Histologically, the greatly diminished GFAP protein expression of OTUD7B-deficient astrocytes gave the impression that these cells might have been eliminated by apoptosis. However, we detected identical presence of SOX2 + Sox9 + astrocytes in both GFAP-cre Otud7b fl/fl and Otud7b fl/fl mice and spatial transcriptomics showed that astrocyte numbers did not differ in the core, rim and peri-lesion between the two genotypes. Also, short and long-term in vitro stimulation of OTUD7B-deficient astrocytes did not result in cell death (data not shown). Thus, OTUD7B-deficient astrocytes with impaired K63-ubiqutination of RIPK1 were highly resilient against TNF-induced cell death. This is in contrast to OTUD7B-deficient dendritic cells, which rapidly undergo apoptosis upon exposure to TNF in murine cerebral malaria 29 . On the contrary, OTUD7B does not regulate apoptosis in T cells but facilitates proximal T cell receptor signaling by deubiquitination of the tyrosine kinase ZAP70 in EAE and murine listeriosis, two diseases characterized by the production of large amounts of TNF. At present, the cell type-specific differences underlying the differential impact of OTUD7B on TNF induced apoptosis are unresolved. Limitations of the study This study illustrates that astrocyte activity is dynamically and interdependently regulated by the concentration of external factors such as TNF and by intrinsic signaling molecules, in particular OTUD7B. Although OTUD7B is a critical factor regulating astrocyte reactivity in EAE and unique in its capacity to regulate both GFAP and RIPK1, the interplay with other external factors including those derived from the microbiome, which also regulate astrocyte intrinsic responses 75–78 , still has to be explored. Thus, it remains to be determined whether OTUD7B has the same function and central role under other experimental conditions and in human CNS disorders. At present the complex network of interdepend external and intrinsic factors regulating astrocyte reactivity cannot be resolved in its kinetic at the spatial level for single astrocytes. Thus, it remains to be determined whether OTUD7B regulates the plasticity of astrocytes or is a determinant for the development of specific astrocyte subpopulations under inflammatory conditions. Materials and Methods Mice OTUD7B fl/fl mice with C57BL/6 background were generated with C57BL/6N- Otud7btm1b(EUCOMM)Wtsi/Wtsi embryonic stem cells purchased from the European Mouse Mutant Archive. First, Otud7b mutant mice were crossed with B6.129S4- Gt(ROSA)26Sor tm1(FLP1)Dym /RainJ (Stock No: 009086, The Jackson Laboratory, Bar Habor, ME, USA) to delete the frt -flanked sequences. Thereafter, otud7b mutants were crossed with C57BL/6 GFAP-cre mice 34 to obtain GFAP-cre Otud7b fl/fl mice. The genotyping was performed by PCR of the tail DNA with primers specific for GFAP-cre and Otud7b fl/fl , respectively. Wildtype C57BL/6 mice were obtained from Janvier (Le Genest-Saint Isle, France). Animals were kept under specific pathogen-free (SPF) conditions in animal facilities of the Otto-von-Guericke University Magdeburg (Magdeburg, Germany) and Hannover Medical School (Hannover, Germany). Animal care and experimental procedures were carried out according to the European animal protection law and approved by local authorities (Landesverwaltungsamt Halle, file number 42502-2-1260). Induction of EAE and clinical assessment For active EAE induction, 8–12 weeks old mice were immunized with 200µg of myelin oligodendrocyte glycoprotein (MOG) 35−55 peptide mixed in complete Freund’s adjuvant containing 800µg of killed Mycobacterium tuberculosis . In addition, 200ng pertussis toxin dissolved in 200 µl PBS was intraperitoneally injected respectively at day 0 and 2 post immunization (p.i.). The symptoms and body weight were monitored daily in a double-blinded way according to a previously published score with increasing severity from 0 to 5 as follows 10 : 0, no signs; 0.5, partial tail weakness; 1, limp tail or slight slowing of righting from supine position; 1.5, limp tail and slowing of righting; 2, partial hind limb weakness; 2.5, dragging of hind limb(s) without complete paralysis; 3, complete paralysis of at least one hind limb; 3.5, hind limb paralysis and slight weakness of forelimbs; 4, severe forelimb weakness; 5, moribund or dead. Daily clinical scores were displayed as the mean of all individual disease scores within each group. Astrocyte isolation from adult mice Spinal cords were isolated from anaesthetized and PBS-perfused naïve and EAE mice at day 15 d p.i. Single-cell suspension was generated using NeuroCult™ Enzymatic Dissociation Kit according to the manufacturer’s instruction. Astrocytes were purified from the single-cell suspension with the anti-ACSA-2 Microbead Kit and the purity was analyzed by flow cytometry with anti-ACSA-2-PE antibody. Isolation of cells from the spinal cord and flow cytometry To obtain leukocytes from the spinal cord of non-immunized mice (d0) and mice with EAE (d15 p.i.) mice were first cardially perfused with 0.1 M PBS (pH 7.4) in deep methoxyflurane anesthesia. Immediately thereafter, spinal cords were removed, minced through 70µm cell strainers followed by Percoll ® gradient centrifugation. Cells were counted with a hemocytometer and stained with fluorochrome-coupled antibodies against CD4, CD3, CD45, CD8, CD19, B220, Ly6C, Ly6G, CD11b and CD11c to differentiate T cells, B cells, inflammatory monocytes, granulocytes, macrophages, and dendritic cells respectively (see STAR Methods table). For intracellular staining, cells were incubated with PMA (50 ng/ml), ionomycin (500 ng/ml), and Brefeldin A (1 µg/ml) in RPMI 1640 medium supplemented with 10% FCS, 1% Non-essential amino acids (NEAA), and 1% L-glutamine at 37 o C for 4 h. Thereafter, cells were stained with CD3, CD4, CD45 antibodies, fixed and permeabilized with Intracellular Fixation/Permeabilization Kit followed by staining with anti-IL-17, anti-GM-CSF, and anti-IFN-γ antibodies, respectively. Flow cytometry was performed on a Cytek Northern Light Flow Cytometer and data were analyzed with the FlowJo software. Histology Mice anesthetized with methoxyflurane were perfused with 0.1 M PBS followed by 4% paraformaldehyde in PBS. After embedding in paraffin, sections of brains and spinal cords were used for hematoxylin & eosin and cresyl violet-luxol fast blue (CV-LFB) staining. Expression of GFAP was demonstrated in an ABC protocol with 3,3’ diaminobenzidine and H 2 O 2 as substrate. For the immunofluorescent staining, the slides were deparaffinized for 1 h at 60°C, followed by a removal of the residual paraffin with 2 × 15 min washing in xylene. Next, the tissue was rehydrated and incubated in 0.5% NaBH4 for 30 min at room temperature to reduce autofluorescence 79 . For antigen retrieval, the slides were incubated in 10 mM citric acid buffer (with 2 mM EDTA, 0.05% Tween20) for 15 min at 95°C and allowed to cool down for 20 min. After washing in PBS-T (1% TritonX-100) for 30 min and PBS for 10 min at room temperature, slices were incubated with the primary antibodies diluted in blocking solution (3% NDS, 0.5% TritonX-100) at 4°C 80 . As primary antibodies rabbit anti-GFAP (1:1000), mouse anti-Sox2 (1:500) goat anti-SOX9 (1:500) and rat anti-Iba-I (1:800) were used. After 2x15 min washing with PBS the slices were incubated with secondary antibodies diluted in blocking solution at 4°C overnight. As secondary antibodies Alexa488-conjugated donkey anti-goat (1:400), Alexa488-conjugated donkey anti-mouse (1:400), Cy3-conjugated donkey anti-rat (1:400) and Cy5-conjugated donkey anti-rabbit (1:400) were used. Subsequently, secondary antibodies were removed, and nuclei were stained using DAPI (1:10000). After 3x10 min washing in PBS, slides were mounted with Aquapolymount solution and stored at 4°C. Images were taken using a Zeiss inverted Axio Observer seven with ApoTome.2 equipped with an Axiocam 503 and a Colibri 7 LED light source, and Zeiss LSM 780 with four lasers (405, 488, 559 and 633 nm) and ×20, ×40 and ×63 objective lenses. For apotome acquisition on Zen2.6 pro software was used. Primary astrocyte cultures and treatment Primary astrocytes were isolated from 1- to 2-day-old newborn mice and cultured in DMEM containing 1% glutamine, 10% FCS, and 1% penicillin/streptomycin as described before 10 . The purity of astrocyte cultures was more than 95%, as assessed by flow cytometry with antibodies against CD11b and ACSA-2. For the analysis of cytokine receptor-activated signaling pathways, astrocytes were stimulated at the indicated concentrations with TNF (10ng/ml), IL-17 (50 ng/ml), and IFN-γ (10 ng/ml), respectively, for the indicated time points. For long term TNF treatment, primary astrocytes were stimulated with increasing concentrations of TNF (10 ng/mL, 20 ng/mL or 50 ng/mL) for 5 days. CHX chase assay- To detect the stability of GFAP protein, cells were stimulated with either TNF alone (50ng/mL) or in combination with α-IL6 antibody (2µg/mL) for 3 days. At d3 post-stimulation, cells were treated with 10µg/mL of cycloheximide (CHX) with/without 10µM of proteasome inhibitor MG132 for 6h. siRNA transfection- For siRNA-mediated knockdown of A20, primary astrocytes were transfected with 5 µM of A20-specific siRNA according to the manufacturer's instructions. Thereafter, the cells were stimulated with 50 ng/mL of TNF for 5 days, followed by protein isolation and WB analysis Western blot Samples from primary astrocytes and mouse organs were lysed on ice in RIPA lysis buffer supplemented with PhosSTOP, phenylmethylsulfonyl fluoride (PSMF) and protease inhibitor cocktail. Cell lysates were pre-cleared by centrifugation at 14,000 rpm for 15mins at 4°C. Supernatant was collected and quantified by BCA assay according to manufacturer’s protocol. Protein samples heated in lane marker reducing sample buffer at 99°C for 5 min. Equal amounts of samples were separated by SDS-PAGE and subsequently transferred to polyvinylidene difluoride (PVDF) membranes, which were blocked with 5% BSA at room temperature for 1h, followed by incubation with mentioned primary antibodies (STAR methods Table) at 4°C overnight. Blots were developed using the ECL Plus Kit and images were captured on Intas Chemo Cam Luminescent Image Analysis system (INTAS). Quantification and analysis of WB images was performed with the LabImage 1D software. Co-immunoprecipitation (Co-IP)- Whole cell lysates from astrocytes were precleared by incubation with GammaBind G Sepharose beads with gentle shaking at 4°C for 2 h. After removal of beads by centrifugation, samples were incubated with specific antibodies under continuous shaking at 4°C overnight. Following day, antibody-protein complexes were captured by incubating samples with GammaBind G Sepharose beads at 4°C for 2 hours. Thereafter, the beads were washed with ice cold PBS thrice, resuspended in 2x lane marker reducing sample buffer and boiled at 99°C for 5 min. Samples were centrifuged and supernatant was collected for WB analysis. Quantitative RT-PCR Total mRNA was isolated from spinal cord tissue or astrocytes in buffer RLT using the RNeasy Mini Kit according to manufacturer’s protocol. mRNA was reverse-transcribed into cDNA with the SuperScript Reverse Transcriptase Kit. Quantitative RT-PCR was performed with a LightCycler 480 system using TaqMan probes (STAR Methods table). Gene expression levels were normalized to internal control Hprt and fold change increase in gene expression over naïve controls was calculated according to the ∆∆ cycle threshold (CT) method (Livak and Schmittgen, 2001 81 ). Transcriptome analysis of isolated astrocytes Astrocytes were isolated by magnetic microbeads from spinal cords of Otud7b fl/fl and GFAP-cre Otud7b fl/fl mice, respectively, at day 15 p.i. Astrocytes isolated from naïve Otud7b fl/fl and GFAP-cre Otud7b fl/fl mice were used as control. Total mRNA was isolated from purified astrocytes and RNA Library was created using a NEBNext® UltraTM II Directional RNA Library Prep Kit. The library was sequenced using a NovaSeq 6000 sequencer. The online platform DAVID Bioinformatics from National Institutes of Health (NIH) was used for the KEGG pathway analysis. Spatial transcriptomics: In situ RNA expression analysis at a single-cell level was performed using the Xenium system (10x Genomics). 