Establishment and characterization of a hUCMSC model of Mycobacterium smegmatis infection

Abstract:

[Background] The formation of dormant Mycobacterium tuberculosis (Mtb) is considered to be the main cause of latent tuberculosis infection (LTBI), the mechanism of which remains to be studied with in vivo and in vitro models. Mtb can infect mesenchymal stem cells (MSC) and survive in the cells in a dormant state for a long time. However, the modeling of Mtb-infected cells requires long time and high laboratory biosafety level. Therefore, it is essential to explore the available MSC for modeling Mtb infection and further deciphering the dormancy mechanism of Mtb. [Objective] To establish and characterize the human umbilical cord MSC (hUCMSC) model of Mycobacterium smegmatis (Ms) infection. [Methods] After hUCMSC were infected with Ms, flow cytometry was employed to identify the surface antigen. The cell membrane was labeled with DiI and then the phagocytosis of the hUCMSC infected with Ms was observed in a fluorescence microscope. The plate counting method was employed to measure the intracellular survival rate of Ms. The formation of lipid droplets in hUCMSC was observed by oil red O staining. Cytoskeletal changes were observed by fluorescence staining with phalloidin. The transcription levels of the genes involved in the lipid metabolism, resting, and differentiation of the hUCMSC infected with Ms were determined by RT-qPCR. [Results] Ms survived in hUCMSC in a non-replicating state, and the infection rate was positively correlated with the multiplicity of infection. Ms simulated the expression of the genes involved in lipid biosynthesis and inhibited the transcription of the genes involved in lipid degradation to promote the formation of lipid droplets in the Ms-infected hUCMSC. Ms promoted the cytoskeleton remodeling of hUCMSC. The RT-qPCR results showed up-regulated expression of the genes related to proliferation and differentiation, indicating that Ms promoted the differentiation of hUCMSC into lipoblasts and osteoblasts. [Conclusion] We successfully established a hUCMSC model of Ms infection, which could mimic the main characteristics of Mtb dormancy and be used in the study of LTBI mechanism.