Soonchunhyang University College of Medicine Bongmyeong-dong, Dongnam-gu, Cheonan-si, South Korea
Sung Hae Chang1, Jeongjun Choe2, Seon Uk Kim3, Jeong Yeon Kim4, Sung Won Lee5, Jeong Seok Lee6 and Eun Young Lee4, 1Soonchunhyang University College of Medicine, Bongmyeong-dong, Dongnam-gu, Cheonan-si, Republic of Korea, 2Sungkyunkwan University, Seoul, Republic of Korea, 3Seoul National University College of Medicine, Jongno-gu, Seoul, Republic of Korea, 4Seoul National University College of Medicine, Seoul, Republic of Korea, 5Sooncheonhyang University College of Medicine, Seoul, Republic of Korea, 6Korea Advanced Institute of Science and Technology, KAIST, Graduate school of Medical Science and Engineering, Daejeon, Republic of Korea
Background/Purpose: Rheumatoid arthritis-associated interstitial lung disease (RA-ILD) is one of the pivotal extrapulmonary conditions. However, the pathophysiology of RA-ILD, including the effect of disease-modifying anti-rheumatoid drugs (DMARDs) is largely unknown. To study the effect of methotrexate and TNFα inhibitor on lung microenvironment of SKG mice using single-nucleus RNA sequencing.
Methods: Male SKG mice of eight to ten weeks received a single intraperitoneal zymosan A (ZyA) injection to induce arthritis and pneumonitis. We gave them twice-weekly intraperitoneal injections of PBS, methotrexate (7.5 mg/kg; MilliporeSigma), or TNFα inhibitor (100 μg/kg; R&D Systems, Inc., Minneapolis, MN, USA). Nuclei were prepared from frozen lung tissue under ribonuclease-free conditions by a method adapted from an existing protocol.
Results: Macroscopically, pneumonitis was most evident in SKG mice with ZyA and methotrexate treatment. A total of 59,860 nuclei were obtained from sixteen mice, four mice from each of the four groups. We classified each cell type using previous reports on the single-nucleus cells of mouse lungs. The most frequently observed cell was the type 2 alveolar (AT2) cell among all four groups. Alveolar epithelial cells were further subclustered into six clusters. Cluster 2 highly expressed type 1 alveolar (AT1) cell marker genes. Interestingly, a distinct cluster 3 was observed after methotrexate treatment. This distinctive alveolar epithelial cell cluster (cluster 3) showed IL-1, TNF, IFNα, and IFNγ perturbation-response signatures using Library of Integrated Network-Based Cellular Signatures (LINCS) 1000 ligand perturbation analysis. In addition, enrichment analysis revealed that cluster 3, as well as cluster 2 (i.e., AT1 cells) significantly enriched genes of BMI1 knockout mice that fail to self-renew their lung cells after damage.
Conclusion: The current study shows that methotrexate exacerbates lung inflammation via attenuation of the regenerative potential of AT2 cells. Disclosures: S. Chang, None; J. Choe, None; S. Kim, None; J. Kim, None; S. Lee, None; J. Lee, None; E. Lee, None.
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