5µm thick sections were placed on a Xenium slide according to the manufacturer's protocol, with drying at 42°C for 3 hours and overnight placement in a desiccator at room temperature, followed by deparaffinization and permeabilization to make the mRNA accessible. The Probe Hybridization Mix was prepared using a pre-designed panel with 247 genes (Xenium Mouse Brain Gene Expression Panel v1) and custom add-on panel with 50 genes (Xenium Custom Gene Expression panel, design ID: QZD68C) according to the user guide (CG000582, Rev D, 10x Genomics). The staining for Xenium was performed using Xenium Nuclei Staining Buffer (10x Genomics product number: 2000762) as a part of the Xenium Slides & Sample Prep Reagents Kit (PN-1000460). Following the Xenium run, Hematoxylin and Eosin (H&E) staining was performed on the same section according to the Post-Xenium Analyzer H&E Staining user guide (CG000613, Rev B, 10x Genomics). Quantification and statistical analysis Quantification of WB was performed using NIH ImageJ software. Statistical analysis and graphic design were performed using GraphPad Prism 10. The two-tailed Student’s t test was used to detect statistical differences in all experiments except for EAE scores, which were analyzed by the Mann-Whitney U test. P values < 0.05 (*) were considered statistically significant. All experiments were performed at least twice. Declarations Acknowledgments The authors thank Birgit Brennecke, Kerstin Ellrott, Elena Fischer, Izabela Plumbon and Nadja Schlüter for expert technical assistance. 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Supplementary Files Supplementarytable1.xlsx SupplementaryFig1.jpg Supplementary Figure 1: OTUD7B in astrocytes does not regulate inflammatory responses under homeostatic conditions A-F) Analysis of Otud7b fl/fl and GFAP-cre Otud7b fl/fl mice under homeostatic conditions. (A) OTUD7B WB of astrocytes isolated from the spinal cords showing efficient deletion of OTUD7b in GFAP-cre Otud7b fl/fl but not in Otud7b fl/fl mice. (B) GFAP staining (left) and Cresyl-violet luxol-fast blue (CV-LFB) staining (right) in spinal cord of unimmunized Otud7b fl/fl and GFAP-cre Otud7b fl/fl mice showing normal architecture and myelination of spinal cords independent of OTUD7B. Scale bars correspond to 100μm. (C) Flow cytometric dots plot showing equal relative numbers of CD3 + CD4 + CD45 + (CD4T cells) and CD3 + CD8 + CD45 + (CD8 T cells) in the spinal cords of Otud7b fl/fl and GFAP-cre Otud7b fl/fl mice. (D-F) Absolute numbers of CD3 + CD4 + CD45 + (CD4 T cells), CD3 + CD8 + CD45 + (CD8T cells), CD19 + B220 + CD45 + (B cells), CD11c + CD45 + (dendritic cells), Ly6C high CD11b + CD45 + (monocytes), and F4/80 + CD11b + CD45 + (macrophages) in spinal cord (D), spleen (E), and lymph nodes (F) of non-immunized Otud7b fl/fl mice and GFAP-cre Otud7b fl/fl mice. SupplementaryFig2.jpg Supplementary Figure 2: Regulation of RIPK1 and GFAP by OTUD7B is time- and concentration dependent (A-C) OTUD7B-sufficient and -deficient primary astrocytes were stimulated with 50ng/mL of TNF (A, C) or increasing concentrations (10,20 and 50ng/mL) of TNF (B) for 5 days. The cells were lysed in RIPA lysis buffer for protein isolation. Proteins were immunoblotted for RIPK1 and GFAP and the expression was normalized to GAPDH and quantified. Quantitative data showing the expression of RIPK1 (A, B) and GFAP (C) relative to GAPDH (n=3). 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5937561","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":420790351,"identity":"f4d79a22-1346-4535-9b4d-11bb45f3f9b0","order_by":0,"name":"Dirk Schlüter","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABIElEQVRIie3QsUrEMBjA8a8UziXVtVBpX+E7Ch6HRR/EpeWgkxXdbhAMHNTl0LUHPsSJi24pgZuiroEO+gA3nLgIHmJTTh0aFTeH/KeQ8CP5AmAy/cuIxVYrZMAiQCBfh66eQE3wg6R/JACM/06C01vGyXDp96h9yYfifq8HTvl8dB0l5x6z5KJNUBzEnAgMN1nnsBSyym7o+sCbiDSZnMV2v9AQlyB3ckwKIFjSRZVNH+boOTkPUUDHI20SFIq84cmK3GVTRsJXRXYVWWqGkYpQjN2GSKbIlrrFR1ITjUCxj+XFLOwWdj0LFYOGbDt56rvCGvXHuh8T3cf5sR+4a6OrJzrbaR5WOXlENsZ2KV8013xmt7cs+hMwmUwm0/e9AwutacdCHPLqAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-1478-3328","institution":"Hannover Medical School","correspondingAuthor":true,"prefix":"","firstName":"Dirk","middleName":"","lastName":"Schlüter","suffix":""},{"id":420790352,"identity":"502d9db7-5884-4d65-8cd8-92a6b93b11ce","order_by":1,"name":"Kunjan Harit","email":"","orcid":"","institution":"Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School","correspondingAuthor":false,"prefix":"","firstName":"Kunjan","middleName":"","lastName":"Harit","suffix":""},{"id":420790353,"identity":"41e422da-b0da-44b0-a6de-f01c0cf73299","order_by":2,"name":"Wenjing Yi","email":"","orcid":"","institution":"Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School","correspondingAuthor":false,"prefix":"","firstName":"Wenjing","middleName":"","lastName":"Yi","suffix":""},{"id":420790354,"identity":"04910e62-690e-4772-bc2e-7e5938e60264","order_by":3,"name":"Andreas Jeron","email":"","orcid":"","institution":"Institute of Medical Microbiology and Hospital Hygiene, Otto-von-Guericke University","correspondingAuthor":false,"prefix":"","firstName":"Andreas","middleName":"","lastName":"Jeron","suffix":""},{"id":420790355,"identity":"cb21e49c-79d5-4763-a53f-61480055a7b5","order_by":4,"name":"Jakob Schmidt","email":"","orcid":"","institution":"Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg","correspondingAuthor":false,"prefix":"","firstName":"Jakob","middleName":"","lastName":"Schmidt","suffix":""},{"id":420790356,"identity":"42384e1d-6b15-4fca-912a-8ae75f2f04c6","order_by":5,"name":"Ruth Beckervordersandforth","email":"","orcid":"https://orcid.org/0000-0002-3617-7567","institution":"Friedrich-Alexander Universität Erlangen-Nürnberg","correspondingAuthor":false,"prefix":"","firstName":"Ruth","middleName":"","lastName":"Beckervordersandforth","suffix":""},{"id":420790357,"identity":"23d542ac-6899-463d-bb66-e9f35c470d32","order_by":6,"name":"Emanuel Wyler","email":"","orcid":"https://orcid.org/0000-0002-9884-1806","institution":"Max-Delbrueck-Center","correspondingAuthor":false,"prefix":"","firstName":"Emanuel","middleName":"","lastName":"Wyler","suffix":""},{"id":420790358,"identity":"dfdb4179-a2b4-464f-8bb8-33f0d94a19a2","order_by":7,"name":"Artür Manukyan","email":"","orcid":"","institution":"Berlin Institute for Medical Systems Biology (BIMSB), Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC)","correspondingAuthor":false,"prefix":"","firstName":"Artür","middleName":"","lastName":"Manukyan","suffix":""},{"id":420790359,"identity":"768a0495-304b-4890-89c5-70e1320f52ba","order_by":8,"name":"Martina Deckert","email":"","orcid":"","institution":"Universität zu Köln","correspondingAuthor":false,"prefix":"","firstName":"Martina","middleName":"","lastName":"Deckert","suffix":""},{"id":420790360,"identity":"1084f9bc-fd13-4b30-a48e-988615e146d1","order_by":9,"name":"Helena Radbruch","email":"","orcid":"https://orcid.org/0000-0001-6941-3397","institution":"Charité - 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(B) The mice were monitored daily for the clinical signs of the disease. Upper panel represents mean clinical score ± SEM (Otud7b\u003csup\u003efl/fl \u003c/sup\u003en=7, GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e n=11) and the lower panel shows the disease incidence, day of disease onset and maximum clinical score of all immunized mice per group. Data represented as mean values ± SEM. (B) The body weight was measured daily up to day 16 p.i. Graph represents percent body weight ± SEM (Otud7b\u003csup\u003efl/fl\u003c/sup\u003e n=7, GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e n=5). All bars represent mean values ± SEM. Student’s t-test *p\u0026lt;0.05, **p\u0026lt;0.01. (C) Cresyl-violet luxol-fast blue staining showing inflammatory infiltrates (marked by dotted line) and myelin staining at day 15 p.i in spinal cord of Otud7b\u003csup\u003efl/fl\u003c/sup\u003e and GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice. (D) H\u0026amp;E staining showing immune cell infiltration (marked by dotted line) in spinal cord sections from Otud7b\u003csup\u003efl/fl\u003c/sup\u003e and GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice at day 15 p.i. (E, F, G) At day 15 p.i., reactive astrocytes of Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice exhibit strong GFAP immunoreactivity close to inflammatory lesions in the spinal cord, whereas GFAP immunoreactivity of astrocytes surrounding inflammatory lesions in spinal cord sections of GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice was largely lost by immunohistochemistry (F) and immunofluorescence (G) Scale bars correspond to 100μm (D-G). (H) Immunofluorescence staining showing presence of SOX2/SOX9\u003csup\u003e+\u003c/sup\u003eGFAP\u003csup\u003e-\u003c/sup\u003e astrocytes surrounding inflammatory lesions (marked by yellow dotted line) in spinal cord sections of Otud7b\u003csup\u003efl/fl\u003c/sup\u003e and GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice at day 15 p.i. Scale bars correspond to 20μm. (I) Dot plot showing GFAP mRNA expression in different lesion compartments at d 15 p.i.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5937561/v1/fe5caacffe2ef246c7bc77b2.jpg"},{"id":77334751,"identity":"a041f39d-887d-41ab-845c-3d06453dd6b1","added_by":"auto","created_at":"2025-02-27 14:04:21","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1238784,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eOTUD7B deficient astrocytes show pro-inflammatory gene signature\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A-F) EAE was induced in Otud7b\u003csup\u003efl/fl\u003c/sup\u003e and GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e by MOG\u003csub\u003e35-55 \u003c/sub\u003epeptide immunization combined with pertussis toxin. Spinal cord and magnetic cell sorted astrocytes were isolated from non-immunized (d0) and immunized mice at day 15 p.i. and processed for RNA sequencing. (A) Venn diagram of 1944 upregulated genes (FC\u0026lt;3) in Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice compared to GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e at d0 and d15 p. i. Absolute and relative values are shown. (B - D) Gene ontology (B, D) and KEGG pathway (C) analysis was performed using ClueGO software with right-sided hypergeometric test with Bonferoni-step-down p-value correction. Only terms with FDR \u0026lt; 0.05 and at least GO-level 6 are shown. Numbers in brackets represent number of enriched genes compared to total number of annotated genes in that term. (E) Heat map showing the expression profile for chemokine genes from astrocytes of non-immunized and immunized mice at day 15 p. i. (F) Spatial transcriptomics analysis of differential gene expression in various lesional compartments of Otud7b\u003csup\u003efl/fl\u003c/sup\u003e and GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice at day 15 p.i. (G) RNA was isolated from the spinal cord of non-immunized and immunized mice (day 15 p.i.). Expression of Cxcl1, Cxcl10, Cxcl11, Ccl2 and Ccl20 was analyzed by qRT-PCR. Data show fold-change increase in gene expression compared to non-immunized mice (n=3 per group, all bars represent mean ± SEM. Student's t-test *p\u0026lt;0.05, **p\u0026lt;0.01).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5937561/v1/11bd1f982097f3f34d566729.jpg"},{"id":77333267,"identity":"ee68d556-bbd8-4f61-8c56-5c1ebcf64c64","added_by":"auto","created_at":"2025-02-27 13:48:21","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":744443,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eReduced leukocyte infiltration and inflammation in the spinal cord of Otud7b\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e mice during EAE.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eEAE was induced in Otud7b\u003csup\u003efl/fl\u003c/sup\u003e (n=8) and GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e (n=8) by MOG\u003csub\u003e35-55 \u003c/sub\u003epeptide and spinal cord was isolated at day 0 and 15 p.i. The infiltrating leukocytes were isolated by Percoll gradient centrifugation and analyzed by flow cytometry. (A) Representative dot plots show the percentage of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells and (B) absolute number of infiltrating leukocytes in the spinal cord of Otud7b\u003csup\u003efl/fl\u003c/sup\u003e and GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice at day15 p.i. (C, D) GM-CSF, IFN-g and IL-17 producing CD4\u003csup\u003e+\u003c/sup\u003e T cells were analyzed by flow cytometry. Representative dot plots (C) and absolute numbers of GM-CSF-, IFN-g- and IL-17-producing CD4\u003csup\u003e+\u003c/sup\u003e T cells (D) are shown. All bars represent mean values ± SEM. Student’s t-test *p\u0026lt;0.05, **p\u0026lt;0.01, ***\u0026lt;0.001. (E) Relative mRNA expression of IFN-g, IL-17, GM-CSF,\u003cstrong\u003e \u003c/strong\u003eAdd a correct gamma! TNF and Nos2 was analyzed by qRT-PCR from spinal cord of Otud7b\u003csup\u003efl/fl\u003c/sup\u003e and GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice at day 15 p.i.