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Optineurin modulates the maturation of dendritic cells to regulate autoimmunity through JAK2-STAT3 signaling - Nature Communications

Results .OPTN is required for DC maturation . To explore the expression pattern of OPTN during DC maturation or activation, human monocyte-derived dendritic cells (MoDCs) were stimulated with bacterial lipopolysaccharide (LPS), a toll-like receptor 4 (TLR4) agonist, for maturation14. Array analysis showed that Optn gene expression was significantly increased in mature MoDCs, similar as the dramatically upregulated DC maturation-related genes, such as CD40 , TNF , IL-1B , CXCL9 , and CXCL10 (Fig.? 1a ). Next, we cultured murine bone marrow-derived dendritic cells (BMDCs) by GM-CSF (50?ng/mL) and interleukin (IL)-4 (20?ng/mL), and then acquired mature BMDCs by LPS stimulation. Be different from the common GM-CSF (20?ng/ml) and IL-4 (5?ng/ml) system that comprised both of DCs and macrophages15, our system acquired quite a low number of CD11c + MHC-II - CD11b high cells and almost no CD115 + macrophages (Supplementary Fig.? 1 ), thus representing an effective BMDCs culture system in vitro. As expected, qRT-PCR verified the elevated expression of Optn in mature BMDCs (Fig.? 1b ). Immunostaining and western blotting analysis also indicated that OPTN was accumulated in CD11c + human MoDCs and BMDCs after LPS or Polyl:C treatment (Fig.? 1c ), suggesting that OPTN may be involved in the regulation of DC maturation or activation.Fig. 1: OPTN expression is required for DC maturation.a Heatmap of the indicated gene expression levels in human MoDCs stimulated with LPS for 8?h or unstimulated from GEO dataset (GSE2706). b qRT-PCR analysis of selected gene expressions in BMDCs stimulated with LPS for 8?h. Red line, relative fold change =2. n ?=?3 independent experiments, P( Nck1 ) ?=?0.0046, P( Sqstm1 ) ?=?0.0015, P( Optn ) ?=?0.0115, P( Tank ) ?=?0.0041, P( Scimp ) ?=?0.0056, P( Sarm1 ) ?=?0.0029, P( Pde4d ) ?=?0.0026, P( Cd40 ) ?=?1.9E?05, P( Tnf ) ?=?0.0022, P( Il-1b ) ?=?0.0056, P( Cxcl9 ) ?=?0.0132, P( Cxcl10 ) ?=?0.0064. c Immunostaining of OPTN (red) and CD11c (green) in LPS treated or control human MoDCs and western blotting for OPTN in BMDCs stimulated with LPS or poly I:C for 8?h. Arrows indicate colabeled cells. Scale bar, 40?μm. n ?=?3 independent experiments. d qRT-PCR and western blotting to verify the deficiency of OPTN in DCs. n ?=?3 independent experiments, P ?=?1.0E?11. e Flow cytometry analysis of the frequency of total DCs (CD11c + ) in the bone marrow and spleen of Ctrl and OptnΔCD11c mice. n ?=?3 independent animals, P(Spleen) ?=?0.3289, P(Marrow) ?=?0.5035. f, g BMDCs from Ctrl and OptnΔCD11c mice were stimulated without ( f ) or with LPS ( g ). Expression levels of CD80, CD86, MHC-I, and MHC-II were analyzed by flow cytometry. n ?=?3 independent experiments, P(CD80) ?=?0.2275, P(CD86) ?=?0.1680 in ( f ); P(CD80) ?=?0.0034, P(CD86) ?=?0.0015, P(MHC-I) ?=?0.0017, P(MHC-II) ?=?6.9E?05 in ( g ). Data are presented as means?±?SD. P ?t -test. Source data are provided in Source data file. Full size image To understand the biological role of OPTN in DC maturation, we generated CD11c + cells conditional Optn knockout mice ( Optnfl/fl ; CD11c -Cre mice, hereafter called OptnΔCD11c mice) and confirmed the total deletion of OPTN in BMDCs by qRT-PCR and western blotting analysis (Fig.? 1d ). The number of CD11c + DCs from bone marrow and spleen were comparable between OptnΔCD11c mice and control mice (Fig.? 1e ), and the expression levels of classic co-stimulatory molecules CD80 and CD86 were also changed faintly in Optn deficient BMDCs (Fig.? 1f ), suggesting that OPTN was dispensable for the generation of DCs. Interestingly, the phenotypic maturation of LPS challenged Optn deficient BMDCs was inhibited obviously compared to that of controls (Fig.? 1g ). These data indicate that Optn deficiency blocks DC maturation.OPTN regulates the T cells priming function of DC . It is well known that mature DCs can secrete anti-inflammatory cytokines to complete immunogenicity. Our results showed that Optn deficient BMDCs secreted more anti-inflammatory cytokines like IL-10 and transforming growth factor (TGF)-β, although failed to inhibit the production of pro-inflammatory cytokines like IL-6 and IL-12 (Fig.? 2a ). Encouraged by these results, we further found that cDCs were decreased, while pDCs were increased in OptnΔCD11c mice (Fig.? 2b ). Meanwhile, the percentages of peripheral tissue-derived migratory DCs (M-DCs) but not resident DCs (R-DCs) were overtly reduced in the DLN of OptnΔCD11c mice (Fig.? 2c ). These data represent a potential role of OPTN in regulating the immunogenic phenotype of DCs, which may further control the T-cell activation.Fig. 2: OPTN regulates the T cells priming function of DC.a ELISA of cytokines in supernatants of LPS treated Ctrl and Optn null BMDCs. n ?