The critical role of autophagy in cadmium-induced immunosuppression regulated by endoplasmic reticulum stress-mediated calpain activation in RAW264.7 mouse monocytes.

Cadmium (Cd) has toxic and suppressive effects on the immune system, but the underlying mechanisms remain poorly understood. Here, we show that autophagy plays a critical role in regulation of Cd-induced immunosuppression in RAW264.7 cells. Cd decreased cell viability in a dose-dependent manner; cleaved caspase-8, caspase-3, and poly (ADP-ribose) polymerase (PARP)-1; increased DNA laddering; induced CCAAT-enhancer-binding protein homologous protein (CHOP); and reduced tumor necrosis factor (TNF)-α expression; indicating that caspase-dependent and endoplasmic reticulum (ER)-mediated apoptosis are involved in Cd-induced immunotoxicity. Furthermore, Cd induced autophagy, as demonstrated by microtubule-associated protein 1 light chain 3B (LC3B) plasmid DNA transfection and its conversion from LC3-I to the LC3-II form by autophagy inhibitors, via AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) signaling. Pharmacological and genetic inhibition of autophagy suppressed Cd-induced apoptosis, as evidenced by inhibition of caspase-8, caspase-3, and PARP-1 cleavage, indicating that autophagy promotes apoptosis. The pan-caspase inhibitor zVAD inhibited Cd-induced apoptosis, but increased autophagy and decreased cell viability, indicating that autophagy can compensate for reduced apoptotic cell death. Calpain inhibitors blocked Cd-induced apoptosis and autophagy, indicating that calpain plays a critical role in Cd cytotoxicity. Treatment with Ca2+ chelators completely recovered Cd-induced cell viability and inhibited Cd-induced apoptosis and autophagy. Treatment with N-acetyl-l-cysteine (NAC) suppressed Cd-induced antioxidant enzyme levels, apoptosis, and autophagy. Collectively, Cd-induced oxidative stress triggers ER stress, leading to Ca2+-dependent calpain activation and subsequent activation of autophagy and apoptosis, resulting in immune suppression.