Foxp3+ Regulatory T Cells in Bone and Hematopoietic Homeostasis

Abstract

The bone represents surprisingly dynamic structures that are subject to constant remodeling by the concerted action of bone-forming osteoblasts and bone-resorbing osteoclasts - two cell subsets of distinct developmental origin that are key in maintaining skeletal integrity throughout life. In general, abnormal bone remodeling due to dysregulated bone resorption and formation is an early event in the manifestation of various human bone diseases, such as osteopetrosis/osteoporosis and arthritis. But bone remodeling is also closely interrelated with lympho-hematopoietic homeostasis, as the bone marrow niche is formed by solid and trabecular bone structures that provide a framework for the long-term maintenance and differentiation of HSCs (>blood lineage cells and osteoclasts) and MSCs (>osteoblasts). Numerous studies in mice and humans have implicated innate and adaptive immune cells in the dynamic regulation of bone homeostasis, but despite considerable clinical relevance, the exact mechanisms of such immuno-bone interplay have remained incompletely understood. This holds particularly true for CD4⁺ regulatory T (Treg) cells expressing the lineage specification factor Foxp3: Foxp3⁺ Treg cells have been shown to play an indispensable role in maintaining immune homeostasis, but may also exert critical non-immune functions, which includes the control of metabolic and regenerative processes, as well as the differentiation of HSCs and function of osteoclasts. Here, we summarize our current knowledge on the T cell/bone interplay, with a particular emphasis on our own efforts to dissect the role of Foxp3⁺ Treg cells in bone and hematopoietic homeostasis, employing experimental settings of gain- and loss-of-Treg cell function. These data make a strong case that Foxp3⁺ Treg cells impinge on lympho-hematopoiesis through indirect mechanisms, i.e., by acting on osteoclast development and function, which translates into changes in niche size. Furthermore, we propose that, besides disorders that involve inflammatory bone loss, the modulation of Foxp3⁺ Treg cell function in vivo may represent a suitable approach to reinstate bone homeostasis in non-autoimmune settings of aberrant bone remodeling.

Keywords: Foxp3+ Treg cells; bone disorders; bone microenvironment; lympho-hematopoiesis; osteoclasts.

Figure 1

Accumulation of Foxp3⁺ Treg cells in the BM. (A) Age-related accumulation of BM-resident Foxp3⁺ Treg cells. Flow cytometry analysis of Foxp3^GFP+ cells within the CD4⁺ T cell population in the BM of wildtype mice of different age groups. Each experiment was performed with at least 3 mice and symbols and lines indicate individual mice and mean values, respectively (young: 4–5 weeks; adult: 8–13 weeks; aged: circles: 20–22 weeks; and squares: 50–70 weeks). (B) Schematic overview of the experimental design. Four-week-old Rag1^−/− mice were adoptively transferred with bulk CD4⁺ T cells (upper plot: Foxp3⁺ Treg cell proportion among total CD4⁺ T cells before adoptively transferred). The distributions of gated Foxp3^DTR−GFP+ cells in various organs of the recipient mice (scLN, subcutaneous lymph nodes; SP, spleen, and BM, bone marrow) were analyzed by flow cytometry 8 weeks after transfer. Numbers in plots indicate the mean percentages ± SD of gated cells within the respective gate. Graph illustrates the accumulation of Foxp3⁺ Treg cells in the BM. Data are collected from two independent experiments with 4–5 mice; symbols and lines denote individual mice and mean values, respectively.

Figure 2

Impact of Foxp3⁺ Treg cell ablation on the BM niche. Foxp3^DTR−GFP mice were i.p. injected with DT on 3 consecutive days and the BM was analyzed at day 6 after the first DT dose by flow cytometry. Representative plots of (A) LSK (Lineage⁻IL7R⁻Sca-1⁺c-Kit⁺) cells and (B) mature/recirculating B cells (B220^highIgM⁺) in the BM of 9-week-old untreated (left) and DT-treated (right) mice.

Figure 3

Identification of a cell population with high in vitro osteoclast differentiation potential. Ly6C and CD11b expression of bead-enriched B220⁻CD3⁻Ter119⁻ BM cells was detected by flow cytometry. Four distinct populations (A: Ly6C^highCD11b^−/low; B: Ly6C⁻CD11b⁻; C: Ly6C^lowCD11b⁺; D: Ly6C⁺CD11b⁺) were FACS-purified and after post-sort analysis in vitro differentiated under osteoclast-forming conditions (cytokines: M-CSF and RANKL). The differentiated cultures were fixed and a TRAP staining was performed to identify mature osteoclasts. Additionally, population A was further subdivided by the expression of c-Kit and c-Fms (A1: c-Kit⁺c-Fms⁻; A2: c-Kit⁻c-Fms⁻; A3: c-Kit⁻c-Fms⁺; A4: c-Kit⁺c-Fms⁺). After FACS-purification and post-sort analysis, the populations were cultured and analyzed for their osteoclastogenic potential.

Figure 4

Detailed characterization of in vitro Foxp3⁺ Treg cell-osteoclast precursor interaction. Representative single cell (upper panels) and cluster (lower panels) analysis by imaging flow cytometry. Stimulated FACS-purified CD4⁺CD25⁺Foxp3^GFP+ Treg cells and freshly isolated irradiated T-cell-depleted splenocytes were added to the BM culture and cultured under osteoclastogenic conditions. After 48 h, Foxp3⁺ Treg cells and CD11b⁺ osteoclast precursors were analyzed for intracellular expression of CD80 and CTLA-4 by imaging flow cytometry. Cell clusters depict physical interaction of CTLA-4⁺Foxp3^GFP+ Treg cells and CD11b⁺ osteoclast precursors. Co-localization of CD80 in Treg cells indicates trans-endocytosis from CD11b⁺ osteoclast precursors. Numbers indicate the interface area and illustrate the overlapping fluorescence of Foxp3 and CD11b.

Figure 5

Hypothesized direct cross-talk between osteoclast precursors and T cells and the indirect effect on hematopoiesis. The expression of the costimulatory molecules CD80 and CD86 on osteoclast precursors has been implicated in the interplay with Foxp3⁺ Treg cells (via the inhibitory molecule CTLA-4), leading to the suppression of the differentiation of osteoclast precursors into mature functional osteoclasts. As a consequence, disturbed bone remodeling results in modulation of the HSC niche. On the other hand, osteoclast precursors are able to upregulate the expression of PD-L1, whose interaction with PD-1 on T cells has immune modulatory functions, thus representing a crucial immune checkpoint. This figure was in part created with modified Servier Medical Art templates, licensed under a Creative Commons Attribution 3.0 Unported license: http://smart.servier.com.