=?3 independent experiments, P(IL-10) ?=?0.0029, P(TGF-β) ?=?0.0239, P(IL-6) ?=?0.6114, P(IL-12) ?=?0.8559. b Flow cytometry analysis of the frequency of total DC (CD11c + ), conventional DC (cDC, CD11c + MHCII + ) and plasmacytoid DCs (pDC, CD11c + PDCA-1 + ) in the spleen of Ctrl and OptnΔCD11c mice. n ?=?3 independent animals, P(DC) ?=?0.4925, P(cDC) ?=?0.0271, P(pDC) ?=?0.0216. c Flow cytometry analysis of the frequency of total DC (CD11c + ), migratory (M-DC, MHCII hi CD11c + ) and resident (R-DC, MHCII int CD11c + ) DCs in the draining lymph nodes of Ctrl and OptnΔCD11c mice. n ?=?3 independent animals, P(DC number) ?=?0.2950, P(DC%) ?=?0.2297, P(M-DC) ?=?0.0394, P(R-DC) ?=?0.0776. d Uptake of FITC-dextran was measured by flow cytometry in Ctrl and Optn null BMDCs. n ?=?3 independent experiments, P ?=?0.0007. e Proliferation rate of CFSE-labeled OT-II CD4 + T cells incubated with Ctrl or Optn null BMDCs pulsed with OVA 323-339 . n ?=?3 independent experiments, P ?=?0.0055. f The percentages of differentiated Th1 and Th17 subsets of OT-II CD4 + T cells incubated with Ctrl or Optn null BMDCs pulsed with OVA 323-339 . n ?=?3 independent experiments, P(IFN-γ) ?=?8.6E-05, P(IL-17) ?=?0.0028. Data are presented as means?±?SD. P ?t -test. Source data are provided in Source data file. Full size image Given that the function of DCs switches from capturing and processing antigens to predominantly presenting them to T cells after maturation16, we next examined if OPTN ablation in DCs affects the ability of T-cell priming in vitro. Flow cytometry results showed that Optn -deficient BMDCs had a much better ability than control cells to ingest more fluorescein isothiocyanate-dextran (FITC-dextran), suggesting that Optn deficiency in DCs improved its ability of antigen uptake (Fig.? 2d ). Then, we applied an in vitro T-cell priming model which involved the activation of OT-II CD4 + T cells co-incubated with DCs pulsed with a specific peptide, chicken OVA 323–339 17. As expected, Optn -deficient DCs inhibited the proliferation of OT-II CD4 + T cells, as measured by CFSE staining (Fig.? 2e ). In addition, Optn -deficient DCs exhibited an impaired ability to initiate the production of autoimmune factors IFN-γ and IL-17 in OT-II CD4 + T cells (Fig.? 2f ). Thus, these results suggest that Optn deletion suppresses the T-cell priming function of DCs in vitro.Mice lacking Optn in CD11c + cells are resistant to autoimmunity . We next employed a classical central nervous system (CNS) autoimmunity animal model EAE to explore the role of OPTN in regulating inflammatory injuries in vivo. Western blotting results showed that BMDCs from EAE mice expressed a higher level of OPTN than those from control mice (Fig.? 3a ). OptnΔCD11c mice displayed substantially decreased sensitivity to EAE induction and milder symptom compared with control mice, as indicated by lower clinical scores (Fig.? 3b ). As expected, OptnΔCD11c mice exhibited a less demyelinated area and an increased number of CC1 + mature oligodendrocytes in the white matter of spinal lesions at the peak of EAE (Fig.? 3c, d ), which could ultimately promote remyelination. In addition, OptnΔCD11c mice displayed a reduced frequency of inflammatory Th1 (CD4 + IFN-γ + ) and Th17 (CD4 + IL-17 + ) cells as well as an increased amount of Th2 (CD4 + IL-4 + ) and Treg (CD4 + Foxp3 + ) cells in both spleen and DLNs (Fig.? 3e, f ). Likewise, the absolute numbers of mononuclear cells (MNCs) infiltrating into the CNS were lower in OptnΔCD11c mice than that in control mice (Fig.? 3g ). Taken together, these data indicate that depletion of Optn in CD11c + cells protects mice from CNS autoimmunity.Fig. 3: Optn deficient mice are resistant to EAE pathogenesis and inflammation.a Western blotting analysis for the expression of OPTN in the BMDCs from four pairs of C57BL/6 Ctrl and EAE mice (8-week-old, female) at day 20 after immunization. n ?=?3 independent experiments. b Mean clinical scores of Ctrl and OptnΔCD11c mice (8-week-old, female) subjected to MOG 35–55 -induced EAE. n ?=?8 independent animals, P ?=?3.3E?09. c (Upper) Luxolfast blue (LFB) staining of mouse spinal cords from Ctrl and OptnΔCD11c EAE mice at day 20 post immunization (dpi 20). Scale bars, 200?