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5937561/v1/97db84328843de9d5b29dc5b.jpg"},{"id":77332863,"identity":"605deeb1-9c66-42b4-9d68-17155f1916c5","added_by":"auto","created_at":"2025-02-27 13:40:22","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1076220,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eOTUD7B impairs TNF mediated pro-inflammatory responses in astrocytes\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A, B) Primary astrocyte cultures were prepared from P0/1 pups of Otud7b\u003csup\u003efl/fl\u003c/sup\u003e and GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice, respectively. (A) Cells were stimulated with TNF (10ng/mL), IFN-g (10ng/mL) or IL-17 (50ng/mL) for 16h and lysed in buffer RLT for RNA isolation. Relative mRNA expression of Cxcl-1, Cxcl-10, Cxcl-11, Ccl-1, Ccl-20, Nos-2 and IL-6 was determined by qRT-PCR (n=3). All graphs represent fold change over unstimulated control. All bars represent mean values ± SEM. Student’s t-test *p\u0026lt;0.05, **p\u0026lt;0.01, ***\u0026lt;0.001.\u0026nbsp; Proteins were harvested and analyzed by WB with the indicated antibodies. Representative WB are shown after (B) TNF (10ng/mL), (C) IFN-g (10ng/mL) and (D) IL-17 (50ng/mL) stimulation, respectively. Blots represent one out of three independent experiments.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5937561/v1/abd646b7165d3c964ab419ee.jpg"},{"id":77334351,"identity":"b88c6433-b4f1-4ff6-b9ee-af4f3b61aa73","added_by":"auto","created_at":"2025-02-27 13:56:22","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1556164,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eTemporal regulation of TNF signaling by OTUD7B\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A) Spatial transcriptome analyzes of TNF mRNA expression in EAE lesions of non-immunized and immunized Otud7b\u003csup\u003efl/fl\u003c/sup\u003e and GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice. (B-G) OTUD7b-sufficient and -deficient primary astrocytes were stimulated with TNF (B, C, E: 50ng/mL, C, E, F, G: indicated concentrations). At indicated timepoints, astrocytes were lysed in RIPA lysis buffer for protein isolation. (B) Proteins were immunoblotted with the indicated antibodies. RIPK1 and TRAF2 expression was normalized to GAPDH and quantified based on WB data. Representative WB from one out of three experiments are shown. (C Co-immunoprecipitation was performed with anti-RIPK1 antibody followed by immunodetection of the indicated proteins by WB. (D) OTUD7B-sufficient and -deficient astrocytes were transfected with non-specific control siRNA or A20 siRNA (5μM) and stimulated with TNF for 5 days. At day 5 post-stimulation, astrocytes were lysed with RIPA buffer and proteins were analyzed by WB using indicated antibodies. RIPK1 and TRAF2 expression was normalized to GAPDH and quantified based on WB data. (E) Astrocytes were treated with TNF for 3 days and MG132 was added on day 3 for 6 hours to inhibit the proteasome. Astrocytes were lysed in RIPA buffer and the protein lysates were analyzed by WB for RIPK1 and GAPDH. (F, G) OTUD7B-sufficient and -deficient primary astrocytes were stimulated with 10 ng/mL, 20 ng/mL, and 50 ng/mL TNF for 5 days. On day 5 post-stimulation, cells were lysed in RIPA lysis buffer and proteins were isolated for (F) analysis of protein expression by WB and (G) identification of interacting proteins by co-immunoprecipitation of protein complexes with anti-RIPK1 antibody and WB. All WB blots are representative of one of three independent experiments.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5937561/v1/945e728f25e3c3103fdb3519.jpg"},{"id":77332861,"identity":"ffd98391-b7b4-4ed4-a3b7-2c7621d6c082","added_by":"auto","created_at":"2025-02-27 13:40:21","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":974429,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eOTUD7B prevents K48-ubiquitination of GFAP and its subsequent proteasomal degradation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A) Spatial transcriptome analyzes of IL-6 mRNA expression in EAE lesions of non-immunized and immunized Otud7b\u003csup\u003efl/fl\u003c/sup\u003e and GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice. (B-E) Primary astrocytes were stimulated with 50 ng/mL of TNF and lysed at the indicated time points in RLT buffer for RNA extraction (C) or RIPA lysis buffer for protein isolation (B, D, E). (B) WB was performed to analyze the expression levels of OTUD7B, p-STAT3, STAT3, GFAP, and GAPDH. (C) Fold change in mRNA expression levels of \u003cem\u003eGfap\u003c/em\u003e over unstimulated control was analyzed by qRT-PCR and normalized to \u003cem\u003ehprt\u003c/em\u003e. (D) OTUD7B-sufficient and -deficient astrocytes were stimulated with 50 ng/mL of TNF alone, or in combination with anti-IL6 neutralizing antibody for 3 days. On day 3 post-stimulation, 10 μg/mL of CHX and/or 10 μM of MG132 were added to the cells for 6 hours, and proteins were harvested for WB. The expression level of GFAP was analyzed by WB using GAPDH as a loading control. Relative protein expression of GFAP normalized to GAPDH is shown. (E) Protein complexes from total protein lysates were immunoprecipitated using anti-GFAP antibody, and precipitates were analyzed for GFAP, OTUD7B, and K48 ubiquitin chains by WB. (F) Schematic representation showing the dual protective function of astrocytic OTUD7B in EAE: OTUD7B (i) prevents hyperinflammation by limiting TNF-induced chemokine production in astrocytes through early K63- and late K48-deubiquitination of RIPK1, and (ii) fosters astrocyte bordering of lesions by preventing proteasomal degradation of GFAP through K48-deubiquitination.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5937561/v1/922c5f63aa8e3d836b644a1d.jpg"},{"id":93996119,"identity":"021bb119-a04d-41ac-8c89-cc43a7d416da","added_by":"auto","created_at":"2025-10-21 07:07:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11540619,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5937561/v1/2b3314f9-82b2-48a2-809a-8da50542c440.pdf"},{"id":77332883,"identity":"6b9ab48b-fb39-4bb8-87eb-702ff72ce4bd","added_by":"auto","created_at":"2025-02-27 13:40:24","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":22819,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5937561/v1/d55cce2b0cb7d3c2626c2293.xlsx"},{"id":77332859,"identity":"87608a68-022d-4244-b10d-681af9758891","added_by":"auto","created_at":"2025-02-27 13:40:21","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1598139,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSupplementary Figure 1: OTUD7B in astrocytes does not regulate inflammatory responses under homeostatic conditions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA-F) Analysis of Otud7b\u003csup\u003efl/fl\u003c/sup\u003e and GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice under homeostatic conditions. (A) OTUD7B WB of astrocytes isolated from the spinal cords showing efficient deletion of OTUD7b in GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e but not in Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice. (B) GFAP staining (left) and \u0026nbsp;Cresyl-violet luxol-fast blue (CV-LFB) staining (right) in spinal cord of unimmunized Otud7b\u003csup\u003efl/fl\u003c/sup\u003e and GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice showing normal architecture and myelination of spinal cords independent of OTUD7B. Scale bars correspond to 100μm. (C) Flow cytometric dots plot showing equal relative numbers of CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003e (CD4T cells) and CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003e (CD8 T cells) in the spinal cords of Otud7b\u003csup\u003efl/fl\u003c/sup\u003e and GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice. (D-F) Absolute numbers of CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003e (CD4 T cells), CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003e (CD8T cells), CD19\u003csup\u003e+\u003c/sup\u003eB220\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003e (B cells), CD11c\u003csup\u003e+\u003c/sup\u003e CD45\u003csup\u003e+\u003c/sup\u003e (dendritic cells), Ly6C\u003csup\u003ehigh\u003c/sup\u003e CD11b\u003csup\u003e+ \u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003e (monocytes), and F4/80\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e+ \u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003e (macrophages) in spinal cord (D), spleen (E), and lymph nodes (F) of non-immunized Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice and GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice.\u003c/p\u003e","description":"","filename":"SupplementaryFig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5937561/v1/e88a9195d4cda0eb6eccaafe.jpg"},{"id":77332862,"identity":"69fe5d61-6146-4dbc-a1d4-bd1b6bd62551","added_by":"auto","created_at":"2025-02-27 13:40:21","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":212001,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSupplementary Figure 2: Regulation of RIPK1 and GFAP by OTUD7B is time- and concentration dependent\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A-C) OTUD7B-sufficient and -deficient primary astrocytes were stimulated with 50ng/mL of TNF (A, C) or increasing concentrations (10,20 and 50ng/mL) of TNF (B) for 5 days. The cells were lysed in RIPA lysis buffer for protein isolation. Proteins were immunoblotted for RIPK1 and GFAP and the expression was normalized to GAPDH and quantified. Quantitative data showing the expression of RIPK1 (A, B) and GFAP (C) relative to GAPDH (n=3).\u003c/p\u003e","description":"","filename":"SupplementaryFig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5937561/v1/c2dea07342f9c6f1a8b43ac4.jpg"},{"id":77332876,"identity":"a25c3d84-f05e-45ed-a443-7b22b145ee8b","added_by":"auto","created_at":"2025-02-27 13:40:22","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":25209,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterialsandmethods.docx","url":"https://assets-eu.researchsquare.com/files/rs-5937561/v1/9d529b841235dc8dea35ac81.