Giardia duodenum is a parasitic organism that causes giardiasis, an intestinal infection especially common in young children with clinical signs of diarrhea. We have previously reported that extracellular G. duodenalis triggers the activation of intracellular oligomerization-like receptor 3 (NLRP3) binding nucleotides and regulates host inflammatory responses through extracellular vesicle (EV) secretion. However, the exact molecular patterns of the pathogen-associated duodenococcal EV (GEV) involved in this process and the role of the NLRP3 inflammasome in giardiasis remain to be elucidated.
        Recombinant eukaryotic expression plasmids pcDNA3.1(+)-alpha-2 and alpha-7.3 giardins in GEV were constructed, transfected into mouse primary peritoneal macrophages, and detected by measuring the inflammation target molecule caspase-1. The p20 expression level was screened. . G. duodenalis alpha-2 and alpha-7.3 giardines were originally identified by measuring NLRP3 inflammasome (NLRP3, pro-interleukin-1 beta [IL-1β], pro-caspase-1 and caspase-1 p20), IL secretion. 1β levels, apoptotic spotted protein (ASC) oligomerization levels, and immunofluorescent localization of NLRP3 and ASC. The role of the NLRP3 inflammasome in the pathogenicity of G. duodenalis was then assessed using mice in which NLRP3 activation was blocked (NLRP3 blocked mice) and pathological changes in body weight, duodenal parasitic load, and duodenal tissue were monitored. In addition, we investigated whether hiardines alpha-2 and alpha-7.3 induce IL-1β secretion in vivo via the NLRP3 inflammasome and determined the role of these molecules in the pathogenicity of G. duodenalis in mice.
        Alpha-2 and alpha-7.3 giardines induce the activation of the NLRP3 inflammasome in vitro. This led to the activation of p20 caspase-1, an increase in the expression levels of NLRP3, pro-IL-1β, and pro-caspase-1 proteins, a significant increase in IL-1β secretion, the formation of ASA spots in the cytoplasm, and the induction of ASA oligomerization. NLRP3 inflammation Penile loss exacerbates the pathogenicity of G. duodenalis in mice. Mice treated with cysts by gavage from NLRP3-blocked mice exhibited an increased number of trophozoites and severe damage to the duodenal villi, characterized by necrotic crypts with shrunken and branching. In vivo experiments have shown that giardines alpha-2 and alpha-7.3 can induce secretion of IL-1β via the NLRP3 inflammasome, and immunization with giardines alpha-2 and alpha-7.3 reduced the pathogenicity of G. duodenalis in mice.
       Taken together, the results of this study suggest that giardia alpha-2 and alpha-7.3 cause upregulation of host NLRP3 inflammation and reduce the infectivity of G. duodenalis in mice, which are promising targets for preventing giardiasis.
        Giardia duodenum is an extracellular protozoan parasite that lives in the small intestine and causes 280 million cases of giardiasis with diarrhea annually, especially among young children in developing countries [1]. People become infected by drinking water or food contaminated with M. duodenum cysts, which then enter the stomach and are excreted in the gastric juices. Giardia duodenum trophozoites attach to the duodenal epithelium, causing nausea, vomiting, diarrhea, abdominal pain, and weight loss. Individuals with immunodeficiency and cystic fibrosis are susceptible to infection. Infection can also occur through oral and anal sex [2]. Drugs such as metronidazole, tinidazole, and nitazoxanide are the preferred treatment options for duodenal infections [3]. However, these chemotherapy drugs cause adverse side effects such as nausea, carcinogenesis, and genotoxicity [4]. Therefore, more effective strategies need to be developed to prevent G. duodenalis infection.
