The protozoan parasite Giardia duodenalis inhabits the upper small intestine of mammals including humans and causes a disease known as giardiasis, which can lead to diarrhea, abdominal cramps, and bloating. G. duodenalis was known as a causative factor of intestinal epithelial cell (IEC) apoptosis. Cyclooxygenase-2 (COX-2) has been identified as an influencing factor of pathogen infection by participating in immune response, while its role in host defense against Giardia infection is not clear. Here we initially observed the involvement of COX-2 in the regulation of Giardia -induced IEC apoptosis. Inhibition of COX-2 activity could promote Giardia - induced reduction of IEC viability, increase of reactive oxygen species (ROS) production, and decrease of nitric oxide (NO) release, which would exacerbate IEC apoptosis. In addition, during Giardia -IEC interactions, COX-2 inhibition was able to accelerate caspase-3 activation and PARP cleavage, and inhibit the expressions of some anti-apoptotic proteins like cIAP-2 and survivin. In contrast, COX-2 over-expression could reduce Giardia -induced IEC apoptosis. We further investigated the regulatory mechanisms affecting COX-2 expression in terms of anti-apoptosis. The results showed that p38/ERK/AKT/NF-κB signaling could regulate COX-2-mediated ROS/NO production and anti-IEC apoptosis during Giardia infection. We also found that COX-2-mediated anti-IEC apoptosis induced by Giardia was related to TLR4-dependent activation of p38-NF-κB signaling. Collectively, this study identified COX-2 as a promoter for apoptotic resistance during Giardia -IEC interactions and determined the potential regulators, furthering our knowledge of anti- Giardia host defense mechanism.

The extracellular protozoan parasite Giardia duodenalis is a well-known and important causative agent of diarrhea on a global scale. Macrophage pyroptosis has been recognized as an important innate immune effector mechanism against intracellular pathogens. Yet, the effects of noninvasive Giardia infection on macrophage pyroptosis and the associated molecular triggers and regulators remain poorly defined. Here we initially observed that NLRP3 inflammasome-mediated pyroptosis was activated in Giardia-treated macrophages, and inhibition of ROS, NLRP3, or caspase-1 could block GSDMD cleavage, IL-1β, IL-18 and LDH release, and the cell viability reduction. We also confirmed that Giardia-induced NLRP3 inflammasome activation was involved in its K63 deubiquitination. Thus, six candidate deubiquitinases were screened, among which A20 was identified as an effective regulator. We then screened TLRs on macrophage membranes and found that upon stimulation TLR4 was tightly correlated to ROS enhancement, A20-mediated NLRP3 deubiquitination, and pyroptotic signaling. In addition, several Giardia-secreted proteins were predicted as trigger factors via secretome analysis, of which peptidyl-prolyl cis-trans isomerase B (PPIB) independently induced macrophage pyroptosis. This was similar to the findings from the trophozoite treatment, and also led to the TLR4-mediated activation of NLRP3 through K63 deubiquitination by A20. Collectively, the results of this study have significant implications for expanding our understanding of host defense mechanisms after infection with G. duodenalis.

Giardia duodenalis, the causative agent of giardiasis, is among the most important causes of waterborne diarrheal diseases around the world. Giardia infection may persist over extended periods with intestinal inflammation, although minimal. Cyclooxygenase (COX)-2 is well known as an important inducer of inflammatory response, while the role it played in noninvasive Giardia infection remains elusive. Here we investigated the regulatory function of COX-2 in Giardia-induced pro-inflammatory response and defense-related nitric oxide (NO) generation in macrophage-like cell line, and identified the potential regulators. We initially found that Giardia challenge induced up-regulation of IL-1β, IL-6, TNF-α, prostaglandin (PG) E2, and COX-2 in macrophages, and pretreatment of the cells with COX-2 inhibitor NS398 reduced expressions of those pro-inflammatory factors. It was also observed that COX-2 inhibition could attenuate the up-regulated NO release and inducible NO synthase (iNOS) expression induced by Giardia. We further confirmed that Giardia-induced COX-2 up-regulation was mediated by the phosphorylation of p38 and ERK1/2 MAPKs and NF-κB. In addition, inhibition of reactive oxygen species (ROS) by NAC was shown to repress Giardia-induced activation of MAPK/NF-κB signaling, up-regulation of COX-2 and iNOS, increased levels of PGE2 and NO release, and up-expressions of IL-1β, IL-6, and TNF-α. Collectively, in this study, we revealed a critical role of COX-2 in modulating pro-inflammatory response and defense-related NO production in Giardia-macrophage interactions, and this process was evident to be controlled by ROS-dependent activation of MAPK/NF-κB signaling. The results can deepen our knowledge of anti-Giardia inflammatory response and host defense mechanisms.

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.

Giardia duodenalis, a cosmopolitan noninvasive protozoan parasite of zoonotic concern and public health importance, infects the upper portions of the small intestine and causes one of the most common gastrointestinal diseases globally termed giardiasis, especially in situations lacking safe drinking water and adequate sanitation services. The pathogenesis of giardiasis is complex and involves multiple factors from the interaction of Giardia and intestinal epithelial cells (IECs). Autophagy is an evolutionarily conserved catabolic pathway that involves multiple pathological conditions including infection. Thus far, it remains uncertain if autophagy occurs in Giardia-infected IECs and if autophagic process is associated with the pathogenic factors of giardiasis, such as tight junction (TJ) barrier defects and nitric oxide (NO) release of IECs. Here Giardia-in vitro exposed IECs showed upregulation of a series of autophagy-related molecules, such as LC3, Beclin1, Atg7, Atg16L1, and ULK1, and downregulation of p62 protein. IEC autophagy induced by Giardia was further assessed by using autophagy flux inhibitor, chloroquine (CQ), with the ratio of LC3-II/LC3-I significantly increased and downregulated p62 significantly reversed. Inhibition of autophagy by 3-methyladenine (3-MA) rather than CQ could markedly reverse Giardia-induced downregulation of TJ proteins (claudin-1, claudin-4, occludin, and ZO-1; also known as epithelial cell markers) and NO release, implying the involvement of early-stage autophagy in TJ/NO regulation. We subsequently confirmed the role of ROS-mediated AMPK/mTOR signaling in modulating Giardia-induced autophagy, TJ protein expression, and NO release. In turn, impairment of early-stage autophagy by 3-MA and late-stage autophagy by CQ both exhibited an exacerbated effect on ROS accumulation in IECs. Collectively, we present the first attempt to link the occurrence of IEC autophagy with Giardia infection in vitro, and provides novel insights into the contribution of ROS-AMPK/mTOR-dependent autophagy to Giardia infection-related downregulation of TJ protein and NO levels.