Spatial Transcriptomics of Joint-Draining Popliteal Lymph Nodes from Wild-type and TNF-Tg Mice with Early and Advanced Inflammatory-Erosive Arthritis

Lymphatic dysfunction is an integral aspect of rheumatoid arthritis (RA) pathogenesis. During "Early" arthritis in tumor necrosis factor transgenic (TNF-Tg) mice, the joint-draining popliteal lymph nodes (PLNs) dramatically expand in volume. After a prolonged expansion phase, at ~8-months-old the PLNs stochastically collapse with reduced bone volume and associated onset of "Advanced" inflammatory-erosive arthritis in affected joints. Through this pathologic process, B-cells are known to translocate into the PLN sinuses. Bulk tissue approaches (i.e. bulk genomics, flow cytometry, and ex vivo cell cultures) have been insufficient to specifically evaluate the transcriptional changes of B-cells within the sinuses of TNF-Tg PLNs. Thus, we utilized spatial transcriptomics technology to evaluate transcriptional changes in sinus B-cells and the functional genomic relationships with arthritic severity. Overall design: A single Gene Expression Slide (10X Genomics) was used to evaluate PLNs for spatial transcriptomics with 3 different groups represented on the 4 capture areas: 1) 5.5-month-old wild-type (n = 5 mice, 10 PLNs), 2) 5.5-month-old TNF-Tg with Early arthritis (n = 3 mice, 6 PLNs), and 8.5-9-month-old TNF-Tg with Advanced arthritis (2 replicates with n = 3 mice, 6 PLNs per replicate). Only male mice were used for the study due to early mortality of TNF-Tg females, and all mice were on a C57BL/6 genetic background. Standard protocols from the Visium Methanol Fixation and H&E Staining Guide and Visium Spatial Gene Expression Kit (10X Genomics) were performed.