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

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.


Introduction
The intestinal protozoan parasite Giardia duodenalis is responsible for 280 million infections annually around the world [1]. Giardia infections remain a major public health burden in developing countries, and giardiasis has been included in WHO's Neglected Disease Initiative [2]. The life cycle of Giardia includes two stages: the infectious cyst and disease-causing trophozoites. Susceptible hosts ingest cysts via contaminated food and water, then trophozoites are released from the cysts upon stimulation by gastric acid and bile and interact with and adhere to intestinal epithelial cells (IECs) via the ventral disc [3]. G. duodenalis poses a serious risk of infection in immunocompromised individuals and children; the infection often causes diarrhea, nausea, vomiting, and weight loss, and may also trigger latent inflammatory bowel disease and irritable bowel syndrome [4,5].
Combined innate and adaptive immune responses are needed to control Giardia infection and replication [6]. Giardia excretory-secretory products and variant-specific surface proteins (VSPs) have been known to cause high levels of serum and salivary IgA in PMs were treated with trophozoites for the indicated time periods. Total RNA was extracted from cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA). The cDNA was synthesized by reverse transcription from 1 µg of total RNA using the Hiscript 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). Amplification was carried out on an LC480 Lightcycler system (Roche, Indianapolis, IN, USA) using the SYBR Green qPCR Master Mix (Bimake, Houston, TX, USA), and β-actin was applied as an endogenous control. The specific qPCR primers used here can be found in Table S1. The relative mRNA expression in the control was taken by us as 1, and fold changes relative to control were shown. The relative expression of mRNA was quantified by the 2 −∆∆Ct method.

Immunofluorescence Assays
J774A.1 cells or PMs were stimulated with trophozoites for the indicated time periods, fixed in 4% paraformaldehyde for 30 min, and permeabilized with 0.25% Triton-X 100 for 10 min at RT. Nonspecific sites were blocked by incubation with Fc receptor blocking solution (Abace, Beijing, China) containing 2% normal rabbit serum (Bioss, Beijing, China) for 30 min. Cells were incubated with primary antibodies against apoptosis-associated speck-like protein containing a CARD (ASC) and TLR4 (1:100 each; ABclonal, Wuhan, China) at 4 • C overnight and subsequently with FITC-AffiniPure goat anti-rabbit IgG (H + L) (1:200; Jackson, West Grove, PA, USA) at 37 • C for 1 h protected from light. Cell nuclei were labeled by DAPI (2 µg/mL; Alphabio, Tianjin, China). Epifluorescence imaging was performed using the Lionheart FX Automated Microscope.

Flow Cytometry for TLR Detection
PMs were treated with parasites for the indicated time periods, harvested by trypsinization, fixed in 2% paraformaldehyde for 10 min, and blocked with Fc receptor blocking solution containing 10% normal rabbit serum for 15 min at RT. All samples were incubated with anti-TLR2/TLR4/TLR5 antibody (1:100 each) for 30 min and then treated with FITC-AffiniPure goat anti-rabbit IgG (H + L) (1:200) for 40 min at RT. Fluorescently-labeled cells were analyzed on a BD FACS Canto II running FACSDiva and FlowJo.

Co-Immunoprecipitation (Co-IP) Analysis
We performed co-IP for analysis of NLRP3 ubiquitination. J774A.1 cells or PMs were treated with trophozoites for the indicated time periods. Total cellular proteins were extracted using the lysis buffer mentioned earlier and incubated with protein A/G magnetic beads (Bimake, Houston, TX, USA) and anti-NLRP3 antibody (1:200) at 4 • C overnight. The immunoprecipitates were collected using a magnetic rack, and the sediments were resuspended in lysis buffer and boiled for 8 min. The eluted proteins were analyzed by Western blotting with the anti-NLRP3 antibody and the antibody recognizing K63-linked polyubiquitin chains (both diluted 1:1000 in PBS for use).

