During the last decade, mesenchymal stem cells (MSCs) have gained much attention in the field of regenerative medicine due to their capacity to differentiate into different cell types and to promote immunosuppressive effects. However, the underlying mechanism of MSC-mediated immunoregulation is not fully understood so far. Macrophages are distinguished in classical activated, pro-inflammatory M1 and alternatively activated M2 cells, which possess different functions and transcriptional profiles with respect to inflammatory responses. As polarization is not fixed, macrophage functional plasticity might be modulated by the microenvironment allowing them to rapidly react to danger signals and maintaining tissue homeostasis.Murine MSCs were preconditioned with IL-1ß and IFN-ɣ to enhance their immunosuppressive capacity regarding macrophage polarization under M1- and M2a-polarizing conditions. Macrophage polarization was analyzed by real-time PCR, flow cytometry, and cytokine detection in culture supernatants. The role of MSC-derived nitric oxide (NO), prostaglandin E2 (PGE2), and IL-6 in this process has been evaluated using siRNA transfection and IL-6 receptor-deficient macrophages, respectively.+ T lymphocytes. Finally, we show that MSC-mediated macrophage polarization strongly depends on IL-6, whereas a minor role for NO and PGE2 was found.Preconditioned, but not unprimed, MSCs secreted high levels of NO, IL-6, and PGE2. Co-culture with macrophages (M0) in the presence of M1 inducers (LPS + IFN-ɣ) led to significant reduction of CD86 and iNOS protein in macrophages and diminished TNF-α secretion. Additionally, CD86 and iNOS protein expression as well as NO and IL-10 secretion were markedly increased under M2a-polarizing culture conditions (IL-4). MSC-dependent macrophage polarization did not depend on direct cell-cell contact. Co-culturing in the presence of LPS and IFN-ɣ resulted in the upregulation of M2a, M2b, and M2c marker genes, whereas in the presence of IL-4 only M2b markers were significantly increased. In turn, IL-10-producing regulatory M2b cells significantly inhibited IFN-ɣ expression in CD4Preconditioning of MSCs highly strengthens their capacity to regulate macrophage features and to promote immunosuppression. Repression of M1 polarization during inflammation and M2b polarization under anti-inflammatory conditions strongly depend on functional IL-6 signaling in macrophages. The potential benefit of preconditioned MSCs and IL-6 should be considered for future clinical treatment.The online version of this article (10.1186/s13287-018-1039-2) contains supplementary material, which is available to authorized users. MSCs activity , 7. The activity –10. The activity  and B ceactivity , among oactivity . M2 macractivity . ThroughIn this study, adult 8–12-week-old C57/Bl6 and IL-6Rα-deficient  C57/Bl6 6/cm2 in MSC culture medium (PAN-Biotech) supplemented with 2.5 ng/ml human basic fibroblast growth factor FGF , 100 U/ml penicillin, and 10 μg/ml streptomycin (Sigma Aldrich) at 37 °C until a proliferative and homogenous MSC population was obtained. To prove MSC status, cells were differentiated along adipogenic, chondrogenic, and osteogenic lineages by means of differentiation medium as previously described [MSCs were isolated from femurs of 10–12-week-old C57Bl/6 mice. Bone marrow cells were obtained by flushing out femurs with PBS. Cells were cultured in a density of 1.5–2 × 10escribed .In addition, MSC-specific surface markers were analyzed by incubation of cells with monoclonal fluorochrome-labeled antibodies against stem-cell antigen 1 , CD29 , CD44 , and CD49e  for 20 min at 4 °C in cellwash buffer (BD) supplemented with 3% FCS. Antibodies against CD11b  and CD45  were used to exclude the expression of these molecules on our MSC population. After washing with 3% FCS in cell wash buffer (BD), cells were analyzed by flow cytometry using a MACSQuant Analyzer (Miltenyi Biotec).For in vitro cell activation of bone marrow-derived MSCs, MSCs were treated with 30 ng/ml recombinant murine IFN-γ (Peprotech) and 3 ng/ml recombinant murine IL-1β (Peprotech) in MSC medium supplemented with 2.5 ng/ml FGF-b for 24 h. MSCs without cytokine treatment served as controls.6) were cultured in six-well plates in RPMI medium supplemented with 20% FCS containing 20 ng/ml recombinant murine M-CSF (Peprotech), 100 U/ml penicillin, and 10 μg/ml streptomycin (Sigma Aldrich) in a humidified incubator at 37 °C. On day 3 and day 6, the medium was changed. Differentiated macrophages (M0) were obtained after 7 days of culture. Expression of the macrophage markers CD11b and F4/80 was proven by flow cytometry using PE- and FITC-labeled antibodies . For in vitro polarization, M0 macrophages were cultured in RPMI medium with 10% FCS supplemented with 20 ng/ml recombinant murine IFN-ɣ (Peprotech) and 100 ng/ml LPS (Sigma Aldrich) to induce a M1-like phenotype or in medium supplemented with 20 ng/ml recombinant murine IL-4 (Peprotech) to induce M2a-like macrophages, respectively. In some experiments, macrophages were additionally treated with 25 ng/ml recombinant murine IL-6 (Peprotech).Bone marrow cells were isolated by flushing femurs of 8–12-week-old C57/Bl6 or IL-6Rα-deficient C57/Bl6 mice, respectively. Cells . The following fluorochrome-labeled antibodies were used: CD11b , F4/80 , CD86 , CD206 , and IL6Rα (PE anti-mouse CD126 (IL-6Rα chain), clone D7715A7, Bio Legend). The macrophage phenotypes were further examined by real-time PCR and ELISA.+-positive lymphocytes were isolated from spleens of C57/Bl6 wild type mice. Single-cell suspensions were obtained by using gentle MACS C tubes and gentle MACS dissociator according to the protocol provided by the company (Miltenyi Biotec). Spleen isolation and cell labeling were performed under sterile conditions. The spleens were isolated and transferred into gentle MACS C tubes containing sterile PBS supplemented with 0.5% BSA and 2 mM EDTA. Using the gentle MACS dissociator, the spleens were segregated. The cell suspension was then transferred into a 50-ml tube using a 70-μm filter and centrifuged at 1000 rpm for 2 min. After that, the cells were washed using 5 ml PBS supplemented with 0.5% BSA and 2 mM EDTA and centrifuged at 400×g for 10 min. Following, residual erythrocytes were removed by resuspending the cell pellet in 6 ml 0.2% NaCl for 45 s. The lysis was then stopped by adding 14 ml 1.2% NaCl. The cell suspension was filtered, centrifuged, and counted, and cells were labeled for negative depletion of CD4+ cells. According to the protocol provided by Miltenyi Biotec, the cells were labeled with biotin-antibody cocktail and incubated for 5 min at 4 °C. After that, anti-biotin conjugated microbeads were added and incubated for 10 min at 4 °C. Finally, the CD4+-positive lymphocytes were separated by negative depletion using autoMACS (Miltneyi Biotec). Purity of CD4+ cells was confirmed by flow cytometry using PE-conjugated CD4 antibody .CD45 MSC and 5 × 105 M) for 24 h. Controls of macrophages and MSCs cultured alone were included. In parallel experiments, MSCs were preconditioned with 30 ng/ml IFN-γ and 3 ng/ml IL-1β for 24 h before culturing with macrophages. After co-culture, cells were separated using magnetic separation  by following the manufacturer’s instructions. In brief, macrophages were labeled with a biotin-conjugated F4/80 antibody (Miltenyi Biotec) for 10 min at 4 °C and then further incubated with monoclonal anti-biotin microbeads UltraPure (Miltenyi Biotec) for 15 min at 4 °C. After washing of cells, cells were loaded onto AutoMACS columns (Miltneyi Biotec) and non-labeled cells (MSCs) were collected at the outlet port “negative” whereas labeled macrophages were eluted at the positive outlet. Cells were immediately examined by flow cytometry.MSCs and macrophages (M0) were suspended in RPMI medium supplemented with 10% FCS, 100 U/ml penicillin, 10 μg/ml streptomycin, and M1 or M2 activating cytokines, respectively. Cells were cultured in six-well plates at a MSC:M ratio of 1:2  and cells were further cultured in the presence of M1 and M2 inducers for 24 h at 37 °C. Supernatants were collected and stored at − 80 °C for further analyses. Cells were immediately analyzed by flow cytometry.+ T cells (1 × 106 cells per well). Anti-mouse CD3e  and anti-mouse CD28  were added to the co-culture system, and cells were incubated for 24 h at 37 °C. CD4+, non-adherent cells were harvest from the co-culture supernatants by gentle pipetting and lysed for RNA extraction.Macrophages pre-cultured in transwells with preconditioned MSCs under M2a polarizing conditions were further cultured with purified CD4iNOS and COX-2 expression, 5 × 104 MSCs were seeded in six-well plates 2 days before transfection. Transfection has been performed using OptiMEM medium (Gibco) and Lipofectamine RNAiMAX Reagent (Invitrogen) according to manufacturer’s instructions. Cells were transfected with 5.5 nM Silencer Select iNOS siRNA, Silencer Select COX-2 siRNA, and Silencer Select negative control siRNA (Ambion), respectively, and incubated for 24 h at 37 °C and 5% CO2. MSCs were further activated by treatment with 30 ng/ml recombinant murine IFN-γ (Peprotech) and 3 ng/ml recombinant murine IL-1β (Peprotech) for additional 24 h. Transfected, preconditioned cells were used for co-culture experiments. Transfection efficiency was proven by real-time PCR, ELISA, and Griess Assay.To knockdown free Kit DNA Removal Kit (Ambion). RNA was reverse transcribed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). For real-time PCR, gene-specific primers for iNOS [Arg I [COX-2 [TNF-α [Fizz1/RELMα, Ym-1 [SPHK1, LIGHT [MertK [IFN-ɣ, IL-2, IL-4 [IL-10 [GATA-3 [IL-6 and 18S RNA expression, the following primers were used: IL-6 forward 5′-CCACTTCACAAGTCGGAGGCTTA-3′; IL-6 reverse 5′-GCAAGTGCATCATCGTTGTTCATAC-3′; 18S RNA forward 5′-CGGCTACCACATCCAAGGAA-3′, 18S RNA reverse 5′-GCTGGAATTACCGCGGCT-3′.Total RNA was extracted using RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Contaminating DNA was removed by DNA-for iNOS , Arg I [S [Arg I , COX-2 [I [COX-2 , TNF-α [2 [TNF-α , Fizz1/RMα, Ym-1 , SPHK1, 1, LIGHT , MertK [T [MertK , IFN-ɣ, -2, IL-4 , IL-10 [4 [IL-10 , and GAT [GATA-3  were use18S RNA gene. Fold expression was calculated using the 2−ΔΔCT method [All samples were run in triplicates. Relative gene expression levels were determined using Power SYBR Green PCR Master Mix (Applied Biosystems) according to the manufacturer’s recommended protocol with following thermal cycling conditions: 10 min 95 °C, 40 cycles of 15 s 95 °C and 60 s 60 °C, and 4 °C hold . Expression of target genes was normalized to the endogenous control T method .Cells were lysed in RIPA buffer supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail . Cell lysates were loaded on 10% polyacrylamide gel and blotted onto nitrocellulose membrane. Membranes were further blocked with blocking buffer and incubated with pSTAT3(Tyr705) antibody  according to the manufacturer’s instructions. Afterwards, membranes were incubated with a HRP-conjugated goat anti-rabbit secondary antibody (Dako) and developed with UptiLight HRP Blot Chemiluminescent ECL Substrate (Uptima). Blots were further stripped and reincubated with anti-STAT3 antibody  and HRP-conjugated goat anti-mouse secondary antibody (Dako).2 ELISA Kit-Monoclonal  following the manufacturers’ instructions.For cytokine quantification in culture supernatants, the following ELISA kits have been used: Human/mouse TGF beta 1 2nd Generation ELISA Ready-SET-Go (eBioscience), Mouse IL-6 DuoSet ELISA (R&D Systems), Mouse IL-10 DuoSet ELISA (R&D Systems), Mouse TNF alpha ELISA Ready-SET-Go (eBioscience), and Prostaglandin E2) in culture supernatants, using a modified Griess reagent (Sigma Aldrich). In brief, 80 μl supernatant was mixed with 80 μl Griess reagent and incubated at room temperature for 15 min. Absorbance at 540 nm was measured using a microplate reader  and nitrite concentrations were estimated using a standard nitrite curve (range 0 μM–100 μM).NO production was quantified by measuring nitrite (NOt test. One-sample t test was used when samples were compared with reference control sample (set as 1). Normally distributed unpaired data of multiple