Assessing the Ability of Auxotrophic Mycobacterium tuberculosis Strains to Trigger Trained Innate Immunity in Mouse Macrophages

Overview

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is once again the world's deadliest infectious disease, after being replaced by COVID-19 for three years. The only approved vaccine for TB, the Bacillus Calmette-Guérin (BCG) vaccine, shows limited efficacy in adults, but promising alternatives are auxotrophic strains of M. tuberculosis; they are avirulent, nonlethal, and BSL-2 approved. However, the ability of auxotrophs of M. tuberculosis to trigger trained innate immunity (TII), a key feature of BCG vaccination, has not, until this study, been researched. TII is a heightening of innate immune cell inflammatory response following heterologous stimuli after training by a primary stimulus. I assessed the ability of auxotrophic M. tuberculosis strains mc27901, mc27902, and mc28755 as well as the fast-growing M. smegmatis mc2155 to trigger TII in wildtype and Stat3-/- RAW 264.7 mouse macrophages. Cells were trained with mycobacterial strains or BCG for 24 hours, rested for six days, and restimulated with lipopolysaccharide or lipoteichoic acid. Cytokine expression (IL-6, IL-10, TNF-?) was analyzed via RT-qPCR following TRIzol RNA extraction. I found mc27902 trained WT macrophages to elicit ~3 times greater IL-6 upregulation compared to BCG training with no IL-10 upregulation, indicating mc27902 can trigger TII with a greater pro-inflammatory cytokine response. Additionally, Stat3-/- macrophages displayed less IL-6 and IL-10 upregulation but similar TNF-? responses compared to WT. Surprisingly, STAT3 loss increased mc27902-trained IL-6 and IL-10 upregulation, but had the opposite effect in BCG, suggesting different TII induction mechanisms. These results underscore mc27902's potential to be a valid and superior BCG replacement.