Specific TLR-mediated HSP70 activation plays a potential role in host defense against the intestinal parasite Giardia duodenalis

Abstract

Giardia duodenalis, an important flagellated noninvasive protozoan parasite, infects the upper small intestine and causes a disease termed giardiasis globally. Few members of the heat shock protein (HSP) family have been shown to function as potential defenders against microbial pathogens, while such information is lacking for Giardia. Here we initially screened and indicated that in vitro Giardia challenge induced a marked early upregulation of HSP70 in intestinal epithelial cells (IECs). As noted previously, apoptotic resistance, nitric oxide (NO)-dependent cytostatic effect and parasite clearance, and epithelial barrier integrity represent effective anti-Giardia host defense mechanisms. We then explored the function of HSP70 in modulating apoptosis, NO release, and tight junction (TJ) protein levels in Giardia-IEC interactions. HSP70 inhibition by quercetin promoted Giardia-induced IEC apoptosis, viability decrease, NO release reduction, and ZO-1 and occludin downregulation, while the agonist celastrol could reverse these Giardia-evoked effects. The results demonstrated that HSP70 played a previously unrecognized and important role in regulating anti-Giardia host defense via attenuating apoptosis, promoting cell survival, and maintaining NO and TJ levels. Owing to the significance of apoptotic resistance among those defense-related factors mentioned earlier, we then elucidated the anti-apoptotic mechanism of HSP70. It was evident that HSP70 could negatively regulate apoptosis in an intrinsic way via direct inhibition of Apaf-1 or ROS-Bax/Bcl-2-Apaf-1 axis, and in an extrinsic way via cIAP2-mediated inhibition of RIP1 activity. Most importantly, it was confirmed that HSP70 exerted its host defense function by downregulating apoptosis via Toll-like receptor 4 (TLR4) activation, upregulating NO release via TLR4/TLR2 activation, and upregulating TJ protein expression via TLR2 activation. HSP70 represented a checkpoint regulator providing the crucial link between specific TLR activation and anti-Giardia host defense responses. Strikingly, independent of the checkpoint role of HSP70, TLR4 activation was proven to downregulate TJ protein expression, and TLR2 activation to accelerate apoptosis. Altogether, this study identified HSP70 as a potentially vital defender against Giardia, and revealed its correlation with specific TLR activation. The clinical importance of HSP70 has been extensively demonstrated, while its role as an effective therapeutic target in human giardiasis remains elusive and thus needs to be further clarified.

ROS-AMPK/mTOR-dependent enterocyte autophagy is involved in the regulation of Giardia infection-related tight junction protein and nitric oxide levels

“…Autophagy and apoptosis are biologically linked as discussed previously ( 80 ). Although our recent studies have demonstrated Giardia -induced TNFR1-mediated extrinsic and ROS-mediated mitochondrial pathways of apoptosis in Caco-2 and HT29 cells ( 43 , 44 , 48 ), the interplay between autophagy and apoptosis in IECs, notably primary epithelial cells, during noninvasive Giardia infections is still unknown and is not elucidated under the present circumtances, and therefore needs to be further investigated.…”

“…Giardia -induced early-stage autophagy was then biologically linked to TJ/NO regulation in IECs, and this link was proven to be closely associated with ROS-dependent AMPK/mTOR signaling. Although in vitro models of interaction between Giardia and IECs (Caco-2 and HT29) has also been widely applied previously ( 46 , 48 , 56 – 59 ), the present observations need to be further clarified by in vivo studies. However, effective in vivo models of Giardia infection by WB isolate are still lacking since modeling human giardiasis in animals is complicated and difficult.…”

“…After cooling in an ice bath, the parasites were harvested and washed, counted by a hemocytometer, diluted to the designated amount using cell culture medium, and used to challenge cells at a ratio of 10 parasites/cell for the indicated time periods as noted ( 43 – 48 ), although other challenge ratios (5 or 20 parasites/cell) have been applied before ( 49 , 50 ). In time-course experiments, the trophozoites were added at different time points and cells harvested together.…”