IL-1β–driven osteoclastogenic Tregs accelerate bone erosion in arthritis

Purpose: The goal of this study is to evaluate Il1rn-/- Tregs features by bulk RNA sequencing (RNA-seq) of Tregs from WT and Il1rn-/- lymph nodes. Methods: mRNA profiles of isolated CD4+Foxp3eGFP+ from lymph nodes of three 35-day-old wild-type (WT) and three Il1rn-/- mice were generated by deep sequencing. RNA was extracted from sorted cells (5X10^4) using a Qiagen RNeasy Micro Kit. RNA-seq was performed using the Smart-Seq2 platform. Smart-Seq2 libraries were prepared by the Broad Technology Labs and sequenced by the Joint Biology Consortium– associated Broad Genomics Platform. Transcripts were quantified by the Broad computational pipeline using Cuffquant version 2.2.1. Results: WT and Il1rn-/- Tregs showed similar expression of multiple genes associated with suppressor function, including Foxp3, Il2ra, Il7r, and Ebi3 (a component of the suppressive cytokine IL-35), and transcription factors including E_o_m_e_s_, Tbx21 (encoding Tbet), and Irf4. Tregs from WT and Il1rn-/- _mice showed similar expression of genes encoding molecules involved in Treg localization and trafficking, including CXCR6, CXCR3, and CCR10. However, gene set enrichment analysis (GSEA) identified enhanced expression of genes encoding proteins involved in fatty acid metabolism (for example, NTHL1 and GABARAPL1) in Il1rn-/- Tregs and oxidative phosphorylation (OxPhos; for example, ATP5H and NDUFB3) in WT Tregs. Conclusions: Il1rn-/- Foxp3+ Tregs exhibited greater glycolytic capacity and reserve that WT Tregs. This finding is consistent with the superiority of Il1rn-/- Tregs with respect to suppression of T cell proliferation, a capacity associated with glycolysis Lymph node mRNA profiles of 35-day old wild type (WT) and Il1rn-/- mice