Soybean lectin-induced autophagy follows P2RX7 activated NF-κB-ROS pathway to kill intracellular mycobacteria

Abstract

Host-directed therapy has now been adopted as a novel anti TB strategy to strangulate the ever threatening global tuberculosis (TB) burden. In past few years, autophagy is one of such strategy that has been manipulated through various inducers to curtail the intracellular Mycobacterium tuberculosis (M. tuberculosis) survival. Amongst various inducers, triggering autophagy through natural compounds is a treasure resource whose potential can be harnessed for better therapeutics against TB. In the present study, we investigated the antimycobacterial role of soybean lectin (SBL), a lectin isolated from Glycine max (Soybean) and mechanistic interplay of autophagy in this response. We first performed time kinetic experiment with the non-cytotoxic dose of SBL (20 µg/ml) in PMA differentiated THP-1 (dTHP-1) cells and observed optimum expression of major autophagic markers like autophagy related genes (Atg) 7, Atg 3 and elevated LC3 puncta formation after 24 h of treatment. We further validated this autophagy induction through MDC staining where SBL treated cells showed more accumulation of autophagosomes in comparison to the control cells. Abrogation of autophagy in the presence of 3-MA and increase in LC3 puncta formation upon Baf-A1 addition clearly elucidated the specific effect on autophagy and autophagic flux in SBL treated dTHP-1 cells. SBL treatment also led to autophagy induction in mycobacteria (M. smegmatis and M. bovis BCG) infected macrophages that restrained the intracellular mycobacterial growth thus emphasizing the host defensive role of SBL induced autophagy. To understand the mechanism of antimycobacterial effect through autophagy, we found increased P2RX7 expression, NF-κB activation and reactive oxygen species (ROS) generation in dTHP-1 cells upon SBL treatment. Inhibition of P2RX7 expression either by KN-62 (P2RX7 antagonist) or P2RX7 siRNA obstructed p65 nuclear translocation thereby suppressing NF-κB dependent ROS level in SBL treated dTHP-1 cells. Moreover, SBL induced autophagy was abrogated in the presence of different inhibitors like KN-62/NF-κB inhibitor/N-acetyl cysteine (NAC) and P2RX7 siRNA leading to more survival of intracellular mycobacteria. Taken together, these results conclude that SBL induced autophagy exerts antimycobacterial effect in P2RX7-NF-κB dependent manner through ROS generation.