Titer dynamic analysis of D29 within MTB-infected macrophages and effect on immune function of macrophages

Abstract

The use of mycobacteriophage D29 to treat Mycobacterium tuberculosis (MTB)-infected macrophages results in significant inhibitory activity. This study aims to explore the novel treatment strategy of intracellular mycobacterial infection from the point of view of phages. We investigated the dynamic phagocytosis and elimination of D29 by macrophages, measured the titer of D29 inside and outside MTB within macrophages by fluorescence quantitative PCR, and detected the levels of interleukin 12 (IL-12) and nitric oxide (NO) in the culture supernatants of D29-infected macrophages by ELISA. Results showed that the activity of D29 phagocytosed by macrophages was significantly lower than that of D29 phagocytosed by MTB-infected macrophages. The titer of D29 that infected intracellular MTB ranged from 10(9) pfu to 10(4) pfu. The titer of D29 inside and outside intracellular MTB transiently increased when MTB-infected macrophages were incubated with D29 for 40 and 50 min; then, a large number of D29 were eliminated by macrophages. The levels of IL-12 and NO had no significant differences versus the negative control but were significantly lower compared with the lipopolysaccharide (LPS) positive control. These results suggest D29 has no effect on the immune function of macrophages and that high phage titer must be administered repeatedly if D29 is applied to treat intracellular MTB infection.

Prophylaxis of Mycobacterium tuberculosis H37Rv Infection in a Preclinical Mouse Model via Inhalation of Nebulized Bacteriophage D29

“…Additionally, with daily prophylactic doses, the likelihood that a specific alveolus receives a sufficient number of phages to eradicate intruding bacteria increases. As it may take days for granulomas to form (51), it is possible that phages delivered soon after M. tuberculosis exposure may still offer protection, but this depends on macrophage uptake dynamics (33) and awaits further exploration.…”

“…As more than 80% of TB cases originate from M. tuberculosis infections in the lungs, aerosol delivery of phage may be an ideal mechanism for enabling activity at the primary site of M. tuberculosis infection (32). Note that phage aerosol delivery without the use of additional vectors is unlikely to have efficacy against M. tuberculosis already harbored within a granuloma; even phage infection of M. tuberculosis within a macrophage, the primary target cell of M. tuberculosis, has low efficiency (33). However, prophylactic delivery of phages to the alveoli may allow the phage to infect the mycobacteria before macrophage uptake (17,27,34).…”

Heparin-binding Hemagglutinin of Mycobacterium tuberculosis Is an Inhibitor of Autophagy

“…It is composed of type I and type II pneumocytes, which form a cell layer that provides a barrier function (Lin et al, 1998 ; Chuquimia et al, 2013 ). Increasing evidence implicated that alveolar epithelium, particularly type II pneumocyte, plays an important role in both host cell defense and bacterial dissemination (Xiong et al, 2014 ; Fine-Coulson et al, 2015 ; Ryndak et al, 2015 ). Mycobacterium smegmatis ( MS ) takes advantage of macropinocytosis for entry into epithelial cells, and internalized MS are killed by A549 cells (Garcia-Perez et al, 2008 ).…”

Mycobacteriophages as Potential Therapeutic Agents against Drug-Resistant Tuberculosis

“…Only a few studies detailing the use of mycobacteriophages to eliminate M. tuberculosis complex are available, with limited data regarding the immunological effects of phage therapeutics [138,242]. Although no documented cases of anaphylaxis have been associated to phage treatment in humans, as opposed to drugs [24,243], it is critical to define the exact immunological responses in order to predict the success of new potential phage therapies to treat drug resistant-TB.…”