μm. (Lower) Immunofluorescent labeling of CC1 (green) and DAPI (blue) in the spinal cord of indicated mice at day 20 after immunization. Scale bars, 20?μm. n ?=?3 independent animals. d The summary graphs of CC1 + cells in ( c ). n ?=?3 independent animals, P ?=?0.0004. e , f Frequency of Th1, Th17, Th2, and Treg cells in the spleen (SP) ( e ) and draining lymph nodes (DLN) ( f ) of MOG 35–55 -immunized Ctrl and OptnΔCD11c mice on dpi 20. n ?=?3 independent animals, P(IFN-γ) ?=?0.0016, P(IL-17) ?=?0.0001, P(IL-4) ?=?0.0082, P(Foxp3) ?=?0.0051 in ( e ); P(IFN-γ) ?=?0.0003, P(IL-17) ?=?0.0075, P(IL-4) ?=?0.0103, P(Foxp3) ?=?0.0088 in ( f ). g The total numbers of MNCs in whole spinal cord and brain were isolated from Ctrl and OptnΔCD11c EAE mice on dpi 20. n ?=?3 independent animals, P ?=?0.0015. Data are presented as means?±?SD. P ?t -test. Source data are provided in Source data file. Full size image OptnΔCD11c mice may have normal functions of T cells and macrophages . Although DNA recombination in CD11c-cre line is mainly present in DCs, previous reports have shown that CD11c -Cre line has germline recombination and off-target problems, which may cause partial knockout of target genes in T cells and macrophages18 , 19. Consistently, we also found a weak deletion of OPTN in CD4 + T cells but similar expression of OPTN in bone marrow-derived macrophages (BMDMs) from OptnΔCD11c mice (Supplementary Fig.? 2a ). Considering the significant role of DCs in T-cell activation, we then want to define the intrinsic impact of T cells in OptnΔCD11c mice. Optn knockout by CD11c -Cre did not influence T cells in the spleen and DLN, as the comparative numbers of CD4 + and CD8 + T cells in OptnΔCD11c and control mice (Supplementary Fig.? 2b ). In addition, no infiltration of lymphocytes in the liver, lung, and spleen from OptnΔCD11c mice was observed (Supplementary Fig.? 2c ), coupled with the similar frequency of activated (CD44 hi CD62L lo ) CD4 + and CD8 + T cells in the spleen (Supplementary Fig.? 2d, e ). These results demonstrate that Optn knockout by CD11c -Cre does not influence T cells homeostasis in the immune system.Given that EAE is mainly driven by CD4 + T-cell activation20, to rule out the role of Optn in CD4 + T cells from OptnΔCD11c mice, we isolated CD4 + T cells from WT and OptnΔCD11c mice, co-incubated with DCs from WT mice pulsed with MOG 35–55 for 2 days. FACS analysis showed that the percentage of Th1, Th2, Th17, and Treg cells were comparable between WT and OptnΔCD11c mice-derived CD4 + T cells (Supplementary Fig.? 2f ), indicating that slight deletion of Optn had no effect on CD4 + T-cell activation. To further clarify the role of OptnΔCD11c mice-derived CD4 + T cells in the in vivo phenotype of EAE mice, we then adoptively transferred CD4 + T cells that were treated by WT DCs pulsed with MOG 35–55 to induce EAE. As expected, results indicated that WT and OptnΔCD11c mice-derived CD4 + T cells had similar abilities to induce EAE pathogenesis (Supplementary Fig.? 2g–i ). Collectively, although we cannot completely rule out the role of OPTN in T cells, our results suggest that CD4 + T cells derived from OptnΔCD11c mice do not affect EAE progression.On the other hand, BMDMs from WT and OptnΔCD11c mice had comparable capacities on M1/M2 polarization and pro/anti-inflammatory cytokine expression (Supplementary Fig.? 3a-g ). Inspired by the findings that BMDMs can also express CD11c21, our flow cytometry results showed that BMDMs from OptnΔCD11c mice had similar expression of CD11c to that of WT mice (Supplementary Fig.? 3h ). Besides, the polarization ability of F4/80 + CD11c + BMDMs from WT and OptnΔCD11c mice was also comparable (Supplementary Fig.? 3i ). Thus, macrophages from OptnΔCD11c mice may have similar function to that from WT mice in vitro. Even so, there are still drawbacks that the potential function of OPTN in CD11c + macrophages in vivo is still unknown and whether CD11c + macrophages in OptnΔCD11c mice influent EAE pathology is also undefined. Nonetheless, our data at least clarify the function of OPTN in DCs and prove that Optn deficiency in CD11c + cells mitigates EAE effectively.STAT3 signaling pathway participates in Optn deficient DC maturation . STAT3, among all the members of the STAT family, has been well documented to promote the abnormal DC differentiation and function22. Consistently, we also found that phosphorylated STAT3 (p-STAT3), a critical transcription factor for STAT3 transduction, was elevated after LPS