Prokaryotic Expression of Giardia-Secreted Proteins
Among Giardia-secreted proteins highly expressed in the trophozoite culture supernatant as described before [31], PNPO (GL50803_5810), PPIB (GL50803_17163), and tenascin-1/2/3 (GL50803_114815/10330/16833) were randomly chosen and expressed in a prokaryotic expression system. The genes encoding these proteins were amplified using the primer sets shown in Table S2, and then cloned into the pCold I vector (TaKaRa, Ohtsu, Japan). The resulting plasmids pCold I-PNPO/PPIB/tenascins were transformed into E. coli strain BL21 (DE3) cells. Protein expression was induced with 1 mM isopropyl β-dthiogalactoside (Solarbio, Beijing, China) at 16 • C for 20 h. The harvested cells were lysed using an ultrasonic cell crusher (XO-650D; Xianou, Nanjing, China). Precipitated proteins and supernatants were subjected to SDS-PAGE analysis. Before treatment, the recombinant proteins were purified with a HisTrap HP nickel column (SMART, Changzhou, China). We then used an endotoxin ELISA kit (Meimian Biotech, Yancheng, China) to measure if the purified proteins were contaminated with endotoxins that may be responsible for the observed activation. If contaminated, proteins were further purified with Endotoxin Removal Beads (Smart-Lifesciences Biotechnology, Changzhou, China).

Statistical Analysis
For the statistical analysis, the data distribution was assumed to be normal. Data are presented as means ± standard deviation (SD). Statistical differences were assessed with Student's t-test for two groups and one-way ANOVA for multiple groups, followed by Fisher's least significant difference test. Statistics were generated using GraphPad Prism 7.0 software. A p value < 0.05 was considered statistically significant.

Giardia-Induced Cell Death and Mitochondrial Damage in Macrophages
We applied Giardia trophozoites to interact with J774A.1 cells and PMs of C57BL/6 mice to create in vitro infection models. Upon trophozoite treatment, LDH release was enhanced in a time-dependent manner in the culture supernatants, especially at 9 h and 12 h (p < 0.05), indicating cell membrane destruction ( Figure 1A). LDH present in the extracellular media has been regarded as a reliable metric of pyroptosis-induced cytotoxicity [34]. When exposed to trophozoites, the population of Annexin V-positive J774A.1 cells and PMs increased in a time-dependent manner (p < 0.01, Figure 1B). The macrophage cell viability was significantly decreased after parasite incubation for >9 h compared with mock cells (p < 0.01, Figure 1C).  The ultrastructure of Giardia-treated PMs was examined by TEM. In Giardia-treated PMs, as indicated by the red arrows in Figure 1D, there was an increase in cristae disruption in some mitochondria. The link between mitochondrial damage and ROS overproduction is well known [35]. As illustrated in Figure 1E, trophozoite stimulation increased ROS production in J774A.1 cells and PMs in a time-dependent manner. In addition, trophozoite stimulation could also induce the MMP loss in macrophages, as indicated by a decrease in the red/green fluorescence intensity ratio ( Figure 1F), which may be implicated in programmed cell death. And more notably, TEM micrographs showed that the plasma membrane integrity of PMs was disrupted at 12 h after treatment (blue arrows, Figure 1D), which is known as an important marker of pyroptosis [36].

