The Journal of Experimental Medicine

Carbon monoxide (CO), a byproduct of heme catabolism by heme oxygenase (HO), confers potent antiinflammatory effects. Here we demonstrate that CO derived from HO-1 inhibited Toll-like receptor (TLR) 2, 4, 5, and 9 signaling, but not TLR3-dependent signaling, in macrophages. Ligand-mediated receptor trafficking to lipid rafts represents an early event in signal initiation of immune cells. Trafficking of TLR4 to lipid rafts in response to LPS was reactive oxygen species (ROS) dependent because it was inhibited by diphenylene iodonium, an inhibitor of NADPH oxidase, and in gp91 phox -deficient macrophages. CO selectively inhibited ligand-induced recruitment of TLR4 to lipid rafts, which was also associated with the inhibition of ligand-induced ROS production in macrophages. TLR3 did not translocate to lipid rafts by polyinosine-polycytidylic acid (poly(I:C)). CO had no effect on poly(I:C)-induced ROS production and TLR3 signaling. The inhibitory effect of CO on TLR-induced cytokine production was abolished in gp91 phox -deficient macrophages, also indicating a role for NADPH oxidase. CO attenuated LPS-induced NADPH oxidase activity in vitro, potentially by binding to gp91 phox . Thus, CO negatively controlled TLR signaling pathways by inhibiting translocation of TLR to lipid rafts through suppression of NADPH oxidase–dependent ROS generation. Footnotes Abbreviations used: CO, carbon monoxide; CTx, cholera toxin; DPI, diphenylene iodonium; GM1, glycosphingolipid 1; HO, heme oxygenase; IP-10, IFN-γ–inducible protein 10; IRF-3, IFN regulatory factor 3; MAPK, mitogen-activated protein kinase; MβCD, methyl-β-cyclodextrin; MDC, monodansylcadaverine; NAC, N -acetyl- l -cysteine; PAMP, pathogen-associated molecular pattern; poly(I:C), polyinosine-polycytidylic acid; RANTES, regulated upon activation, normal T expressed, and presumably secreted; ROS, reactive oxygen species; TLR, Toll-like receptor; TRAF, TNF receptor–associated factor; TRIF, TIR domain–containing adaptor-inducing IFN-β. K. Nakahira and H.P. Kim contributed equally to this work. Submitted: 21 April 2006 Accepted: 31 August 2006

doi: 10.1084/jem.20061469pmid: 16954371

Cellular cardiomyoplasty is an attractive option for the treatment of severe heart failure. It is, however, still unclear and controversial which is the most promising cell source. Therefore, we investigated and examined the fate and functional impact of bone marrow (BM) cells and embryonic stem cell (ES cell)–derived cardiomyocytes after transplantation into the infarcted mouse heart. This proved particularly challenging for the ES cells, as their enrichment into cardiomyocytes and their long-term engraftment and tumorigenicity are still poorly understood. We generated transgenic ES cells expressing puromycin resistance and enhanced green fluorescent protein cassettes under control of a cardiac-specific promoter. Puromycin selection resulted in a highly purified (>99%) cardiomyocyte population, and the yield of cardiomyocytes increased 6–10-fold because of induction of proliferation on purification. Long-term engraftment (4–5 months) was observed when co-transplanting selected ES cell–derived cardiomyocytes and fibroblasts into the injured heart of syngeneic mice, and no teratoma formation was found ( n = 60). Although transplantation of ES cell–derived cardiomyocytes improved heart function, BM cells had no positive effects. Furthermore, no contribution of BM cells to cardiac, endothelial, or smooth muscle neogenesis was detected. Hence, our results demonstrate that ES-based cell therapy is a promising approach for the treatment of impaired myocardial function and provides better results than BM-derived cells. Footnotes Abbreviations used: α-MHC, α–myosin heavy chain; ASMAC, α–smooth muscle actin; EB, embryoid body; EGFP, enhanced GFP; ES cell, embryonic stem cell; LCA, left coronary artery; LVEF, left ventricular ejection fraction; LVF, left ventricular function; MEA, microelectrode array; PECAM, platelet/endothelial cell adhesion molecule. E. Kolossov, T. Bostani, W. Roell, and M. Breitbach contributed equally to this work. Submitted: 11 July 2006 Accepted: 10 August 2006