treatment for 16?h (Fig.? 4a ). However, a subsequent western blotting analysis showed that Optn deficiency activated STAT3 transduction prominently after LPS treatment for just 8?h (Fig.? 4b ), which was further confirmed by the increased p-STAT3 level in nuclear sub-fractions of Optn null BMDCs (Fig.? 4c ). Although STAT3 in macrophages was found to limit the inflammation of EAE mice23, our results showed that BMDMs from OptnΔCD11c mice had similar expression of (p-)STAT3 to WT mice, and the level of p-STAT3 was elevated fairly on IL-4 stimulation, further indicating that OptnΔCD11c may have similar macrophage function (Supplementary Fig.? 3j ). Generally, STAT3 can transcriptionally regulate the expression of many classical target genes like Vegfa , Hif1a , Hgf , Ptgs2 , and Socs3 , as well as many cytokine genes like Il-10 , Il-6 , Il-13 , and Tgfb 24 , 25 , 26 , 27. And many of them, such as VEGF, HIF-1α, HGF, SOCS3, IL-10, and IL-13, have been verified in previous studies to inhibit LPS-dependent DC maturation or induce the differentiation of DCs into tolerogenic DCs28 , 29 , 30 , 31 , 32. Interestingly, we found that most of these target genes were significantly enriched in Optn knockout BMDCs (Fig.? 4d ), suggesting that STAT3 signaling pathway was activated in an early stage in Optn deficient DC maturation.Fig. 4: STAT3 participates in OPTN-regulated DC maturation.a Expression of p-STAT3 in BMDCs stimulated with LPS for different time. n ?=?3 independent experiments. b Expression of (p)-STAT3 in Ctrl or Optn knockout BMDCs stimulated with LPS for 8?h. n ?=?3 independent experiments. c Cytoplasmic and nuclear proteins from Ctrl and Optn knockout BMDCs were analyzed by western blotting. The nuclear marker Lamin B and the cytoplasmic marker GAPDH were used to demonstrate the purity of the fractions. n ?=?3 independent experiments. d qRT-PCR analysis of STAT3 target genes in Ctrl and Optn knockout BMDCs. n ?=?3 independent experiments, P( Vegfa ) ?=?0.0238, P( Hif1a ) ?=?0.0023, P( Hgf ) ?=?1.8E?05, P( Ptgs2 ) ?=?2.1E?06, P( Socs3 ) ?=?0.0205, P( Il-10 ) ?=?0.0137, P( Il-13 ) ?=?0.0076, P( Tgfb2 ) ?=?8.9E?07, P( Tgfb3 ) ?=?3.1E?05. e , f Ctrl BMDCs were transfected with STAT3-overexpression (OE) plasmid. mRNA level of Cd80 , Cd86 , H2-k1 , and Hl-dra were analyzed by qRT-PCR. n ?=?3 independent experiments, P( Cd80 ) ?=?0.0056, P( Cd86 ) ?=?0.0031, P( H2-k1 ) ?=?6.4E?05, P( Hl-dra ) ?=?0.0034. g – j Ctrl and Optn knockout BMDCs were treated with stattic (10?μM) before LPS stimulation. Expression of Cd80 , Hl-dra ( h ) and CD86, MHC-II ( i , j ) were analyzed by qRT-PCR and flow cytometry, respectively. n ?=?3 independent experiments, P( Optn ΔDC-Ctrl) ?=?0.9612, P(Ctrl+stattic-Ctrl) ?=?0.0025, P( Optn ΔDC+stattic- Optn ΔDC) ?=?0.9157, P( Optn ΔDC+stattic-Ctrl+stattic) ?=?0.0030 of Cd80 in ( h ); P( Optn ΔDC-Ctrl) ?=?0.9902, P(Ctrl+stattic-Ctrl) ?=?0.0031, P( Optn ΔDC+stattic- Optn ΔDC) ?=?0.3368, P( Optn ΔDC+stattic-Ctrl+stattic) ?=?0.0208 of Hl-dra in ( h ); P( Optn ΔDC-Ctrl) ?=?0.00105, P(Ctrl+stattic-Ctrl) ?=?0.0153, P( Optn ΔDC+stattic- Optn ΔDC) ?=?0.0057, P( Optn ΔDC+stattic-Ctrl+stattic) ?=?0.0024 of CD86 in ( j ); P( Optn ΔDC-Ctrl) ?=?6.5E?06, P(Ctrl+stattic-Ctrl) ?=?0.0002, P( Optn ΔDC+stattic- Optn ΔDC) ?=?2.2E?05, P( Optn ΔDC+stattic-Ctrl+stattic) ?=?4.7E?05 of MHC-II in ( j ). Data are presented as means?±?SD. P ?t -test for ( d , f ); one-way ANOVA Tukey’s post hoc analysis for ( h , j ). Source data are provided in Source data file. Full size image We next explored if STAT3 participates in OPTN-mediated DC maturation and activation. Notably, STAT3 overexpression significantly reduced the mRNA levels of DC co-stimulatory molecules and MHC molecules (Fig.? 4e, f ), consistent with previous findings that STAT3 is crucial for the tolerant function of DCs and DCs conditional deletion of Stat3 resulted in T-cell activation and autoimmunity in mice33. On the contrary, a pharmacological inhibitor of STAT3 phosphorylation, stattic, treatment reduced the p-STAT3 level in both control and Optn -deficient DCs (Fig.? 4g ). qRT-PCR and flow cytometry analysis indicated that stattic treatment not only promoted mature DCs markers CD80 and MHC-II expression in control BMDCs, but also rescued the impaired maturation and activation in Optn knockout BMDCs (Fig.? 4h–j ). These data suggest that STAT3 has an important role in Optn deficient DC maturation.The well-characterized functions of OPTN are autophagy and ubiquitin binding11, so we then checked if these functions may be related to