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"The deubiquitinase OTUD7B ameliorates central nervous system autoimmunity by inhibiting degradation of glial fibrillary acidic protein and astrocyte hyperinflammation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAstrocytes are a highly abundant cell population present in all regions of the central nervous system (CNS) and play an important role in the maintenance of CNS homeostasis and health. Under physiological conditions, astrocytes provide physical and metabolic support for neurons and blood-brain barrier (BBB) functions\u003csup\u003e1\u0026ndash;3\u003c/sup\u003e. Upon CNS damage, astrocytes are rapidly activated and undergo structural and functional changes, by a process known as reactive astrogliosis which critically regulates the pathogenesis, development and outcome of CNS disorders including autoimmune diseases, infections, neurodegenerative diseases and trauma\u003csup\u003e4\u003c/sup\u003e. Reactive astrogliosis is characterized by astrocyte hypertrophy and increased STAT3-mediated expression of the glia fibrillary acidic protein (GFAP)\u003csup\u003e5\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDepending on the underlying disease and the inflammatory milieu astrocytes can either support or suppress CNS inflammation, contribute to CNS damage but also to regeneration. This functional plasticity of astrocytes is based on the activation of different signaling pathways leading to the respective production and secretion of disease-modifying proteins. In multiple sclerosis (MS), a human inflammatory demyelinating disease and its murine model experimental autoimmune encephalitis (EAE), reactive astrocytes upregulate GFAP protein expression and are the major producers of chemokines \u003csup\u003e6,7\u003c/sup\u003e. These chemokines foster the recruitment of immune cells, primarily autoimmune CD4\u003csup\u003e+\u003c/sup\u003e T cells to the site of inflammation. The CNS infiltrating CD4\u003csup\u003e+\u003c/sup\u003e T cells orchestrate an attack on the myelin sheath resulting in demyelination and neurodegeneration\u003csup\u003e8,9\u003c/sup\u003e. The production of chemokines by astrocytes in MS and EAE is driven by several signaling pathways, including NF-κB, MAPK and JAK-STAT pathways\u003csup\u003e10\u0026ndash;12\u003c/sup\u003e. Experimental studies have also shown that astrocyte-specific ablation of NF-κB signaling molecules attenuates EAE\u003csup\u003e11,12\u003c/sup\u003e. In addition to disease promoting functions, astrocytes can exert protective roles, in particular, at later stages of EAE and MS. These protective mechanisms include the local restriction and resolution of neuroinflammation and the promotion of remyelination and axonal repair\u003csup\u003e13\u0026ndash;15\u003c/sup\u003e. In this regard, STAT3-mediated astrocyte survival, proliferation and up-regulation of GFAP expression ameliorates EAE by limiting the spread of encephalitogenic T cells and bordering of inflammatory lesions\u003csup\u003e16\u003c/sup\u003e. Depending on the subtype of MS and the EAE model, active and resolving inflammatory lesions exist in parallel throughout the CNS. In these different lesions astrocyte reactivity is diverse, shows a high plasticity, differs regionally and may also change over time\u003csup\u003e17\u0026ndash;19\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAstrocyte reactivity is significantly determined by the differential activation of signaling pathways which regulate inflammatory reactions and glia scar formation. These signaling pathways are critically modulated by ubiquitination, a posttranslational modification mediated by the covalent linkage of ubiquitin, a 76-aa large protein, to substrates. Ubiquitination is catalyzed sequentially by an ubiquitin-activating enzyme (E1), an ubiquitin-conjugating enzyme (E2), and an ubiquitin ligase (E3). Ubiquitin molecules can be added to proteins in the form of monomers and polyubiquitin chains, in which ubiquitins are linked through the N-terminal methionine residue (M1) or one of the seven lysine residues K6, K11, K27, K33, K48, K63)\u003csup\u003e20\u003c/sup\u003e. The fate of ubiquitinated substrates is determined by the type of ubiquitin linkage. K48- and K11 chains lead to proteasomal degradation of substrates, whereas K63-linked ubiquitin chains modify protein function and can trigger signal transduction\u003csup\u003e21\u0026ndash;24\u003c/sup\u003e. Ubiquitination is reversible and can be counteracted by deubiquitinating enzymes (DUBs). As fine-tuning modulators of cell signaling and activities, DUBs have emerged as important regulators of astrocytes and CNS autoimmunity mediating both protective and disease-promoting astrocyte functions\u003csup\u003e25\u003c/sup\u003e. Of note, the function of DUBs in CNS autoimmunity is also cell type-specific as illustrated by EAE promoting function of the DUB A20 (TNFAIP3) in T cells and its EAE inhibitory function in astrocytes\u003csup\u003e26,27\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eOTUD7B is a DUB belonging to the OTU subfamily and is expressed in all human and murine tissues. OTUD7B is expressed in all regions of the CNS, in particular in astrocytes and oligodendrocytes but not in neurons and microglia (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.proteinatlas.org/ENSG00000264522-OTUD7B\u003c/span\u003e\u003cspan address=\"https://www.proteinatlas.org/ENSG00000264522-OTUD7B\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). OTUD7B can hydrolyze K11-\u003csup\u003e28\u003c/sup\u003e, K48-\u003csup\u003e29\u003c/sup\u003e and K63-\u003csup\u003e30\u003c/sup\u003e linked ubiquitin chains from distinct substrates and regulates pro-inflammatory signaling by inhibiting TNF-mediated NF-κB activation through K63 deubiquitination of RIPK1 and TRAF6\u003csup\u003e30,31\u003c/sup\u003e. The regulation of TNF signaling by OTUD7B is cell type-specific since OTUD7B prevents TNF-mediated apoptosis in dendritic cells \u003cem\u003ein vitro\u003c/em\u003e and in murine cerebral malaria by cleaving K48-ubiquitin chains from the E3 ubiquitin ligase TRAF2\u003csup\u003e29\u003c/sup\u003e. In T cells, OTUD7B inhibits T cell receptor (TCR) mediated activation by deubiquitination of ZAP70, a central molecule of proximal TCR signaling\u003csup\u003e32\u003c/sup\u003e. Consequently, OTUD7B-deficient mice are protected from myelin oligodendrocyte glycoprotein (MOG)-induced EAE due to impaired activation of encephalitogenic CD4\u003csup\u003e+\u003c/sup\u003e T cells\u003csup\u003e32\u003c/sup\u003e further illustrating cell type- and disease-specific functions of OTUD7B in inflammatory diseases.\u003c/p\u003e \u003cp\u003eTo determine the unresolved astrocyte function of OTUD7B in CNS autoimmunity and its impact on astrocyte plasticity, we analyzed expression data of \u003cem\u003eOtud7b\u003c/em\u003e in MS data sets and explored the \u003cem\u003ein vivo\u003c/em\u003e astrocyte function of OTUD7B in murine EAE. We identified that OTUD7B is upregulated in inflammatory lesions of both MS patients and mice with EAE as compared to healthy CNS tissue. Murine studies revealed that astrocytic OTUD7B was not required for normal CNS development but alleviated EAE. Mechanistically OTUD7B inhibited proteasomal degradation of GFAP in reactive astrocytes by cleaving K48-polyubiquitin chains from GFAP and reducing GFAP mRNA production. In addition, OTUD7B suppressed TNF-induced chemokine production of astrocyte by sequential K63- and K48 -deubiquitination of RIPK1. The simultaneous upregulation of GFAP and inhibition of pro-inflammatroy TNF-signaling by OTUD7B resulted in reduced neuroinflammation, local containment of inflammatory lesions and reduced demyelination. Thus, deubiquitination of GFAP and RIPK1 by OTUD7B are key processes regulating astrocyte reactivity and inhibiting astrocyte-dependent damage in CNS autoimmunity.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eUpregulation of astrocytic Otud7b during CNS autoimmunity\u003c/h2\u003e \u003cp\u003eTo investigate the role of OTUD7B in CNS autoimmunity, we first compared the expression of \u003cem\u003eOtud7b\u003c/em\u003e in the astrocytes of patients with a fulminant, anti-MOG-mediated acute form of MS and healthy individuals. Analysis of public available microarray dataset (No. GSE32915) revealed that \u003cem\u003eOtud7b\u003c/em\u003e mRNA expression was upregulated in the inflammatory lesions and normal appearing white matter of MS patients as compared to healthy individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Next, we determined the expression of \u003cem\u003eOtud7b\u003c/em\u003e in MOG-induced EAE, which is characterized by a peak of disease around day 15 post immunization (p.i.) followed by a gradual decline of clinical symptoms\u003csup\u003e33\u003c/sup\u003e. In EAE, \u003cem\u003eOtud7b\u003c/em\u003e mRNA expression was upregulated and highest in spinal cords at day 15 p.i. and declined up to day 22 p.i. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, analysis of \u003cem\u003eOtud7b\u003c/em\u003e mRNA expression of astrocytes isolated from the normal and EAE-diseased spinal cord revealed a prominent upregulation of \u003cem\u003eOtud7b\u003c/em\u003e mRNA expression in astrocytes of mice with EAE (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). For a detailed spatial analysis of the expression of astrocytic \u003cem\u003eOtud7b\u003c/em\u003e in EAE, we performed spatial transcriptomics with single-molecule resolution on the Xenium platform. Upon immunization with MOG\u003csub\u003e35\u0026ndash;55\u003c/sub\u003e peptide and a pertussis toxin boost, cell types were identified based on the transcriptome data using the pre-designed mouse brain panel with 247 genes in combination with a custom panel including additional 50 genes (Suppl. Table\u0026nbsp;1). We defined inflammatory lesions in the sections characterized by the accumulation of CD3\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e and CD3\u003csup\u003e+\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). In addition, we defined a lesion rim in direct proximity to the lesions, with fewer T cells (200\u0026micro;m), and as a third region defined peri-lesion with no T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, black lines are defined lesion regions). We identified that \u003cem\u003eOtud7b\u003c/em\u003e mRNA is upregulated about 1.5-fold in Sox2\u003csup\u003e+\u003c/sup\u003e Sox9\u003csup\u003e+\u003c/sup\u003e Aqp4\u003csup\u003e+\u003c/sup\u003e astrocytes within the lesion core and lesion rim, but not in the peri-lesions as compared to non-immunized mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). As expected, the number of identified cells per section increased almost two-fold upon immunization (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), with a strong influx of CD68\u003csup\u003e+\u003c/sup\u003e microglia and macrophages into the lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). We observed a strong induction of genes associated with inflammation including Isg15, the chemokines CXCL9, Ccl2, CXCL10 and the cytokines IL-6 and IL1β at d15 p.i. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), with the induction being particularly strong within the lesion core and a gradual decline in the lesion rim and peri-lesion. Taken together, astrocytes upregulate \u003cem\u003eOtud7b\u003c/em\u003e expression in CNS autoimmunity with the highest expression in the T cell-enriched lesions and a gradual decline with increasing distance from the lesions core, suggesting that astrocytic OTUD7B is a regulator of neuroinflammation.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAstrocytic OTUD7B-deficiency aggravates EAE\u003c/h3\u003e\n\u003cp\u003eTo determine the astrocyte-specific function of OTUD7B in EAE, we crossed GFAP-cre mice with Otud7b\u003csup\u003efl/fl\u003c/sup\u003e to generate GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice with deletion of \u003cem\u003eOtud7b\u003c/em\u003e in astrocytes. The GFAP-cre strain used here expresses Cre late during embryonic development (day 14.5\u003csup\u003e34\u003c/sup\u003e), has a high deletion efficacy in all spinal cord and brain astrocytes but only low deletion in neurons\u003csup\u003e10,11,16,34\u003c/sup\u003e, and allows analysis of astrocyte functions in inflammatory CNS disorders \u003csup\u003e10,11,16,17,35\u003c/sup\u003e. \u003cem\u003eIn vivo\u003c/em\u003e deletion of \u003cem\u003eOtud7b\u003c/em\u003e in spinal cord astrocytes was validated by spatial transcriptomics showing a strong upregulation of \u003cem\u003eOtud7b\u003c/em\u003e in astrocytes in EAE of Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice, which was absent in GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice, \u003cem\u003eOtud7b\u003c/em\u003e was most strongly expressed in astrocytes, much weaker in neurons and marginally in pericytes, microglia, oligodendrocytes and endothelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Additional analysis of cultured astrocytes by Western blot (WB) also demonstrated efficient deletion of OTUD7B in astrocytes of GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice (Suppl. Figure\u0026nbsp;1A). GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice were born in a normal mendelian ratio and grew normally (data not shown). Histologically, spinal cords of GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice were normal with intact astrocytes and showed no signs of neurodegeneration and inflammation (Suppl. Figure\u0026nbsp;1B). Major leukocyte populations in the spinal cord, spleen, and lymph node were comparable between Otud7b\u003csup\u003efl/fl\u003c/sup\u003e and GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice (Suppl. Figure\u0026nbsp;1C-F), consolidating that OTUD7B expression in astrocytes does not regulate inflammatory responses under homeostatic conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo explore the role of astrocytic OTUD7B in EAE, we immunized Otud7b\u003csup\u003efl/fl\u003c/sup\u003e and GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice with MOG peptide. Although the two genotypes had similar disease onset at around day 12 p.i.,, GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice showed significantly increased clinical scores with higher maximal clinical scores and disease incidence (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), and reduced body weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eIn accordance with the aggravated clinical symptoms, demyelination was more pronounced in GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e as compared to control mice at d15 p.i. