TLR9 Priming Promotes Proliferation Of Mesenchymal Stem Cells and Restores The Immunosuppressive Activity Impaired By TLR4 Priming: Potential Involvement Of Non-Canonical NF-Kb Signaling

Mesenchymal stem cells (MSC) represent a promising cell-based therapy for Graft Versus Host Disease (GVHD), for they can suppress the proliferation of activated lymphocytes in vitro, through either direct contact or secretion of anti-inflammatory cytokines. However, clinical trials performed in the past decade evaluating the effectiveness of infused MSC to improve the outcome of GVHD patients had variable results so far. Since the presence of associated infections are frequent in patients with GVHD, and given that priming to different Toll-like receptors (TLR) may elicit opposite immunosuppressory proprieties in MSC, in this study we investigated the modulation of the immune suppressive potential of bone marrow MSC (BM-MSC) by different TLR ligands. Thereby, BM-MSC (n=3) were primed for 24h with LPS (1µg/ml) and/or the CpG oligonucleotide CpG ODN DSP30 (1µM), to stimulate the TLR4 and TLR9, respectively. After that, peripheral blood CD3+ lymphocytes were labeled with the fluorescent dye CFSE, activated with antibody-loaded beads anti CD2, CD3 and CD28, and cocultured for 5 days with treated or untreated BM-MSC. Suppression of lymphocyte proliferation, by BM-MSC in different conditions, was evaluated by flow cytometry. As compared to untreated control MSC, the immunosuppressive capacity of TLR4 primed BM-MSC was inhibited by 30% (p<0.05), while that of TLR9 primed BM-MSC was enhanced by 32% (p<0.05). As a result, simultaneous priming of MSC with both ligands kept immunosuppression at levels comparable to that of untreated BM-MSC. In order to identify the possible mechanisms involved in this modulatory activity, BM-MSC (n=3) in same conditions were evaluated for changes in proliferation, using an assay of EdU incorporation into DNA and by direct cell count of fluorescently labeled cells, using an automated High Content Screening (HCS) imaging device. As results, we observed that TLR9 priming significantly promoted the proliferation of BM-MSC, as demonstrated by higher (p<0.05) EdU incorporation and cell count; while TLR4 priming had no effect in these parameters. Since priming by TLR4 and TLR9 may lead to the respective activation of canonical and non-canonical NF-kB pathways (which are mediated by the transcription factors RelA and RelB, respectively); we used chromatin immunoprecipitation (ChIP) to evaluate the binding of RelA and RelB to the gene promoter of the vascular cell adhesion molecule-1 (VCAM-1) in BM-MSC (n=3) primed of TLR4 and/or TLR9. In addition, expression of RelA or RelB in BM-MSC (n=3) in the same conditions, was quantified using a HCS device. Priming of TLR4 led, exclusively, to the binding of RelA, (three fold enrichment, compared to unprimed BM-MSC). Moreover, simultaneous priming of TLR4 and TLR9 resulted in an increased binding of both RelA and RelB. Finally, increased staining of RelB was also verified by HCS, in simultaneously primed with ligands for TLR4 and TLR9. Altogether, our results indicates that BM-MSC had its immunosuppressory capacity hampered by priming of TLR4 by LPS (possibly due to the activation of canonical NF-Kb pathway), while TLR9 priming by CpG oligonucleotide enhances the immune suppression, which may be related to an increase in proliferation and, possibly, due to the activation of non-canonical NF-Kb pathway in MSC simultaneously primed for TLR4 and TLR9. These observations may help to elucidate the cellular and molecular mechanisms underlying the heterogeneous results in clinical studies of GVHD patients receiving BM-MSC infusions. As patients subjected to bone marrow transplantation are more prompt to infections by different pathogens (potentially exposing BM-MSC to different TLR ligands), the identification of the CpG oligonucleotide as a TLR9 ligand capable of enhancing and protecting the immunosuppressive activity of BM-MSC, may lead to the establishment of a laboratory protocol aiming the priming to TLR9, or the specific activation of non-canonical NF-Kb pathways, in order to boost BM-MSC efficiency in clinical settings aiming at the treatment of GVHD patients.