groups were analyzed using one-way ANOVA with Newman Keuls post hoc test. p value less than 0.05 was considered as statistically significant.Data were analyzed with GraphPad Prism 5 software. Experimental data are presented as means with standard error of the mean (SEM). Unpaired data of two groups were analyzed using unpaired iNOS gene expression in MSCs , CD44 (65.55 ± 7.4%), CD49e (66.35 ± 6.4%), and Sca-1 (89.3 ± 4.5) and were negative for CD11b and CD45. Further on, differentiation of bone marrow-derived MSCs into adipocytes, chondrocytes, and osteoblasts was evidenced . Transwell experiments were conducted in parallel to avoid direct cross-talk between MSCs and macrophages. Percentage of cells expressing co-stimulatory CD86 was significantly downregulated in the presence of MSCs, whereas the expression of the M2a marker CD206 was not influenced in co-cultures Fig. . Becauseund Fig. , despiteTo elaborate MSC-triggered effects on M2 polarization, same experiments as described above were performed in the presence of IL-4, which favors M2a polarization. The number of CD86 positive cells was significantly upregulated by primed MSCs when compared to unstimulated MSCs, but no alterations regarding CD206 expression were found Fig. . In addiTNF-α and IL-6 gene expression was confirmed under M1-polarizing conditions  under same conditions. However, MSC-derived PGE2 seemed to promote M2c polarization, as MertK was significantly reduced in macrophages co-cultured with COX-2 transfected MSCs   Fig. b. The laIL-10 gene expression did not differ between wild type and IL-6Rα-deficient M2-polarized macrophages (not shown). As shown in Fig. IL-10 compared to M2a-polarized control macrophages. Recombinant IL-6 also increased the expression of IL-10 in macrophages; however, this increase was significantly reduced when compared to MSC-mediated IL-10 upregulation. Correspondingly, macrophages from IL-6Rα-deficient mice showed strong reduction in IL-10 expression after co-culturing with preconditioned MSCs (versus wild type macrophages), arguing against an M2b phenotype.To further confirm that MSCs trigger M2b polarization, we investigated the expression of IL-10 in cells previously co-cultured with MSCs. In this regard, it is already known that M2b macrophages produce high levels of IL-10 when compared to other macrophage subtypes . IL-10 g+ T lymphocytes. To this end, we cultured M2a-polarized and macrophages pre-cultured with preconditioned MSCs (M2b phenotype) with CD4+ T cells , demonstrating that the therapeutic effectiveness of MSCs needs to be investigated. Among all MSC-based studies, there is a substantial proportion dealing with the treatment of cardiovascular diseases. Because ischemic heart diseases and heart failure remain major causes of morbidity and mortality, much effort is expanded to repair cardiac tissue with stem cell transplantation. The therapeutic properties of MSCs are largely related to their anti-inflammatory and immunomodulatory abilities which have been confirmed by in vivo and in vitro studies , 10, 32.Impaired pro-inflammatory response of macrophages and changes in the M1/M2 balance in the presence of MSCs has already been evidenced. Macrophage polarization is critical for the resolution of inflammation and tissue remodeling . During SPHK1 and LIGHT in macrophages in the presence of the M2a inducer IL-4. In turn, all M2 markers were upregulated under M1-polarizing co-culture conditions. Notably, although unprimed MSCs were able to reduce the expression of pro-inflammatory IL-6 and TNF-α in macrophages, these cells failed to modulate M2 macrophage marker expression, thus being in line with recently reported data [In this study, we found that preconditioning of murine MSCs with IFN-ɣ and IL-1ß highly improves their potential to promote immunomodulation. In fact, preconditioning of MSCs resulted in significantly elevated NO, IL-6, and PGE2 production, which have been already described to represent prominent immunosuppressive molecules , 37, altted data .IFN-ɣ expression in CD4+ T lymphocytes and also slight increase in expression of the Th2 marker GATA-3 [We also showed that macrophages co-cultured with preconditioned MSCs in the presence of IL-4 displayed highest IL-10 production, which could be attributed to the M2b phenotype initiated by preconditioned MSCs. In this regard, when compared with other macrophage subtypes, M2b cells are known to represent the main cellular source for IL-10, playing an important role in the resolution of inflammation . Direct r GATA-3 , indicatIL-6 and TNF-α suppression under M1-polarizing conditions. Hence, MSCs failed to upregulate almost all M2 marker genes in cells cultured in the presence of M1 inducers lacking IL-6 signaling. Most importantly, IL-6Rα-deficient macrophages showed upregulated TNF-α expression and significantly reduced SPHK1, LIGHT, and MertK expression after co-culturing with preconditioned MSCs, suggesting that IL-6 represents an essential regulator of M2b and probably M2c polarization. Indeed, after co-culture with preconditioned MSCs, macrophages deficient in IL-6Rα expressed strongly suppressed levels of IL-10 when compared to wild type cells, most likely due to impaired IL-6 signaling as evidenced by suppressed STAT3 activation. Although there is already evidence for the role of IL-6 for the polarization of alternatively activated macrophages [COX-2 knockdown indeed strongly reduced PGE2 secretion, without affecting NO or IL-6 secretion by preconditioned MSCs, but this impaired PGE2 production did not diminish the expression of M2 marker genes, except for FIZZ/RELMα. These discrepancies between results might be based on different experimental setups used. In this regard, in vitro polarized macrophage subsets [Previous studies argue for a prominent role of PGE2 as a key immunosuppressive mediator derived from MSCs , 41, 43.rophages , 45, in rophages , 46. Nevrophages , 43, PGE subsets  have bee subsets . In addi subsets .Altogether, our results presented here strongly suggest that MSCs, beside their potency to inhibit T cell proliferation  and to pPreconditioning of MSCs with IL-1ß and IFN-ɣ strongly improves the capacity of MSCs to modulate macrophage plasticity, mainly by IL-6 secretion. MSC-dependent polarization toward the M2 phenotype in a pro-inflammatory microenvironment and toward the regulatory M2b phenotype in the presence of anti-inflammatory IL-4 should be considered for future therapeutic approaches to treat cardiovascular diseases.Additional file 1:Figure S1. Characterization of bone-marrow derived MSCs. (a) As assessed by flow cytometry, MSCs were positive for the well-established MSCs markers CD29, CD44, CD49e and Sca-1 and negative for CD11b and CD45. (b) Adipogenic, chondrogenic and osteogenic differentiation of MSCs. Scale bar, 100 μm. (TIF 9562 kb)Additional file 2:Figure S2. Characterization of M0, M1- and M2a-like macrophages. (a) CD11b and F4/80 expression on bone marrow-derived differentiated M0 macrophages. M0 macrophages were further polarized in vitro to M1-like and M2a-like cells by treatment with 20 ng/ml IFN-ɣ and 100 ng/ml LPS (M1) or 20 ng/ml IL-4 (M2), respectively, for 24 h. (b) Gene expression of iNOS and Arg I was determined by real-time PCR. n = 3. (c) TNF-α and IL-6 levels in culture supernatants were quantified by Elisa. n = 5, *p < 0.05, **p < 0.001. (TIF 1040 kb)Additional file 3:Figure S3. Characterization of IL-6Rα-deficient macrophages. (a) IL-6Rα expression on bone marrow-derived M0 macrophages from WT and IL-6Rα-deficient mice. n = 3, **p < 0.01. (b) IL-6Rα-deficient and wild  type macrophages were differentiated into M1- and M2a-like cells as described in Methods. Gene expression of IL-6, TNF-α and the subtype-specific markers Ym-1, FIZZ1/RELMα (M2a markers), SPHK1, LIGHT (M2b markers) and MertK (M2c marker) was determined by real-time PCR. n = 4–6, *p < 0.05, ***p < 0.001. (TIF 2221 kb)Additional file 4:Figure S4. siRNA knockdown of iNOS and COX-2 expression in preconditioned MSCs. MSCs were transfected with siRNA specific for iNOS, COX-2 or control siRNA (each 5.5 nM), respectively. After 24 h, cells were preconditioned with IFN-ɣ and IL-1ß. (a) Transfection efficiency was proven by real-time PCR. n = 5, **p < 0.01 vs. control siRNA (Ctrl). ##p < 0.01, ###p < 0.001. (b) Levels of NO, IL-6 and PGE2 secreted by transfected MSCs were determined by Griess assay and Elisa. n = 5–6, *p < 0.05, ***p < 0.001, n.s. not significant (TIF 1510 kb)Additional file 5:Figure S5. Flow cytometric analysis of CD4 expression on isolated T lymphocytes. (TIF 429 kb)


The cationic one-dimensional ribbon consists of two copper(I) centers each ligated by one acetonitrile molecule and interconnected through two bridging 1,3-dithiolane ligands. One S-donor site of each ligand is κ1-bound to Cu, whereas the second S atom acts as a four-electron donor, bridging two Cu atoms in a κ4-bonding mode. The positive charge of each copper cation is compensated for by a hexafluoridophosphate counter-ion. In the crystal, the polymer chains are linked by a series of C—H⋯F hydrogen bonds, forming a supramolecular framework.The polymeric title compound, [Cu Notably, for the soft coinage metal ions copper(I), silver(I) and gold(I), numerous structurally characterized examples coordinated by terminal or bridging THT ligands have been documented  4][PF6] as reactant to obtain the title polymeric ionic salt-like material, which could be interesting for electrochemical investigations.In a comparative study with respect to our previous work on the coordination chemistry of the open-chain dithioether analogues et al., 20172-Br)}(μ2-L1)]n and [{Cu(μ2-Cl)}(μ2-L1)]n, formed upon treatment of CuBr and CuCl with 1,3-dithiolane (L1). The title complex salt, a ribbon of composition [Cu(MeCN)]n+ (CP1) also results from the reaction of [Cu(MeCN)4][PF6] with L1, but its architecture is quite different.We have previously described (MeCN)]n+  contains three unique copper(I) centers, that of CP1  contains only two unique CuI atoms. Each displays a CuNS3 four-coordinate environment; see Table 1L—Cu—L angles: 99.97 (7) to 119.47 (11)° for Cu1, and 99.29 (11) to 118.69 (4)° for Cu2]. The τ4 descriptor for fourfold coordination is = 0.89 for both atoms Cu1 and Cu2, indicating that each have a trigonal-pyramidal geometry –2.3367 (11) Å, the mean Cu—S bond length of 2.314 (12) Å is quite similar to that in [Cu(MeCN)]n+. In addition, the mean Cu—N bond distance matches well with that of [Cu(MeCN)]n [1.979 (4) versus 1.984 (7) Å]. The three dithiolane ligands each have one S atom that is a two-electron donor and one S atom that is a μ2-four-electron donor. The Cu⋯Cu separations of ca 3.689–3.852 Å are far above the sum of the van der Waals radii of two Cu atoms (2.8 Å), excluding any bonding interaction. These two bonding modes lead to the formation of a ribbon-like coordination polymer, which runs parallel to the a axis, where each copper(I) center is bonded to two μ2-S atoms and one μ1-S atom trisilver(I)] (nitrato-O)silver(I)]  phenyl](diphenyl)phosphine}ruthenium(II) chloroform solvate  in CH2Cl2 (10 ml) was added an equimolar amount of 1,3-dithiolane (L1) via a syringe. The solution was stirred at 293 K for 2 h, then layered with Et2O (10 ml) and stored in a refrigerator for 2 days. Colourless block-like crystals formed progressively .The reaction scheme for the synthesis of the title compound is illustrated in Fig. 410H18Cu2F12N2P2S4: C, 16.88; H, 2.54; N, 3.94; S, 18.03%. Found: C, 16.44; H, 2.28; N, 3.44; S, 17.81%. IR : 2280 w (weak) (CN), 835 vs (very strong) (PF6).Elemental analysis calculated for CUiso(H) = 1.5Ueq(C-methyl) and 1.2Ueq(C) for other H atoms. The structure was refined as a two-component inversion twin; BASF = 0.121 (12). In the final cycles of refinement three reflections were omitted; one was affected by the backstop and two were most disagreeable reflections.