Giardia-Induced NLRP3/CASP1/GSDMD-Mediated Macrophage Pyroptosis
In addition to an increase in LDH release due to plasma membrane rupture, CASP1mediated activation and the release of IL-1β and IL-18 are also important indicators of pyroptosis [37,38]. We found that the mRNA expression of IL-1β and IL-18 was notably upregulated in Giardia-treated PMs (p < 0.01, Figure 2A). The Western blot analysis revealed that trophozoite stimulation could promote pro-IL-1β cleavage to its mature and biologically active form IL-1β, which is then released into the extracellular environment (p < 0.01, Figure 2B,C). Similarly, treated macrophages showed a strikingly increased production of IL-18 in the supernatant (p < 0.01, Figure 2B,C).
Proinflammatory cytokine release is mediated by inflammasome activation, which is essential for host defense against enteropathogens [39]. Four major inflammasome sensors, NLRP1a, AIM2, NLRP3, and NLRC4, were analyzed for their expression levels in treated and untreated cells, only NLRP3 expression was significantly upregulated at both the mRNA and protein levels (p < 0.01, Figure 2D-F). NLRP3 can initiate the canonical inflammasome assembly by recruiting pro-CASP1 with the ASC adaptor, which induces pyroptosis via cleavage and activation of CASP1 and GSDMD [17]. Our study noted Giardia-induced cleavage of CASP1 and the formation of N-GSDMD ( Figure 2E,F); the latter would facilitate pyroptotic pore formation and IL-1β and IL-18 release. An increase in the fluorescence intensity of ASC was observed in treated cells ( Figure 2G).

Giardia-Induced ROS/NLRP3/CASP1/GSDMD-Mediated Macrophage Pyroptosis
To identify the mediators leading to Giardia infection-associated GSDMD cleavage, we used N-acetylcysteine (NAC) and Ac-YVAD-CMK (AYC) to inhibit ROS generation and CASP1 activation, respectively. Pretreatment of PMs with NAC before exposure to trophozoites could inhibit the activation of NLRP3, cleavage of CASP1 and GSDMD, and the secretion of IL-1β and IL-18 (p < 0.01, Figure 3A,B). In comparison with NAC, AYC had similar inhibitory effects except that it could not block NLRP3 activation ( Figure 3A,B). In addition, the suppression of NLRP3 by its inhibitor MCC950 can disturb the subsequent pyroptotic signaling (p < 0.01, Figure 3C,D). As visualized by immunofluorescence staining with an anti-ASC antibody, NAC and MCC950 pretreatment could suppress the upregulation of ASC expression in Giardia-stimulated PMs, while AYC did not exert a similar effect ( Figure 3E). Taken together, the upstream regulator (ROS) and downstream effectors (ASC, CASP1, and GSDMD) of NLRP3 were determined. In addition, the reduced cell viability and increased LDH release in Giardia-treated PMs can both be alleviated in the presence of NAC, MCC950, or AYC (p < 0.01, Figure 3F,G).
Proinflammatory cytokine release is mediated by inflammasome activation, which is essential for host defense against enteropathogens [39]. Four major inflammasome sensors, NLRP1a, AIM2, NLRP3, and NLRC4, were analyzed for their expression levels in treated and untreated cells, only NLRP3 expression was significantly upregulated at both the mRNA and protein levels (p < 0.01, Figure 2D-F). NLRP3 can initiate the canonical inflammasome assembly by recruiting pro-CASP1 with the ASC adaptor, which induces pyroptosis via cleavage and activation of CASP1 and GSDMD [17]. Our study noted Giardia-induced cleavage of CASP1 and the formation of N-GSDMD ( Figure 2E,F); the latter would facilitate pyroptotic pore formation and IL-1β and IL-18 release. An increase in the fluorescence intensity of ASC was observed in treated cells ( Figure 2G).  subsequent pyroptotic signaling (p < 0.01, Figure 3C,D). As visualized by immunofluorescence staining with an anti-ASC antibody, NAC and MCC950 pretreatment could suppress the upregulation of ASC expression in Giardia-stimulated PMs, while AYC did not exert a similar effect ( Figure 3E). Taken together, the upstream regulator (ROS) and downstream effectors (ASC, CASP1, and GSDMD) of NLRP3 were determined. In addition, the reduced cell viability and increased LDH release in Giardia-treated PMs can both be alleviated in the presence of NAC, MCC950, or AYC (p < 0.01, Figure 3F,G).

