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

Optineurin (OPTN) has important functions in diverse biological processes and diseases, but its effect on dendritic cell (DC) differentiation and functionality remains elusive. Here we show that OPTN is upregulated in human and mouse DC maturation, and that deletion of Optn in mice via CD11c-Cre attenuates DC maturation and impairs the priming of CD4+ T cells, thus ameliorating autoimmune symptoms such as experimental autoimmune encephalomyelitis (EAE). Mechanistically, OPTN binds to the JH1 domain of JAK2 and inhibits JAK2 dimerization and phosphorylation, thereby preventing JAK2-STAT3 interaction and inhibiting STAT3 phosphorylation to suppress downstream transcription of IL-10. Without such a negative regulation, Optn-deficient DCs eventually induce an IL-10/JAK2/STAT3/IL-10 positive feedback loop to suppress DC maturation. Finally, the natural product, Saikosaponin D, is identified as an OPTN inhibitor, effectively inhibiting the immune-stimulatory function of DCs and the disease progression of EAE in mice. Our findings thus highlight a pivotal function of OPTN for the regulation of DC functions and autoimmune disorders.

Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.  Fig. 7j and Supplementary Fig. 6 are available in the NCBI GEO database accession code GSE27161 [https://www.ncbi.nlm.nih.gov/geo/ query/acc.cgi?acc=GSE27161]. The data generated in Fig. 1, 2, 3a-b, 3d-g, 4a-h, 4j, 5a-b, 5d-f, 6, 7a-i, 7k, 8a-b, 8d-e, 8g-k and Supplementary Fig. 2a 4d-e, 4g-h, 5a, 5c, 5e, 7 are provided in the Source Data file. The reporting summary for this Article is available in the Supplementary Information file. All the other data supporting this study are available within this Article, Supplementary  No data were excluded.
The experimental findings were reliably reproduced, for representative data used for statistical analysis, the number of animals or experiments is described in corresponding figure legends.
All samples were randomly allocated into experimental groups.
For in vivo experiments, the investigators were blinded to group allocation during data collection and analysis.
For cell-based experiments, immunostaining image analysis, collection of samples were not blinded sometimes due to the cell isolated from the specific animals or the tissue section gained from the specific EAE mice which have obvious differences between groups. The samples were then processed and analysed blindingly in all experiments.

March 2021
Eukaryotic cell lines Note that full information on the approval of the study protocol must also be provided in the manuscript.

Human research participants
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Recruitment
Ethics oversight Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.
BMDCs and BMDMs were prepared from the bone marrow of 6~8-week-old C57BL/6 mice; CD11c+ DCs and CD4+ T cells were isolated from the spleen and lymph nodes of 6~8-week-old C57BL/6 mice; human monocyte-derived dendritic cells and peripheral blood mononuclear cells were isolated from human blood samples; HEK293 cells were purchased from Invitrogene.
DCs were authenticate by Flow cytometry analysis for CD11c staining. BMDMs were authenticate by Flow cytometry analysis for F4/80 and CD11b staining. CD4+ T cells were authenticate by Flow cytometry analysis for CD4 staining. Other cells were used without modification once received from supplier and therefore were not authenticated.
All cell lines tested negative for mycoplasma contamination.
No commonly misidentified lines were used.
As reported in Methods section of "Mice" for the information of animal species and strains. The mouse strains used in this study were generated and maintained on a mixed C57Bl/6 background. C57BL/6 mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. CD11c-Cre mice were acquired from The Jackson Laboratory. Optnfl/fl mice were gifted by Pro. Ronggui Hu. Stat3fl/fl mice were obtained from the Shanghai Model Organisms Center.
OT-II mice were given by Pro. Lie Wang as gift.
All mice were housed in specific pathogen-free environment at 21 ± 1°C and 60 ± 5% humidity, with a 12-h light/dark cycle. Experimental and control animals were bred separately. All mice were used at 6-8 weeks of age. Female mice were used for EAE model, female or male mice were used for in vitro experiment.
Wild animals were not involved in this study.
Field-collected samples were not involved in this study.
All animal use and studies were performed in compliance with all relevant ethical regulations, and were approved by the Institutional Animal Care and Use Committee (IACUC) at Zhejiang University.
Healthy donor are 10~30 years old males and females.
Blank human peripheral blood was collected from the Second Affiliated Hospital of School of Medicine, Zhejiang University. Samples from healthy people who showed no history of major diseases and had normal biochemical indicators and blood routine after physical examination were involved in this cohort. Ailing peripheral blood was excluded in the study to avoid the influence of the diseases to their peripheral blood or MoDCs.
Volunteers provided informed consent for this study. All experiments were approved by the Human Subject Research Ethics Committee of the Second Affiliated Hospital of School of Medicine, Zhejiang University (No. Yan2016-003).