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S205698901901627X/su5530sup1.cifCrystal structure: contains datablock(s) Global, I. DOI: 10.1107/S205698901901627X/su5530Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S205698901901627X/su5530Isup3.cdxSupporting information file. DOI: 1969688CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The influence of the substituents in the crystals of the title compounds has not made any significant effect on the crystal packing and intermolecular hydrogen bonds. The validity of chlorine–methyl exchange rule is confirmed. H-pyrrolothiazine-1,3-dicarboxylate, C26H29Cl2NO6S, (I), and its isomorphous pair diethyl 1-(4-methylbenzyl)-4-(4-methylphenyl)-2,2-dioxo-3,4,6,7,8,8a-hexahydro-1H-pyrrolothiazine-1,3-dicarboxylate, C28H35NO6S, (II), are described. The molecular aggregation patterns appear to be strikingly similar despite changes in the substituents, with a Cl atom in (I) being replaced by a methyl group in (II). Inspite of the chemical modifications, the structures of (I) and (I) are isomorphous, isostructural and found to obey the chlorine–methyl exchange rule. Both the structures feature C—H⋯O hydrogen bonding. However, a distinguishing feature between (I) and (II) is observed in the conformation of the pyrrole rings where the twist occurs on different C—N bonds. Hirshfeld analysis of both structures is presented and discussed.Accurate studies on the effect of substituents on the crystal packing are essential for understanding the intermolecular interactions and thus paving the way to crystal structure prediction. The crystal structures of diethyl 1-(4-chlorobenzyl)-4-(4-chlorophenyl)-2,2-dioxo-3,4,6,7,8,8a-hexahydro-1 In this context, the display of chlorine–methyl and benzene–thiophene exchange rules in the close-packing model of organic molecules Q(2) = 0.3604 (19) Å and φ = 191.2 (4)°. However, in compound (II)Q(2) = 0.377 (2) Å and φ(2) = 169.3 (4)°. The Cremer and Pople puckering parameters of the six-membered heterocyclic ring in (I)Q = 0.6441 (15) Å, θ = 8.51 (14)° and φ = 95.8 (8)°, close to a chair conformation (1C4), which is comparable with the values of Q = 0.6511 (16) Å, θ = 9.53 (15)° and φ = 97.5 (7)° for (II)The molecular structures of the title compounds differ from each other only by a chlorine atom in (I)viz. O1. The non participation of the other oxygen atom (O2) cannot be explained from the viewpoint of intermolecular interactions whereas the absence of such interactions involving O3 and O5 may be attributed to steric factors arising from an unfavourable packing geometry. In both crystals, molecules are connected into inversion dimers via pairs of weak C—H⋯O hydrogen bonds, forming via weak C—H⋯O interactions into chains running along [011]. A parallel-displaced π–π stacking interaction is observed in both compounds between the C21–C26 benzyl rings. In (I)Cg⋯Cg = 4.0485 (13) Å, with a slippage of 1.749 Å  where Cg is the ring centroid.The crystal packing of both compounds Figs. 3 and 4 ▸ CrystalExplorer 3.0 software  as eluent sulfonyl]acetate (1.6 mmol), aromatic aldehyde (3.2 mmol) and pyrrolidine (1.6mmol) was dissolved in ethanol (10 mL), heated until the solution turned yellow and stirred at room temperature for 2–5 days. After completion of the reaction, the crude product was purified using flash column chromatography on silica gel (230–400 mesh) with petroleum ether and ethyl acetate mixture (95:5 Uiso(H) set at 1.2–1.5Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989018011416/xu5934sup1.cifCrystal structure: contains datablock(s) I, II, global. DOI: 10.1107/S2056989018011416/xu5934Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989018011416/xu5934IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989018011416/xu5934Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989018011416/xu5934IIsup5.cmlSupporting information file. DOI: 1861314, 1861313CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Nature Communications (2018) 9, 4854; 10.1038/s41467-018-07405-5, published online 19 November 2018Correction to: +IL-7Rα+ ILC1’ incorrectly read ‘CD49a+IL-7Rα− ILC1’. In Fig. 6b, the label ‘LNx (e)/ILN-deficient mice (c)’ incorrectly read ‘LNx (c)/ILN-deficient mice (e)’. These errors have now been corrected in both the PDF and HTML versions of the Article.The original version of this Article contained errors in Figs. 1 and 6. In Fig. 1c, the label ‘CD49a


Lactobacillus reuteri glucansucrases, with lactose and sucrose as substrates. Here, we show that these GL34 compounds are largely resistant to the hydrolytic activities of common carbohydrate-degrading enzymes. Also, the ability of single strains of gut bacteria, bifidobacteria, lactobacilli, and commensal bacteria, to ferment the GL34 compounds was studied. Bifidobacteria clearly grew better on the GL34 mixture than lactobacilli and commensal bacteria. Lactobacilli and the commensal bacteria Escherichia coli Nissle and Bacteroides thetaiotaomicron only degraded the F2 compound α-D-Glcp-(1 → 2)-[β-D-Galp-(1 → 4)-]D-Glcp, constituting around 30% w/w of GL34. Bifidobacteria digested more than one compound from the GL34 mixture, varying with the specific strain tested. Bifidobacterium adolescentis was most effective, completely degrading four of the five GL34 compounds, leaving only one minor constituent. GL34 thus represents a novel oligosaccharide mixture with  synbiotic properties towards B. adolescentis, synthesized from cheap and abundantly available lactose and sucrose.Previously we structurally characterized five glucosylated lactose derivatives (F1–F5) with a degree of polymerization (DP) of 3–4 (GL34), products of The online version of this article (10.1007/s00253-018-9473-8) contains supplementary material, which is available to authorized users. The human gut microflora has drawn increasing attention in recent years. It constitutes a very interesting ecosystem that varies in density and functionality in the different gut compartments   Bode , β-galacOS) Bode . All of OS) Bode . IsomaltOS) Bode , xylo-olOS) Bode , resistaOS) Bode , and soyOS) Bode , althougOS) Bode .Bifidobacterium breve 26M2 has been reported  using the –F5 usingBacteroides thetaiotaomicron VPI-5482, Lactobacillus acidophilus ATCC 4356, B. adolescentis ATCC 15703, and Bifidobacterium longum subsp. infantis ATCC 15697 were purchased from ATCC. B. breve DSM 20213 was obtained from the DSMZ culture collection. L. reuteri strain 121 was obtained from TNO Quality of Life, Zeist, the Netherlands. Lactobacillus casei W56 was provided by Winclove Probiotics B.V. (Amsterdam). All reagents, chemicals, or medium components were purchased from Sigma , or as stated otherwise. The purified GOS mixture TS0903  was provided by FrieslandCampina Domo; its detailed GOS composition was published elsewhere  mixture of the purified TS0903 GOS and long-chain Inulin , also serving as a control for the current prebiotic formula added to infant nutrition . The bacterial pellets were washed twice with sterile 10% NaCl and diluted 25-fold in 2× modified MRS   or by autoclaving solutions (glucose). Cultures were inoculated by mixing 1:1 diluted bacterial suspensions with sterilized carbohydrate solutions in sterile microtiter plates to obtain an initial OD600 of 0.01. Inoculation of microtiter plates was carried out in an anaerobic glove box  with a constant nitrogen flow, the microtiter plates sealed air tightly and transferred into a plate reader placed under constant N2 flow. Glucose was used as positive control to compare growth of these lactobacilli on the mixture of GL34 compounds. Media without an added carbon source was used as negative control. Bacterial growth was followed at 37 °C by measuring the optical density at 600 nm (OD600 nm) in 5-min intervals. OD values of the negative control samples (no carbohydrate added) were deducted while measuring their corresponding samples and positive controls.Lactobacilli were pre-cultured in MRS medium  anaerobically ) under an NBifidobacterium strains were sub-cultured (from stocks stored at − 80 °C) in 10 mL of Bifidobacterium medium (BM) supplemented with 1% glucose . After autoclaving, BM was supplemented with 0.05% (w/v) filter-sterilized cysteine-HCl , 15 mM NaCl, 8.5 mM (NH4)2SO4, 4 mM L-cysteine, 1.9 M hematin, 200 M L-histidine, 100 nM MgCl2, 1.4 nM FeSO4·7 H2O, 50 M CaCl2, 1 g mL−1 vitamin K3, 5 ng mL−1 vitamin B12, and individual carbon sources   or by autoclaving solutions (lactose). Growth was carried out in fermentation tubes at 37 °C under anaerobic conditions maintained by GasPak EZ anaerobe container system . Cell suspensions from overnight cultures were prepared in 3 mL of BM supplemented with different carbon source in a final concentration of 5 mg mL−1. BM without an added carbon source was used as negative control. OD600 nm measurements of the fermentation tubes were carried out manually at 1-h intervals and data used to generate growth curves. OD values of the negative control samples (no carbohydrate added) were deducted while measuring their corresponding samples and positive controls.The Escherichia coli Nissle was cultured using M9 medium as previously described, at 37 °C under aerobic conditions  were deducted while measuring their corresponding samples and positive controls.−1) was incubated for 24 h with different carbohydrate degrading enzymes (5 U mL−1): α-amylase 1 (porcine pancreas); α-amylase 2 (Aspergillus oryzae); α-glucosidase (yeast); Iso-amylase ; β-galactosidase 1 (A. oryzae) and β-galactosidase 2 (Kluyveromyces lactis) . The harvested cell pellets were washed twice with 0.1 M potassium phosphate buffer (pH 6.6) and then suspended in 1 mL of this buffer into 2.0-mL screw-cap microtubes containing 400 mg of 0.1-mm-diameter glass beads (Biospec Products). Cell disruptions were carried out by homogenization by a mini bead-beater (Biospec Products) at 4200 rpm for six 1-min cycles with 40-s cooling on ice in between. The cytoplasmic extracts were harvested by centrifugation at 10,000×g for 5 min to remove cell wall fragments, and then concentrated to one fifth of the initial volume using Amicon Ultra-4 units as above.After growth with GL34 as their only carbon source, the three tested −1 of the GL34 mixture. All reactions were performed in Milli-Q at 37 °C for 24 h. The progress of the reactions was followed by high-performance anion-exchange chromatography (HPAEC).The concentrated cell-free supernatants and cytoplasmic extracts (10 μg protein for each) were incubated separately with 5 mg mL−1 using a complex gradient of eluents A (100 mM NaOH); B (600 mM NaOAc in 100 mM NaOH); C (Milli-Q water); and D 50 mM NaOAc. The gradient started with 10% A, 85% C, and 5% D in 25 min to 40% A, 10% C, and 50% D, followed by a 35-min gradient to 75% A, 25% B, directly followed by 5-min washing with 100% B and reconditioning for 7 min with 10% A, 85% B, and 5% D. External standards of lactose, glucose, and fructose were used for calibration. For the determination of glucosylated lactose compounds with a degree of polymerization (DP) of 3, maltotriose was used as external standard.Samples were analyzed on an ICS-3000 workstation  equipped with an ICS-3000 pulse amperometric detection (PAD) system and a CarboPac PA-1 column . The analytical separation was performed at a flow rate of 0.25 mL minB. adolescentis ATCC 15703, B. longum subsp. infantis ATCC 15697, B. breve DSM 20123, and B. breve UCC 2003 used in this study were extracted from the National Center for Biotechnology Information (NCBI) database. Database searches used the non-redundant sequence database accessible at the NCBI website (http://www.ncbi.nlm.nih.gov) using BLASTP and global align search. The BLASTP searches and multiple-sequence alignments were used to find similarity between the characterized glucosidases of B. breve UCC 2003 and annotated glucosidases encoded by the studied bifidobacterial strains. Annotation of carbohydrate-active enzymes encoded by the genome sequences of L. reuteri 121 and L. acidophilus ATCC 4356 was carried out using dbCAN (http://csbl.bmb.uga.edu/dbCAN).All protein sequences from p-(1 → 4)-β-D-Galp-(1 → 4)-D-Glcp; F2 (2-glc-lac): α-D-Glcp-(1 → 2)-[β-D-Galp-(1 → 4)-]D-Glcp; F3 (3′-glc-lac): α-D-Glcp-(1 → 3)-β-D-Galp-(1 → 4)-D-Glcp; F4 : α-D-Glcp-(1 → 4)-β-D-Galp-(1 → 4)-[α-D-Glcp-(1 → 2)-]D-Glcp; and F5 : α-D-Glcp-(1 → 3)-β-D-Galp-(1 → 4)-[α-D-Glcp-(1 → 2)-]D-Glcp (4). Four types of glycosidic linkages thus occur in this mixture, namely α(1 → 2), α(1 → 3), α(1 → 4), and β(1 → 4). Only the F2 2-glc-lac compound had