Involvement of A20-Mediated NLRP3 Deubiquitination in Giardia-Induced Macrophage Pyroptosis
Emerging evidence demonstrates that NLRP3 inflammasome activation involves its deubiquitination [40]. It was indicated here in co-IP analysis that NLRP3 was decorated by K63-linked polyubiqutin chains, while trophozoite treatment significantly reduced the amounts of K63-linked polyubiquitin chains on NLRP3 (p < 0.01, Figure 4A,B). Protein ubiquitination can be reversed through the removal of ubiquitin from target proteins by deubiquitinating enzymes (DUBs) [41]. We examined the mRNA expression levels of six candidate DUBs (A20, CYLD, ABRO1, BRCC3, USP47, and USP7), only A20 expression exhibited a time-dependent increase in treated PMs (p < 0.01, Figure 4C). The upregulation of A20 expression was then confirmed by Western blot analysis (p < 0.01, Figure 4D,E). The siRNA-mediated knockdown of A20 was conducted to elucidate the potential role of A20 in regulating NLRP3 deubiquitination and the resulting pyroptosis.
As predicted, the A20 mRNA and protein expression were effectively decreased by 48% (p < 0.01, Figure 4F) and 35% (p < 0.01, Figure 4G,H), respectively, after transfection with siA20. The enhanced A20 mRNA and protein expression in Giardia-treated PMs were inhibited by 70% (p < 0.01, Figure 4F) and 44% (p < 0.01, Figure 4G,H) by A20 knockdown, respectively. More importantly, the A20 knockdown could significantly alleviate Giardiainduced K63 deubiquitination of NLRP3 (p < 0.01, Figure 4G,H). In follow-up experiments, we found that Giardia-induced cleavage of CASP1 and GSDMD and the secretion of IL-1β and IL-18 could both be suppressed in A20-knockdown PMs (p < 0.01, Figure 4I,J). The A20 knockdown could also affect the expression of ASC in Giardia-treated PMs since a weaker fluorescence was observed in the group pretreated with siA20 than in the untreated group ( Figure 4K). The A20 knockdown could also rescue the reduced cell viability induced by Giardia (p < 0.01, Figure 4L), as well as block Giardia-induced LDH release enhancement (p < 0.01, Figure 4M).

Involvement of TLR4 Activation in Giardia-Induced Macrophage Pyroptosis
TLRs play vital roles in the innate reaction to microbial products and transmit specific immune responses against pathogens [22]. Here we evaluated the mRNA expression levels of TLR2, TLR3, TLR4, and TLR5 in Giardia-treated and untreated PMs ( Figure 5A), among which only TLR2 and TLR4 showed a marked increase in expression (p < 0.01). Flow cytometry analysis indicated that TLR2 and TLR4 were strongly upregulated at 12 h posttreatment (p < 0.01, Figure 5B,C). Similar results to those seen in flow cytometry were also seen in Western blotting (p < 0.01, Figure 5D,E). The data imply that trophozoite stimulation activated TLR2 and TLR4 in PMs. We then used the TLR2 inhibitor TLR2-IN-C29 (C29) and the TLR4 inhibitor TAK-242 (TAK) to pretreat PMs to explore the potential association of TLR2 or TLR4 with Giardia-induced pyroptosis. TAK showed an inhibition of LDH release enhancement and recovered the reduced cell viability during Giardia-macrophage in vitro interactions (p < 0.01), while this was not the case for C29 ( Figure 5F,G). Giardiaactivated TLR2 and TLR4 can both be suppressed by their specific inhibitors (p < 0.01, Figure 5H,I); however, we found that only TLR4 inhibition by TAK could block CASP1 activation and IL-1β and IL-18 secretion (p < 0.01, Figure 5H,I). In addition, TAK also had obvious inhibitory effects on the K63 deubiquitination of NLRP3 and GSDMD cleavage (p < 0.01, Figure 5J,K). It is noteworthy that Giardia-induced ROS overproduction could be alleviated by TLR4 inhibition by TAK ( Figure 5L), and pretreatment with TAK and NAC both suppressed the A20 upregulation in Giardia-treated PMs (p < 0.01, Figure 5M,N). Collectively, TLR4-mediated ROS production seems to regulate A20 expression and control pyroptotic outcomes in response to Giardia infection.