OPTN-regulated DC maturation. First, although Optn deletion inhibited the rapamycin (RAPA) activated GFP + mCherry + autophagic puncta (Supplementary Fig.? 4a, b ), Optn knockout cannot rescue RAPA mediated depressed expression of mature DCs markers CD80 and CD86 (Supplementary Fig.? 4c, d ). On the other hand, we constructed a whole OPTN plasmid and an OPTN plasmid with UBD deletion (OPTN ΔUBD ), and transfected them into Optn null BMDCs (Supplementary Fig.? 4e ). Flow cytometry results showed that both OPTN and OPTN ΔUBD overexpression restored the percentage of CD11c + CD80 + and CD11c + CD86 + cells in LPS-stimulated Optn null BMDCs (Supplementary Fig.? 4f, g ). Moreover, OPTN and OPTN ΔUBD had similar ability to reverse the mRNA level of Il-10 in Optn deficient BMDCs (Supplementary Fig.? 4h ). In conclusion, these results suggest that OPTN-mediated autophagy or ubiquitination has no contribution to OPTN-regulated DC maturation.STAT3 is crucial for the anti-inflammatory effects of OptnΔCD11c mice . Given the essential role of STAT3 in DC maturation, we generated CD11c + cells conditional Stat3 knockout mice ( Stat3fl/fl ; CD11c -Cre mice, hereafter called Stat3ΔCD11c mice) to investigate if STAT3 in CD11c + cells limits EAE. Adoptive transfer study showed that Stat3 deficient BMDCs aggravated MOG 35-55 induced EAE progression when compared with control BMDCs (Supplementary Fig.? 5 ). To further determine whether the immune tolerance of OptnΔCD11c mice is dependent on STAT3 signaling, we crossed the OptnΔCD11c mice with Stat3fl/fl mice ( Optnfl/fl ; Stat3fl/fl ; CD11c -Cre mice, hereafter called DKO mice) and verified the knockout efficiency by western blotting (Fig.? 5a ). Results showed that DKO mice displayed much more severe autoimmune symptoms than OptnΔCD11c mice, as evidenced by higher clinical score, more severe demyelination, and less CC1 + mature oligodendrocytes (Fig.? 5b–d ). In addition, the percentages of Th1 and Th17 cells were much higher in both the spleen and DLN of DKO mice during EAE than those of OptnΔCD11c mice (Fig.? 5e, f ). Conversely, the amounts of Th2 and Treg cells in DKO mice were significantly decreased when compared with OptnΔCD11c mice (Fig.? 5e, f ). Together, these results suggest that STAT3 in CD11c + cells is a negative regulator of autoimmune response, and OPTN can manipulate STAT3 activity in CD11c + cells to control autoimmune progress.Fig. 5: Deletion of Stat3 aggravates autoimmunity in OptnΔCD11c mice.a Western blotting to verify the deficiency of OPTN and STAT3 in DKO DCs. n ?=?3 independent experiments. b Mean clinical scores of Ctrl, OptnΔCD11c and DKO mice (8-week-old, female) subjected to MOG 35–55 -induced EAE. n ?=?8 independent animals, P(Ctrl) ?=?5.1E?08, P(DKO) ?=?1.6E?07. c (Upper) Luxolfast blue (LFB) staining of spinal cords from indicated EAE mice at day 20. Scale bars, 200?μm. (Lower) Immunofluorescent labeling of CC1 (green) and DAPI (blue) in the spinal cord of indicated mice at day 20. Scale bars, 20?μm. n ?=?3 independent animals. d The summary graphs of CC1 + cells in ( c ). n ?=?3 independent animals, P(Ctrl) ?=?0.0003, P(DKO) ?=?0.0003. e , f Frequency of Th1, Th17, Th2, and Treg cells in the spleen (SP) ( e ) and draining lymph nodes (DLN) ( f ) from indicated EAE mice on day 20 after immunization. n ?=?3 independent animals, P(Ctrl) ?=?2.3E?05, P(DKO) ?=?2.8E?05 of IFN-γ in ( e ); P(Ctrl) ?=?0.0018, P(DKO) ?=?0.0003 of IL-17 in ( e ); P(Ctrl) ?=?0.0059, P(DKO) ?=?0.0020 of IL-4 in ( e ); P(Ctrl) ?=?7.1E?05, P(DKO) ?=?1.5E?05 of Foxp3 in ( e ); P(Ctrl) ?=?0.0002, P(DKO) ?=?0.00102 of IFN-γ in ( f ); P(Ctrl) ?=?0.0215, P(DKO) ?=?0.0018 of IL-17 in ( f ); P(Ctrl) ?=?0.0002, P(DKO) ?=?3.2E?05 of IL-4 in ( f ); P(Ctrl) ?=?0.0026, P(DKO) ?=?0.0155 of Foxp3 in ( f ). Data are presented as means?±?SD. P ?Source data are provided in Source data file. Full size image OPTN restrains the phosphorylated activation of JAK2 in DCs . Encouraged by the findings, we proceeded to study how OPTN regulates STAT3 signaling pathway. We found JAK2, the classic upstream signal of STAT3, was also activated observably after LPS treatment for only 8?h (Fig.? 6a ). To find out whether OPTN modulates JAK2 phosphorylated activation, we overexpressed OPTN in control and Optn deficient BMDCs. Results showed that OPTN abundance could inhibit the expression of p-JAK2 in both control and Optn knockout BMDCs stimulated with LPS or poly I:C (Fig.? 6b, c ). Considering that IL-6 and IL-10, classical stimulators of JAK2, are rapidly released in response to TLR4/NF-κB