Chloroquine modulates the redox-sensitive signalling via inhibiting the AMPK-ULK1 under LPS induced state in murine splenic macrophages

Chloroquine (CQ) is known for anti-inflammatory and autophagy-inhibitory properties; however, its impact on endotoxin-induced macrophage response remains unclear. This study aims to investigate how CQ acts as a signalling modulator in the oxidative stress-autophagy-apoptosis axis in presence of LPS and how the modulation of TLR4 and NLRP3 affects that axis in murine splenic macrophages. The isolated macrophages were pretreated with TLR4 Ab and NLRP3 inhibitor along with CQ for 30 min and further for an additional 60 min after LPS addition. From the in vitro stimulated cells several functional and cellular parameters along with expression of key signalling protein markers were analysed. Result shows that CQ administration in presence of LPS enhances the intracellular ROS which may contribute to alteration of AMPK-ULK1 signalling. The downregulation of pAMPK-pULK1 confirms that CQ + LPS is associated with alterations in upstream autophagy-regulatory signalling markers and consistent with impaired autophagy-associated processing. Additionally, the accumulation of p62 along with LC3-II cautiously acts as a critical indicator of autophagosome degradation inhibition in response to CQ and LPS. The NLRP3 activation, p62 accumulation further corroborate with the Bax and cleaved caspase 3 overexpression in CQ and LPS administered cells which confirm the apoptosis induction. Restoration of pAMPK-pULK1 reduces the mTOR level and restarts the autophagic phenomenon in the case of dual inhibition of TLR4 and NLRP3 in presence of CQ, LPS. The blocking of TLR4 and NLRP3 is able to mitigate the inflammation by restarting the autophagy and limiting apoptosis in murine splenic macrophages.