Abstract
Introduction
Immune homeostasis requires effective host defense while preventing excessive inflammation and immune-mediated tissue damage. Regulatory T-cells (Tregs) preserve this balance through a Foxp3-directed suppressive program, yet inflammatory stress threatens their lineage stability. Although bone morphogenetic protein (BMP) signaling influences immune cell differentiation, the role of BMP receptor type 1A (BMPR1α) in maintaining mature Treg identity remains unknown. We hypothesized that BMPR1α signaling preserves Treg lineage stability by limiting proinflammatory reprogramming.
Methods
To investigate BMPR1α function in Tregs, we generated mice with tamoxifen-inducible, Treg-specific deletion of Bmpr1a; wild-type (WT) littermates served as controls. Lymph node CD4⁺ T-cells from both genotypes were phenotyped by flow cytometry before adoptive transfer. Yellow fluorescent protein (YFP) permanently marked cells expressing Foxp3 during tamoxifen treatment, whereas green fluorescent protein (GFP) identified current Foxp3 expression. This approach distinguished YFP⁺GFP⁻ former Tregs, YFP⁺GFP⁺ lineage-traced Tregs retaining Foxp3, and YFP⁻GFP⁺ newly generated Tregs. Activation status and cytokine-receptor expression were compared between genotypes across all three populations. Recipients are monitored for weight loss, after which donor-derived Tregs will be recovered from lymph nodes and reanalyzed for Foxp3 stability, activation, and cytokine-receptor expression. In parallel, bulk and single-cell RNA sequencing and assay for transposase-accessible chromatin with sequencing (ATAC-seq) analyses are in progress to characterize BMPR1α-dependent molecular changes under homeostatic and inflammatory conditions.
Results
Preliminary analyses indicated enhanced IL-18Rα/IL-1 signaling following BMPR1α loss, consistent with activation of inflammatory pathways capable of promoting Treg effector reprogramming. Before adoptive transfer, flow-cytometric analysis revealed increased proportions of activated cells relative to naïve cells across all three fate-mapped populations, YFP⁺GFP⁻, YFP⁺GFP⁺, and YFP⁻GFP⁺, in BMPR1α-deficient mice compared with their WT counterparts. Enhanced IL-18Rα/IL-1 signaling provides a potential mechanistic basis for these activation changes. Because YFP permanently records prior Foxp3 expression, whereas GFP reflects current Foxp3 expression, the concordant findings suggest that BMPR1α deficiency affects multiple Treg fate states, including former Tregs that have lost Foxp3, lineage-traced Tregs that retain Foxp3, and newly generated Tregs. We are comparing expression of cytokine and chemokine-receptors modulating inflammatory responses of treg cells between genotypes within each population in BMPR1α-deficient cells across all three fate states. Together, these findings suggest that BMPR1α deletion broadly alters Treg activation and cytokine-receptor profiles rather than selectively affecting a single fate-defined population. Adoptive-transfer experiments are underway to determine how BMPR1α-deficient Tregs respond to a Treg-depleted inflammatory environment, while bulk RNA-seq, single-cell RNA-seq, and ATAC-seq analyses are in progress to characterize the associated transcriptional and chromatin-accessibility changes related to the genetic reprogramming of Tregs.
Conclusion
These preliminary findings associate BMPR1α deficiency to decreased phenotypic stability Treg cell population and altered genetic reprogramming in response to inflammation. Defining how BMPR1α signaling regulates Treg stability during inflammation may uncover molecular mechanisms that could be targeted to develop new therapeutic strategies to alter Treg suppressor functions in autoimmune and chronic inflammatory diseases.