During

infection, the ability of macroautophagy to remove large cytoplasmic structures with selectivity enables this pathway to be used to clear intracellular bacteria, parasites, and viruses (i.e., xenophagy) [1, 8, 9]. Several medically important human pathogens are degraded in vitro by xenophagy, including bacteria (e.g., group A streptococcus, Mycobacterium tuberculosis, Shigella flexneri, Navitoclax concentration Salmonella enterica, Listeria monocytogenes, and Francisella tularensis), viruses such as herpes simplex virus type 1 (HSV-1) and chikungunya virus, and parasites such as Toxoplasma gondii[9]. We therefore wondered whether induction of autophagy could affect the growth of E. coli in infected HMrSV5 cells. We found that stimulation of autophagy by LPS in infected HMrSV5 cells could lead to degradation of E. coli within autophagosomes. Furthermore, we observed that 3-MA or Wm blockade of autophagy markedly attenuated the co-localization of E. coli with autophagosomes, leading to a defect in bactericidal activity. To more specifically determine whether autophagy affect the elimination of E.coli, Beclin-1 siRNA was employed to inhibit autophagy. As expected, fewer E.coli were targeted to the autophagosomes, and consequently more remaining

E.coli were observed in cells deficient in Beclin-1. Taken together, these data demonstrated that the effect of LPS on bactericidal Everolimus ic50 activity was dependent on the induction of autophagy. LPS is the ligand for TLR4, and it also exerts multiple cellular

effects by inducing signaling through TLR4 [10]. The activation of TLR4 by LPS in peritoneal mesothelial cells might result in a massive influx of leukocytes in the peritoneal cavity, leading to the development of peritoneal dysfunction or peritoneal fibrosis [28]. It was demonstrated that TLR4 served as a previously unrecognized environmental sensor for autophagy [10]. Therefore we further investigated whether TLR4 played roles in LPS-induced autophagy in HMrSV5 cells. Our results showed that the LPS treatment increased the expression of TLR4 protein significantly in a dose-dependent and time-dependent way. ROS1 Moreover, the increased expression of TLR4 protein occurred earlier than the increase of LC3-II protein. Pretreated with PMB, a TLR4 inhibitor, displayed defective autophagy activation as indicated by the significantly decreased expression of both Beclin-1 and LC3-II protein as well as the decreased GFP–LC3 aggregation in cells. Consistent with the pharmacological inhibition of TLR4, knockdown of TLR4 with TLR4 siRNA also led to reduction of autophagy-associated proteins. Importantly, LPS-induced bactericidal activity in HMrSV5 cells was significantly decreased after knockdown of TLR4. To sum up, these results demonstrated that upregulation of autophagic response by LPS was dependent on TLR4 signaling in HMrSV5 cells.