Extracellular vesicles released by J774A.1 macrophages reduce the bacterial load in macrophages and in an experimental mouse model of tuberculosis

Abstract

Background

Tuberculosis is the leading cause of death by an infectious microorganism worldwide. Conventional treatment lasts at least six months and has adverse effects; therefore, it is important to find therapeutic alternatives that reduce the bacterial load and may reduce the treatment duration. The immune response against tuberculosis can be modulated by several mechanisms, including extracellular vesicles (EVs), which are nano-sized membrane-bound structures that constitute an efficient communication mechanism among immune cells.

Methods

The EVs released by the J774A.1 mouse macrophage cell line, both spontaneously (S-EV) and after infection with Mycobacterium tuberculosis H37Rv (Mtb-EV), were purified by ultra-centrifugation and size-exclusion chromatography. The size distribution and chemical composition of these EVs were evaluated, and their effect on the bacterial load and the production of cytokines was determined in both in vitro and in vivo models of M. tuberculosis infection.

Results

Mtb-EV are larger than S-EV, they contain M. tuberculosis-specific antigens (not detected in EVs released from M. fortuitum-infected J774A.1 cells) and are rich in phosphatidylserine, present in their outer membrane layer. S-EV, but not Mtb-EV, reduced the bacterial load and the production of MCP-1 and TNF-α in M. tuberculosis-infected macrophages, and these effects were reversed when phosphatidylserine was blocked with annexin V. Both S-EV and Mtb-EV significantly reduced the lung bacterial load in mice infected with M. tuberculosis after 60 days of treatment, but they had no effect on survival or on the lung pneumonic area of these mice.

Conclusion

J774A.1 macrophages infected with M. tuberculosis H37Rv released EVs that differed in size and phosphatidylserine content from spontaneously released EVs, and these EVs also had different biological effects: S-EV reduced the mycobacterial load and the cytokine production in vitro (through a phosphatidylserine-dependent mechanism), while both EVs reduced the lung bacterial load in vivo. These results are the basis for further experiments to evaluate whether EVs improve the efficiency of the conventional treatment for tuberculosis.

Keywords:

• tuberculosis
• extracellular vesicles
• phosphatidylserine
• macrophages
• lung disease

Supplementary material

Figure S1 EVs are stable at 4 ºC for a week. EVs were purified from J774A.1 cells (S-EV), from J774A.1 cells that had been infected with M. fortuitum for 4 h (Mf-EV) or from J774A.1 cells that had been infected with M. tuberculosis H37Rv for 4 h (Mtb-EV) and stored at 4 ºC for one or two weeks. The proteins were purified with trichloroacetic acid and loaded into a 5/12% polyacrylamide gel, which was visualized with silver staining

Acknowledgments

We thank Raúl Borja-Urbi from Centro de Nanociencias, Micro y Nanotecnologías, Instituto Politécnico Nacional, for his assistance in the use of the JEM-2100 microscope. We thank M. Teresa Horn for English language copy-editing. Funding was provided by Consejo Nacional de Ciencia y Tecnología (CONACYT grant 221002to IEG and FC 2015-1/115 to RHP) and by Secretaría de Investigación y Posgrado (SIP), Instituto Politécnico Nacional (IPN). This work was partially carried out with equipment of “Laboratorio Nacional para Servicios Especializados de Investigación, Desarrollo e Innovación (I+D+i) para Farmoquímicos y Biotecnológicos”, LANSEIDI-FarBiotec-CONACyT, which is part of Unidad de Desarrollo e Investigación en Bioprocesos (UDIBI)-IPN. MGM, LVF, and VDAJ were recipients of CONACYT fellowships. MIH, LEST, SEP, RCS, JSL and IEG are fellows of the Comisión de Operación y Fomento de Actividades Académicas–IPN. MIH, LEST, SEP, RCS, JSL, IWB and IEG are fellows of Estímulo al Desempeño de los Investigadores–IPN.

Disclosure

The authors report no conflicts of interest in this work.