been described before  common intestinal bacteria in more detail.Subsequent incubation of the GL34 mixture with the β-galactosidase enzymes from is Table  however,L. casei W56, L. acidophilus ATCC 4356, and L. reuteri 121 and observed that all three strains showed limited growth on media with the GL34 mixture as the only carbon source (5 mg mL−1): the final (relative) OD600 values reached were 3.8%, 10.4%, and 26.5%, respectively, compared to a 100% control grown on glucose  showed different stimulatory effects on the growth of B. breve DSM 20123, B. adolescentis ATCC 15703, and B. longum subsp. infantis ATCC 15697. B. adolescentis grew very well on GL34, its final OD600 value reached 80% of that of a 100% control growing on lactose, the purified TS0903 GOS mixture, and the GOS/FOS mixture , was relatively minor with final OD600 values of 0.19 ÷ 0.35 after 24-h of incubation, compared to growth on lactose and glucose with final OD600 values of 0.66 and 0.74, respectively  linked to glucosyl residue in F2 2-glc-lac. Many putative α-glucosidases were found encoded in the genome sequence of this strain, however. In view of the results obtained, these enzymes apparently are inactive on the GL34 compounds, or the GL34 mixture is unable to induce their expression. This was not investigated further.HPAEC profiles of ded Fig. . When seLactobacillus acidophilus ATCC 4356 and L. reuteri 121 strains only showed hydrolytic activity with F2 2-glc-lac in the GL34 mixture. L. reuteri 121 degraded this compound to a higher level than L. acidophilus ATCC 4356    linkage in sucrose and kojibiose   Gänzle .This Lac%) Table . The obs−1 GL34 were harvested at 36 h  transporters. Further utilization may be carried out by cytoplasmic GHs such as α/β-glucosidases and α/β-galactosidases to produce monosaccharides which are used for growth , namely AglB; AglA and BAD-0971. AglB exhibited a preference for hydrolyzing α(1 → 4) linkages in maltose  and B. breve UCC 2003 . Agl3 from B. breve UCC 2003, as mentioned above, has a broad hydrolytic specificity towards α(1 → 2), α(1 → 3), α(1 → 4), α(1 → 5), and α(1 → 6) in kojibiose, turanose, maltose, leucrose, and palatinose, respectively  and α(1 → 6) glucosidic linkages, namely α-1,4 glucosidase (locus tag BLIJ-0129); putative amylase (locus tag BLIJ-0286); putative iso-amylase (locus tag BLIJ-0286 and BLIJ-2315); and oligo-1,6-glucosidase (locus tag BLIJ-2526) (www.cazy.org/). However, BLAST analysis showed very low similarity in their protein sequence between the annotated glucosidases from this strain and Alg3  (partly) remained unutilized in culture supernatants after growth  and α(1 → 6) glucosidic linkages present in starch, amylopectin and pullulan  linked to the galactosyl residues occurring in F3 3′-glc-lac and F5 3′,2-glc-lac but not the glucose units α(1 → 4) linked to the galactosyl residues in F1 4′-glc-lac and F4 4′,2-glc-lac.In case of wth Fig. , panel 3ion Fig. , panel 3B. breve DSM 20213 genome, however, are annotated to act mainly on α(1 → 4) and α(1 → 6) glucosidic linkages; namely α-1,4 glucosidase (locus tag BBBR-0095); putative amylase  and oligo-1,6-glucosidase (locus tags BBBR-0484 and BBBR-1863) (www.cazy.org/). This uncharacterized α-1,4 glucosidase (locus tag BBBR-0095) was found to have 99% similarity with Alg3 of B. breve UCC 2003  linkage to kojibiose. Following growth on GL34 as only carbon source, all three strains exhibited clear intracellular activity with F2 2-glc-lac. The data showed that individual Bifidobacterium strains have preference for degradation of glucosylated lactose compounds with α(1 → 3) and α(1 → 4) glucosidic linkages.The annotated β-galactosidases (family GH2) of these three A. oryzae. These are endo-acting enzymes (EC 3.2.1.1), degrading α(1 → 4) glucosidic linkages in polysaccharides such as starch or glycogen  debranching enzyme  glucosidic linkages in pullulan and starch  linkage makes F3 3′-glc-lac more selective than F1 and F4: F3 was utilized by only two out of three studied Bifidobacterium strains: B. adolescentis ATCC 15703 and B. breve DSM 20213. Also, F5 3′,2-glc-lac with both α(1 → 2) and α(1 → 3) glucosidic linkages showed similar stimulatory effects on all three Bifidobacterium strains.There is abundant clinical evidence for the important roles of B. adolescentis. Four out of five compounds in this GL34 mixture exerted high and selective growth stimulatory effects towards health-beneficial probiotic bifidobacteria. The combination of monomer composition and linkage type clearly determines the fermentable properties of the GL34 compounds. Individual gut bacteria were able to utilize only specific compounds in the GL34 mixture. Synergistic activities between bacterial species thus are likely to be essential for the utilization of the whole GL34 mixture. In future work, this will be studied in more detail, e.g., by using fecal bacterial cultures. Only B. adolescentis was able to utilize almost all structures, providing a potential synbiotic combination.In conclusion, the GL34 mixture promotes growth of the tested bacteria to different extents. The bifidobacteria tested generally were better at degrading GL34 compounds than the lactobacilli and commensal bacteria. The stronger metabolic toolset of bifidobacteria in comparison with lactobacilli also has been observed when comparing their growth on human milk oligosaccharides and other prebiotic oligosaccharides as primary carbon source


We have revealed that diacylglycerol kinase η (DGKη)-knockout (KO) mice display bipolar disorder (BPD) remedy-sensitive mania-like behaviors. However, the molecular mechanisms causing the mania-like abnormal behaviors remain unclear. In the present study, microarray analysis was performed to determine global changes in gene expression in the DGKη-KO mouse brain. We found that the DGKη-KO brain had 43 differentially expressed genes and the following five affected biological pathways: “neuroactive ligand-receptor interaction”, “transcription by RNA polymerase II”, “cytosolic calcium ion concentration”, “Jak-STAT signaling pathway” and “ERK1/2 cascade”. Interestingly, mRNA levels of prolactin and growth hormone, which are augmented in BPD patients and model animals, were most strongly increased. Notably, all five biological pathways include at least one gene among prolactin, growth hormone, forkhead box P3, glucagon-like peptide 1 receptor and interleukin 1β, which were previously implicated in BPD. Consistent with the microarray data, phosphorylated ERK1/2 levels were decreased in the DGKη-KO brain. Microarray analysis showed that the expression levels of several glycerolipid metabolism-related genes were also changed. Liquid chromatography-mass spectrometry revealed that several polyunsaturated fatty acid (PUFA)-containing phosphatidic acid (PA) molecular species were significantly decreased as a result of DGKη deficiency, suggesting that the decrease affects PUFA metabolism. Intriguingly, the PUFA-containing lysoPA species were markedly decreased in DGKη-KO mouse blood. Taken together, our study provides not only key broad knowledge to gain novel insights into the underlying mechanisms for the mania-like behaviors but also information for developing BPD diagnostics.  •mRNA levels of prolactin and growth hormone were strongly increased in DGKη-KO mice.•An annotation of the results identified several affected biological pathways.•Phosphorylated ERK1/2 levels were decreased in the DGKη-KO brain.•PUFA-containing phosphatidic acid (PA) species were decreased by DGKη-KO.•PUFA-containing lysoPA species were decreased in DGKη-KO mouse blood. CurrentDGKH (diacylglycerol kinase (DGK) η gene) is associated with the etiology of BPD. Moreover, DGKH is located within the BPD linkage region on 13q14 [DGKH is one of the few replicated risk genes for BPD and is attracting much attention as a BPD-associated gene [Recently, several genome-wide association studies (GWASs) conducted by Baum et al. , Weber eon 13q14 ,7. Furthon 13q14 . Therefoted gene .DGK phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA) . To dateRecently, we generated DGKη-knockout (KO) mice and performed behavioral tests . IntriguPrl (prolactin), Gh (growth hormone), Foxp3 (forkhead box P3), Glp1r (glucagon-like peptide 1 receptor) and Il1b (interleukin 1β), which have previously been implicated in BPD. Regarding glycerolipid metabolism, the expression levels of several glycerolipid-related genes were changed. We found that polyunsaturated fatty acid (PUFA)-containing PA species, such as 18:1/18:2-, 18:0/20:3-, 18:0/22:5-, 20:0/20:4- and 18:1/22:2-PA, were significantly decreased in the DGKη-KO mouse brain and that 18:2- and 20:5-lysoPA (LPA) were markedly decreased in DGKη-KO mouse blood.The purpose of the present work was to elucidate differences between the brains of DGKη-KO mice and wild-type (WT) sibling controls. To determine this, we performed a microarray analysis. The analysis revealed that the deficiency of DGKη appears to modulate gene expression in five biological pathways including “neuroactive ligand-receptor interaction”, “positive regulation of transcription by RNA polymerase II”, “positive regulation of cytosolic calcium ion concentration”, “Jak-STAT signaling pathway” and “Positive regulation of ERK1 and ERK2 cascade”. Notably, all five biological pathways include at least one gene among 22.1dgkh–/–, Accession No. CDB0606K) were generated as previously described [This study received approval from the Animal Experiment Committee of Chiba University (permission number: 28–77 and 29–195 and 30–185). All procedures relating to animal care and treatment were conducted in compliance with the National Institutes of Health: Guide for the Care and Use of Laboratory Animals. The mice  were housed in groups of 3–4 in standard cages at 24 ± 2 °C under a 12 h light–dark cycle (lights on from 7:00 to 19:00) with ad libitum access to food and water. DGKη-KO mice , and total RNA was isolated with the Direct-zol™ RNA Miniprep . Microarray hybridization  was performed at the TaKaRa Bio . Three experiments using independent cohorts were carried out.21 = 0)) to obtain the fold-change values of all genes. The values are presented as the mean (log2) ± SEM of three independent experiments. Statistical comparisons were performed using Student's t-tests. A cut-off P-value <0.05 and fold-change ≥ absolute 2 (log22 = 1 or log21/2 = −1) were applied.Differential gene expression in DGKη-KO mice was normalized to WT data (WT = 1 (log2.3https://david.ncifcrf.gov/).Functional annotation lists of Gene Ontology: Biological Process (GO:BP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) categories were generated in the Database for Annotation, Visualization and Integrated Discovery -specific oligonucleotide primers in the outlined conditions: Prl, forward primer (5′-ATGACCATGAACAGCCAGGGGT-3′) and reverse primer (5′-TTCCTCAATCTCTTTGGCTCTTGATAGGAT-3′) with PCR conditions of 94 °C for 3 min, then 30 (DGKη-KO) or 35 (WT) cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min, and finally 72 °C for 5 min; Gh, forward primer (5′-ATGGCTACAGACTCTCGGACCTC-3′) and reverse primer (5′-CTGCATCAGAGCCTGGATGCC-3′) with PCR conditions of 94 °C for 3 min, then 34 (DGKη-KO) or 39 (WT) cycles of 94 °C for 30 s, 53 °C for 30 s, and 72 °C for 1 min, and finally 72 °C for 5 min; GAPDH, forward primer (5′-TCGGTGTGAACGGATTTGGCCGTATT-3′) and reverse primer (5′-CATGTAGGCCATGAGGTCCACCAC-3′) with PCR conditions of 94 °C for 3 min, then 35 cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min; and finally 72 °C for 5 min. The amplified PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide .Total RNA isolation, reverse transcription and PCR amplification were performed as previously described . PCR amp2.52) containing cOmplete™ EDTA-free protease inhibitor ) and centrifuged at 1,000 g for 5 min. The tissue lysates (20 μg of protein) were separated on SDS-PAGE (10% acrylamide gel). The separated proteins were transferred to a polyvinylidene difluoride membrane . The membrane was blocked with 5% skim milk and incubated with an anti-ERK1/2 antibody , an anti-phospho-ERK1/2 antibody  or an anti-β-actin polyclonal antibody  overnight at 4 °C. The immunoreactive bands