Autofluorescence contributes to false-positive intracellular Foxp3 staining in macrophages: A lesson learned from flow cytometry

• Published in: Journal of immunological methods 2012-12, Vol.386 (1-2), p.101-107
• Main Authors: Li, Fuqiang, Yang, Mingjie, Wang, Lai, Williamson, Ian, Tian, Fang, Qin, Minghui, Shah, Prediman K., Sharifi, Behrooz G.
• Format: Article
• Language: English
• Subjects: animal models; Animals; antibodies; Autofluorescent; bone marrow; bone marrow cells; cell culture; Cell Separation; Cells, Cultured; Disease Models, Animal; False Positive Reactions; Flow cytometry; Flow Cytometry - methods; fluorescence microscopy; Forkhead Transcription Factors - genetics; Forkhead Transcription Factors - metabolism; Foxp3 transcription factor; gene expression; Humans; hyperlipidemia; Hyperlipidemias - diagnosis; Hyperlipidemias - immunology; liver; lymph nodes; Macrophage; macrophages; Macrophages - immunology; Macrophages - metabolism; messenger RNA; Mice; Mice, Inbred C57BL; Mice, Knockout; Optical Imaging; protein synthesis; quantitative polymerase chain reaction; RNA, Messenger - analysis; spleen; splenocytes
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2012.08.014

Forkhead box P3 (Foxp3) is well known for its highly restricted expression in T regulatory cells (Tregs). A recent study suggested the existence of a Foxp3 positive macrophage subpopulation in mouse bone marrow, spleen, liver, lymph nodes, and thymus that exhibited immune regulatory effect similar to Tregs. Before this report was retracted, we attempted to study the function of this macrophage subpopulation in a mouse model of hyperlipidemia. Bone marrow and spleen cells isolated from C57BL/6 apo E−/− mice were stained with anti-CD11b, anti-F4/80 and anti-Foxp3 and analyzed by flow cytometry. Our results showed that 3.06–8.08% of CD11b+F4/80+ macrophages from bone marrow cells and 0.24–2.21% from splenic were Foxp3-positive. Unexpectedly, unstained or isotype stained controls also showed strong autofluorescence and similar percentages of these cells fell within the same FL1 channel that counted the anti-Foxp3 stained population. Back gating of the autofluorescent population onto a SSC/FSC plot showed that this population of cells had a higher side scatter. The peritoneal macrophages (PMø) exhibited similar autofluorescence. We used qPCR to further evaluate the expression of Foxp3 mRNA in PMø that were treated with M-CSF, M-CSF+IL-4, M-CSF+TGFβ1 or in BMDM treated with TGFβ1 in the presence of anti-CD3 and CD28 antibody co-stimulators. No expression of Foxp3 mRNA was detected in either cell culture systems, whereas robust Foxp3 gene expression was induced in naïve CD4+ cells stimulated with TGFβ1. Consistent with these findings, fluorescence microscopy showed no Foxp3 protein expression in PMø, however Foxp3 expression was easily detected in induced Tregs. We conclude that the reported expression of Foxp3 in macrophages is likely an artifact and that a stringent multimodality approach is critical to demonstrate candidate gene expression in any cell type. ► Foxp3 has been reported to be expressed in macrophages. ► We determined the macrophage phenotype in bone marrow and spleen of apo E−/− mice. ► Flow cytometry analysis showed that the Foxp3-positive macrophages are an artifact. ► qPCR analysis confirmed flow cytometry data ► Foxp3 expression in macrophages is an artifact of autofluorescence.