        Inflammasomes are a class of cytosolic protein complexes that are part of the innate immune response, helping to defend against pathogen invasion and mediate inflammatory responses [5]. Among these inflammasomes, nucleotide-binding oligomerization (NOD) receptor 3 (NLRP3) nucleotide-binding oligomerization (NLRP3) nucleotide-binding-like inflammasome has been extensively studied because it can be detected by various pathogen/damage-associated molecular patterns (PAMP/DAMP), recognizes, activates the innate immune system. and regulates intestinal homeostasis in many inflammatory diseases [6,7,8]. It consists of the pattern recognition receptor (PRR) NLRP3, an adapter apoptotic spotted protein (ASC), and an effector procaspase-1 or procaspase-11. The NLRP3 inflammasome acts as a host against pathogen invasion, as observed in Neospora caninum [9], Paracoccidioides brasiliensis [10], and Leishmania studies. [11], but it has also been reported that activation of the NLRP3 inflammasome limits protective immune responses and exacerbates disease progression, for example, in worms [12]. Based on our previous findings, we reported that extracellular G. duodenalis triggers intracellular activation of NLRP3 inflammation and modulates host inflammatory responses by secreting extracellular vesicles (EVs) [13]. However, the role of the NLRP3 inflammasome in G. duodenalis infection in vivo remains to be determined.
        Giardins were originally described as structural components of the G. duodenalis cytoskeleton and play an important role in trophozoite motility and epithelial cell attachment in the small intestine. To better adapt to the environment and increase their pathogenicity, G. duodenalis trophozoites developed a unique cytoskeletal structure consisting of 8 flagella, 1 middle body, and 1 ventral disc [14]. The trophozoites of Giardia duodenum use their cytoskeleton to penetrate the upper small intestine, especially the duodenum, and attach to enterocytes. They constantly migrate and attach to epithelial cells using cell metabolism. Therefore, there is a close relationship between their cytoskeleton and virulence. Giardines specific for Giardia duodenum are components of the cytoskeleton structure [15] and are divided into four classes: α-, β-, γ-, and δ-giardines. There are 21 members of the α-giardin family, all of which have a calcium-dependent ability to bind phospholipids [16]. They also connect the cytoskeleton to the cell membrane. In individuals with diarrhea caused by G. duodenalis, α-giardins are highly expressed and immunoreactive during infection [17]. Heterologous vaccines based on Giardia alfa-1 protected against giardiasis in mice and are potential candidate antigens for vaccine development [18]. Alpha-8 giardin, localized in the plasma membrane and flagella, but not in the ventral disc, enhances the motility and growth rate of trophozoites in G. duodenalis [19]. Alpha-14 giardin attaches to microtubule structures on flagella and affects the viability of G. duodenalis [20]. Alpha-11 giardine is present in abundance throughout the life cycle, and overexpression of alpha-11 giardine damages G. duodenalis itself [21]. However, it is unclear whether alpha-2 giardine and alpha-7.3 giardine are protective against G. duodenalis infection and their underlying mechanisms.
        In this study, recombinant eukaryotic expression plasmids pcDNA3.1(+)-alpha-2 giardine and pcDNA3.1(+)-alpha-7.3 giardine were transfected into mouse primary peritoneal macrophages to activate host NLRP3. Inflammasome targets were then screened. We also assessed the role of the NLRP3 inflammasome in the pathogenicity of G. duodenalis, investigated whether alpha-2 and alpha-7,3 giardines induce activation of the NLRP3 inflammasome in vivo, and determined that these two roles of giardines in the pathogenicity of G. duodenalis. Our common goal was to develop promising targets for the prevention of G. duodenalis infection.
        Wild type (WT) C57BL/6 female mice aged 5–8 weeks were purchased from Liaoning Changsheng Experimental Animal Center (Liaoning, China). Mice had free access to water, received sterilized food and were kept in a 12/12 hour light/dark cycle. Before infection, mice received antibiotics ad libitum in drinking water supplemented with ampicillin (1 mg/mL), vancomycin (1 mg/mL), and neomycin (1.4 mg/mL) (all purchased from Shanghai, China, artificial organisms) [ 22]. ]. Mice that lost the ability to eat and drink for > 24 hours and lost ≥ 20% body weight were humanely euthanized by cervical dislocation.