were visualized using a peroxidase-conjugated anti-rabbit IgG antibody  and the ECL Western Blotting Detection System .Western blotting of brain tissues were performed as previously described . The cer2.62 and cOmplete™ EDTA-free protease inhibitor, followed by centrifugation at 1000×g for 5 min at 4 °C. Total lipids were extracted from the mouse brain  according to the method of Bligh and Dyer [2 gas, and the extracted lipids were reconstituted in 100 μl of chloroform/methanol .The cerebral cortexes of mice were homogenized in ice-cold lysis buffer  containing 40 pmol of the 14:0/14:0-PA internal standard  were analyzed by LC-MS using an Accela LC system  equipped with a Unison UK-Silica column  and coupled online to an Exactive Orbitrap MS (Thermo Fisher Scientific) equipped with an electrospray ionization source as described previously . A binar2.8Total lipids were extracted from mouse serum according to the method of Bligh and Dyer . LPAs in2.9® software . A binary gradient consisting of two solvents: solvent A (chloroform/methanol (89:10) containing 0.28% ammonia) and solvent B (chloroform/methanol/water (55:39:5) containing 0.28% ammonia). The gradient elution program was 20% B for 5 min, 20%–30% B for 10 min, 30%–60% B for 25 min, 60% for 5 min, 60%–20% B for 1 min, followed by 20% B for 14 min. The flow rate was 0.3 ml/min, and chromatography was performed at 25 °C.).For the identification of fatty acid residues in PA molecular species by LC-MS/MS. Total lipids were extracted from the cerebral cortexes as described above. The extracted lipids (10 μl) were separated on a LC system  using a Unison UK-Silica column  as described previously . This LCThe LC system was coupled online to Triple Quad™ 4500 (AB SCIEX), a triple-quadrupole tandem mass spectrometer equipped with turbo spray ionization source. The experimental conditions are: ion spray voltage −4500 v, curtain gas 30 psi, collision gas 6 psi, temperature 300 °C, declustering potential −160 v, entrance potential −10 v, collision energy −42 v, collision cell exit potential −11 v, ion source gas I 70 psi and ion source gas II 30 psi. PA molecular species were detected in a multiple reaction monitoring (MRM) mode.2.10t-test or one-way ANOVA followed by Tukey's post hoc test . No sample size calculation was performed. No test for outliners was conducted.The Kolmogorov-Smirnov test was carried out to assess the normality of data. Statistical comparisons were performed using a two-tailed 33.1P-value <0.05 and fold-change ≥ absolute 2 (log2 1 or log2 –1)) in DGKη-KO mice were analyzed. There was a total of 43 (26 up- and 17 downregulated) differently regulated genes in DGKη-KO mice ), we performed a microarray analysis. To obtain a broad overview of differential gene expression, all genes meeting the cut-off values  and Gh (growth hormone), which were the most upregulated (log2 5.4 ± 1.7 (absolute 42.2 ± 3.2) fold, P = 0.031 and log2 5.3 ± 1.8 (absolute 39.4 ± 3.5) fold, P = 0.042, respectively) (Prl and Gh (log2 5.4 ± 1.5 (absolute 42.2 ± 2.8) fold, P = 0.004 and log2 4.7 ± 0.8 (absolute 26.0 ± 1.7) fold, P = 0.001, respectively)  , using R) Suppl. .Fig. 1WePrl [Gh [Il1b (interleukin 1β) [Foxp3 (forkhead box P3), Glp1r (glucagon-like peptide 1 receptor) [Arg1 (arginase) [We analyzed the relationship between BPD and individual genes affected in DGKη-deficient mice . Prl [25Prl [Gh , Il1b (iukin 1β) , Foxp3 (eceptor) ,29 and Arginase) , which w3.2To gain some insight into the biological role of the affected genes found  and how 3.3Glp1r, Gh and Prl, previously implicated with BPD.We next analyzed the relationship between BPD and biological pathways affected in DGKη-KO mice. As mentioned above, all five biological pathways include at least one gene previously implicated in BPD . “NeuroaGh and Prl  receptor 1) and Ces2c (carboxylesterase 2C) (Glp1r (rase 2C)  are glyc) Glp1r , Table 2)  fold, Prl (prolactin), which is an anterior pituitary hormone, was intensively increased by DGKη-KO ((log2 5.3 ± 1.8 (absolute 39.4 ± 3.5) fold, B receptor agonist [B receptor agonists for mania and DGKη-KO.Gh (growth hormone), which is an anterior pituitary hormone, similarly to prolactin, was also strongly increased by DGKη deficiency (log agonist . There mIl1b (interleukin 1β), which was significantly increased in DGKη-KO mouse brains , is comm+ CD25+ FoxP3+) [Foxp3 (forkhead box P3), which was significantly increased in DGKη-KO mouse brains , is list FoxP3+) ,38.The expression of Glp1r (glucagon-like peptide 1 receptor), which is annotated in “positive regulation of transcription by RNA polymerase II”, “positive regulation of cytosolic calcium ion concentration” and “neuroactive ligand-receptor interaction” , was sigArg1 (arginase) was substantially increased in DGKη-KO mouse brains . Plasma There are several BPD-related proteins including GSK3β and Clock. Contrary to DGKη, transgenic mice of GSK3β exhibited mania-like behaviors ,57. As eWe found that the deficiency of DGKη decreased PUFA-containing PA species, 36:3 (18:1/18:2)-, 38:3 (18:0/20:3)-, 40:5 (18:0/22:5)-, 40:4 (18:0/22:4)- and 40:3 (18:0/22:3)-PA, in the mouse cerebral cortex . Thus, tin vivo. Alternatively, there could also be changes in the membrane environment that affects the specificity of DGKη as has recently been found with DGKε [To date, ten DGK isozymes have been identified . We previth DGKε . DG specith DGKε . Therefoith DGKε , DGKη woLC-MS analysis revealed that the PUFA-containing LPA species, 18:2- and 20:5-LPA, were significantly decreased in DGKη-KO mouse serum . ParticuTaken together, our study provides not only key broad knowledge to gain novel insights into the underlying mechanisms of how BPD-related mania-like behaviors were caused by DGKη-KO but also information for developing BPD diagnostics. However, the molecular mechanisms by which DGKη-KO induces mania-like behaviors are not yet fully understood. Further studies will be necessary to explore this issue in more detail.MEXT/JSPS KAKENHI Grant Numbers 26291017 (FS), 15K14470 (FS), 17H03650 (FS), and 16J06865 (SKo); the Futaba Electronic Memorial Foundation (FS); the Ono Medical Research Foundation (FS); the Japan Foundation for Applied Enzymology (FS); the Food Science Institute Foundation (FS); the Skylark Food Science Institute (FS); the Asahi Group Foundation (FS); the Japan Milk Academic Alliance (FS); the Japan Food Chemical Research Foundation (FS) and the SENSHIN Medical Research Foundation (FS).This work was supported in part by grants from S.Ko. designed and conducted the experiments, analyzed the data and wrote the manuscript. Y.S., C.M., F.H., M.A., S.C. and S.Ka. performed the experiments and analyzed the data. D.T. designed the study and revised it critically for important intellectual content. F.S. conceived the study and wrote the manuscript.The authors declare that they have no conflicts of interest with the contents of this article.


Thus, our studies delineate a mechanism connecting signaling related polyubiquitination of Malt1 and Stat3, leading to NF-kB activation and RORγt expression, to pathogenic Th17 cell function in EAE.Polyubiquitination promotes proteasomal degradation, or signaling and localization, of targeted proteins. Here we show that the E3 ubiquitin ligase Hectd3 is necessary for pathogenic Th17 cell generation in experimental autoimmune encephalomyelitis (EAE), a mouse model for human multiple sclerosis. Hectd3-deficient mice have lower EAE severity, reduced Th17 program and inefficient Th17 cell differentiation. However, Stat3, but not RORγt, has decreased polyubiquitination, as well as diminished tyrosine-705 activating phosphorylation. Additionally, non-degradative polyubiquitination of Malt1, critical for NF-κB activation and Th17 cell function, is reduced. Mechanistically, Hectd3 promotes K27-linked and K29-linked polyubiquitin chains on Malt1, and K27-linked polyubiquitin chains on Stat3. Moreover, Stat3 K180 and Malt1 K648 are targeted by Hectd3 for non-degradative polyubiquitination to mediate robust generation of RORγt Ubiquitination may control protein stability or function. Here the authors show that an ubiquitination enzyme, Hectd3, ubiquitinates Stat3 and Malt1 to modulate their function but not degradation in T cells, and thereby promoting the differentiation of pathogenic Th17 cells and susceptibility to a mouse model of multiple sclerosis. Th17 cells have recently come into sharp focus in relation with their role in autoimmunity, including experimental autoimmune encephalomyelitis (EAE)3, multiple sclerosis (MS)5, collagen-induced arthritis6, Crohn’s disease7, and rheumatoid arthritis8. Key cytokines and transcription factors are critical for the differentiation and function of Th17 cells. Following T cell receptor (TCR) stimulation, the transcription factors BATF9 and IRF410 are upregulated and cooperatively pre-pattern the chromatin landscape for Th17 cell specification11. In addition, the cytokines IL-6 and TGF-β are required for initiation of Th17 differentiation12. Specifically, IL-6 signaling engenders phosphorylation and activation of Stat3, which is another key transcription factor in Th17 cell differentiation15. The master transcription factor controlling Th17 cell identity, RORγt, acts synergistically with activated Stat3 to maximize the transcription of Il17a, Il17f, Il23r, and Ccr616. The terminal differentiation and induction of pathogenic Th17 cells requires IL-23R17. IL-23R signals through Stat3, to maintain and stabilize the expression of RORγt, IL-17A, and IL-23R itself17. In addition, in MS and its mouse model EAE19, IL-23R signaling drives the co-expression of the highly pro-inflammatory cytokines GM-CSF and IFNγ through RORγt, activated Stat3, and Blimp-120.T helper 17 (Th17) cells are a distinct subset of CD421. RORγt is additionally regulated by ubiquitination via TRAF5 through K63-linked polyubiquitin chains, which promotes its stability and IL-17A production in in vitro polarized human CD4+ T cells22. Furthermore, the UBR box E3 ligase UBR5 and the deubiquitinase DUBA regulate RORγt-associated colonic inflammation in a cell-intrinsic manner23. In addition to RORγt, Stat3 was found to be ubiquitinated and degraded in Th17 cells by the E3 ubiquitin ligase SLIM/PDLIM2 also in a cell-intrinsic manner24. TRIM21 also influences T cell differentiation by targeting IRF3 to inhibit ex vivo Th1 and Th17 differentiation in CD4+ T cells isolated from inflammatory bowel disease (IBD) patients25. Numerous deubiquitinases have also been shown to influence Th17 cells. USP426, USP1527, and USP1728 promote RORγt activity in in vitro polarized Th17 cells and enhance the recruitment of the co-activator SRC1 or stabilize RORγt via deubiquitination. USP18 promotes Th17 cell differentiation by limiting production of IL-2 and Stat5 activation through deubiquitination of TAK1 in EAE29. However, the E3 ubiquitin ligase mediating TAK1 ubiquitination is unknown. Thus, no E3 ubiquitin ligase has been shown to regulate Th17 cells in a cell-intrinsic manner in vivo through a signaling type of polyubiquitination that does not target its substrate for proteasomal degradation. Given the regulatory roles of E3 ubiquitin ligases, this is of outstanding importance in the context of autoimmunity in EAE and MS.Ubiquitination is known to play a key role in the regulation of Th17 cells. RORγt was shown to be targeted for proteasomal degradation via K48-linked polyubiquitination by the HECT E3 ubiquitin ligase Itch to suppress colonic inflammation30. In addition, Hectd3 was found to ubiquitinate several caspases, regulating survival of cancer cell lines32. Recently, Hectd3 was found to promote type I interferon (IFN) response in bone-marrow-derived macrophages through polyubiquitination of TRAF3, and this correlated with altered antibacterial response33. Malt1, an essential component of the Carma1-Bcl10-Malt1 (CBM) complex34, was found to be essential for Th17 pathogenicity in EAE through promoting nuclear translocation of p65 and inhibiting nuclear translocation of RelB36. Given that Hectd3 was detected in the molecular core regulating Th17 cell differentiation11, we asked the questions whether Hectd3 ubiquitinates Malt1 to regulate Th17 cell