Giardia-Secreted PPIB Independently Induced Macrophage Pyroptosis
It can be concluded from the statements above that Giardia can induce macrophage pyroptosis via TLR4 signaling. Since noninvasive Giardia trophozoites cannot interact directly with macrophages in the gut lumen [5], we hypothesized that the secreted virulence factors probably function as a trigger to activate TLR4. As mentioned earlier, five secreted proteins PNPO, PPIB, and tenascin-1/2/3 are highly expressed in the culture supernatants of Giardia trophozoites [31,32]. We initially obtained five recombinant proteins of them using prokaryotic expression systems. The recombinant His6-tagged PNPO and PPIB were expressed as soluble proteins in E. coli, while the expressed tenascins were present in the form of inclusion bodies. After endotoxin removal if contaminated, these proteins were used to treat PMs, meanwhile, the Giardia-treated group was set as a positive control. We found that PPIB had a significantly negative effect on macrophage viability in a dosedependent manner (p < 0.01, Figure 6A). PPIB stimulation could also induce LDH release from macrophages as was seen in trophozoite stimulation experiments (p < 0.01, Figure 6B). However, other tested proteins had no such effect ( Figure 6A,B). Cells 2021, 10, x FOR PEER REVIEW 13 of 20   We then assessed the relevance of PPIB to the pyroptosis occurrence. PPIB also increased the protein levels of TLR4 similarly to those induced by Giardia ( Figure 6C). The effects of PPIB stimulation on the expression of pyroptosis-related proteins are similar to those of trophozoite stimulation. For example, PPIB stimulation could promote CASP1 and GSDMD cleavage and IL-1β and IL-18 secretion in macrophages (p < 0.01, Figure 6D,E), and the related inhibitors of the upstream signal molecules including TAK, NAC, MCC950, or AYC could reverse these changes (p < 0.01, Figure 6D,E). In addition, PPIB-induced upregulation of A20 could be significantly repressed by TLR4 inhibition via TAK or by ROS inhibition via NAC, rather than by the inhibition of its downstream signaling molecule NLRP3 via MCC950 or CASP1 via AYC ( Figure 6D,E). The co-IP assay revealed that PPIB stimulation induced K63 deubiquitination of NLRP3 and this process could be blocked by TAK, NAC, or MCC950 (p < 0.01, Figure 6F,G), while inhibition of CASP1 via AYC had no such blocking effects on its upstream molecule NLRP3. All the inhibitors just referred to could induce an increase in cell viability and a decrease in LDH release in PPIB-stimulated PMs (p < 0.01, Figure 6H,I).
The knockdown of A20 by a specific siRNA could significantly decrease the levels of cleaved CASP1 and GSDMD and extracellularly released IL-1β and IL-18 in PPIBtreated PMs, but had little effect on PPIB-activated TLR4 ( Figure 6J,K). Importantly, A20 knockdown could suppress the activation of NLRP3 by modulating its deubiquitination (p < 0.01, Figure 6L,M), as well as reverse the decreased cell viability and increased LDH release induced by PPIB (p < 0.01, Figure 6N,O).

