Expansion of Regulatory T Cells Promotes the Clearance of Apoptotic Cells by Macrophages in Murine Atherosclerotic Plaques
Authors: Proto Jonathan D , Sozen Erdi , Subramanian Manikandan , Tabas Ira 
Published: November 2023

A key process in inflammation resolution is efferocytosis, the clearance of apoptotic cells (AC) by phagocytes. Defective efferocytosis has been implicated in diseases characterized by chronic, nonresolving inflammation, including atherosclerosis. In atherosclerotic plaques, the accumulation of uncleared apoptotic cells leads to necrotic core formation, thereby increasing plaque instability, likelihood of rupture, and risk for acute cardiovascular events such as heart attack and stroke. Expansion of regulatory T cells (Tregs) in murine atherosclerosis decreases aortic root lesion area through mechanisms that are not fully understood. Given that Treg coordination of macrophage function has been reported, we hypothesized that expansion of the Treg compartment during advanced atherosclerosis promotes lesional efferocytosis. First, we used zymosan induced peritonitis as a proof‐of‐concept model to interrogate how Tregs may affect the efferocytic capacity of macrophages. Using mice in which the human diphtheria toxin (DT) receptor sequence is knocked into the 3′ UTR of the Treg lineage‐specific gene, Foxp3, we used DT to deplete Tregs in the days following injection with 0.1 mg Zymosan. Twelve days after injury, mice were ip injected with fluorescently labelled apoptotic neutrophils. After 45 minutes, the peritoneal cavity was lavaged and efferocytosis was measured by flow cytometry. Interestingly, uptake of labelled AC by macrophages in Treg‐depleted mice was significantly reduced compared to PBS‐injected control mice (30% vs 52%, p<0.05). Similarly, when assayed ex vivo, efferocytosis was again found to be defective in macrophages harvested from Treg‐depleted mice (p<0.05). Next, to determine whether Tregs could promote efferocytosis in atherosclerotic lesions, low density lipoprotein receptor deficient (Ldlr−/−) mice were placed on a western diet for a total of 15 weeks. Starting at week 12, mice were treated with with an IL‐2/anti‐IL2 antibody complex (IL2C) that is known to specifically expand Foxp3+ regulatory T cells. After three weeks of treatment, Tregs were significantly increased in the spleen and blood of treated, but not control, mice. We investigated efferocytosis in aortic root lesions by immunofluorescence staining of tissue sections to identify the association of macrophages (F4/80+) and apoptotic cells (TUNEL+). Quantification of the ratio of macrophage‐associated to “free” AC revealed that efferocytosis was significantly increased in aortic root lesions of IL2C treated mice (p<0.05). Interleukin‐10 has been reported to be elevated during Treg expansion in athero‐prone mice and is known to be a Treg effector molecule in other settings. To identify whether IL‐10 is an important player in the promotion of efferocytosis during Treg expansion, we treated mice with an IL‐10 neutralizing antibody or IgG during the expansion period. Strikingly, IL‐10 neutralization abrogated the improvement in lesional efferocytosis without affecting Treg expansion. Cumulatively, these data demonstrate a positive relationship between Foxp3+ Tregs and efferocytosis. Future investigations aim to elucidate the signaling mechanism through which this occurs.Support or Funding InformationThis work was supported in part by a T32 training grant from the NIH (HL007343)