Abstract
Background Mesenchymal stromal cells (MSCs) are a heterogeneous cell population endowed with multi-lineage differentiation potential and extensive immunomodulatory properties. MSCs have been successfully used for prevention and treatment of immune disorders such as graft-versus-host disease. Emerging preclinical studies suggest that MSCs might also protect against infectious challenge.
Aims This study aimed to rule out the potential mechanism of human MSCs against Toxoplasma gondii (T. gondii).
Methods Human bone marrow-derived MSCs (hMSCs) were pretreated for 24h with a series of concentrations of IFN-γ and then infected with T. gondii strains of variant virulences (virulent RH and avirulent ME49). RNA-seq and westernblots were used to analyze gene and protein expression patterns of hMSCs in IFN-γ-stimulated and unstimulated conditions. The intracellular parasites (with fluorescence labeled) were counted microscopically at multiple time points postinfection. The short hairpin RNA (shRNA) expression was used to generate RNAi of GBP-1, GBP-2 and GBP-5.
Results Human MSCs stimulated with IFN-γ were capable to inhibit the growth of T. gondii (eg: at IFN-γ 10ng/ml, the inhibition rates are 26.5% (RH) and 37.5% (ME49) 12hr postinfection) in a dose-dependent manner. Compared with the unstimulated MSCs (controls), IFN-γ treatment at 5, 10, 20ng/ml inhibited T. gondii (ME49) growth by percent of 27.1±7.9, 37.5±6.2, 47.0±7.6 (mean±SD, n=4) 12 hr postinfection and the inhibition rates are 54.5±2.1%, 62.5±4.9% and 78.5±2.1 at 24 hr postinfection, respectively. After 48 hr postinfection, the ratio between parasites per parasitophorous vacuole (PV) containing rosettes and single paraites in IFN-γ-stimulated MSCs was significantly reduced compared with that in the unstimulated MSCs (p<0.01, p<0.01, p<0.001 for ME49 at IFN-γ 5, 10, 20ng/ml, respectively). Furthermore, There was no significant effect of conditioned medium (CM) from IFN-γ-stimulated MSCs on T. gondii growth in comparison with CM from unstimulated MSCs (p=0.74 for RH and p=0.69 for ME49). We observed that the resistance in hMSCs does not depend on IDO (p=0.85 for RH and p=0.79 for ME49). RNA-seq data showed that IFN-γ-inducible p65 guanylate-binding proteins (GBPs) might play pivotal roles in the inhibition of T. gondii growth. Reads per kilobase-pairs per million (RPKM) mean values of GBP1, 2, 5 in IFN-γ-stimulated MSCs are 1093.3, 443.3, 348.2, respectively. By RNAi knockdown, the results showed that silencing of GBP1 (but not GBP2, GBP5) in hMSCs resulted in recovery of T. gondii growth inhibition at 12 hr and 24 hr postinfection (p<0.05 and p<0.001 for ME49).
Conclusion: Human MSCs pre-stimulated with IFN-γ inhibited the growth of T. gondii in a dose-dependent manner via up-regulation of GBP-1 expression.
Disclosures
Liu: the project of the Zhujiang Science & Technology Star of Guangzhou city (2013027): Research Funding; the Technology Plan of Guangdong Province of China (2012B031800403): Research Funding; the project of health collaborative innovation of Guangzhou city (201400000003-4, 201400000003-1): Research Funding; Natural Science Foundation of Guangdong Province (S2012010009299): Research Funding; National Public Health Grand Research Foundation (201202017): Research Funding; National High Technology Research and Development Program of China (863 Program) (2011AA020105): Research Funding; National Natural Science Foundation of China (81270647, 81300445, 81200388): Research Funding.
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