signals stimulated by LPS34 , 35, we therefore suspected that IL-6/IL-10 may be involved in the regulation of JAK2-STAT3 in DCs. Despite the fact that both anti-IL6 and anti-IL10 eliminated LPS induced JAK2-STAT3 activation in Optn -deficient DCs (Fig.? 6d ), there was no direct interaction between TLR4 and JAK2 or STAT3 in DCs after LPS treatment and no marked difference in NF-κB signals between control and Optn -deficient DCs (Fig.? 6e, f ). In consequence, these data indicate that LPS activated JAK2/STAT3 is dependent on the IL-6/IL-10 but not the recruitment of JAK2 or STAT3 to TLR4, and OPTN deletion cannot interfere the NF-κB signals which manipulate the initial transcription of IL-6/IL-10 in LPS treatment.Fig. 6: OPTN restrains the IL-6/IL-10 activated JAK2/STAT3 phosphorylation in DCs.a Expression of (p)-JAK2 in Ctrl or Optn deficient BMDCs stimulated with LPS for 8?h. b , c Ctrl and Optn deficient BMDCs were stimulated with LPS ( b ) or poly I:C ( c ) after transfection with empty or OPTN expression vector. The expressions of OPTN and p-JAK2 were confirmed by western blotting. d Western blotting for expression of (p)-JAK2 and (p)-STAT3 in Ctrl and Optn deficient BMDCs stimulated with anti-IL6 or anti-IL10 for 48?h. e Interaction between endogenous TLR4 and JAK2 or STAT3 in BMDCs was analyzed by IP with anti-JAK2 or anti-STAT3 using normal rabbit IgG as a control, followed by immunoblotting with anti-TLR4, anti-JAK2, and anti-STAT3. f Expression of (p-) signaling proteins in whole-cell lysates of Ctrl or Optn deficient BMDCs stimulated with LPS. g HEK293 cells were transfected with si-Optn or Optn plasmid before IL-6 stimulation. The expressions of OPTN and p-JAK2 were confirmed by western blotting. h Western blotting for expression of p-JAK2 and p-STAT3 in Ctrl and Optn deficient BMDCs stimulated with RAPA. All data are representative of three independent experiments. Source data are provided in Source data file. Full size image We then asked whether Optn knockdown could cause the constitutive activation of JAK2 and employed IL-6 to activate the IL-6 receptor/JAK2 signaling pathway. As a result, OPTN overexpression decreased the phosphorylation level of JAK2, while Optn depletion augmented the JAK2 activation (Fig.? 6g ). More importantly, this effect was strengthened after IL-6 treatment (Fig.? 6g ). Finally, the treatment of RAPA did not alter the level of p-JAK2 and p-STAT3 (Fig.? 6h ), proving that the activation of JAK2/STAT3 in Optn knockout BMDCs was independent of the OPTN-mediated autophagy deficiency. Overall, our results confirm that OPTN negatively modulates JAK2 phosphorylated activation in DCs.OPTN inhibits JAK2 dimerization and subsequent STAT3 activation . Next, we wonder how OPTN regulates JAK2/STAT3 signaling. Immunoprecipitation analysis showed that ectopically expressed OPTN was reciprocally precipitated with JAK2, but not JAK1 or JAK3, from HEK293 cells overexpressing the proteins (Fig.? 7a ). A subsequent co-immunoprecipitation further confirmed the direct interaction between endogenous OPTN and JAK2 in mouse primary BMDCs (Fig.? 7b ). JAK2 kinase is composed of 7 JAK homology (JH) domains, termed JH1-7. The JH1 region functions as the kinase domain of JAK2, while JH2 can physically interact with JH1 and inhibit its kinase activity. And the JH3-7 region of JAK2 is essential for receptor interactions36. Accordingly, we generated a series of truncated JAK2 mutants to represent these domains. Analysis of the interaction between OPTN and the JAK2 mutants identified JH1, rather than JH2 or JH3-7, as the site for OPTN binding (Fig.? 7c ). Besides, the interaction between OPTN and JH1 did not rely on its UBD, as OPTN ΔUBD can also interact with JAK2 normally (Fig.? 7d ). Therefore, these results suggest that OPTN may regulate JAK2 directly.Fig. 7: OPTN inhibits the dimerization of JAK2 and subsequent STAT3 activation.a HA-OPTN expression plasmid was transfected with Flag-JAK1, Flag-JAK2, or Flag-JAK3 expression plasmid into HEK293 cells. Expression of OPTN and JAKs was confirmed by immunoblotting. Interaction between OPTN and JAKs was determined by immunoprecipitation (IP) with anti-HA antibody followed by immunoblotting with anti-Flag antibody. b Interaction between endogenous OPTN and JAK2 in BMDCs was analyzed by IP. c Schematic illustration of the truncated JAK2. Interactions between OPTN and truncation JAK2 in transiently transfected HEK293 cells were determined by IP. d Interactions between JAK2 with OPTN or OPTN ΔUBD in transiently transfected HEK293 cells were