differentiation during EAE and whether there are additional Malt1-independent targets by which Hectd3 controls pathogenic Th17 cell differentiation.The HECT E3 ubiquitin ligase Hectd3 was shown to promote non-K48-linked polyubiquitination on Malt1, enhancing its stability in cancer cell linesHectd3−/− mice, which correlates with diminished IL-17A and GM-CSF production, RORγt levels, and reduced phosphorylated (p)Stat3 Y705 in Hectd3−/− CD4+ T cells. Related to this phenotype, Stat3 polyubiquitination is decreased in Hectd3−/− CD4+ T cells, while that of RORγt remains unchanged. In addition, we show that Hectd3 promotes non-degradative K27 polyubiquitination at Stat3 K180, which is essential for robust generation of RORγt+IL-17Ahi Th17 cells. In addition, Malt1 is targeted for non-degradative K27 and K29 polyubiquitination by Hectd3 at K648, which is also essential for RORγt+IL-17Ahi Th17 cell generation. Our study thus demonstrates that non-degradative polyubiquitination of Malt1 and Stat3 by Hectd3, leading to NF-kB activation and RORγt upregulation, respectively, delineates a mechanism connecting ubiquitination to Th17 differentiation and function in the context of autoimmunity and EAE.Here we show that Hectd3 promotes pathogenic Th17 cell differentiation in EAE, in a cell-intrinsic manner, through non-degradative polyubiquitination of Malt1 and Stat3. We find that EAE severity is attenuated in 30 and plays a role in Th17 cell-associated pathogenicity in EAE35. Additionally, Hectd3 was recently detected in the gene network regulating Th17 lineage, as reported by Ciofani et al.11 . We thus investigated Hectd3 expression in T helper subsets and found that Hectd3 protein was expressed in naïve and in vitro polarized CD4+ T cells, including in the Th17 subset  CD4+ T cells in vitro under Th1, Th2, and Treg conditions and showed no differences in the Th1 transcription factor T-bet and cytokine IFNγ, Th2 transcription factor Gata3 and cytokine IL-4, and Treg transcription factor Foxp3, respectively  of EAE Hectd3−/− and WT control mice. The frequencies and absolute numbers of CD4+ T cells, as well as that of IL-17A+GM-CSF+CD4+ T cells, were reduced in the CNS of Hectd3−/− mice  only ubiquitin mutants where all seven lysine residues were mutated to arginine, except the indicated lysine residue, and two-step immunoprecipitation of Flag-tagged Malt1A, a splice variant of Malt1 expressed in activated CD4+ T cells40, we found that the HA-Ub K27 only and HA-Ub K29 only mutants were sufficient for Hectd3-mediated ubiquitination of Malt1A  only ubiquitin mutants and two-step immunoprecipitation of Flag-tagged Stat3, we observed that the HA-Ub K27 only (HA-Ub K27) mutant was sufficient to promote ubiquitination of Stat3 in the presence of Hectd3 at the same level as WT HA-Ub Fig. . Convers+ T cells and further show that they interact in transfected HEK293T cells as well , and TRAF333. We found that Hectd3 mediates K27-linked polyubiquitin chains on Stat3 and identified K180, within the Stat3 coiled-coil domain, as the target for polyubiquitination, which was important for proper Stat3 Y705 phosphorylation and generation of RORγt+IL-17Ahi Th17 cells. One study found that TRAF6-mediated ubiquitination of Stat3 at six lysine residues in the SH2 domain promoted phosphorylation of Stat3 Y705 in mouse embryonic fibroblasts infected with Salmonellatyphimurium51; however, the in vivo significance of this TRAF6-mediated ubiquitination on Stat3 activation remains to be determined. We showed that Stat3 uses its linker region, located between DNA binding domain and SH2 domain, to associate with the Hectd3 DOC domain.Hectd3 did not promote RORγt polyubiquitination, but exerted control over RORγt level through regulating Stat3 activating phosphorylation at Y705, and thus promoting Th17 pathogenicity. We found Stat3 as a target of Hectd3 for non-degradative polyubiquitination in agreement with previous studies showing that Hectd3 mediates types of polyubiquitination not associated with proteasomal degradation but with signaling, controlling substrate activity on caspase-852. Cross talk between ubiquitination and phosphorylation is well characterized in antiviral innate immunity53, but is limited to proximal TCR signaling and CBM complex signal transduction in T cell biology54. We present a mechanism wherein pStat3 Y705 is reduced in the absence of Hectd3 in vivo during EAE, as well as in Stat3 K180R mutant in Th17 cells, thus linking ubiquitination by an E3 ubiquitin ligase to phosphorylation and activation of a critical Th17 transcription factor. Hectd3-Stat3 ubiquitination-phosphorylation interplay may be similar to the ubiquitination of IRF7 by TRAF6 in promoting phosphorylation and activation of IRF7 in innate antiviral pathways55. Stat3 polyubiquitination by Hectd3 at K180 may promote pStat3 Y705 through recruitment of a kinase, protection from a phosphatase, or a novel mechanism.The regulation of Stat3 Y705 phosphorylation and activation by Hectd3-mediated polyubiquitination is similar to the regulation of PLCγ-1 phosphorylation by Cbl-b-mediated ubiquitination. In both cases, the cross talk between ubiquitination and phosphorylation is not associated with proteasomal degradation. However, whereas Hectd3-mediated polyubiquitination of Stat3 promotes phosphorylation and activation of Stat3, Cbl-b ubiquitination of PLCγ-1 inhibits phosphorylation and activation of PLCγ-154. The deubiquitinase DUBA and the E3 UBR5 were shown to negatively regulate Th17 response. Specifically, DUBA deubiquitinates and stabilizes UBR5, then UBR5 ubiquitinates and targets RORγt for proteasomal degradation23. The deubiquitinases USP426 and USP1728 stabilize RORγt level through deubiquitination, preventing its proteasomal degradation. Itch was also shown to polyubiquitinate and target RORγt for proteasomal degradation, thus inhibiting colitis-associated colorectal cancer and colon inflammation21. USP15 was found to deubiquitinate RORγt at K446 to promote co-activator recruitment27. The E3 PDLIM2 was found to induce polyubiquitination and proteasomal degradation of Stat3, inhibiting the Th17 granulomatous response24. A theme emerges from these studies, namely, ubiquitination negatively impacts Th17 function. In contrast, we have demonstrated a positive regulation of Th17 function by the E3 ubiquitin ligase Hectd3. Moreover, Hectd3-mediated polyubiquitination of Stat3 at K180 does not target Stat3 for proteasomal degradation, but rather promotes the activating Y705 phosphorylation to enhance RORγt and IL-23R expression, as well as IL-17A and GM-CSF production. Similarly, polyubiquitination of Malt1 by Hectd3 at K648 does not promote Malt1 degradation, but positively regulates generation of RORγt+IL17hi Th17 cells.Ubiquitination and deubiquitination have been shown to play crucial roles in regulation of T cell immunity31 and caspase 932, respectively. K27-linked polyubiquitination has been shown to be involved in signaling-complex assembly in T cells56 and innate immunity57, and K29-linked polyubiquitin chains mediated by TRIM13 on TRAF6 promotes TLR2 signaling in macrophages58. Along the same line, Hectd3-mediated K27- and K29-linked polyubiquitin chains on Malt1 and K27-linked polyubiquitin chains on Stat3 play a role in promoting Malt1 and Stat3 signaling, respectively. It is known that the C-lobe of HECT domain controls linkage specificity and that linkage-specific chain formation is dependent on ubiquitin residues adjacent to particular acceptor lysines60. Hectd3 was found to bind both Malt130 and Stat3 through its DOC domain. Malt1 interacts with Hectd3 through N-terminal DD30 and Stat3 interacts with Hectd3 through a linker domain, between the DNA-binding domain and SH2 domain. The targeted lysine by Hectd3 in Malt1, K648, is at the C-terminus, while in Stat3, K180, is located in the coiled-coil domain. Steric hindrance between Hectd3 domains and Malt1, Stat3, and other substrate domains may play a role in determining the linkage specificity of Hectd3-mediated polyubiquitination on a particular substrate. Malt1 also uses DD to associate with BCL10 in the formation of CBM complex49, thus raising the possibility that Hectd3 competes with BCL10 for Malt1 DD, and this competition may affect Hectd3 linkage specificity.We have characterized that Hectd3 mediates K27- and K29-linked polyubiquitin chains on Malt1 and K27-linked polyubiquitin chains on Stat3. Previous studies have shown that Hectd3 mediates K63-linked and K27/29-linked polyubiquitin chains on caspase 8+IL-17Ahi cells. It is unknown if Malt1 and Stat3 are in a common ubiquitination signalosome with Hectd3, but mass spectrometry analysis of Flag-Malt1A and Flag-Stat3 in HEK293T cells did not yield Stat3 peptides in the Flag-Malt1A, or Malt1 peptides in the Flag-Stat3 immunoprecipitates. A recent mouse lung cancer model study found that Malt1 controls Stat3 activation in vivo downstream of EGFR through NF-κB-dependent autocrine/paracrine IL-6 production61, raising the possibility that the Malt1 pathway may intersect the Stat3 pathway in Th17 cells. Exploiting the ubiquitination system for therapeutic intervention is gaining prominence due to the specificity conferred by E3 ubiquitin ligases and the benefit of modulating molecular switches in favoring beneficial outcome62, as well as the possibility of designing selective E3 inhibitors63. Therefore, Hectd3 may be a favorable target for MS, other Th17-related diseases such as psoriasis, or cancer therapy since Stat3 activation is instrumental in their pathogenesis.In the context of Th17 cells, the Malt1 and Stat3 pathways have not been previously shown to intersect. We have identified a common post-translational mechanism, namely, non-degradative ubiquitination mediated by Hectd3, that controls both Malt1 and Stat3 pathways in Th17 cells. Abolition of polyubiquitination at Malt1A K648 or Stat3 K180 was independently sufficient to reduce IL-17A production by Th17 cells, which suggests that Hectd3-mediated polybubiquitination of Malt1 and Stat3 are independent from each other and both are non-redundant in the generation of RORγtHectd3 on a segregating 129/SvEv-C57BL/6 background were obtained from Taconic Biosciences  (Cat#TF2706), and backcrossed to C57BL/6NTac (Taconic Biosciences) for at least ten generations (Hectd3−/−). All experiments were performed using 10–20-week-old male and female mice. However, only female mice were used for experiments requiring EAE disease scoring64. Hectd3−/− mice are born in normal Mendelian ratios, retain fecundity, and are healthy. Malt1−/− mice were obtained from Dr. Robert Rickert  with the approval of Genentech 65. B6.129S1-Stat3tm1Xyfu/J (Stat3flox) mice were purchased from Jackson Laboratory . Stat3f/f mice were crossed with Tg(Cd4-cre)1Cwi/BfluJ(CD4-cre) mice  to generate Stat3 conditional knockout mice. All mice were housed under SPF conditions in the Animal Care Service facilities of the University of Florida College of Medicine (UFCOM), on 12-h light and 12-h dark cycles and 25±2 °C. All protocols were approved by the University of Florida Institutional Animal Care and Use Committee (UF IACUC).Mice with a targeted deletion of + T cells  from dLNs during EAE or CD4+ T , CD8+ T , and CD11b+ myeloid  cells from spleen and peripheral lymph nodes at steady state were purified following manufacturer’s instructions, after mashing through a 40-μm nylon filter . Isolated CD4+ T cells were stimulated at 37 °C and 5% CO2 for 30 min in 10% RPMI containing PMA (20 ng/ml) and Ionomycin (1 µg/ml) for Malt1 and Stat3 ubiquitination analysis.Primary mouse CD4+ T cells were purified from steady state Hectd3−/−, Malt1−/−, and WT mice as above. Anti-CD3  was diluted in PBS to 5 μg/ml and plated on flat-bottom 96-well plates  at 4 °C overnight to bind anti-CD3 to plate. Purified CD4+ T cells at 2 × 106 cells/ml were cultured in wells of anti-CD3 plates with the following antibodies and cytokines for each T helper subset polarizing condition in 10% RPMI: Th0, 2 μg/ml anti-CD28 , 100 U/ml IL-2 ; Th1, 2 μg/ml anti-CD28, 100 U/ml IL-2, 10 μg/ml anti-IL-4 , 10 ng/ml IL-12 ; Th2, 2 μg/ml anti-CD28, 100 U/ml IL-2, 10 μg/ml anti-IFNγ , 10 μg/ml anti-IL-12 , 10 ng/ml IL-4 ; Th17, 2 μg/ml