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 Foxp(3+) 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 Foxp(3+) 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-gamma, transforming growth factor-beta, 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. Treg 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.