determined by IP. e Interactions between HA-JAK2 and FLAG-JAK2 in transiently transfected HEK293 cells was determined by IP. f , g HEK293 cells were transfected with FLAG-JAK2 and HA-OPTN or empty vector ( f ). BMDCs were transfected with HA-OPTN or empty vector ( g ). Whole-cell lysates were incubated with disuccinimidyl suberate (DSS). JAK2 antibody marked two bands: the upper band referring to the dimer and the lower band representing the monomer of JAK2. h Interaction of FLAG-JAK2 and HA-STAT3 in transiently transfected HEK293 cells was determined by IP. i BMDCs were transfected with HA-OPTN or empty vector. Interaction between endogenous STAT3 and JAK2 in BMDCs was analyzed by IP. j ChIP-seq analysis of the binding between STAT3 and Il-10 or Il-6 from GSE27161. kIl-10 and Il-6 mRNA level in Ctrl and Optn deficient BMDCs stimulated with LPS for different times. n ?=?3 independent experiments, P(4) ?=?3.2E?06, P(8) ?=?0.0497, P(12) ?=?1.2E?05, P(16) ?=?3.9E?07, P(24) ?=?0.0060 of Il-10 . Data in ( a – i ) are representative of three independent experiments. Data are presented as means?±?SD. P ?t -test. Source data are provided in Source data file. Full size image Previous studies suggested that JH1-JH1 trans-interaction mediates the dimerization and activation of JAK237, which prompted us to investigate whether OPTN inhibits JAK2 dimerization. The results showed that the ability of FLAG-tagged JAK2 to co-immunoprecipitate with HA-tagged JAK2 was markedly hampered by the overexpression of OPTN (Fig.? 7e ). Moreover, the disuccinimidyl suberate-mediated cross-linking assays indicated that the dimer/monomer ratio of JAK2 was decreased in the presence of OPTN in both HEK293 and BMDCs (Fig.? 7f, g ), further showing the inhibitory ability of OPTN to JAK2 dimerization. Given that once activated, JAK2 recruits and phosphorylates STAT3, we then wondered whether OPTN could affect JAK2 downstream activation. As expected, the JAK2-STAT3 interaction was abrogated by OPTN overexpression in both HEK293 and BMDCs (Fig.? 7h, i ), implying that OPTN antagonized the binding of JAK2 with STAT3, further interfering STAT3 activation.STAT3, a classical transcription factor, can transfer to the nucleus and bind to its target genes like Il-10 after phosphorylated activation27. To further explore the transcriptional regulatory role of STAT3, we analyzed the public STAT3 ChIP-seq data from GEO dataset38. Peak tracking results revealed that STAT3 had quite high bindings with Il-10 gene in DCs, while STAT3 showed weak bindings with genes like Il-6 , Cd80 , Cd86 , Mhc-i ( H2-K1 ), Mhc-ii ( H2-Aa ), Vegfa , Hif1a , Hgf , Ptgs2 , Il-13 , Tgfb2 , or Tgfb3 (Fig.? 7j and Supplementary Fig.? 6 ), suggesting IL-10 might be the effective target gene of STAT3 in DCs. Consistent with the previous studies35 , 39, we found that the expression of Il-6 and Il-10 was increased and then went down after LPS treatment in control BMDCs (Fig.? 7k ). Inspiringly, knocking out of Optn in BMDCs facilitated and maintained the expression of IL-10 but not IL-6 (Figs.? 2 a and 7k ), thus exaggerating the immunosuppressive function of IL-1031, and resulting in the evident activation of JAK2/STAT3 in Optn knockout BMDCs. Collectively, our data demonstrate that OPTN negatively regulates JAK2/STAT3 activation by interacting with JAK2 and inhibiting its dimerization, and Optn deficiency hinders DC maturation via activating IL-10/JAK2/STAT3/IL-10 positive feedback loop.In addition to Il-10 target, we also found STAT3 could bind to its canonical target Socs3 gene in DCs by STAT3 ChIP-seq data peak tracking (Supplementary Fig.? 6 ), and our qPCR results showed the gene expression of Socs3 was significantly upregulated in Optn deficient BMDCs (Fig.? 4d ). Given that SOCS3 is found to inhibit DC maturation together with subsequent Th17 response32, these data indicate that Optn deficiency can inhibit DC maturation at least partially via activating STAT3 target Socs3 .SSD impairs DC function and alleviates EAE by reducing OPTN expression . The promising effect of Optn deficiency on DCs dictated immune activation shed creative lights on finding treatments for autoimmune diseases. We screened the natural product library for small molecule inhibitors of OPTN expression and found that Saikosaponin D (SSD), a triterpenoid saponin derived from Bupleurum falcatum L, significantly downregulated OPTN in BMDCs after LPS stimulation (Fig.? 8a ). To investigate how SSD inhibits OPTN expression, we next analyzed the binding profiles of SSD and OPTN by using surface plasmon resonance (SPR) assay. Results revealed that SSD specifically binds to OPTN (KD?