Apoptotic mesenchymal stromal cells induce prostaglandin E2 in monocytes: implications for the monitoring of mesenchymal stromal cell activity

Extensive experience with mesenchymal stromal cells (MSC) in the treatment of inflammatory disorders has provided convincing evidence to support their clinical efficacy in selected cases, especially in steroid-resistant acute graft-versus-host disease (GvHD). Although MSC can induce immunosuppression directly through the secretion of soluble factors and contact-mediated mechanisms, it is becoming clearer that the recruitment of recipient immunoregulatory networks is fundamental for delivering the therapeutic effects of these cells. We recently identified a novel mechanistic pathway by which MSC mediate immunosuppression in GvHD. Following contact with recipient cytotoxic cells, MSC undergo in vivo apoptosis and are efferocytosed by phagocytes which are then induced to upregulate indoleamine 2,3-dioxygenase (IDO). Therefore, to accomplish MSC immunosuppression, patients must have activated cytotoxic cells and competent phagocytes. While the magnitude of MSC apoptosis induced by recipient cytotoxic cells can provide a biomarker predictive of clinical responses to MSC, there are no tools to monitor the immunological effects of MSC following efferocytosis. In order to identify potential molecules for monitoring MSC immunological effects in patients, we investigated the molecular and functional profile of human monocytes exposed to apoptotic MSC (ApoMSC). We isolated CD14 monocytes from the peripheral blood of healthy donors (Online Supplementary Methods) because of their recognized role in engulfing dead MSC. MSC were isolated and expanded as previously described, and induced to undergo apoptosis using antiFas monoclonal antibody (CH11, Merck). ApoMSC were characterized by annexin-V (BD Biosciences) staining and immunoblotting for activated caspase-3 (Cell Signaling) (Online Supplementary Methods and Online Supplementary Figure S1A-C). CellTraceTM Violet (Thermo Fisher Scientific)-labeled (staining concentration: 1 μM) monocytes were cultured with ApoMSC labeled with 20 ng/mL pH-sensitive fluorescent dye pHrodoTM Red (Thermo Fisher Scientific). Efferocytosis was determined by quantitating pHrodoTM Red fluorescence (mean fluorescence intensity, MFI) in monocytes using flow cytometry or CSU-X1 Spinning-Disk Confocal. Confocal data were processed with NIS Elements AR (Nikon) software. After 2 h, monocytes rapidly engulfed ApoMSC but not the non-apoptotic MSC (Online Supplementary Figure S2AB). ApoMSC efferocytosis was enhanced in the presence of lipopolysaccharide (LPS, 100 ng/mL, Sigma-Aldrich) to model inflammation (Figure 1A). When exposure to ApoMSC was extended to 24 h, the magnitude of efferocytosis was increased (Figure 1B,C). Apoptotic cell clearance is generally regarded as immune-silencing, although under particular circumstances, it can elicit immune responses. To examine the immunomodulatory activity of ApoMSC-conditioned monocytes (ApoMSC-Mono), we measured their ability to inhibit CD3/CD28-induced T-cell proliferation. CD3 T cells were isolated from the peripheral blood of healthy donors (Online Supplementary Methods). Monocytes were cultured with ApoMSC for 8 h, and then added to