ABSTRACT
To investigate the effects and mechanisms of Mycobacterium avium MAV-5183 protein on apoptosis in mouse Ana-1 macrophages. A pET-21a-MAV-5183 recombinant plasmid was constructed. The recombinant MAV-5183 protein was cloned, expressed, purified, and identified using an anti-His-tagged antibody. Rabbits were immunized to obtain antiserum, and its potency and immunoreactivity were assessed through WB. Mouse Ana-1 macrophages were incubated with varying concentrations of MAV-5183 protein. Flow cytometry, following ANNEXIN V-FITC/PI double staining, detected apoptosis. Western Blot analysis was conducted to identify apoptosis-related molecules Caspase-9/8/3 and vesicle-related molecules ASC, NLRP3, and Cleaved-casp1. ELISA measured TNF-α and IL-6 levels in the culture supernatant. LDH activity and ROS levels were analyzed separately. RT-qPCR measured mRNA levels of Caspase-9/8/3, ASC, NLRP3, Caspase-1, IL-1ß, Bax, MAPK-p38, Bcl-2, TNF-α, and IL-6. MAV-5183 protein was successfully cloned, purified, and identified. In in vitro studies on Ana-1 macrophages, MAV-5183 protein increased the expression of Caspase-9/8/3, ASC, NLRP3 (P < 0.01), induced ROS secretion (P < 0.05), and promoted inflammatory cytokine secretion (TNF-α, IL-6, P < 0.0001); however, it did not significantly affect LDH (P > 0.05). MAV-5183 also induced apoptosis in Ana-1 macrophages (P < 0.05). RT-qPCR results indicated a significant increase in mRNA expression of Caspase-9/8/3, ASC, NLRP3, TNF-α, IL-6, MAPK-p38, and pro-apoptotic factor Bax (P < 0.01), with no significant effect on Bcl-2 and IL-1ß mRNA (P > 0.05). The data indicate that MAV-5183 induces macrophage apoptosis through a caspase-dependent pathway and promotes inflammatory cytokine secretion via ROS.
ABSTRACT
Murine Natural Killer cells were cultivated in vitro to isolate NK-derived exosomes. Subsequent quantification via qPCR confirmed enrichment of miR-1249-3p. Ana-1 murine macrophages were cultured in vitro and subsequently inoculated with Mycobacterium tuberculosis (MTB) strain H37Rv. NK-exo and NK-exo miR-1249-3p were separately applied to the infection model, followed by immunological assays conducted post-48-hour co-culture. Western blot analyses corroborated that NK-exo exhibited exosomal marker proteins Granzyme A (GzmA), Granzyme B (GzmB), and Perforin (PFN), alongside a notable enrichment of miR-1249-3p. Functionally, NK-exo augmented the expression levels of Caspase-9,-8, and -3, as well as PARP, while attenuating the expression of NLRP3, ASC, and Cleaved-Caspase-1. Furthermore, qPCR demonstrated an up-regulation of Caspase-9, -8, and -3, along with pro-apoptotic factors Bax and Bid, and a concomitant down-regulation of the anti-apoptotic factor Bcl-2. The expression levels of inflammatory markers ASC, NLRP3, Cleaved-Caspase-1, and IL-1ß were concomitantly decreased. ELISA findings indicated diminished levels of TNF-α and ROS secretion. NK-exo miR-1249-3p specifically targeted and attenuated the expression of SKOR-1, engendering up-regulation of apoptosis-associated proteins and down-regulation of inflammation-related proteins, consequently affecting cellular fate.Our empirical evidence substantiates that NK-exo induces macrophage apoptosis, thereby mitigating MTB survival. Furthermore, NK-exo miR-1249-3p directly targets and inhibits SKOR-1 expression, leading to macrophage apoptosis and consequently hampering the proliferation of MTB. The data implicate the potential therapeutic relevance of NK-exo and miR-1249-3p in managing drug-resistant tuberculosis.