        WB G. duodenalis trophozoites (American Type Culture Collection, Manassas, USA) were supplemented with 12.5% ​​fetal bovine serum (FBS; Every Green, Zhejiang, China) and 0.1% bovine bile (Sigma-Aldrich, St. Missouri, USA). USA) under microaerobic conditions. Confluent trophozoites were collected on ice and passaged at a ratio of 1:4 for further reproduction.
        Giardia duodenum cysts were induced as described previously [23], trophozoites were harvested in logarithmic phase and then diluted with encapsulation inducing medium, pH 7.1 (modified TYI-S-33) to a final concentration of 1 × 106 trophozoites/mL. bile concentration 0.05% medium). Trophozoites were cultured under anaerobic conditions at 37°C until the logarithmic growth phase. Change the medium to cyst inducing medium (pH 7.8; modified TYI-S-33 medium with 1% bile concentration) and culture G. duodenalis at 37°C for 48–96 hours, during which the formation cysts were observed under a microscope. After most of the trophozoites had been induced to form cysts, the culture mixture was harvested and resuspended in sterile deionized water to lyse the remaining trophozoites. Cysts were counted and stored at 4°C for subsequent analyzes through a gastric tube in mice.
        Giardia extracellular vesicles (GEVs) were enriched as described previously [13]. Trophozoites in logarithmic growth phase were resuspended in modified TYI-S-33 medium prepared with exosome-depleted FBS (Biological Industries, Beit-Haemek, Israel) to a final concentration of 1 × 106 parasites/mL and incubated for 12 hours. were isolated from the culture supernatant by centrifugation at 2000 g for 10 min, 10,000 g for 45 min, and 100,000 g for 60 min. Precipitates were dissolved in phosphate buffered saline (PBS), quantified using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) and stored at -80° C. or used directly for further analyses.
        Primary mouse peritoneal macrophages were prepared as described previously [24]. Briefly, mice (aged 6-8 weeks) were injected (intraperitoneally [ip]) with 2.5 ml of 2.98% Difco liquid thioglycol medium (BD, Franklin Lakes, NJ, USA) and fed 3-4 palates. A suspension of macrophages was collected from the abdominal cavity of mice after euthanasia and centrifuged 3 times at 1000 g for 10 min. Harvested cells were detected by flow cytometry using the CD11b marker until cell purity was >98%, then added to 6-well cell culture plates (4.5 x 106 cells/well) and incubated with 10% FBS (Bioindustry) at 37°C. and 5% CO2.
       RNA was extracted from 1 × 107 trophozoites in 1 ml of TRIzol reagent (Vazyme, Nanjing, China), genomic DNA was extracted from total G. duodenalis RNA using MonScript dsDNase (Monad, Wuhan, China) and complementary DNA (cDNA) was synthesized using MonScript RTIIII Super Mix (Monad) according to the manufacturer’s instructions.
        CDS sequence information for the target G. duodenalis gene was obtained from the NCBI GenBank. Use Primer 5.0 to design specific seamless cloning primers for each target gene. The forward primer (5′-3′) consists of three parts: an overlapping sequence with a linearized vector pcDNA3.1(+) EcoRV (TGGTGGAATTCTGCAGAT) and start codons ATG and GNN (if the first base is not G). This is done to improve the efficiency of the expression. In addition, at least 16 b.p. combined bases (GC content 40–60%/Tm approx. 55 °C). The reverse primer (5′-3′) consists of two parts, an overlapping sequence with an EcoRV-linearized vector pcDNA3.1(+) (GCCGCCACTGTGCTGGAT) and a combined base of at least 16 bp. (excluding the last two stops). bases) a codon such as AA or GA to allow recombinant plasmids to express their labeled proteins). The primer sequences are listed in Table 1 and were synthesized by Kangmet Biotechnology Co., Ltd. (Changchun, China).