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). In addition, inflammatory infiltrates were larger, more confluent and infiltrated deeper in the spinal cord tissue in GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). In EAE, astrocyte morphology and reactivity was strongly altered. Adjacent to and bordering inflammatory lesions astrocytes of Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice strongly expressed GFAP and showed prolonged GFAP\u003csup\u003e+\u003c/sup\u003e astrocytic processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). On the contrary, astrocytes of the GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice associated with inflammatory infiltrates only weakly expressed GFAP and, thus, did not form a GFAP\u003csup\u003e+\u003c/sup\u003e border surrounding the inflammatory infiltrate (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). In both control and GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice, equal numbers of SOX2\u003csup\u003e+\u003c/sup\u003e SOX9\u003csup\u003e+\u003c/sup\u003e astrocytes were present indicating that the reduction of GFAP expression was not caused by a loss of OTUD7B-deficient astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Interestingly, a comparison of GFAP mRNA levels by spatial transcriptomics showed upregulation particularly in the lesion core and the lesion rim, which was higher in Otud7b\u003csup\u003efl/fl\u003c/sup\u003e than in GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI) indicating that the diminished transcription of GFAP may contribute to the reduced GFAP protein in GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-H).\u003c/p\u003e\n\u003ch3\u003eOTUD7B suppressed chemokine production, and recruitment of encephalitogenic CD4 T cells to the CNS\u003c/h3\u003e\n\u003cp\u003eReactive astrocytes contribute to the development of EAE by producing pro-inflammatory mediators, including chemokines, which induce the recruitment of encephalitogenic autoimmune CD4\u003csup\u003e+\u003c/sup\u003e T cells into the CNS \u003csup\u003e36,37\u003c/sup\u003e. Since GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice exhibited increased CNS inflammation, we next determined the impact of OTUD7B on the transcriptome of astrocytes isolated from the spinal cords of non-immunized and MOG-immunized (d15 p.i.) Otud7b\u003csup\u003efl/fl\u003c/sup\u003e and GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice. We detected 1944 genes differentially regulated between OTUD7B-deficient and -sufficient astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Of these 1994 genes, 689 genes were differentially regulated under homeostatic conditions, 1090 genes were regulated at d15 p.i. and 165 genes were regulated under both conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Clustering of the genes using the K-means clustering algorithm resulted in 10 clusters. Ontology and KEGG pathway enrichment analysis showed that the pathways related to chemokine and cytokine signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) were upregulated in cluster 6 and 2 of OTUD7B-deficient astrocytes upon EAE. In addition, OTUD7B-deficient astrocytes of cluster 6 and 2 had increased expression of genes regulating transendothelial migration of leukocytes, pro-inflammatory cytokine and chemokine signaling, cell adhesion and angiogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). A detailed analysis of chemokine genes showed that upon induction of EAE, expression of CCL and CXCL chemokines was upregulated in both genotypes but expression of CCL2, 3, 4, 5, 6, 7, 8, 11, 19 and CXCL 1, 2, 9, 10, 12, 13, 16 were higher in OTUD7B-deficient astrocytes. This suggests that the increased disease pathology in GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice was due to increased chemokine and cytokine mediated infiltration of leukocytes into the spinal cord, (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Only Ccl22, Cxcl16, and Cxcl12, which are stronger expressed by microglia and brain macrophages as compared to astrocytes (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.proteinatlas.org\u003c/span\u003e\u003cspan address=\"https://www.proteinatlas.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were increased upregulated in OTUD7B-comptetent astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSpatial transcriptome analysis of spinal cord tissue provided further information on the anatomic distribution of chemokines and cytokines. Chemokine mRNA expression was higher in OTUD7B-deficient astrocytes in the lesion cores and in the rims surrounding the lesion in relation to peri-lesion areas where the chemokine levels were comparable (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Among the cytokines studied, only IL-6 and IL-1β but not TNF, TGF-β1 were highly expressed in OTUD7B-deficient astrocytes in EAE. In contrast to astrocytes, chemokine and cytokine expression of microglia was equal between the two genotypes.\u003c/p\u003e \u003cp\u003eConsistent with the astrocyte RNA-sequencing data, RT-PCR-based quantification of chemokine mRNA in spinal cords of mice with EAE revealed that GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice had significantly higher levels of CXCL1, CXCL10, CXCL11, Ccl2, and Ccl20 mRNA at day 15 p.i., which are major attractants for lymphoid and monocytic cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG).\u003c/p\u003e\n\u003ch3\u003eIncreased recruitment of encephalitogenic CD4 T cells in GFAP-cre Otud7b mice\u003c/h3\u003e\n\u003cp\u003eTo determine whether the increased chemokine production of OTUD7B-deficient astrocytes resulted in an enhanced recruitment of leukocytes to the CNS in MOG-immunized mice, we performed a flow cytometry analysis of leukocytes isolated from the spinal cords. In contrast to non-immunized mice (Suppl. Figure\u0026nbsp;1E), relative and absolute numbers of CD4\u003csup\u003e+\u003c/sup\u003e but not of CD8\u003csup\u003e+\u003c/sup\u003e T cells were significantly increased in Otud7b\u003csup\u003efl/fl\u003c/sup\u003e and GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice with EAE (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Additionally, numbers of CD11c\u003csup\u003e+\u003c/sup\u003e dendritic cells, Ly6C\u003csup\u003ehigh\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e inflammatory monocytes and F4/80\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e macrophages/microglia but not of CD19\u003csup\u003e+\u003c/sup\u003e B cells were higher in GFAP-cre OTUD7b\u003csup\u003efl/fl\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Among CD4\u003csup\u003e+\u003c/sup\u003e T cell subsets, IFN-producing Th1 cells, IL-17-producing Th17 cells, and GM-CSF-producing CD4\u003csup\u003e+\u003c/sup\u003e T cells are the major effectors in EAE and each of them can induce EAE independently \u003csup\u003e38\u0026ndash;40\u003c/sup\u003e. We identified that the absolute but not relative numbers of infiltrating GM-CSF\u003csup\u003e+\u003c/sup\u003e, IFN-γ\u003csup\u003e+\u003c/sup\u003e, and IL17\u003csup\u003e+\u003c/sup\u003e CD4 cells were significantly increased in the spinal cord of GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice with EAE (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). This indicates that astrocyte-specific OTUD7B limits the recruitment of encephalitogenic CD4\u003csup\u003e+\u003c/sup\u003e T cell subsets to the CNS but does not influence the differentiation and composition of the recruited CD4\u003csup\u003e+\u003c/sup\u003e T cell subsets during EAE. In accordance with the increased recruitment of encephalitogenic CD4\u003csup\u003e+\u003c/sup\u003e T cells, inflammatory monocytes and macrophages into the CNS of GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice with EAE, mRNA production of IFN-γ,TNF, IL-17, GM-CSF and NOS2, which all contribute to demyelination in EAE\u003csup\u003e41\u0026ndash;45\u003c/sup\u003e, was also increased,\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCollectively, OTUD7B expression in astrocytes suppressed astrocyte chemokine production, recruitment of encephalitogenic leukocytes, demyelination and disease symptoms in EAE illustrating the important neuroprotective function of astrocytic OTUD7B.\u003c/p\u003e\n\u003ch3\u003eOTUD7B suppressed early TNF-induced pro-inflammatory signaling in astrocytes\u003c/h3\u003e\n\u003cp\u003eThe increased astrocyte activation and chemokine production of GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice under inflammatory but not under homeostatic conditions indicates that pro-inflammatory cytokines induced OTUD7B-dependent immunoregulatory astrocyte function. To identify whether astrocyte chemokine and cytokine production induced by the encephalitogenic cytokines TNF, IFN-γ and IL-17, respectively, is regulated by OTUD7B, we cultured OTUD7B-deficient and -competent astrocytes, stimulated them with the cytokines and determined chemokine and cytokine production by RT-PCR. Upon stimulation with TNF, mRNA expression of CXCL1, CXCL11, CCL20, IL-6, CCL2 and NOS2 were significantly increased in OTUD7B-deficient astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In contrast, OTUD7B did not regulate IFN-γ- and IL-17-induced chemokine and cytokine production (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) indicating that TNF signaling is the major pathway regulated by OTUDB in astrocytes. Thus, both increased TNF mRNA expression in EAE (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE) and TNF-induced astrocytic cytokine and chemokine production are under control of astrocytic OTUD7B.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo directly assess the effect of OTUD7B on TNF, IFN-γ and IL-17 signaling, we stimulated cultivated astrocytes with these cytokines and analyzed activation of the respective signaling pathways by WB. Upon stimulation with TNF, activation of both the NF-κB pathway, indicated by increased phosphorylation of p65 and degradation of IκBα, and the phosphorylation of ERK, p38 and JNK in the MAPK pathways were stronger in OTUD7B-deficient astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Of note, these differences were detectable as early as 10 min post TNF stimulation. In contrast, stimulation with IFN-γ did not result in increased STAT1 phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) and activation with IL-17 had no effect on phosphorylation of IκBα, ERK, p38 and JNK (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Thus, OTUD7B is an inhibitor of TNF but not IFN-γ and IL-17 signaling in astrocytes.