Discussion
Macrophages are primary effector cells of the innate immune system that form the first line of defense against microbial pathogens [42]. In this study, we found that Giardia and its secreted PPIB were able to induce macrophage pyroptosis and promote the release of the proinflammatory cytokines IL-1β and IL-18. Mechanistically, TLR4 was the first upstream molecule that was activated by PPIB or trophozoites, which triggered ROS overproduction and A20 upregulation. A20 promoted inflammasome activation by deubiquitinating NLRP3, driving pyroptotic cell rupture and IL-1β and IL-18 release. The resulting inflammatory reactions promote antimicrobial host defense as noted in [43].
Giardia trophozoites primarily colonize the proximal small intestine and do not invade host cells [44]. Giardia-secreted giardipain-1 and cysteine proteases have been identified as potent inducers of intestinal epithelial barrier breakdown [28,29], and the broken IEC barrier facilitates the process of PAMP (perhaps via certain secreted proteins) recognition by innate immune cells, notably macrophages that populate the intestinal lamina propria, to initiate immune responses required for the maintenance of intestinal homeostasis. Besides, Giardia trophozoite-secreted proteins like cathepsin B proteases and tenascins can disrupt the normal physiological function of IECs [31]. Peptidyl prolyl cis/trans isomerases (PPIases) are a superfamily of proteins ubiquitously distributed in both prokaryotes and eukaryotes [45]. Most characterized PPIases were previously considered to be involved in the folding and structuring of unfolded and partially folded polypeptide chains and proteins [46], whereas some were later identified as virulence-associated proteins upregulated during infection [47]. The roles of the highly variable PPIases in microbial pathogenesis remain little known [48]. Here Giardia-secreted PPIB was proved as a virulence protein that can trigger macrophage pyroptosis via TLR4 signaling. Actually, it has been reported that the secreted PPIase of Helicobacter pylori, HP0175, is able to induce apoptosis of the gastric epithelial cells in a TLR4-dependent manner [49], and it can also drive Th17 inflammation in gastric adenocarcinoma as described in [50]. Yet, the detailed role of PPIB in giardiasis remains to be explored.
Innate immune cells have complex signaling pathways for sensing pathogens, among which the most significant one is the TLR-mediated recognition of extracellular stimuli [51]. TLRs function as the effectors of immune surveillance and induce the transcriptional activation of proinflammatory factors [52]. It was reported that Giardia infection can enhance the production of proinflammatory cytokines, such as TNF-α, IFN-γ, IL-6, and IL-12 p40 in PMs, through the TLR2-mediated activation of MAPK signaling [13]. A recent study found that Giardia EVs trigger IL-1β, IL-6, and TNF-α secretion from mouse PMs via promoting the nuclear translocation of the NF-κB p65 subunit [53]. Another recent study identified TLR2 as a mediator for pyroptotic signaling in mouse PMs induced by Giardia EVs [25]. In contrast, our study revealed a significant increase of both TLR2 and TLR4 expression in Giardia-treated macrophages. We further found that TLR4 inhibition exerted a significant effect on impeding Giardia-induced NLRP3 activation and macrophage pyroptosis, while this is not the case for TLR2 inhibition. It is of interest to note that in macrophages NLRP3 can be activated by up to a 4-h stimulation with LPS and then ATP, while a prolonged (12 to 24 h) exposure dampens NLRP3 activation [54]. By contrast, our study showed that Giardia and its secreted PPIB could trigger the upregulation of NLPR3 in macrophages within 12 h of treatment; however, more data are needed to determine if any changes occur when subjected to a prolonged stimulation. We also noted a relatively high level of TLR5 expression in a small population of Giardia-treated macrophages, which might be attributed to the stimulus trophozoite flagellin [55]. The pyroptosis executioner GSDMD is known as a substrate for CASP1/4/5/11 [17]. Our study showed that Giardia was able to activate CASP1 to promote the cleavage of GSDMD. It has been demonstrated that CASP3-dependent cleavage of GSDME could be induced by chemotherapy [56], while no obvious cleavage of CASP3 was observed here. CASP1 is the best-described inflammatory caspase that requires inflammasome sensors like NLRP1a, AIM2, NLRP3, or NLRC4 for its activation [21]. Of the candidate sensors tested in this study, NLRP3 was