        Targets were amplified using Pfu DNA polymerase (Tiangen, Beijing, China) or Ex-taq (Takara Biomedical Technology [Beijing] Co., Ltd., Beijing, China) using prepared G. duodenalis cDNA as a template. The eukaryotic expression vector plasmid pcDNA3.1(+) was linearized with restriction enzyme EcoRV and dephosphorylated using Fast AP (Thermo Fisher Scientific). Linearized pcDNA3.1(+) fragments and amplified target gene fragments were purified using a DNA gel purification kit (Tiangen) and quantified using a Nanodrop ND-2000 (Thermo Fisher Scientific). The pcDNA3.1(+) fragment and each target gene fragment were recombined using MonClone single assembly cloning mix (Monad Biotech Co., Ltd., Suzhou, China) and confirmed by DNA sequencing using Comate Bioscience Company Limited (Changchun, China) . .
        Endotoxin-free plasmids pcDNA3.1(+)-alpha-2 and pcDNA3.1(+)-alpha-7.3 were generated using the SanPrep Endotoxin-free Plasmid Mini Kit (Sangon Biotech). The concentration was maintained above 500 ng/µl to ensure that the EDTA in the elution buffer did not interfere with the transfection assay. Primary mouse peritoneal macrophages were cultured in 6-well plates with complete RPMI 1640 medium (Biological Industries) for 12 hours, then the cells were washed 3 times in warm PBS to remove penicillin and streptomycin, and then in medium supplemented with complete medium. Endotoxin-free plasmids pcDNA3.1(+)-alpha-2 and pcDNA3.1(+)-alpha-7.3 (2.5 μg) were diluted in 125 μl of Opti-MEM reduced serum medium (Gibco, Thermo Fisher Scientific) . . Then 5 µl of Lipofectamine 2000 transfection reagent (Invitrogen, Thermo Fisher Scientific) was diluted in 125 µl of low serum Opti-MEM medium. Prepare liposome-DNA complexes by mixing the diluted endotoxin-free plasmid with Lipofectamine 2000 and allowing the mixture to stand at room temperature for 5 minutes. Transfer the complexes separately to cells in each well and mix slowly. After 4 hours, the cell culture medium was replaced with 2 ml of complete RPMI 1640 medium and culture was continued for 24 hours. Fresh cell culture medium was added to the cells and incubated for various time points depending on the assay design.
        Protein samples from supernatants and cell lysates were prepared as described previously [25]. Membrane transfer parameters for pro-IL-1β, pro-caspase-1, caspase-1 p20, NLRP3, β-actin, and His-tag were 200 mA/90 min. For interleukin 1β (IL-1β; R&D Systems, Minneapolis, Minnesota, USA), caspase-1 (p20) (Adipogen, Switzerland) and NLRP3 (Adipogen SA, Epalinges, Switzerland) and 1:5000 targeting His tag ( Amylet Scientific, Wuhan, China) and β-actin (Proteintech, Wuhan, China).
        Cross-linking with disuccinimide suberate (DSS) was performed as described previously [26]. Cells were washed 3 times with cold PBS and completely lysed with a 27 gauge needle in 50 µl ASC reaction buffer (pH 8.0) containing 25 mM Na2PO4, 187.5 mM NaCl, 25 mM HEPES and 125 mM NaHCO3. The mixture was centrifuged at 5000 g for 3 min and the pellet was sutured with 10 µl DSS (25 mM in DMSO) and 40 µl ASC reaction buffer for 30 min at 37°C. After centrifugation at 5000 g for 10 min, the pellet was dissolved in a solution of 40 µl of ASC reaction buffer and 10 µl of 6x protein loading buffer (TransGen, Beijing, China), and then the solution was quenched at room temperature for 15 min. , Then boil 10 minutes. Protein samples were then subjected to Western blotting using primary anti-ASC antibodies (Wanleibio, Shenyang, China) at a dilution ratio of 1:500.