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eOTUD7B regulates TNF signaling by sequential K63- and K48- deubiquitination of RIPK1\u003c/h2\u003e \u003cp\u003eSince chemokine production of OTUD7B-deficient astrocytes was highest in the lesion core (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) and OTUD7B-regulated TNF-dependent chemokine production, we next analyzed the spatial distribution of TNF mRNA in Otud7b\u003csup\u003efl/fl\u003c/sup\u003e and GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice with EAE. In accordance with the chemokine data, expression of TNF was highest in the lesion core with a gradual decline in the lesion rim and the peri-lesional tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). These data show a gradient of TNF production and also indicate that astrocytes will be exposed to TNF for several days based on the persistence of the lesions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTNF signaling is dynamically regulated by K63 and K48 poly-ubiquitination of RIPK1. Rapidly after TNF stimulation, RIPK1 is K63 poly-ubiquitinated by TRAF2 and cIAP1 leading to RIPK1-dependent activation of NF-κB and MAPKs \u003csup\u003e29,46\u003c/sup\u003e. The activation of NF-κB induces expression of A20 (TNFAIP3), which cleaves K63 chains from RIPK1 and can also induce K48-dependent proteasomal degradation of RIPK1\u003csup\u003e47\u003c/sup\u003e, which also leads to degradation of TRAF2 and termination of NF-κB and MAPK signaling\u003csup\u003e48,49\u003c/sup\u003e. Thus, we determined the impact of OTUD7B on RIPK1, TRAF2 and cIAP1 over a period of 5 days to reflect that under \u003cem\u003ein vivo\u003c/em\u003e conditions astrocytes are exposed to TNF for several day. WB analysis showed that RIPK1, TRAF2 and cIAP1 protein levels remained unchanged in both genotypes but that phosphorylation of p65, p38 and JNK was increased in OTUD7B-deficient astrocytes at days 1 and 2 of TNF stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). From day 3 to 5, RIPK1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, Suppl. Figure\u0026nbsp;2B) and from day 4 to 5 TRAF2 gradually declined in OTUD7B-deficient but not OTUD7B-competent astrocytes, whereas cIAP1 remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). The decline of RIPK1 protein in OTUD7B-deficient astrocytes was preceded by a strong increase of A20, which was much weaker in OTUD7B-competent astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). In OTUD7B-deficient astrocytes the decline of RIPK1 and TRAF2 protein was paralleled by strong reduction of p65, p38 and JNK phosphorylation, which was not detectable in OTUD7B-expressing astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eSince RIPK1 activity and stability are regulated by its K63 and K48 poly-ubiquitination, respectively, we analyzed next whether the DUB OTUD7B regulates the RIPK1 ubiquitination status. Immunoprecipitation of RIPK1 at the various time points up to day 5 of TNF stimulation, showed that the activating K63-ubiqutination of RIPK1 was higher in OTUD7B-deficient astrocytes at days 1 and 2 but reduced at days 3 to 5 as compared to OTUD7B-expressing astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). On the opposite, K48 poly-ubiquitination of RIPK1 strongly increased from days 3 to 5 in OTUD7B-deficient but to a much lesser extent in OTUD7B-competent astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). In the RIPK1 complexes, higher amounts of A20 and TRAF2 were present in OTUD7B-deficient as compared to OTUD7B-expressing astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eTo validate that OTUD7B counteracts A20-mediated proteasomal degradation of RIPK1, we silenced A20 by siRNA in TNF-stimulated astrocytes. Inhibition of A20 restored RIPK1 protein in OTUD7B-deficient astrocytes to the same level as in OTUD7B-competent astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). In parallel to the increased RIPK1 protein, TRAF2 increased in A20 siRNA-treated OTUD7B-deficient astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), which is explained by the stabilizing role of RIPK1 for TRAF2\u003csup\u003e49\u003c/sup\u003e. In addition, proteasome inhibition by MG132 prevented degradation of RIPK1 in OTUD7B-deficient astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE), which is in line with both the K48-dependent proteasomal degradation of RIPK1 in OTUD7B-deficient astrocytes and the prevention of proteasomal degradation by K48-deubiquitination of RIPK1 by OTUD7B in OTUD7B-competent astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eSince spatial transcriptomics revealed an increase of TNF mRNA from the healthy tissue to the core of EAE lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), we stimulated cultivated astrocytes with increasing TNF concentrations for 5 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). These studies revealed that TNF-stimulation reduced RIPK1 proteins in OTUD7B-deficient but not in OTUD7B-competent astrocytes in dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). In parallel TRAF2 levels declined, whereas cIAP1 protein levels were not affected by OTUD7B-deficiency. In addition, the decrease of downstream p65, p38 and JNK phosphorylation in OTUD7B-deficient astrocytes was higher upon stimulation with increased amount of TNF (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). In both OTUD7B-competent and -deficient astrocytes, A20 was induced by TNF-stimulation and A20 protein levels were independent of the TNF concentration. Importantly and in agreement with the analysis of the kinetic of A20 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), A20 levels were strongly increased in OTUD7B-deficient astrocytes, and, thus, were not affected by the reduced NF-κB activation. K48 poly-ubiquitination of RIPK1 increased in OTUD7B-deficient astrocytes stimulated with higher concentrations of TNF and this was paralleled by a TNF-dependent increase of A20/RIPK1 complex formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, Suppl. Figure\u0026nbsp;2B). Collectively, these data identify that OTUD7B regulates both K63- and K48-ubiquitination of RIPK1 in a time- and TNF dose-dependent manner.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eOTUD7B mediates GFAP-stability by its K48 deubiquitination\u003c/h3\u003e\n\u003cp\u003eIncreased GFAP protein expression is a hallmark of reactive astrocytes but the mechanism regulating GFAP protein levels, i.e. the impact of GFAP mRNA transcription and, in particular, GFAP protein stability and turnover are largely unresolved. Histological analysis of GFAP showed that OTUD7B was required for GFAP protein expression in EAE (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-H). Moreover, the reduced GFAP mRNA of GFAP-Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice with EAE (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI) indicates that the reduced GFAP transcription may contribute to the reduction of GFAP protein in GFAP-cre OTUD7b\u003csup\u003efl/fl\u003c/sup\u003e mice.\u003c/p\u003e \u003cp\u003eSince IL-6 activates STAT3 and both are important for the induction of GFAP mRNA, we analyzed first whether IL-6 mRNA production was reduced in GFAP-cre OTUD7b\u003csup\u003efl/fl\u003c/sup\u003e mice with EAE. On the contrary, spatial transcriptomics showed an increase in the levels of IL-6 mRNA in GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), therefore, we next analyzed whether OTUD7B might regulate IL-6 signaling, in particular STAT3 activation. In TNF-stimulated OTUD7B-deficient astrocytes, the phosphorylation of STAT3 was increased in the first two days and subsequently strongly reduced as compared to OTUD7B-competent astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). GFAP mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC) was regulated in the same kinetic indicating that OTUD7B-dependent STAT3 phosphorylation is an important factor regulating GFAP mRNA expression. Of note, total STAT3 protein levels were OTUD7B-independent (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB) demonstrating that OTUD7B did not regulate STAT3 stability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine whether GFAP protein stability might be additionally regulated by OTUD7B, we stimulated astrocytes with TNF, inhibited IL-6, blocked new protein synthesis by CHX and prevented proteasomal degradation of proteins by MG132 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, E). We limited these experiments until day three post TNF stimulation, since from that time point onwards the impaired p38 and JNK activity of OTUD7B-deficient astrocytes might impact on GFAP mRNA expression. These experiments revealed that (i) TNF-stimulation increased GFAP protein in OTUD7B-competent but not in OTUD7B-deficient astrocytes, (ii) additional inhibition of IL-6 reduced GFAP protein in both genotypes, (iii) additional inhibition of protein synthesis by CHX further reduced GFAP and that (iv) inhibition of proteasomes by MG132 restored GFAP levels in OTUD7B-deficient astrocytes to the same level as in OTUD7B-competent astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). This uncovers that OTUD7B stabilizes GFAP by preventing its proteasomal degradation. In good agreement, K48 poly-ubiquitination of GFAP was strongly increased in TNF-stimulated OTUD7B-deficient astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Collectively, OTUD7B supported GFAP protein levels/abundance by two independent but synergistic mechanisms: STAT3-mediated GFAP mRNA expression based on sustained RIPK1, p38 and JNK activation leading to continued STAT3 phosphorylation and by K48-deubiquitination of GFAP critical for stabilization and increased GFAP proteins in reactive astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAstrocytes are an important regulator of CNS inflammation, which can both limit and augment neuroinflammation\u003csup\u003e10,11,27,50\u0026ndash;52\u003c/sup\u003e. However, the astrocyte intrinsic molecular mechanisms determining the pro- and anti-inflammatory function of astrocytes in CNS autoimmunity are incompletely understood. Analysis of public available transcriptome data of MS tissue (\u003csup\u003e53\u003c/sup\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and spatial transcriptome analysis of mice with EAE (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG) identified that OTUD7B is expressed in astrocytes under homeostatic conditions and upregulated in astrocytes associated with the inflammatory lesions. Data presented here identify that the upregulation of OTUD7B is a protective astrocyte-intrinsic mechanism ameliorating CNS autoimmunity by dynamic modulation of pro-inflammatory TNF signaling through RIPK1 deubiquitination and by upregulation of GFAP protein levels.\u003c/p\u003e \u003cp\u003eA hallmark of reactive astrocytes in most CNS pathologies is an increased expression of GFAP protein. Mice with OTUD7B deletion in astrocytes had normal GFAP expression under homeostatic conditions but greatly reduced or even absent GFAP protein proximal to inflammatory lesions in EAE. Mechanistically, prolonged stimulation with TNF induced direct interaction of OTUD7B with GFAP and prevented K48-dependent proteasomal degradation of GFAP. This shows for the first time that GFAP protein abundance is under direct control of the ubiquitin system. Thus, in addition to cleavage of GFAP by caspase-3\u003csup\u003e54\u003c/sup\u003e, the deubiquitinating function of OTUD7B plays a non-redundant role for GFAP stability in reactive astrocytes.\u003c/p\u003e \u003cp\u003eIn addition, sustained GFAP mRNA expression was dependent on OTUD7B \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. STAT3 is the major transcription factor inducing GFAP mRNA expression and can be activated by TNF and IL-6, respectively. Deletion of STAT3 or the cognate receptors for IL-6 family cytokines leads to strongly diminished GFAP protein expression and impaired containment of inflammatory lesions by reactive astrogliosis \u003csup\u003e5,33,55\u0026ndash;57\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSince IL-6 mRNA levels did not differ in inflammatory lesions of OTUD7b\u003csup\u003efl/fl\u003c/sup\u003e and GFAP-cre OTUD7b\u003csup\u003efl/fl\u003c/sup\u003e mice but p38 and JNK were less activated due to impaired RIPK1 stability in OTUD7B-deficient astrocytes, OTUD7B might regulate GFAP mRNA expression indirectly. Key factors for the phosphorylation of STAT3 are the MAPK kinases p38 and JNK \u003csup\u003e58\u0026ndash;63\u003c/sup\u003e, which can be activated by RIPK1 upon TNF stimulation. Our analysis of the impact of OTUD7B on the activation of p38 and JNK had revealed that OTUD7B regulated RIPK1 activity and downstream p38 and JNK activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) in the identical kinetic as STAT3 phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Thus, OTUD7B might regulate GFAP mRNA expression indirectly via the RIPK1-p38/JNK-STAT3 axis. Since OTUD7B-deficiency did not regulate STAT3 protein stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), a direct K48-deubiquitinating activity of OTUD7B on STAT3 leading to reduced GFAP mRNA production can be ruled out. In addition, a K63-deubiquitination of STAT3 by OTUD7B would impair STAT3 activity and, thus, cannot underly the increased \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e GFAP mRNA expression of OTUD7B-competent astrocytes.