confirmed to be active. Neospora caninum infection has also been shown to initiate NLRP3 inflammasome activation [57]. NLRP3 inflammasome activation is also present in macrophages affected by some other protozoan parasites, such as Plasmodium, Leishmania, Trypanosoma, Toxoplasma, and Entamoeba [20]. The NLRP3 inflammasome pathway contributes to host defense against infectious pathogens due to subsequent IL-1β and IL-18 secretion. IL-1β is a potent proinflammatory mediator implicated in recruiting immune cells to the site of infection and stimulating adaptive immune responses, and IL-18 induces the sequential activation of natural killer cells and cytotoxic T cells [58]. Mouse macrophages seem dispensable for anti-Giardia protective immunity [59], yet they could at least initiate an inflammatory response as reflected here.
NLRP3-mediated macrophage pyroptosis is associated with various kinds of infections, but the mechanisms of activation are complex [20]. Post-translational modifications of proteins such as ubiquitination are important for many biological processes [60]. NLRP3 is ubiquitinated with K63-and K48-linked polyubiquitin chains [61], among which the K63-linked polyubiquitin chain presumably serves as a molecular platform for various signaling pathways. Protein ubiquitination can be reversed by DUBs as indicated earlier. It has been known that BRCC3, in combination with ABRO1, can form a cytosolic BRISC complex and modulate K63-linked ubiquitination of NLRP3 [40]. Yet here we found that Giardia trophozoite stimulation modified the ubiquitination status of NLRP3 by upregulation of A20. The A20-promoted K63 deubiquitination of TRAF6 in macrophages induced by intracellular Leishmania leads to a remarkable reduction in TLR2-mediated IL-12 and TNF-α production [62]. In the present study, we confirmed the involvement of A20-mediated NLRP3 deubiquitination in inflammasome activation, CASP1 and GSDMD cleavage, and IL-1β and IL-18 release during Giardia infection. ROS production has been widely described as one of the earliest cellular responses following pathogen infection [63]. Interestingly, here we confirmed the positive relation between TLR4-induced ROS production and A20-mediated NLRP3 deubiquitination. In addition, it is also worth identifying the signaling mechanisms that affect Giardia-induced ROS production and inflammasome activation in macrophages, although this was not seen in this study. Actually, a recent study has demonstrated that LPS-induced macrophage ROS accumulation is associated with TLR4-dependent MYD88/PI3K signaling [64].
Bone marrow-derived macrophages, resident or induced PMs, or macrophages from the spleen, liver, and lung isolated from mice are usually variable in their cytokine production, migratory capacity, and antimicrobial activity [65]. Our study investigated the murine macrophage cell line J774A.1 and thioglycollate broth-induced mouse PMs for their potential to produce an anti-Giardia pyroptotic inflammatory response. Although sound, the current findings need to be confirmed further by using intestinal resident macrophages. Intestinal macrophages are essential players in the regulation of immune homeostasis and host defense against Giardia in the gut [14,59]. While macrophage pyroptosis has been widely identified as an important innate immune effector mechanism against intracellular pathogens, this is not the case for Giardia, widely known as a kind of extracellular parasite. We indicated here the detailed recognition and response mechanisms required for Giardia-macrophage interactions and the occurrence of pyroptosis, and determined PPIB as the potential trigger that implemented this process. The findings lead the way to a better understanding of the host defense mechanisms against Giardia.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/cells10123425/s1, Table S1: Primer pairs used in qPCR analysis, Table S2: Primer pairs used for plasmid construction. Funding: This work was supported by the National Natural Science Foundation of China (no. 32172885) and the Natural Science Fund of Heilongjiang Province for Excellent Young Scholars (no. YQ2020C010) (to W.L.). This work was also funded by the Deutsche Forschungsgemeinschaft DFG grants (no. TRR241-B05 and SFB1181-C05) and the Interdisciplinary Center for Clinical Research, Erlangen, Germany (no. IZKF-J68) (to J.V.P.).

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Care and Use of Laboratory Animals, and approved by the Institutional Animal Care and Use Committee of the Northeast Agricultural University (no. NEAUEC20210316).