mTORC1 links pathology in experimental models of Still’s disease and macrophage activation syndrome

Still’s disease is a severe inflammatory syndrome characterized by fever, skin rash and arthritis affecting children and adults. Patients with Still’s disease may also develop macrophage activation syndrome, a potentially fatal complication of immune dysregulation resulting in cytokine storm. Here we show that mTORC1 (mechanistic target of rapamycin complex 1) underpins the pathology of Still’s disease and macrophage activation syndrome. Single-cell RNA sequencing in a murine model of Still’s disease shows preferential activation of mTORC1 in monocytes; both mTOR inhibition and monocyte depletion attenuate disease severity. Transcriptomic data from patients with Still’s disease suggest decreased expression of the mTORC1 inhibitors TSC1/TSC2 and an mTORC1 gene signature that strongly correlates with disease activity and treatment response. Unrestricted activation of mTORC1 by Tsc2 deletion in mice is sufficient to trigger a Still’s disease-like syndrome, including both inflammatory arthritis and macrophage activation syndrome with hemophagocytosis, a cellular manifestation that is reproduced in human monocytes by CRISPR/Cas-mediated deletion of TSC2. Consistent with this observation, hemophagocytic histiocytes from patients with macrophage activation syndrome display prominent mTORC1 activity. Our study suggests a mechanistic link of mTORC1 to inflammation that connects the pathogenesis of Still’s disease and macrophage activation syndrome.


March 2021
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Antibodies
Antibodies used Data Availability: Source data are provided with this paper. The Single cell RNA sequencing data used in this study are available in the NCBI Gene Expression Omnibus database under accession code GSE181243. Datasets analyzed in this study include GSE80325, GSE80060, GSE7753, GSE21521, GSE17590, GSE147608, GSE112057, and GSE113645.
Sample sizes were not predetermined based on statistical methods. Sample sizes were determined based on preliminary experiments and historical data. n= 3 was chosen as the minimal replicate number for each condition.
No data was excluded from the analyses.
With the exception of single cell RNA sequencing (with multiple animals pooled per group) and bone marrow immunohistochemistry studies on rare patient samples, all studies were independently replicated 2 to 3 times with consistent results.
Mice were randomly assigned to treatment groups, and efforts were made to achieve equal representation of males and females in each experiment.
Blinding was performed for measurement of arthritis and ankle erosion assessment. For other experiments, the investigators were not blinded during experiments and outcome assessment as the same investigators are involved in general animal husbandry and colony maintenance. Data analyses were performed independently by the principle investigators to minimize bias. Note that full information on the approval of the study protocol must also be provided in the manuscript.

Human research participants
Policy information about studies involving human research participants Population characteristics

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

Flow Cytometry
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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.

Methodology
Sample preparation Instrument Software Cell population abundance neutrophils after withdrawal of estrogen as described in Lee et al. Sci Immunol. 2017 May 26;2(11):eaam6641.
Cell lines was tested for mycoplasma contamination and result were negative.
No commonly misidentified cell lines were used.
No wild animals were used in this study.
No field-collected samples were used in this study.
Animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) at Brigham and Women's Hospital and Boston Children's Hospital.
1) Children with newly diagnosed systemic juvenile idiopathic arthritis. 8 patients were recruited with median age of 5 years (4 males and 4 females) 2) adult and children diagnosed with macrophage activation syndrome and with bone marrow biopsy specimen available.
Recruitment was completed prior to the studies. All patients with sJIA from the recruitment period were included without additional selection.
Approval for human subject research and waiver of consent were granted by Institutional Review Board (IRB) of Massachusetts General Hospital (Protocol: 2017P000255) and Boston Children's Hospital (Protocol: P00005723).
Whole blood from the mice was collected by the heart puncture or tail incision. Bone marrow cells were obtained from the femurs. For phospho-flow cytometry, bone marrow cells were flushed from the femur with 4% paraformaldehyde. Common myeloid progenitor , granulocyte-monocyte progenitors , common monocyte progenitor, and monocytes from from ubiquitin ERT2-Cre Tsc2 fl/fl mice were sorted by a FACSAriaTM Fusion Cell Sorter (BD).