        Following a previously described procedure [13], cell culture supernatants were harvested and secretion of the pro-inflammatory cytokine IL-1β was determined using the mouse IL-1 Beta ELISA kit (Invitrogen, Thermo Fisher Scientific). Convert OD450nm values ​​to protein concentrations using the IL-1β standard curve.
        Cells coated on coverslips were gently washed 3 times in warm PBS, fixed in tissue cell fixative (Biosharp, Beijing, China) for 10 min at room temperature (RT), in 0.1% Triton X-Permeabilize at 100 ( diluted in PBS; Biosharp) for 20 minutes at room temperature and block in 5% bovine serum albumin (in PBS) for 2 hours at room temperature. Cells were then incubated overnight at 4°C with primary antibodies against ASC (1:100 dilution) or NLRP3 (1:100 dilution), respectively, and Cy3 labeled goat anti-rabbit IgG(H+L) (1:400; EarthOx, San Francisco, CA, USA) or FITC-conjugated goat anti-mouse IgG (1:400; Earthox) overnight at 37°C in the dark for 1 hour. The nuclei were stained with Hoechst 33258 (10 μg/ml; UE, Suzhou, China) for 5 minutes and observed under a fluorescence microscope (Olympus Corporation, Tokyo, Japan).
        Mice were divided into four groups (n = 7 in each group): (i) PBS-treated negative control group (PBS only; gavage 100 µl/mouse PBS followed by daily intraperitoneal injection 100 µl/mouse PBS 3 hours later) . , continuously for 7 days); (ii) negative control group treated with MCC950 inhibitor [27] (100 µl/mouse via PBS gavage, 3 hours later, 10 mg/kg body weight [BW] MCC950 [in PBS] was administered intraperitoneally daily, duration 7 days); (iii) G. duodenalis cyst infection group (1.5 x 106 cysts/mouse by gavage, 3 hours later, 100 μl/mouse PBS intraperitoneally administered daily for 7 days); (iv) G. duodenalis cyst combined infection group MCC950 inhibitor treatment group (1.5×106 cysts/mouse via gavage, 10mg/kg body weight MCC950 intraperitoneally daily for 7 days at 3h). The body weight of each mouse was monitored daily and all mice were euthanized on the 7th day. Harvested duodenum (3 cm long) was cut into small pieces in 1 ml PBS, cysts were destroyed overnight in PBS at 4°C, and G. duodenalis trophozoites. Fresh duodenum (1 cm long) was isolated for hematoxylin and eosin (H&E) staining.
        Mice were divided into two groups: (i) MOCK control group and (ii) MCC950 inhibitor group. There were five treatments in each group (n = 7/treatment group): (i) PBS treatment negative control group (PBS only; 100 µl/mouse PBS, intramuscular (IM) injection (tibialis anterior) [28, 29] ;(ii) pcDNA3.1(+) plasmid negative control group (100 µg/mouse DNA, via intramuscular injection); (iii) G. duodenalis cyst infection positive control group (1.5 x 106 cysts/mouse, via gavage) (iv) a group treated with plasmid pcDNA3.1(+)-alpha-2 (100 μg/mouse DNA, by intramuscular injection), and (v) a group treated with plasmid pcDNA3.1(+)-alpha-7.3 (100 µg/mouse DNA, after 12 hours of passage, mice in the MCC950 inhibitor group received a daily intraperitoneal injection of MCC950 (10 mg/kg body weight) for 7 days, while mice in the MOCK group received an equal volume of PBS treatment.Blood samples were collected from the eyeballs mice and left overnight at 4 °C. Serum samples were isolated using enzyme-linked immunosorbent assay (ELISA) for and measurements of IL-1β levels.