anti-CD28, 10 μg/ml anti-IFNγ, 10 μg/ml anti-IL-12, 10 μg/ml anti-IL-4 , 5 ng/ml TGF-β3 , 10 ng/ml IL-6 , 10 ng/ml IL-1β , 20 ng/ml IL-21 , 50 ng/ml IL-23 ; Treg, 2 μg/ml anti-CD28, 100 U/ml IL-2, 10 μg/ml anti-IFNγ, 20 ng/ml TGF-β1 . Cells were polarized at 37 °C, 5% CO2 for 3 days, then fresh medium with polarizing cytokines and antibodies was added, and cells were polarized at 37 °C, 5% CO2 for 3 additional days. In vitro polarized CD4+ T cells were then stimulated with 20 ng/ml PMA and 1 µg/ml ionomycin for 4 h with 10 µg/ml Brefeldin A added 1 h into the 4-h stimulation. Following PMA/ionomycin stimulation, cells were surface stained, permeabilized, then stained for intranuclear transcription factors and intracellular cytokines66.CD435–55 peptide with amino acid sequence MEVGWYRSPFSRVVHLYRNGK was purchased from Mimotopes  and resuspended in 0.09% saline solution at a concentration of 2 mg/ml. Fresh complete Freund’s adjuvant (CFA) was reconstituted with 4 mg/ml non-viable, desiccated Mycobacterium tuberculosis H37 Ra  in incomplete Freund’s adjuvant . Pertussis toxin from Bordetella pertussis in glycerol  was freshly diluted in PBS to 3 μg/ml. MOG35–55/CFA emulsion was freshly reconstituted by mixing equal volume of 2 mg/ml MOG35–55 with CFA and vortexed for 30 min at room temperature. For each mouse, a total of 200 μl MOG35–55/CFA emulsion was subcutaneously injected at flank and scruff sites; 100 μl of 3 μg/ml pertussis toxin was administered intraperitoneally 1 h and 24 h following MOG35–55/CFA emulsion. Clinical scoring was performed starting 7 days following active EAE induction, every day until the completion of the experiment. Clinical scoring was established as follows: score 0.5: weakness of tail; score 1: flaccid tail; score 1.5: flaccid tail and hind limb inhibition marked by hind limbs falling through cage bars; score 2: flaccid tail, weak hind limbs marked by hind limbs held close together when picked up at the base of the tail; score 2.5: flaccid tail, both hind limbs are dragging when moving or complete paralysis of one hind limb but not the other; score 3: flaccid tail, hind limb paraparesis; score 3.5: flaccid tail, hind limb paraparesis and one forelimb paralysis; score 4: flaccid tail, quadriplegia; score 5: moribund, euthanasia.MOG35–55 peptide , 10 µg/ml anti-mouse IFNγ , 20 ng/ml recombinant mouse IL-23  at 3 million cells/ml for 72 h at 37 °C, 5% CO2, for in vitro reactivation. In vitro reactivated mouse CD4+ T cells were purified as above, and 20 million in vitro reactivated CD4+ T cells were transferred into recipient mice intraperitoneally. Clinical scoring of recipient mice was performed starting 5 days following adoptive/passive transfer of in vitro reactivated CD4+ T cells, every day until the completion of the experiment.Donor mice were induced with active EAE as described above, but without pertussis toxin. Eleven days after active EAE induction, dLN cells were collected, RBC lysed, and incubated in 10% RPMI containing 10% FBS  with 20 µg/ml MOG66. Mice were euthanized with CO2, hepatic portal vein was severed, and left ventricle of heart was perfused with at least 10 ml of ice-cold PBS. Perfused brain and spinal cord were dissected out and placed in gentleMACS C tubes  containing 3 ml of RPMI-1640 supplemented with 1% l-glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 1% penicillin/streptomycin, 0.01 M HEPES, 220 μM 2-mercaptoethanol, and 1% FBS (1% RPMI). Brain and spinal cord tissue were then dissociated using gentle MACS Dissociator  setting m_brain_03.01, m_brain_02_02, and m_brain_01_02 for C tube. Dissociated brain and spinal cord were pressed through 70-μm nylon mesh  with 1% RPMI up to a volume of 7 ml total; 100% isotonic Percoll was prepared by mixing 46.25 ml Percoll  with 3.6 ml 10× HBSS , and 0.6 ml 7.5% sodium bicarbonate. 100% isotonic Percoll was diluted to 70% isotonic Percoll with 1% RPMI; 3 ml of 100% isotonic Percoll was added to the 7 ml brain and spinal cord homogenate for a final 30% Percoll brain and spinal cord homogenate. The 10 ml 30% Percoll brain and spinal cord homogenate was then underlaid with 2 ml 70% isotonic Percoll using a glass Pasteur pipette. The 30–70% layered Percoll was centrifuged for 30 min, 500 × g at room temperature without interruption. The 30/70% interphase was collected with a transfer pipette following centrifugation, and washed with at least threefold volume of 1% RPMI, then centrifuged for 10 min at 350×g, 4 °C for collecting leukocytes.Leukocytes were isolated from brain and spinal cord2 for 4 h in IMDM media containing PMA (20 ng/ml) and Ionomycin (1 µg/ml). Brefeldin A (10 µg/ml) was added after 1 h. After incubation, the cells were washed and stained with Fixable Viability Dye (Affymetrix) and surface markers. Cells were then fixed and permeabilized with Foxp3 Fix/Perm kit , then subsequently stained for cytokines.For detection of cytokines by intracellular flow cytometry, cells were cultured at 37 °C and 5% COFlow cytometry was performed on a BD LSR II, upgraded by Cytek Biosciences, with data acquired using BD FACS DIVA software. All data were analyzed using FlowJo (TreeStar).Cells were stained with the following antibodies: 0.1 µl/100 μl Fixable Viability Dye , 0.25 µg/100 μl CD4 , 0.5 µg/100 μl RORγt , 0.4 µg/100 μl T-bet , 0.5 µg/100 μl IL-17A , 0.25 µg/100 μl GM-CSF , 0.25 µg/100 μl IFNγ , 0.5 µg/100 μl CD25 , 0.06 µg/100 μl Foxp3 , 0.5 µg/100 μl CD8α , 5 µg/100 μl IL-23R , 0.5 µg/100 μl CCR6 , 1 µg/100 μl CD29 , 2 µl/100 μl Malt1 , 5 µg/ml Alexa Fluor 488 goat anti-rabbit IgG (H + L) , 2 µl/100 μl pStat3 Y705 , 0.25 µg/100 μl CD44 , 0.5 µg/100 μl CD62L , 0.5 µg/100 μl CD69 , 0.25 µg/100 μl Ki-67 , 1 µl/100 μl NF-κB p65 , 1 µl/100 μl RelB , 5 µg/ml GFP , 0.125 µg/100 μl CD90.1 .Spleen and lymph nodes were pressed through 40-μm nylon mesh . Cells were surface stained with anti-B220 antibodies  and sorted using BD FACSAriaIII .+ T cells isolated from WT or Hectd3−/− mice induced with EAE were stimulated with PMA (20 ng/ml) and Ionomycin (1 µg/ml) for 60 min. Following stimulation, cells were collected in 0.1% BSA/PBS, centrifuged at 4 °C, 400 × g, 8 min, then blocked with 0.1% BSA/PBS/2.4G2. Cells were stained with surface CD4 and intracellular p65, RelB, and DAPI markers. Cells were stained with CD4 for 20 min on ice, then washed twice with 0.1% BSA/PBS. Cells were then fixed with 1% PFA for 30 min at room temperature, then washed with 0.1% BSA/PBS, collected at 500 × g for 5 min at 4 °C. Cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature, collected at 500 × g for 5 min at 4 °C, then blocked with 0.1% Triton X-100 in PBS with 2.4G2 blocking buffer. This was followed by intracellular staining with anti-p65 (1:100) or -RelB (1:100) antibodies, followed by incubation at room temperature for 25 min. Cells were washed with 0.1% BSA/PBS, then stained with Alexa Fluor 488 goat anti-rabbit IgG (H + L) (1:1000) for 20 min. at room temperature. Cells were washed with 0.1% BSA/PBS, then fixed with 1% PFA/PBS for 10 min at room temperature and collected at 500 × g for 5 min at 4 °C. Cells were resuspended in 1% PFA/PBS with 0.2 µg/ml DAPI and incubated on ice for 5 min, followed by image acquisition immediately. Imaging flow cytometry was performed using an Amnis ImageStreamX MKII imaging flow cytometer and data were analyzed using IDEAS 6.1 software (Amnis).CD4l-glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 1% penicillin/streptomycin, 0.01 M HEPES, 55 μM 2-mercaptoethanol, and 10% FBS (10% DMEM). Platinum-E (Plat-E) retroviral packaging cells  were grown in 10% DMEM. EL4 cells  or male and female primary CD4+ T cells, isolated as above, were cultured in RPMI-1640 supplemented with 1% l-glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 1% penicillin/streptomycin, 0.01 M HEPES, 220 μM 2-mercaptoethanol, and 10% FBS (10% RPMI). Generation of MALT1KO Jurkat cells was performed as described40. All mammalian cell cultures were grown at 37 °C and 5% CO2. Escherichia coli strain MAX Efficiency DH5α competent cells  were used for plasmid transformation and preparation. Transformed E. coli DH5α were grown at 37 °C and 230 rpm shaking.HEK293T cells , were grown in DMEM supplemented with 1% 40. Mouse Stat3 was cloned into MSCV . PCR product was obtained using Premix Taq DNA Polymerase (Ex Taq Version 2.0) according to manufacturer’s protocol  with the Hectd3 primers listed in Supplementary Table All mutants were generated using Q5 site-directed mutagenesis kit  following manufacturer’s protocol or standard cloning. All primers used are listed in Supplementary Table 67. Plat E cells were seeded on 10-cm cell cultures plates such that 80% confluence was reached overnight. Media was aspirated off Plat E cell culture and 5 ml of fresh 10% DMEM was added; 9 μg MSCV-MALT1A or MSCV-Stat3 or 9 μg MSCV-MALT1A K648R or MSCV-Stat3 K180R vectors were added into serum-free DMEM along with 4.5 μg pVPack-VSV-G and 9 μg pVPack-GP to a total volume of 450 μl, then incubated at room temperature for 5 min. Lipofectamine 2000 (36 μl) was added to 414 μl of serum-free DMEM and incubated at room temperature for 5 min. The Lipofectamine solution was added to the DNA vectors tube, mixed by flicking, then incubated at room temperature for 20 min. The DNA-Lipofectamine 2000 complex with a total volume of 900 μl was added dropwise to Plat E cells. The transfected Plat E cells were incubated overnight at 37 °C, 5% CO2. The medium was aspirated from transfected Plat E cells; then 5.5 ml of pre-warmed (37 °C) 10% RPMI was added to the edge of the plate slowly, then the transfected Plat E cells in 10% RPMI were incubated overnight at 37 °C, 5% CO2. Retroviral particles in 10% RPMI media were collected and filtered through a 0.45-μm syringe filter  and collected at 48 h and 72 h post-transfection. Retrovirus-containing 10% RPMI medium was stored at −80 °C until use for reconstitution assay.Retroviral particles were produced as followsMalt1−/− or Stat3f/fCD4-cre CD4+ T cells were isolated following manufacturer’s protocol . Primary mouse CD4+ T cells were transduced as follows70. Following activation with anti-CD3/anti-CD28 antibody-bounded beads  at 2 × 106 cells/ml of 10% RPMI supplemented with 20 U/ml IL-2, and incubation at 37 °C, 5% CO2 overnight, cells were transduced on retronectin-coated wells. Retronectin  in PBS was used to coat 24-well cell culture plates for 3 h at room temperature, just before transduction, and then aspirated and wells were washed with 0.5% FBS in PBS. Retrovirus-containing 10% RPMI (250 µl) was added to each well of a 24-well plate, and activated primary mouse CD4+ T cells were added to the retrovirus-containing medium immediately. The 24-well plate was then centrifuged at 2000 × g at 30 °C for 1 h, then incubated at 37 °C and 5% CO2 for 24 h. The supernatant (250 μl) was then carefully aspirated off each well, then 250 μl of freshly thawed retrovirus-containing 10% RPMI was added to each well. For a second time, the 24-well plate was then centrifuged at 2000 × g at 30 °C for 1 h, then incubated at 37 °C, 5% CO2 for an additional 24 h. The retrovirus-transduced CD4+ T cells were then subjected to in vitro Th17 polarizing conditions as described above. Th17 in vitro polarized retrovirus-transduced CD4+ T cells were then stimulated with 20 ng/ml PMA and 1 µg/ml ionomycin for 4 h with 10 µg/ml Brefeldin A added 1 h before harvesting cells. Cells were then surface stained, permeabilized, then stained for intranuclear transcription factors and intracellular cytokines.Primary mouse spleen and peripheral lymph node WT or Retrovirus-containing 10% RPMI (250 µl) along with 8 µg/ml Polybrene was added to each well of a 24-well plate, and EL4 or MALT1KO Jurkat cells were added to the retrovirus-containing medium immediately.40. For reconstitution, cDNAs of MALT1 isoform A WT and mutant K648R linked to a C-terminal Flag-Strep-Strep tag were cloned into a pHAGE lentiviral expression vector in frame with hΔCD2 and the co-translational processing site T2A. For lentivirus production, HEK293T cells were transfected with 1.5 µg of psPAX2 , 1 µg of pMD2.G , and 2 µg of pHAGE transfer vector. Lentivirus particles containing MALT1 were collected 72 h after transfection, filtrated