Our results indicate that donor MI-induced inflammatory response leads to the therapeutic impairment of BMCs, making them less able to prevent a decline in cardiac function when implanted into post-MI recipient hearts. We and others have demonstrated that BMCs from healthy donor animals exert a therapeutic effect (3,4,9,23,24
). However, in human autologous BMC therapy clinical trials (6,7
), in which the donor BMCs and recipient heart belong to the same individual, the effect of MI on the (donor) BMCs cannot be distinguished from the effect of MI on the recipient heart. Notably, the experiments described here with inbred mice used different animals as donors and recipients, which enabled us to vary the disease state of the donors while keeping the recipient conditions constant throughout. As a result, any differences between groups were entirely due to differences in the donor
BMCs, a situation that cannot be assessed in patients receiving autologous cells because they are their own donors. Our results reveal that one reason that clinical autologous cell trials have shown less obvious therapeutic effects than most rodent experiments may be that the BMCs used in the human acute MI trials are impaired by the post-MI condition of the patients.
Literature suggests that chronic disease conditions can cause reductions in functional properties of cells from the bone marrow and peripheral blood (10,25,26
). However, the acute nature of the BMC response to donor MI is unlikely to be explained thoroughly by these functional declines. Because MI is a highly inflammatory event that occurs rapidly and causes inflammatory changes in the bone marrow (20,21,27
), we hypothesized that these inflammatory changes underlie the reduction in BMC therapeutic efficacy. It is well-known that acute MI activates innate immune mechanisms immediately initiating the host inflammatory response that increases the number or activation state of inflammatory cells in the circulation and the infarcted heart itself (13
), as well as distant sites such as the spleen (28
). Moreover, donor bone marrow on day 3 post-MI showed several characteristics consistent with an inflammatory reaction. Notably, there is a consistent correlation between the time courses post-MI of impairment and subsequent restoration of BMC therapeutic efficacy, appearance of inflammatory infiltrate in the heart, and elevation and subsequent reduction in levels of many pro-inflammatory cytokines, which supports a link between the inflammatory response and the therapeutic changes in the BMCs ().
Moreover, the therapeutic impairment induced by donor MI is prevented by injection of either a broad-spectrum anti-inflammatory drug or an IL-1 inhibitor into the infarcted donor mice, which suggests the existence of an inflammatory cytokine signaling link between the infarcted heart and the bone marrow. Following MI, dying myocytes trigger an exuberant systemic and loco-regional inflammatory response through activating the nuclear factor-κB system, which induces activation and upregulation of chemokines, as well as release of many pro-inflammatory cytokines such as IL-1β, IL-6, and tumor necrosis factor-α. IL-1 signaling is known to play a significant role in initiating and regulating the inflammatory response post-MI (13
). Therefore, based on our finding that administration of exogenous IL-1 receptor antagonist (IL-1Ra) to the infarcted donor mice prevents the therapeutic impairment of BMCs and reduces the IL-6 serum level of the infarcted donors, we conclude that MI-induced IL-1-mediated inflammatory response leads to pro-inflammatory changes in donor bone marrow composition and reduced BMC therapeutic efficacy. A caveat to this experiment is that measurement of cytokines in serum rather than plasma can be confounded by cytokines released during the clotting process, so absolute cytokine levels as measured may be inaccurate. However, the relative change in levels over time normalized to the healthy baseline value clearly shows the expected increase and decrease of members of the IL-1/6 pathway in agreement with several published reports (29-31
). Because the more general anti-inflammatory drug dexamethasone is more effective than IL-1Ra in preventing the BMC impairment, other pathways may also play a role. It should be emphasized that we do not propose anti-inflammatory drug treatment as a clinical solution due to known dangers of anti-inflammatory drugs in post-MI patients (32
), but these experiments clearly illuminate a mechanistic link between inflammation and the reduced BMC efficacy.
This might result in an increased number or activation state of inflammatory cells among the BMCs that get implanted into the infarcted recipient heart. In support of this reasoning, the day 3 post-MI BMCs include more Ly6Chigh
inflammatory myeloid cells. Ly6Chigh
monocytes are reported to be the pro-inflammatory arm of the monocyte response to MI (33
), and might impair healing when implanted into the infarcted myocardium. The decrease in MHC-II expression was also intriguing. This has been reported in post-MI human patients (19
) and would be consistent with the increase in the immunosuppressive cytokine IL-10 observed at day 3 in post-MI mice. Alternatively, local signals from the MI that trigger the egress of potentially beneficial cells from the bone marrow could also reduce therapeutic efficacy of the remaining BMCs. Accordingly, while most cytokines peaked on day 3 post-MI, a small number of cytokines induced by MI such as G-CSF and MCP-1 showed an increase beginning on day 1. G-CSF and MCP-1 have the potential to enhance cell migration and have been reported to mediate mobilization of bone marrow stem cells in experimental studies (34-36
) and clinical trials (37
). Hence, it is possible that the BMCs on day 3 post-MI are less therapeutic due to mobilization of stem cells and other cells away from the bone marrow. A third possibility is that, because we transfer a constant total number of cells, the local increase in inflammatory cells may dilute, out-compete, or even suppress the beneficial cells. While it is currently unclear which of these explanations, which are not mutually exclusive, predominates in the decreased efficacy of post-MI BMCs, it is clear that the composition of implanted BMCs is altered by the MI-induced inflammatory response. Future studies will focus on elucidating the exact changes that occur within this cellular compartment and how each of those changes influence the therapeutic effects of the implanted BMCs.
In summary, we have shown that acute MI results in specific changes in the bone marrow, such that implantation of BMCs from these mice into infarcted hearts of other mice is less able to prevent the decline in cardiac function. The impairment in therapeutic efficacy caused by donor MI correlates well with the development and resolution of the acute inflammatory response, and is prevented by injection of IL-1Ra to the donor MI mice. Based on these results, we propose that the donor MI increases the inflammatory state of the BMCs, which alters their composition and/or activation state to leave them less therapeutic. Of clinical relevance, it has been reported in human trials that autologous BMC therapy is more effective if performed several days after acute MI (6,7
), but our findings suggest that the optimal time for post-MI harvest of autologous BMCs may be different than the optimal time for the administration of such BMCs to the heart. Optimal therapy may require either that one consideration be prioritized over the other, or that steps are taken to prevent these changes in the bone marrow.