\u003c/p\u003e \u003cp\u003eIn EAE and other inflammatory diseases of the CNS, reactive astrocytes can form borders around inflammatory lesions, which contributes to the local restriction of the neuroinflammation and CNS damage \u003csup\u003e64\u0026ndash;66\u003c/sup\u003e. Functionally important, studies in GFAP-deficient mice revealed that deletion of GFAP leads to a more widespread inflammation and more severe disease in EAE and bacterial and parasitic CNS infections\u003csup\u003e64,67\u0026ndash;69\u003c/sup\u003e. This contributed to the concept that bordering of inflammatory lesions by reactive astrocytes with increased GFAP expression contributes to the local containment of inflammation (reviewed by Sofroniew\u003csup\u003e9\u003c/sup\u003e). Of note, a low level of K48 ubiquitination and GFAP degradation were also detected in OTUD7B-competent astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). This may be an important mechanism to prevent pathological GFAP accumulation in reactive astrocytes. In this regard, excessive accumulation of GFAP in astrocytes induced by gain-of-function mutations in the GFAP gene and by impaired proteasomal GFAP degradation is toxic and underlies human Alexander disease and its corresponding mouse models\u003csup\u003e70\u003c/sup\u003e. Perspectival, it would be interesting to explore OTUD7B in this astrocytopathy and to determine whether GFAP degradation induced by OTUD7B inhibition might have a therapeutic effect. In addition, it remains to be determined which E3 ligases mediate K48 ubiquitination of GFAP. Collectively, these data imply that the regulation of GFAP abundance by OTUD7B-dependent deubiquitination is an important general mechanism potentially regulating the function of reactive astrocytes independent of the underlying disease.\u003c/p\u003e \u003cp\u003eIn addition to reduced GFAP protein, the dominant phenotype of OTUD7B-deficient astrocytes was increased chemokine production in the core and rim of inflammatory EAE lesions, which was associated with an increased recruitment of encephalitogenic T cells, more widespread inflammation and demyelination. Chemokine production of astrocytes occurs also in other CNS disorders and is regarded as a key function of astrocytes leading to the recruitment of leukocytes to the CNS and neuroinflammation \u003csup\u003e71,72\u003c/sup\u003e. Detailed analysis of the molecular functions of OTUD7B identified that OTUD7B rapidly interacted with RIPK1 upon TNF exposure. RIPK1 is a central signaling molecule regulating the activation of pro-inflammatory NF-κB and MAPK signaling as well as cell death pathways\u003csup\u003e73,74\u003c/sup\u003e. The ubiquitination status of RIPK1 is critical to induce or inhibit these individual cellular pathways\u003csup\u003e29,46\u003c/sup\u003e. OTUD7B limited NF-κB and MAPK activation by reducing RIPK1 K63-ubiquitination, which is important for NF-κB - and MAPK-dependent cytokine and chemokine production of TNF-stimulated astrocytes. The reduced chemo- and cytokine expression of OTUD7B-competent astrocytes was evident in vitro, in the bulk RNAseq analysis of ex vivo isolated astrocytes and spatial transcriptomic analysis of mice with EAE. In TNF activated cells, NF-κB activation leads to the expression of A20 which subsequently interacts with and inhibits RIPK1. In this negative feedback loop, A20 inhibits sustained RIPK1 activation by RIPK1 K63-deubiqutination and by inducing K48-dependent proteasomal degradation of RIPK1. Here, we identified that the A20-mediated dynamic change of RIPK1 ubiquitination, resulted in a shift of the targeted ubiquitin chains of OTUD7B from K63 to K48 of RIPK1, and that OTUD7B interacted with A20 and diminished K48 polyubiquitination of RIPK1. In the absence of OTUD7B, A20 induced K48 ubiquitination of RIPK1 induced its proteasomal degradation. Functionally important and in agreement with the present study deletion of A20 in astrocytes results in an augmented and sustained NF-κB and also STAT1 activation leading to aggravation of EAE\u003csup\u003e27\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOf note, the dominant in vivo phenotype of OTUD7B-deficient astrocytes was increased activation and chemokine production at day 15 p.i., i.e. when clinical symptoms of EAE already existed for several days. Thus, the OTUD7B-regulated in vitro shift from TNF-induced increased to reduced astrocyte activation was not detectable in vivo. In this regard, it should be stressed that (i) other NF-κB and MAPK activating pathways, in particular IL-17 signaling, were not regulated by OTUD7B (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), and (ii) IL-17 was increased expressed in the CNS of GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). In addition, disturbance of GFAP protein expression induces endoplasmic reticulum stress, increased activation of MAPK kinases and neuroinflammation as observed in murine models of Alexanders diseases\u003csup\u003e70\u003c/sup\u003e. Thus, the \u003cem\u003ein vitro\u003c/em\u003e diminished RIPK1 signaling upon prolonged TNF exposure might be in vivo compensated by other pro-inflammatory signaling pathways contributing to the sustained increased chemokine production by astrocytes.\u003c/p\u003e \u003cp\u003eTNF can induce RIPK1-dependent cell death, if K63 ubiquitination of RIPK1 is impaired. Histologically, the greatly diminished GFAP protein expression of OTUD7B-deficient astrocytes gave the impression that these cells might have been eliminated by apoptosis. However, we detected identical presence of SOX2\u003csup\u003e+\u003c/sup\u003e Sox9\u003csup\u003e+\u003c/sup\u003e astrocytes in both GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e and Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice and spatial transcriptomics showed that astrocyte numbers did not differ in the core, rim and peri-lesion between the two genotypes. Also, short and long-term \u003cem\u003ein vitro\u003c/em\u003e stimulation of OTUD7B-deficient astrocytes did not result in cell death (data not shown). Thus, OTUD7B-deficient astrocytes with impaired K63-ubiqutination of RIPK1 were highly resilient against TNF-induced cell death. This is in contrast to OTUD7B-deficient dendritic cells, which rapidly undergo apoptosis upon exposure to TNF in murine cerebral malaria\u003csup\u003e29\u003c/sup\u003e. On the contrary, OTUD7B does not regulate apoptosis in T cells but facilitates proximal T cell receptor signaling by deubiquitination of the tyrosine kinase ZAP70 in EAE and murine listeriosis, two diseases characterized by the production of large amounts of TNF. At present, the cell type-specific differences underlying the differential impact of OTUD7B on TNF induced apoptosis are unresolved.\u003c/p\u003e \u003cp\u003eLimitations of the study\u003c/p\u003e \u003cp\u003eThis study illustrates that astrocyte activity is dynamically and interdependently regulated by the concentration of external factors such as TNF and by intrinsic signaling molecules, in particular OTUD7B. Although OTUD7B is a critical factor regulating astrocyte reactivity in EAE and unique in its capacity to regulate both GFAP and RIPK1, the interplay with other external factors including those derived from the microbiome, which also regulate astrocyte intrinsic responses \u003csup\u003e75\u0026ndash;78\u003c/sup\u003e, still has to be explored. Thus, it remains to be determined whether OTUD7B has the same function and central role under other experimental conditions and in human CNS disorders. At present the complex network of interdepend external and intrinsic factors regulating astrocyte reactivity cannot be resolved in its kinetic at the spatial level for single astrocytes. Thus, it remains to be determined whether OTUD7B regulates the plasticity of astrocytes or is a determinant for the development of specific astrocyte subpopulations under inflammatory conditions.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMice\u003c/h2\u003e \u003cp\u003eOTUD7B\u003csup\u003efl/fl\u003c/sup\u003e mice with C57BL/6 background were generated with C57BL/6N-\u003csup\u003eOtud7btm1b(EUCOMM)Wtsi/Wtsi\u003c/sup\u003e embryonic stem cells purchased from the European Mouse Mutant Archive. First, \u003cem\u003eOtud7b\u003c/em\u003e mutant mice were crossed with B6.129S4-\u003cem\u003eGt(ROSA)26Sor\u003c/em\u003e\u003csup\u003e\u003cem\u003etm1(FLP1)Dym\u003c/em\u003e\u003c/sup\u003e/RainJ (Stock No: 009086, The Jackson Laboratory, Bar Habor, ME, USA) to delete the \u003cem\u003efrt\u003c/em\u003e-flanked sequences. Thereafter, \u003cem\u003eotud7b\u003c/em\u003e mutants were crossed with C57BL/6 GFAP-cre mice\u003csup\u003e34\u003c/sup\u003e to obtain GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice. The genotyping was performed by PCR of the tail DNA with primers specific for GFAP-cre and Otud7b\u003csup\u003efl/fl\u003c/sup\u003e, respectively. Wildtype C57BL/6 mice were obtained from Janvier (Le Genest-Saint Isle, France). Animals were kept under specific pathogen-free (SPF) conditions in animal facilities of the Otto-von-Guericke University Magdeburg (Magdeburg, Germany) and Hannover Medical School (Hannover, Germany). Animal care and experimental procedures were carried out according to the European animal protection law and approved by local authorities (Landesverwaltungsamt Halle, file number 42502-2-1260).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eInduction of EAE and clinical assessment\u003c/h2\u003e \u003cp\u003eFor active EAE induction, 8\u0026ndash;12 weeks old mice were immunized with 200\u0026micro;g of myelin oligodendrocyte glycoprotein (MOG)\u003csub\u003e35\u0026minus;55\u003c/sub\u003e peptide mixed in complete Freund\u0026rsquo;s adjuvant containing 800\u0026micro;g of killed \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e. In addition, 200ng pertussis toxin dissolved in 200 \u0026micro;l PBS was intraperitoneally injected respectively at day 0 and 2 post immunization (p.i.). The symptoms and body weight were monitored daily in a double-blinded way according to a previously published score with increasing severity from 0 to 5 as follows\u003csup\u003e10\u003c/sup\u003e: 0, no signs; 0.5, partial tail weakness; 1, limp tail or slight slowing of righting from supine position; 1.5, limp tail and slowing of righting; 2, partial hind limb weakness; 2.5, dragging of hind limb(s) without complete paralysis; 3, complete paralysis of at least one hind limb; 3.5, hind limb paralysis and slight weakness of forelimbs; 4, severe forelimb weakness; 5, moribund or dead. Daily clinical scores were displayed as the mean of all individual disease scores within each group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAstrocyte isolation from adult mice\u003c/h2\u003e \u003cp\u003eSpinal cords were isolated from anaesthetized and PBS-perfused na\u0026iuml;ve and EAE mice at day 15 d p.i. Single-cell suspension was generated using NeuroCult\u0026trade; Enzymatic Dissociation Kit according to the manufacturer\u0026rsquo;s instruction. Astrocytes were purified from the single-cell suspension with the anti-ACSA-2 Microbead Kit and the purity was analyzed by flow cytometry with anti-ACSA-2-PE antibody.