and added to MALT1KO Jurkat T cells in the presence of polybrene (8 µg/ml). After 24 h, the medium containing viral particles was replaced by viral particle-free RPMI medium. Culture of cells for 1 week revealed >95% ΔCD2-positive cells by FACS analysis using an Attune Flow Cytometer (Applied Biosystems).Generation of MALT1-deficient Jurkat T cells has been describedTransfection of HEK293T cells was performed using Lipofectamine 2000  according to manufacturer’s protocol. HEK293T cells were seeded on either 6-well or 10-cm plates overnight at 70% confluence. Transfection of HEK293T cells seeded on 6-well plates was performed as follows: (1) a total of 4.5 μg of plasmid DNA was diluted with serum-free DMEM up to 50 μl volume per well and incubated at room temperature for 5 min; (2) 10 μl of Lipofectamine 2000 was added to 40 μl serum-free DMEM for a total of 50 μl volume per well and incubated at room temperature for 5 min; (3) the diluted Lipofectamine 2000 solution was then added to the diluted DNA tube, flicked for thorough mixing, and incubated at room temperature for 20 min; (4) the DNA-Lipofectamine 2000 complex was then added to each well dropwise and evenly. Transfection of HEK293T cells seeded on 10-cm plates was performed as follows: (1) a total of 24 μg of plasmid DNA was diluted with serum-free DMEM up to 250 μl volume per well and incubated at room temperature for 5 min; (2) 60 μl of Lipofectamine 2000 was added to 190 μl of serum-free DMEM for a total of 250 μl volume per well and incubated at room temperature for 5 min; (3) the diluted Lipofectamine 2000 solution was then added to the diluted DNA tube, flicked for thorough mixing, and incubated at room temperature for 20 min; (4) the DNA-Lipofectamine 2000 complex was then added to each 10-cm plate dropwise and evenly.MG132 (20 μM) or cycloheximide (15 μg/ml) was added 4 h prior to protein extraction in experiments examining Stat3 ubiquitination and protein stability in the presence of Hectd3.70. P8340 protease inhibitor cocktail , cOmplete EDTA-free protease inhibitor cocktail , 1 mM NaF phosphatase inhibitor, 1 mM Na3VO4·2H2O phosphatase inhibitor, 1 mM β-glycerophosphate phosphatase inhibitor, and PR-619 deubiquitinase inhibitor  were added to cytoplasmic protein extraction buffer BN  and nuclear protein extraction buffer NEB . Primary mouse CD4+ T cells, CD8+ T cells, B220+ B cells, and CD11b+ cells or transfected HEK293T cells were vortexed in BN buffer, incubated on ice for 5 min, centrifuged at 3500 rpm at 4 °C, and then the supernatants were collected as cytoplasmic protein extract. The pellets were then vortexed in NEB buffer, incubated at 4 °C with rotation for 30 min, centrifuged at 13,000 rpm for 15 min at 4 °C, and then the supernatants were collected as nuclear protein extract. Cytoplasmic and nuclear protein extracts were combined as total protein for enrichment of ubiquitinated protein, immunoprecipitation, and immunoblot analysis.Protein extraction of cells was performed as follows40. For cellular analyses, 2–3 × 106 Jurkat T cells were lysed in co-immunoprecipitation (co-IP) buffer .For Malt1 substrate cleavage and CBM complex formation experiments, the cell lysis methods were as followsThe concentration of the protein samples was determined using DC Protein Assay  according to the manufacturer’s protocol.+ T cells was enriched for ubiquitinated protein with Ubiquitinated Protein Enrichment Kit  or TUBE2 Agarose , following the manufacturer’s protocol.At least 700 μg of total protein from mouse primary CD470. At least 700 μg of total protein was used for immunoprecipitation. Protein A Sepharose (PAS) CL-4B  was washed ten times with TD150 buffer  and resuspended in equal volume of TD150 to obtain 50% PAS. A total of 100 μl of 50% PAS was needed for each sample. For pre-clearing of each sample, 50 μl of 50% PAS was added to each total protein sample, rotated at 4 °C for 1 h, centrifuged at 5000 rpm for 5 min at 4 °C, and then the supernatant was collected as pre-cleared samples. A total of 2 μg of Anti-Flag M2 antibody  or Anti-Xpress antibody  or Stat3 (124H6)  or Malt1  was added to pre-cleared samples, and then incubated at 4 °C with rotation for 30 min. A total of 40 μl of 50% PAS was then added to antibody-pre-cleared sample mix and incubated at 4 °C with rotation from 4 h to overnight. Immunoprecipitated protein samples were centrifuged at 5000 rpm for 5 min at 4 °C, washed with TD150 three times, then denatured using 4× Laemmli Sample Buffer  with 10% 2-mercaptoethanol and boiled for 5 min. Denatured samples were subjected to immunoblot analysis.Immunoprecipitation were performed as followsTwo-step immunoprecipitation and ubiquitination assays were performed as the following. For the first-round immunoprecipitation, protein extracts were prepared by using Lysis buffer  supplemented with cOmplete EDTA-free protease inhibitor cocktail . Lysates were incubated with the anti-Flag (M2)-agarose affinity gel  for 2 h. The immunoprecipitates were washed three times with Lysis buffer. For the second-round immunoprecipitation, the immunoprecipitates were denatured by boiling for 5 min in the Lysis buffer containing 1% SDS. The elutes were diluted 1:10 with Lysis buffer. The diluted elutes were re-immunoprecipitated with the anti-Flag (M2)-agarose affinity gel. After extensive wash, the immunoprecipitates were subjected to immunoblot analysis.40. CBM complex formation was monitored by StrepTactin pulldown (ST-PD): 2 × 107 cells were lysed in co-IP buffer, lysate controls taken, mixed with 4× SDS-loading buffer and boiled for 5 min at 95 °C. StrepII-tagged proteins were pulled down with StrepTactin Sepharose beads (15 µl 1:1 suspension) at 4 °C overnight. StrepTactin beads were washed with co-IP buffer, 22 µl 2× SDS-loading buffer added, and boiled for 8 min at 95 °C. Lysates and precipitated proteins were separated by SDS polyacrylamide gel electrophoresis and analyzed by western blot.For CBM complex analysis, the following precipitation was used as followsTM West Femto Maximum Sensitivity Substrate  using a ChemiDoc Touch Gel Imaging System . Immunoblot images were analyzed with Image Lab software .Denatured protein samples were subjected to gel electrophoresis by loading into 7.5%  or gradient gel . Gel electrophoresis was performed at 100 V for 10 min, and then at 200 V for at least 40 min. Proteins were then transferred to 0.2 μm PVDF membrane  using the Trans-Blot Turbo Transfer System (Cat#1704150). PVDF membrane with transferred protein was washed with 1× TBST for 5 min at room temperature and shaking, blocked with 5% skim milk in 1× TBST for 30 min at room temperature and shaking, then washed with 1× TBST for 5 min at room temperature and shaking. The following antibodies for immunoblot were diluted in 4% BSA-TBST as indicated: 1:1000 Anti-Flag M2 antibody ; 1:2500 Anti-Xpress antibody ; 1:2000 Anti-HA (12CA5) antibody ; 1:3000 GAPDH (14C10) ; 1:2000 Stat3 (79D7) ; 1:2000 Phospho-Stat3 (Tyr705) (D3A7) ; 1:1000 Malt1 ; 1:1000 Hectd3 ; 1:1000 ubiquitin (P4D1) ; 5 μg/ml RORγt (AFKJS-9) . Diluted antibodies were added as appropriate to the blocked blot and incubated at 4 °C with tilting rotation overnight. Immunoblots were washed with 1× TBST three times for 5 min at room temperature and shaking, and then blocked with 5% skim milk in 1× TBST for 10 min at room temperature and shaking. The following secondary antibodies for immunoblot were diluted 1:5000 in 5% skim milk-TBST: Peroxidase AffiniPure Goat Anti-Mouse IgG (H + L) ; Peroxidase AffiniPure Goat Anti-Rabbit IgG (H + L) ; Peroxidase AffiniPure Goat Anti-Rat IgG (H + L) . Diluted secondary HRP-conjugated antibodies were added to appropriate immunoblots, and incubated at room temperature for 1 h with shaking. Immunoblot were washed once with 1× TBST, then twice with 1× TBS. Immunoblots were visualized with Lumi-Light Western Blotting Substrate  and SuperSignalFor Malt1 KO Jurkat reconstitution experiments, the following protocol was used. For αCD3/CD28 stimulation of Jurkat T cells, the following antibodies were used: mouse anti-human CD3 (1.0 µg/ml), mouse anti-human CD28 (3.3 µg/ml), rat anti-mouse IgG1 (1.7 µg/ml), and rat anti-mouse IgG2a (1.7 µg/ml) . For western blot and FACS studies, the following antibodies were used (dilution 1:1000 except when stated otherwise): anti-CARD11 (ID12) ; anti-BCL10 (H-197), anti-HOIL-1 (H-1), anti-MALT1 (B12), anti-β-Actin , anti-CYLD (E-10) ; anti-hCD2-APC ; horseradish peroxidase (HRP)-conjugated secondary antibodies . For immunodetection, the proteins were transferred onto PVDF membranes using an electrophoretic semi-dry blotting system. Prior to primary antibody addition, membranes were blocked with 5% BSA or milk in PBS-T (0.1% Tween) for 1 h at RT. Membranes were incubated with primary antibody overnight at 4 °C as indicated above in 2.5% BSA or milk in PBS-T. For detection of primary antibodies, HRP-coupled secondary antibodies (in 1.25% BSA or milk in PBS-T) were applied to the membranes for 1 h at room temperature, and HRP visualized by enhanced chemiluminescence (ECL) with LumiGlo reagent  according to the manufacturer’s protocol.g for 1 min at 4 °C, and then washed three times with 1× TBS. Immunoprecipitated samples were eluted with 0.1 M glycine HCl, pH 3.5, following the manufacturer’s protocol .Protein extracts from HEK293T cells co-transfected with HA-Ub and Flag-Stat3 vectors, or HA-Ub, Flag-Stat3 and Xpress-Hectd3 vectors, or HA-Ub and Flag-Malt1A vectors, or HA-Ub, Flag-Malt1A, and Xpress-Hectd3 vectors were subjected to immunoprecipitation using ANTI-FLAG M2 Affinity Gel  according to the manufacturer’s protocol. Anti-FLAG Affinity Gel was washed with 1× TBS ten times and then added to protein samples, and the protein sample-Anti-FLAG Affinity Gel suspension was incubated at 4 °C with rotation for 4 h. The immunoprecipitated samples were centrifuged at 8200×4HCO3 at 25 °C for 30 min, followed by 5 mM iodoacetamide (IAA) at 25 °C for 30 min in the dark. Protein was digested with 1:100 (w/w) lysyl endopeptidase (Wako) at 25 °C for 2 h and further digested overnight with 1:50 (w/w) trypsin (Promega) at 25 °C. Resulting peptides were desalted with a Sep-Pak C18 column (Waters) and dried under vacuum.Samples were treated with 1 mM dithiothreitol (DTT) in 40 mM NHm/z range, 200,000 AGC, 50 ms maximum ion time) were collected at a resolution of 120,000 at m/z 200 in profile mode and the HCD MS/MS spectra  were detected in the ion trap. Dynamic exclusion was set to exclude previous sequenced precursor ions for 30 s within a 10-ppm window. Precursor ions with +1 and +8 or higher charge states were excluded from sequencing.Derived peptides were resuspended in 10 µl of loading buffer . Peptide mixtures (2 µl) were separated on a self-packed C18  fused silica column ; New Objective, Woburn, MA) by a Dionex Ultimate 3000 RSLCNano and monitored on a Fusion mass spectrometer . Elution was performed over a 120-min gradient at a rate of 300 nl/min with buffer B ranging from 3 to 80% . The mass spectrometer cycle was programmed to collect at the top speed for 3-s cycles. The MS scans . Searching parameters included fully tryptic restriction and a parent ion mass tolerance (±20 ppm). Methionine oxidation (+15.99492 Da), asparagine and glutamine deamidation (+0.98402 Da), lysine ubiquitination (+114.04293 Da), and protein N-terminal acetylation (+42.03670) were variable modifications ; cysteine was assigned a fixed carbamidomethyl modification (+57.021465 Da). Percolator was used to filter the peptide spectrum matches to a false discovery rate of 1%. In-house software was used to compile the data across all project runs. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD011756.t test assuming unequal variance and p < 0.05 was considered significant. For comparison of EAE clinical scores, Mann–Whitney two-tailed test was performed and p < 0.05 was considered significant.Measurements were taken from distinct samples in the case of cells in culture and from individual mice in case of mice. Differences between groups were determined by a two-tailed Student’s Further information on experimental design is available in the Supplementary InformationReporting SummarySource Data