Giardia duodenalis and Its Secreted PPIB Trigger Inflammasome Activation and Pyroptosis in Macrophages through TLR4-Induced ROS Signaling and A20-Mediated NLRP3 Deubiquitination

Abstract

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.

COX-2 is required to mediate crosstalk of ROS-dependent activation of MAPK/NF-κB signaling with pro-inflammatory response and defense-related NO enhancement during challenge of macrophage-like cell line with Giardia duodenalis

“…TNF-α and IL-6 have been reported to be essential for effective clearance of Giardia [ 12 – 15 ]. There is also a significant pyroptosis-related IL-1β and IL-18 increase induced by Giardia as described recently [ 16 ]. ROS contributes to a key mechanism in which phagocytic cells induce inflammation or clear pathogens [ 17 ], and it has also been recognized as an important player in Giardia -induced intestinal epithelial cell (IEC) apoptosis [ 1 , 18 ].…”

“…In reality, recombinant Treponema pallidum protein Tp0768 has been shown to promote generation of IL-1β, IL-6, and IL-8 by macrophages through ER stress and ROS-NF-κB pathway [ 50 ]. In addition, it was known that ROS can be activated in J774A.1 cells, peritoneal macrophages, and IECs as early as 3 h after Giardia exposure [ 16 , 18 ], and inhibition of ROS by NAC can affect macrophage pyroptotic outcome after a 12-h exposure [ 16 ] and IEC apoptotic outcome after a 6-h exposure [ 18 ]. In contrast, here we demonstrated that ROS inhibition was able to influence macrophage pro-inflammatory and defense-related responses after a 3-h or prolonged Giardia exposure.…”

“…It is also of interest to note the function of ROS in regulating MAPK/NF-kB/COX-2 signaling as early as 3 h after Giardia exposure here. Giardia -secreted peptidyl-prolyl cis-trans isomerase B has been verified as a trigger for TLR4-dependent ROS activation and pyroptotic cell death in macrophages [ 16 ]. Likewise, continued efforts are needed to identify the potential trigger for ROS-dependent regulation observed here.…”

The Pathogenesis of Giardia Intestinalis

“…Several Giardia excretions may be involved in host pathogenesis, including metabolic enzymes released by Giardia, soluble substances of different sizes and unknown cysteine proteases. Giardia has been found to release arginine deaminase (ADI) and ornithine carboxyltransferase (OTC), both of which may be involved in the metabolism of L-arginine, a nutrient that is the preferred energy source of the growth and reproductive stages of Giardia [14]. Although a single molecule of L-arginine produces one molecule of ATP, Giardia intestinalis uses this pathway to obtain ATP more rapidly than the glycolytic pathway [15].…”

Advances in the Study of the Ubiquitin-Editing Enzyme A20

“…A20 can mediate K48-Linked polyubiquitination modification of RNF138 for proteasomal degradation to affect NF-κB activation and lymphomagenesis ( Yu et al, 2021 ); K63-Linked and M1-Linked polyubiquitination modification is involved in serving as a scaffolding network for the formation of signaling complexes. Studies indicated that A20 can downregulate the K63-Linked polyubiquitination modification of TRAF6, which is involved in the anti-neuroinflammatory effects of bavachin ( Wang H et al, 2021 ); In addition, A20 can also play an anti-microbial host defense role through affecting the K63-Linked polyubiquitination modification of different substrate proteins ( Liu L. et al, 2020 ; Liu et al, 2021 ); Polyubiquitination at K6 is correlated with the development of nonalcoholic steatohepatitis; K29-Linked polyubiquitination is associated with the innate cellular immunity of macrophages ( Wang et al, 2020 ; Gao et al, 2021 ; Tracz and Bialek, 2021 ).…”