FRI0275 In vivo treatment with hydroxichloroquine interferes with ex vivo expansion of mesenchymal stromal cells in a murine model of systemic lupus erythematosus
Authors: S. Vagnani, C. Tani, L. Carli, F. Querci, R. Talarico, C. Baldini, A. Della Rossa, S. Bombardieri, M. Mosca
Journal: Annals of the Rheumatic Diseases (2013)

<h3>Background</h3> Mesenchymal stromal cells (MSCs) are now considered a new therapeutic option for SLE patients refractory to conventional therapies. MSCs autologous transplantation showed less therapeutic efficacy than the allogenic one and morphologic and functional defects observed in MSCs from SLE patients are considered the main cause. However, it has also been postulated that other patient-related factors could interfere with MSC functions and, among these, underlying therapies could represent an important issue to be considered. <h3>Objectives</h3> The present study was designed to explore whether an <i>in vivo</i> treatment with Hydroxichloroquine (HCQ) might interfere with bone marrow MSC frequency and <i>ex vivo</i> expansion in a murine model of SLE. <h3>Methods</h3> Ten NZB/WF1 mice (NZ) were daily treated with oral hydroxychloroquine (3 mg/Kg body weight) starting from 12 weeks of age and compared to 10 age-matched untreated NZ mice. The mice were observed until sacrifice at 30 weeks of age. At sacrifice, bone marrow was harvested from tibias and femurs from each mouse and mononuclear cells were plated at the same density of 300000 cell/cm<sup>2</sup>. Bone-marrow derived MSCs (bmMSCs) were isolated by plastic adherence and expanded in standard medium for murine MSC culture. Clonogenic potential was evaluated by colony-forming-unit-fibroblasts- assay (CFU-f) and population doubling (PD) analysis was performed to evaluate the culture growth curve. To confirm the mesenchymal nature of the cells obtained, differentiation tests (osteogenic and adipogenic) as well as immunophenotyping were performed; the cell sorting analysis evaluated the expression of the following surface marker antigens: Sca-1, CD90.2, CD45, CD106, CD34. <h3>Results</h3> The mean CFU frequency 7 and 14 days after seeding resulted 4.5 x10<sup>-7</sup> (±2.3 x10<sup>-7</sup>) and 27x10<sup>-7</sup> (±9.1 x 10<sup>-7</sup>) respectively in treated mice and 16 x10<sup>-7</sup>(±3.3 x10<sup>-7</sup>) and 38 x 10 <sup>-7</sup>(±14 x 10<sup>-7</sup>) in untreated mice; in treated mice CFU frequency was significantly lower at 7 days (p=0.01) while no statistically significant differences were observed at 14 days (p=0.4). As expressed by linear regression analysis, the cumulative PD was significantly lower in treated mice and the growth curve low steep (y=0.0423x-0.2565, r<sup>2</sup>=0.9 in treated and y=0,1023x-9.7 R² = 0,9 in untreated mice, respectively). Moreover, while in untreated mice the growth curve demonstrated exponential rate until 240 days after seeding (for a cumulative PD of 14), in treated mice the growth curve tended to be flatter and reached the plateau around 130 days after seeding (for a cumulative PD of 6). No differences have been observed between groups in surface marker antigen expression or in differentiation capabilities. <h3>Conclusions</h3> Hydroxichloroqine is frequently used in SLE as background treatment both in inactive and active disease. Our data show that an <i>in vivo</i> treatment with hydroxichloroquine for 18 weeks in NZ mice altered the frequency and clonogenicity of <i>ex vivo</i> expanded bmMSC and the proliferation rate. If confirmed in future studies, this observation might suggest an important issue to be considered when evaluating the functional properties of bmMSC from patients with systemic autoimmune diseases under treatment with hydroxichloroquine. <b>Disclosure of Interest</b>: None Declared