Effector memory regulatory T cells were most effective at suppressing RANKL but their frequency was downregulated in tibial fracture patients with delayed union.

Delayed union and nonunion occur in a minor subject of bone fractures, presenting ongoing challenges to treatment. RANKL, which promotes the differentiation of bone-resorbing osteoclasts, is thought to negatively impact bone healing. In this study, we recruited patients with isolated closed tibial fracture, who were later categorized into normal healing and delayed healing groups based on their healing progression. The regulatory T cell (Treg) compartment was then investigated in each patient. Based on CD45RA and CD62L expression, we distinguished circulating Treg cells into CD45RA+CD62L+ naive (N), CD45RA-CD62L+ central memory (CM), and CD45RA-CD62L- effector memory (EM) subsets. Compared to normal patients, delayed patients presented significantly lower EM Treg proportion and significantly higher N Treg proportion. Among the N, EM, and CM Treg cells, the EM Treg cells were the most potent at suppressing RANKL expression in T conventional (Tconv) cells. This functionality of EM Treg cells was present in both normal healing patients and delayed healing patients, and was dependent on IL-10, as neutralization of IL-10 resulted in significantly elevated RANKL expression. EM Treg cells presented the highest IL-10 and TGF-β expression directly ex vivo, as well as after anti-CD3/anti-CD28/IL-2 stimulation. CM Treg cells did not present high expression of inhibitory cytokines ex vivo, but was capable of upregulating cytokine expression upon stimulation. N Treg cells, on the other hand, presented limited capacity to upregulate inhibitory cytokines. In summary, our study identified that, while the EM Treg cells were the most effective at suppressing RANKL, they in delayed union patients were present at lower frequencies with functional impairment, resulting in decreased RANKL suppression. Hence, bone-resorbing osteoclast formation may be favored in these patients thus suggesting a possible mechanism for delayed bone healing.