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RNA-seq of lymph node CD4+ cells from WT mice and Pggt1bfl/flLys-Cre mice.

ABSTRACT: Conditional knockout of protein geranylgeranyltransferase type I (GGTase-I) in macrophages (GLC) activates RHO-GTPases and causes arthritis in mice. Knocking out Rag1 in GLC mice alleviates arthritis which indicates that lymphocytes are required for arthritis development in those mice. To study GLC dependent changes in the adaptive immunity, we isolated CD4+ T cells from GLC mice (CD4+ GLCs). Spleen and joint draining lymph nodes (dLN) CD4+ GLCs exhibited high expression of Cdc42 and Rac1, which repressed the caudal HOXA proteins and activated the mechanosensory complex to facilitate migration. These Cdc42/Rac1 rich CD4+ GLCs presented a complete signature of GARP+NRP1+IKZF2+FOXp3+ thymic Tregs. Activation of the ß-catenin/Lef1 axis in CD4+ GLCs contributed to a pro-inflammatory Th1 phenotype of Tregs, which was strongly associated with arthritis severity. Knockout of Cdc42 in macrophages of GLC mice affected CD4+ cell biology and development enlarging population of non-thymic Tregs. Knockout of Rac1 and RhoA had no such effects on CD4+ cells although it alleviated arthritis in GLC mice. Disrupting macrophage and T cell interaction with CTLA4 fusion protein reduced the Th1-driven inflammation and enrichment of thymic Tregs into dLNs. Antigen challenge reinforced the CD4+ GLC phenotype in non-arthritic heterozygote GLC mice and increased accumulation of RHO protein–expressing thymic Tregs in dLNs. Our study demonstrates an unexpected role of macrophages in stimulating the development of pro-inflammatory thymic Tregs and reveal activation of RHO-proteins behind their arthritogenic phenotype.

ORGANISM(S): Mus musculus

PROVIDER: GSE190881 | GEO | 2021/12/16

REPOSITORIES: GEO

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Project description:The mechanism of egress of mature regulatory T cells (Tregs) from the thymus to the periphery remains enigmatic, as does the nature of those factors expressed in the thymic environment. Here, we examined the fate of thymic Tregs in TNFÎ±/RelA double-knockout (TA-KO) mice, because TA-KO mice retain a Treg population in the thymus but have only a small Treg population at the periphery. Transplantation of whole TA-KO thymus to under the kidney capsule of Rag1 null mice failed to induce the production of donor-derived splenic Tregs expressing neuropilin-1 (Nrp1), which was reported to be a marker of naturally occurring Tregs, indicating that TA-KO thymic Tregs either do not leave the thymus or are lost at the periphery. We next transplanted enriched TA-KO thymic Tregs to the peripheries of TA-KO mice and traced mouse survival. Transplantation of TA-KO thymic Tregs rescued the lethality in TA-KO mice, demonstrating that TA-KO thymic Tregs remain functional at the periphery. The TA-KO thymic Treg population had highly demethylated CpG motifs in the foxp3 locus, indicating that the cells were arrested at a late-mature stage. Also, the population included a large subpopulation of Tregs expressing IL-7RÎ±, which is a possible marker of late-mature Tregs. Finally, TA-KO fetal liver chimeric mice developed an Nrp1+ splenic Treg population from TA-KO cells, suggesting that Treg arrest is caused by a lack of RelA in the thymic environment. Together, these results suggest that egress of mature Tregs from the thymus depends on RelA in the thymic environment. For the isolation of thymic Tregs, CD4+CD8Î±-CD25hi thymocytes were isolated from five 1.5- to 2-week-old TNFÎ±-KO or TA-KO mice by using a FACSAria cell sorter. For the isolation of thymic stromal cells, 10 thymi from 1.5- to 2-week-old TNFÎ±-KO or TA-KO mice were minced with scissors and treated with RPMI 1640 supplemented with 2% FCS, 0.2 mg/ml collagenase (Roche, Basel, Switzerland), 0.2 mg/ml dispase I (Roche), and 100 U/ml DNase I (Life Technologies) for 30 min with stirring. Digested thymi were centrifuged in a Percoll (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) gradient (density, 1.115, 1.065, and PBS) at 1400g for 30 min. Cells in the upper layer were collected, and the CD45-EpCAM+ (thymic epithelial cells) and CD45+EpCAM- populations (enriched thymic stromal cells containing macrophages or dendritic cells) were sorted.

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RNA-seq of lymph node CD4+ cells from WT mice and Pggt1bfl/flLys-Cre mice.

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