Targeting chondrocytes for arresting bony fusion in ankylosing spondylitis

Bony fusion caused by pathological new bone formation manifests the clinical feature of ankylosing spondylitis (AS). However, the underlying mechanism remains elusive. Here we discovered spontaneous kyphosis, arthritis and bony fusion in mature CD4-Cre;Ptpn11f/f mice, which present the pathophysiological features of AS. A population of CD4-Cre-expressing proliferating chondrocytes was SHP2 deficient, which could differentiate into pre-hypertrophic and hypertrophic chondrocytes. Functionally, SHP2 deficiency in chondrocytes impeded the fusion of epiphyseal plate and promoted chondrogenesis in joint cavity and enthesis. Mechanistically, aberrant chondrocytes promoted ectopic new bone formation through BMP6/pSmad1/5 signaling. It is worth emphasizing that such pathological thickness of growth plates was evident in adolescent humans with enthesitis-related arthritis, which could progress to AS in adulthood. Targeting dysfunctional chondrogenesis with Smo inhibitor sonidegib significantly alleviated the AS-like bone disease in mice. These findings suggest that blockade of chondrogenesis by sonidegib would be a drug repurposing strategy for AS treatment.


March 2021
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We found bone deformation in five female CD4-CKO mice by accident. To explore the morbidity and process of bone deformation in CD4-CKO mice, we monitored ten female and male mice from birth to 12-month-old. Considering mortality of old CD4-CKO mice radiation damage, we used ten mice in each group in the experiment of bone marrow transplant and sonidegib treatment in young mice. At late stage, we chose six mice in each group in the experiment of sonidegib treatment in matured mice after we clearly know the effect of bone deformation and sonidegib on mortality of CD4-CKO mice.
Owing to the deficiency of bone deformation data at initial stage, the data of five female mice found by accident were excluded from analysis.
Experimental findings were reliably reproduced. The number of independent biologic replicates is indicated in each Figure Legend.
The bone deformation in CD4-CKO mice were compared by littermate CD4-Ctrl. The experiment of bone marrow transplant, the mice were allocated based on genotype of mice. In the experiment of sonidegib treatment, the CD4-Ctrl and CD4-CKO mice were grouped randomly to administered drugs.
The investigators were blinded to group allocation during data collection and analysis. Note that full information on the approval of the study protocol must also be provided in the manuscript.

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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. The cell line was not authenticated.
The results of mycoplasma contaimination test of ATDC5 cell and the cell culture facility were negative.
No commonly misidentified cell lines were used.
The animal use and the experimental protocols were reviewed and approved by the Animal Care Committee of the Nanjing University in accordance with the Institutional Animal Care and Use Committee guidelines. Female Ptpn11f/f mice; CD4-Cre;Ptpn11f/f mice; Lck-Cre;Ptpn11f/f mice; CD4-Cre;Ptpn11f/f;Rosa26-mTmG mice were monitored from birth to 12-month-old. The mice were sacrificed at 2-month-old, 7-month-old or 12-month-old at indicated in the figure legends. 3-week-old and 7-month-old female CD4-CKO mice and the littermate control were treatment with sonidegib for three time and 4 months respectively. 4-month-old female NCG mice were transferred with CD3+ T cells isolated from 4-month-old female mice CD4-CKO mice or littermate CD4-Ctrl mice. All mice were given food and water ad libitum and housed in temperature-, moisture-, and light-controlled (12h light/dark cycle).
No wild animals were used in the study.
No field-collected samples were used in the study.
The animal use and the experimental protocols were reviewed and approved by the Animal Care Committee of the Nanjing University (Approval No: IACUC-2009005) in accordance with the Institutional Animal Care and Use Committee guidelines. Animal welfare and experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, USA) and the related ethical regulations of our university. All efforts were made to reduce the number of animals used and to minimize animal suffering.
Patients with Juvenile Idiopathic Arthritis (JIA) and patients with AS. To Patients with JIA were Patients with AS were from three patients (male, 23-52 years old) Patients with ERA and non-ERA (13-14 years old, female and male) Between 2018 and 2020, the patients with JIA that aged 13-14 years old and received treatment in Nanjing University of Chinese Medicine were recruited. The patients diagnosed with AS according to Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) were recruited.
The study and protocols were carried out in accordance withthe ethical guidelines of the 1975 Declaration of Helsinki Principles, and were approved by the Ethics Institutional Review Board of Affiliated Hospital of Nanjing University of Chinese Medicine (study number 2018NL-106-02) and Children's Hospital of Nanjing Medical University (study number 202008041-1). Written informed consents were obtained from all patients.

March 2021
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. Quantification of cell populations are provided as total number of tissue or percentage of reference population as described in the figure and figure legends.

Methodology
Gating schemes were established with fluorescence-minus-one (FMO: staining with all fluorophores except one) controls The MRI images were used to show the bone deformation of patients with AS or ERA The thickness of epiphysis plate in age, sex-matched patients with ERA and no-ERA were chose to compare.
The patient was placed in supine position with 1.5 T high field intensity magnetic resonance (HFMRI) scanning, and body coil was used to obtain rapid spin echo at the bone lesion site.
Structural diffusion 1.5 tesla 1) Diffusion-weight imaging (DWI) with b= 0/900s/mm 3 mm thick axial slices with corresponding apparent diffusion coefficient mapping; 2) T1 sequences (TE 22.00 ms, TR 623.00 ms); 3) T2 sequences (TE 105.00 ms, TR 4710.00 ms) 4) T2 weighed fat saturated (T2-FS) sequence (TE 35.00 ms, TR 5740.00 ms) Whole body scan for patients with AS, Sacroiliac joint and knee joint scan for patients with ERA b value=0/900s/mm, multi shell The images of MRI results were not preprocessed and directly presents the original result.
If data were normalized/standardized, describe the approach(es): specify linear or non-linear and define image types used for transformation OR indicate that data were not normalized and explain rationale for lack of normalization.
Describe the template used for normalization/transformation, specifying subject space or group standardized space (e.g. original Talairach, MNI305, ICBM152) OR indicate that the data were not normalized.
Describe your procedure(s) for artifact and structured noise removal, specifying motion parameters, tissue signals and physiological signals (heart rate, respiration).