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of cells from the spinal cord and flow cytometry\u003c/h2\u003e \u003cp\u003eTo obtain leukocytes from the spinal cord of non-immunized mice (d0) and mice with EAE (d15 p.i.) mice were first cardially perfused with 0.1 M PBS (pH 7.4) in deep methoxyflurane anesthesia. Immediately thereafter, spinal cords were removed, minced through 70\u0026micro;m cell strainers followed by Percoll\u003csup\u003e\u0026reg;\u003c/sup\u003e gradient centrifugation. Cells were counted with a hemocytometer and stained with fluorochrome-coupled antibodies against CD4, CD3, CD45, CD8, CD19, B220, Ly6C, Ly6G, CD11b and CD11c to differentiate T cells, B cells, inflammatory monocytes, granulocytes, macrophages, and dendritic cells respectively (see STAR Methods table). For intracellular staining, cells were incubated with PMA (50 ng/ml), ionomycin (500 ng/ml), and Brefeldin A (1 \u0026micro;g/ml) in RPMI 1640 medium supplemented with 10% FCS, 1% Non-essential amino acids (NEAA), and 1% L-glutamine at 37 \u003csup\u003eo\u003c/sup\u003eC for 4 h. Thereafter, cells were stained with CD3, CD4, CD45 antibodies, fixed and permeabilized with Intracellular Fixation/Permeabilization Kit followed by staining with anti-IL-17, anti-GM-CSF, and anti-IFN-γ antibodies, respectively. Flow cytometry was performed on a Cytek Northern Light Flow Cytometer and data were analyzed with the FlowJo software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eHistology\u003c/h2\u003e \u003cp\u003eMice anesthetized with methoxyflurane were perfused with 0.1 M PBS followed by 4% paraformaldehyde in PBS. After embedding in paraffin, sections of brains and spinal cords were used for hematoxylin \u0026amp; eosin and cresyl violet-luxol fast blue (CV-LFB) staining. Expression of GFAP was demonstrated in an ABC protocol with 3,3\u0026rsquo; diaminobenzidine and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e as substrate. For the immunofluorescent staining, the slides were deparaffinized for 1 h at 60\u0026deg;C, followed by a removal of the residual paraffin with 2 \u0026times; 15 min washing in xylene. Next, the tissue was rehydrated and incubated in 0.5% NaBH4 for 30 min at room temperature to reduce autofluorescence\u003csup\u003e79\u003c/sup\u003e. For antigen retrieval, the slides were incubated in 10 mM citric acid buffer (with 2 mM EDTA, 0.05% Tween20) for 15 min at 95\u0026deg;C and allowed to cool down for 20 min. After washing in PBS-T (1% TritonX-100) for 30 min and PBS for 10 min at room temperature, slices were incubated with the primary antibodies diluted in blocking solution (3% NDS, 0.5% TritonX-100) at 4\u0026deg;C\u003csup\u003e80\u003c/sup\u003e. As primary antibodies rabbit anti-GFAP (1:1000), mouse anti-Sox2 (1:500) goat anti-SOX9 (1:500) and rat anti-Iba-I (1:800) were used. After 2x15 min washing with PBS the slices were incubated with secondary antibodies diluted in blocking solution at 4\u0026deg;C overnight. As secondary antibodies Alexa488-conjugated donkey anti-goat (1:400), Alexa488-conjugated donkey anti-mouse (1:400), Cy3-conjugated donkey anti-rat (1:400) and Cy5-conjugated donkey anti-rabbit (1:400) were used. Subsequently, secondary antibodies were removed, and nuclei were stained using DAPI (1:10000). After 3x10 min washing in PBS, slides were mounted with Aquapolymount solution and stored at 4\u0026deg;C. Images were taken using a Zeiss inverted Axio Observer seven with ApoTome.2 equipped with an Axiocam 503 and a Colibri 7 LED light source, and Zeiss LSM 780 with four lasers (405, 488, 559 and 633 nm) and \u0026times;20, \u0026times;40 and \u0026times;63 objective lenses. For apotome acquisition on Zen2.6 pro software was used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePrimary astrocyte cultures and treatment\u003c/h2\u003e \u003cp\u003ePrimary astrocytes were isolated from 1- to 2-day-old newborn mice and cultured in DMEM containing 1% glutamine, 10% FCS, and 1% penicillin/streptomycin as described before\u003csup\u003e10\u003c/sup\u003e. The purity of astrocyte cultures was more than 95%, as assessed by flow cytometry with antibodies against CD11b and ACSA-2. For the analysis of cytokine receptor-activated signaling pathways, astrocytes were stimulated at the indicated concentrations with TNF (10ng/ml), IL-17 (50 ng/ml), and IFN-γ (10 ng/ml), respectively, for the indicated time points. For long term TNF treatment, primary astrocytes were stimulated with increasing concentrations of TNF (10 ng/mL, 20 ng/mL or 50 ng/mL) for 5 days.\u003c/p\u003e \u003cp\u003eCHX chase assay-\u003c/p\u003e \u003cp\u003eTo detect the stability of GFAP protein, cells were stimulated with either TNF alone (50ng/mL) or in combination with α-IL6 antibody (2\u0026micro;g/mL) for 3 days. At d3 post-stimulation, cells were treated with 10\u0026micro;g/mL of cycloheximide (CHX) with/without 10\u0026micro;M of proteasome inhibitor MG132 for 6h.\u003c/p\u003e \u003cp\u003esiRNA transfection-\u003c/p\u003e \u003cp\u003eFor siRNA-mediated knockdown of A20, primary astrocytes were transfected with 5 \u0026micro;M of A20-specific siRNA according to the manufacturer's instructions. Thereafter, the cells were stimulated with 50 ng/mL of TNF for 5 days, followed by protein isolation and WB analysis\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eSamples from primary astrocytes and mouse organs were lysed on ice in RIPA lysis buffer supplemented with PhosSTOP, phenylmethylsulfonyl fluoride (PSMF) and protease inhibitor cocktail. Cell lysates were pre-cleared by centrifugation at 14,000 rpm for 15mins at 4\u0026deg;C. Supernatant was collected and quantified by BCA assay according to manufacturer\u0026rsquo;s protocol. Protein samples heated in lane marker reducing sample buffer at 99\u0026deg;C for 5 min. Equal amounts of samples were separated by SDS-PAGE and subsequently transferred to polyvinylidene difluoride (PVDF) membranes, which were blocked with 5% BSA at room temperature for 1h, followed by incubation with mentioned primary antibodies (STAR methods Table) at 4\u0026deg;C overnight.\u003c/p\u003e \u003cp\u003eBlots were developed using the ECL Plus Kit and images were captured on Intas Chemo Cam Luminescent Image Analysis system (INTAS). Quantification and analysis of WB images was performed with the LabImage 1D software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCo-immunoprecipitation (Co-IP)-\u003c/h2\u003e \u003cp\u003eWhole cell lysates from astrocytes were precleared by incubation with GammaBind G Sepharose beads with gentle shaking at 4\u0026deg;C for 2 h. After removal of beads by centrifugation, samples were incubated with specific antibodies under continuous shaking at 4\u0026deg;C overnight. Following day, antibody-protein complexes were captured by incubating samples with GammaBind G Sepharose beads at 4\u0026deg;C for 2 hours. Thereafter, the beads were washed with ice cold PBS thrice, resuspended in 2x lane marker reducing sample buffer and boiled at 99\u0026deg;C for 5 min. Samples were centrifuged and supernatant was collected for WB analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative RT-PCR\u003c/h2\u003e \u003cp\u003eTotal mRNA was isolated from spinal cord tissue or astrocytes in buffer RLT using the RNeasy Mini Kit according to manufacturer\u0026rsquo;s protocol. mRNA was reverse-transcribed into cDNA with the SuperScript Reverse Transcriptase Kit. Quantitative RT-PCR was performed with a LightCycler 480 system using TaqMan probes (STAR Methods table). Gene expression levels were normalized to internal control \u003cem\u003eHprt\u003c/em\u003e and fold change increase in gene expression over na\u0026iuml;ve controls was calculated according to the ∆∆ cycle threshold (CT) method (Livak and Schmittgen, 2001\u003csup\u003e81\u003c/sup\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptome analysis of isolated astrocytes\u003c/h2\u003e \u003cp\u003eAstrocytes were isolated by magnetic microbeads from spinal cords of Otud7b\u003csup\u003efl/fl\u003c/sup\u003e and GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice, respectively, at day 15 p.i. Astrocytes isolated from na\u0026iuml;ve Otud7b\u003csup\u003efl/fl\u003c/sup\u003e and GFAP-cre Otud7b\u003csup\u003efl/fl\u003c/sup\u003e mice were used as control. Total mRNA was isolated from purified astrocytes and RNA Library was created using a NEBNext\u0026reg; UltraTM II Directional RNA Library Prep Kit. The library was sequenced using a NovaSeq 6000 sequencer. The online platform DAVID Bioinformatics from National Institutes of Health (NIH) was used for the KEGG pathway analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eSpatial transcriptomics:\u003c/h2\u003e \u003cp\u003eIn situ RNA expression analysis at a single-cell level was performed using the Xenium system (10x Genomics). 5\u0026micro;m thick sections were placed on a Xenium slide according to the manufacturer's protocol, with drying at 42\u0026deg;C for 3 hours and overnight placement in a desiccator at room temperature, followed by deparaffinization and permeabilization to make the mRNA accessible. The Probe Hybridization Mix was prepared using a pre-designed panel with 247 genes (Xenium Mouse Brain Gene Expression Panel v1) and custom add-on panel with 50 genes (Xenium Custom Gene Expression panel, design ID: QZD68C) according to the user guide (CG000582, Rev D, 10x Genomics). The staining for Xenium was performed using Xenium Nuclei Staining Buffer (10x Genomics product number: 2000762) as a part of the Xenium Slides \u0026amp; Sample Prep Reagents Kit (PN-1000460). Following the Xenium run, Hematoxylin and Eosin (H\u0026amp;E) staining was performed on the same section according to the Post-Xenium Analyzer H\u0026amp;E Staining user guide (CG000613, Rev B, 10x Genomics).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eQuantification and statistical analysis\u003c/h2\u003e \u003cp\u003eQuantification of WB was performed using NIH ImageJ software. Statistical analysis and graphic design were performed using GraphPad Prism 10. The two-tailed Student\u0026rsquo;s t test was used to detect statistical differences in all experiments except for EAE scores, which were analyzed by the Mann-Whitney U test. P values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (*) were considered statistically significant. All experiments were performed at least twice.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThe authors thank Birgit Brennecke, Kerstin Ellrott, Elena Fischer, Izabela Plumbon and Nadja Schl\u0026uuml;ter for expert technical assistance. This work was supported by a grand from the DFG (CRC 854, project A5) and the Cluster of Excellence-Resolving Infection Susceptibility (RESIST), (EXC 2155), Hannover Medical School, 30625 Hannover, Germany.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAllen, N.J., and Lyons, D.A. (2018). 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Methods \u003cem\u003e25\u003c/em\u003e, 402\u0026ndash;408. 10.1006/meth.2001.1262.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5937561/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5937561/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAstrocytes are central to the pathogenesis of multiple sclerosis; however, their regulation by intrinsic post-translational ubiquitination and deubiquitination is unresolved. This study shows that the deubiquitinating enzyme OTUD7B in astrocytes confers protection against murine experimental autoimmune encephalomyelitis, a model of MS, by limiting neuroinflammation. RNA-sequencing of isolated astrocytes and spatial transcriptomics showed that in EAE OTUD7B downregulates the expression of chemokines in astrocytes of inflammatory lesions, which is associated with reduced recruitment of encephalitogenic CD4\u0026thinsp;+\u0026thinsp;T cells. Furthermore, OTUD7B was essential for GFAP protein expression of astrocytes bordering inflammatory lesions. Mechanistically, OTUD7B (i) restricted TNF-induced chemokine production of astrocytes by sequential K63- and K48-deubiquitination of RIPK1 limiting NF-κB and MAPK activation and (ii) enabled GFAP protein expression by supporting GFAP mRNA expression and preventing its proteasomal degradation through K48-deubiquitination of GFAP. This dual action on TNF signaling and GFAP identifies astrocyte-intrinsic OTUD7B as a central inhibitor of astrocyte-mediated inflammation.\u003c/p\u003e","manuscriptTitle":"The deubiquitinase OTUD7B ameliorates central nervous system autoimmunity by inhibiting degradation of glial fibrillary acidic protein and astrocyte hyperinflammation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-27 13:40:17","doi":"10.21203/rs.3.rs-5937561/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"68cf60dd-427d-4825-bdd4-3d809c687ccf","owner":[],"postedDate":"February 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":44854497,"name":"Biological sciences/Immunology/Autoimmunity"},{"id":44854498,"name":"Biological sciences/Biochemistry/Proteins/Ubiquitylated proteins"}],"tags":[],"updatedAt":"2025-10-21T07:07:29+00:00","versionOfRecord":{"articleIdentity":"rs-5937561","link":"https://doi.org/10.1038/s41467-025-65093-4","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-10-20 04:00:00","publishedOnDateReadable":"October 20th, 2025"},"versionCreatedAt":"2025-02-27 13:40:17","video":"","vorDoi":"10.1038/s41467-025-65093-4","vorDoiUrl":"https://doi.org/10.1038/s41467-025-65093-4","workflowStages":[]},"version":"v1","identity":"rs-5937561","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5937561","identity":"rs-5937561","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}