Distinct cellular and spatial niches within the inflamed synovium of childhood arthritis [bulk RNA-Seq]

recision use of targeted therapies is urgently needed to improve long-term clinical outcomes for children affected by inflammatory arthritis, known as Juvenile Idiopathic Arthritis. Progress has been obstructed by a lack of understanding of the cellular basis of joint inflammation in children, given the difficulties in obtaining and studying synovial tissue itself. To this end, we combine single-cell RNA-sequencing, multiplexed immunofluorescence imaging and spatial transcriptomics to define the cellular and transcriptomic landscape of the synovium in children with Juvenile Idiopathic Arthritis. We identify spatial niches of resident and infiltrating cell populations that correlate with the degree of inflammation, and gene programs associated with arthritis severity. Combined with analyses of synovial fluid and peripheral blood from the same children, we distinguish differences in cellular composition, signalling pathways and transcriptional programs across anatomical compartments. Whilst we identify several pathogenic populations shared with adult-onset arthritis, our analyses highlight increased vascularity of the inflamed developing joint and TGFb-driven stromal subsets that upregulate expression of disease risk-associated genes. Overall, these findings illustrate the need for treatment algorithms informed by a tissue-based classification of arthritis. Overall design: RNA was extracted using ARCTURUS® PicoPure® RNA Isolation Kit (Thermo Fisher Scientific) according to the manufacturers' instructions. Library prep was completed using Novogene NGS RNA Library Prep Set and sequenced on a NovaSeq 6000 or NovaSeq X Plus using Illumina PE-150 strategy. Sequenced reads were aligned to the GRCh38 human genome using Bowtie2 (v2.4.4). PCR duplicates were removed using SMAtools (v1.15.1). Count matrices were generated using Subread (v2.0.1).