Lung Angiogenesis requires CD4⁺ forkhead homeobox protein-3⁺ regulatory T cells

Angiogenesis in ischemic organs is modulated by immune cells. Systemic neovascularization of the ischemic lung requires macrophages, with chemokines playing a central role in new vessel growth. Because regulatory T (T_reg) cells modulate tumor-induced neovascularization, we questioned whether this CD4⁺ lymphocyte subset impacts blood vessel growth during ischemia. In a model of left lung ischemia, an increase in CD4⁺ CD25⁺ forkhead homeobox protein-3 (Foxp3)⁺ cells was observed 3-5 days after the onset of ischemia in wild-type C57Bl/6 mice. Using transgenic mice where Foxp3⁺ T_reg cells can be depleted with diphtheria toxin (DT; Foxp3^DTR), we unexpectedly found that Foxp3⁺ T_reg depletion led to markedly reduced lung angiogenesis (90% reduction from Foxp3^gfp controls). Adoptive transfer studies using CD4⁺ CD25⁺ splenocytes from congenic CD45.1 mice into Foxp3⁺T_reg-depleted mice showed an almost complete recovery of the angiogenic phenotype (80% of Foxp3^gfp controls). A survey of lung gene expression of angiogenic (lipopolysaccharide-induced CXC chemokine [LIX], IL-6, IL-17) and angiostatic (IFN-γ, transforming growth factor-β, IL-10) cytokines showed T_reg-dependent differences only in LIX (CXCL5) and IL-6. Protein confirmation demonstrated a significant reduction in LIX in T_reg-deficient mice compared with controls 5 days after the onset of ischemia. Phenotyping other inflammatory cells in the lung by multicolor flow cytometry demonstrated a significantly reduced number of macrophages (major histocombatibility complex class II [MHCII]^int, CD11C⁺) in T_reg-deficient lungs compared with T_reg-sufficient lungs. T_reg cells are essential for maximal systemic angiogenesis after pulmonary ischemia. One likely mechanism responsible for the decrease in angiogenesis in T_reg-depleted mice was the decline in the essential CXC chemokine, LIX.