=?5.115?×?e ?5 ?M), with rapid on-rate and off-rate (Supplementary Fig.? 7a, b ), but not ubiquitin-like modifier activating enzyme 3 (UBA3) (Supplementary Fig.? 7c, d ). Then we performed a cycloheximide (CHX) chase assay in SSD-treated BMDCs to determine whether SSD affects the stability of OPTN protein. Western blotting results indicated that the stability of OPTN was decreased in the presence of SSD (Fig.? 8b ), suggesting that the interaction of SSD with OPTN promotes the degradation of OPTN, thus reducing OPTN expression.Fig. 8: OPTN inhibitor SSD impairs DC function and attenuates EAE.a Western blotting analysis of BMDCs that treated with SSD of different concentrations for 24?h before LPS stimulation. n ?=?3 independent experiments. b Western blotting analysis of 293T cells that treated with cycloheximide (CHX) for different time after SSD (1?μM) treatment. n ?=?3 independent experiments. c , d BMDCs were cultured with SSD (1?μM) for 24?h before LPS stimulation. Flow cytometry analysis for the expression of CD80, CD86, and MHC-II. n ?=?3 independent experiments, P(CD80) ?=?0.0004, P(CD86) ?=?0.0049, P(MHC-II) ?=?0.0050. e Proliferation of CFSE-labeled OT-II CD4 + T cells that incubated with BMDCs treated with or without SSD and then pulsed with OVA 323–339 . n ?=?3 independent experiments, P ?=?0.0009. f , g Differentiation of OT-II CD4 + T cells that incubated with BMDCs treated with or without SSD and then pulsed with OVA 323-339 . n ?=?3 independent experiments, P(IFN-γ) ?=?2.6E?05, P(IL-17) ?=?0.0028. h C57BL/6 mice (8-week-old, female) were immunized with MOG 35–55 and administered daily with SSD or placebo solution intragastrically from the day of immunization. Mean clinical scores are shown. n ?=?8 independent animals, P(SSD-10) ?=?8.9E?08, P(SSD-20) ?=?3.1E?11, P(SSD-40) ?=?1.6E?13. i (Upper) Luxolfast blue (LFB) staining of spinal cords from vehicle and SSD (40?mg/kg) treated EAE mice at day 20 after immunization. Scale bars, 200?μm. (Lower) Immunofluorescent labeling of CC1 (green) in the spinal cord of indicated mice at day 20 after immunization. Scale bars, 20?μm. n ?=?3 independent animals, P ?=?0.0047. j , k Flow cytometry analysis of Th1 and Th17 cells in spleen (SP) ( j ) and draining lymph nodes (DLN) ( k ) from vehicle and SSD treated EAE mice at day 20. n ?=?3 independent animals, P(IFN-γ) ?=?0.0148, P(IL-17) ?=?0.0041 in ( j ); P(IFN-γ) ?=?0.0095, P(IL-17) ?=?0.0004 in ( k ). Data are presented as means?±?SD. P ?t -test. Source data are provided in Source data file. Full size image Next, the effect of SSD on DC activation was examined, and flow cytometry results showed that SSD markedly reduced the expression of CD80, CD86, and MHC-II in BMDCs (Fig.? 8c, d ). In addition, SSD-treated BMDCs exhibited an impaired ability to initiate the proliferation and differentiation of OT-II CD4 + T cells (Fig.? 8e–g ). These data indicate that SSD inhibits DC maturation and activation, which may be further linked to the potential therapeutic role of SSD on autoimmune disease. Our results showed that SSD treatment daily protected mice against the EAE challenge in a dose-dependent way (Fig.? 8h ). Luxolfast blue (LFB) and CC1 staining further confirmed that demyelination was markedly decreased and mature oligodendrocytes were dramatically increased after SSD treatment (Fig.? 8i ). Elsewise, the amount of the inflammatory Th1 and Th17 cells in peripheral lymphoid organs of SSD-treated mice were reduced significantly during disease development (Fig.? 8j, k ). All together, these results suggest that SSD effectively inhibits not only the immune-stimulatory function of DCs but also the disease progression of EAE.We then asked whether SSD specifically depends on OPTN to regulate DC maturation and EAE progression. Western blotting results showed that SSD had no effect on TLR4 signaling, as shown by the equivalent expression of TLR4, MyD88, (p-)NF-κB, (p-)IRF3, and (p-)p38 MAPK upon SSD treatment (Supplementary Fig.? 7e ). Meanwhile, qRT-PCR analysis revealed the comparable mRNA levels of Cd80 and Cd86 in Optn knockout BMDCs with or without SSD incubation (Supplementary Fig.? 7f ). Finally, SSD administration exhibited a similar therapeutic effect as Optn deletion in effectively inhibiting the progression of EAE, and had no effect on OptnΔCD11c mice undergoing EAE progress (Supplementary Fig.? 7g ). In aggregate, these results suggest that SSD ameliorates experimental autoimmunity through OPTN modulation. .