        Thirty-five mice were divided into five groups (n=7/group). Group 1 was a negative control group treated with PBS: mice received 100 μl of PBS intramuscularly and 3 days later by gavage. Group 2 is a positive control group infected with G. duodenalis cysts: mice were injected with 100 μl of PBS, and 3 days later 1.5 x 106 cysts/mouse were injected intragastrically. Third group – plasmid immunization with pcDNA3.1(+) in combination with a control group for duodenal cyst infection: mice received 100 μg of plasmid DNA pcDNA3.1(+)(im) orally, 1.5×106 cysts/mouse 3 for several days. Groups 4 and 5 were recombinant pcDNA3.1(+)-alpha-2 giardine plasmid or pcDNA3.1(+)-alpha-7.3 giardine plasmid in combination with G. duodenalis cyst infection. Experimental group: mice received 100 µg of pcDNA3. 1(+)-giardine plasmid DNA (im), then 3 days later, 1.5 × 106 cysts/mouse were injected via gavage. The body weight of each mouse was monitored after the introduction of the G. duodenalis cyst through the tube. Fresh duodenum was collected for parasitic load measurements and HE staining analysis.
        Histopathological changes were analyzed according to a previously published procedure [30]. Fresh duodenum was fixed with tissue cell fixative, embedded in paraffin, cut into 4 μm sections, stained with H&E and analyzed under a light microscope. Representative pathological changes in seven tissue sections from seven independent mice were evaluated by a pathologist unaware of the treatment and were captured at 200x magnification. The length of the villi and the depth of the crypts were measured in accordance with the previously described methods.
        The results in vitro and in vivo were obtained in triplicate. Graphs were generated using GraphPad Prism 7.00 (GraphPad Software Inc., La Jolla, CA, USA). Differences between two groups were analyzed by t-test, while differences between ≥3 groups were analyzed by one-way analysis of variance (ANOVA) using SPSS software (version 22.0; SPSS IBM Corp., Armonk, NY, USA) . Data were analyzed for homogeneity of variance using Levene’s test followed by Bonferroni’s post hoc test (B). Significance is expressed as P<0.05, P<0.01, and P<0.001 (not significant [ns]) (P>0.05).
        Our previous analysis of GEV proteomics in the Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that many targets may be involved in the activation of inflammatory signaling pathways [13]. We selected two promising targets, alpha-2 and alpha-7.3 giardins, amplify these molecules and use them to construct the pcDNA3.1(+) eukaryotic expression vector. After sequencing, recombinant pcDNA3.1(+)-alpha-2 and alpha-7.3 giardine expression plasmids were transfected into primary mouse peritoneal macrophages, and the caspase-1 p20 signature protein of inflammation (a fragment of activated caspase-1) was identified as elucidating key molecules that can trigger inflammation. The results showed that alpha-2 and alpha-7.3 giardines can induce p20 caspase-1 expression similar to GEV. No effect on caspase-1 activation was found in the untreated negative control (PBS only) and plasmid control pcDNA3.1(+) (Figure 1).
        Measurement of p20 caspase-1 activation by pcDNA3.1(+)-alpha-2 and alpha-7.3 giardins. Recombinant eukaryotic expression plasmids pcDNA3.1(+)-alpha-2 and alpha-7.3 giardines (above each lane) were transfected into primary mouse peritoneal macrophages and culture supernatants were harvested 24 hours later. Western blotting was used to measure expression levels of the signature caspase-1 p20 inflammasome protein. The PBS-only treatment group (lane C) and the pcDNA3.1(+) monotherapy group (pcDNA3.1 lane) were used as a negative control, and the GEV treatment group was used as a positive control. Expression of the recombinant protein was confirmed by detecting a histidine tag in each protein, and the expected protein bands were alpha-2 giardine (38.2 kDa) and alpha-7.3 giardine (37.2 kDa). GEV, Giardia duodenum extracellular vesicles, pcDNA3.1(+), EcoRV-linearized vector, SUP, supernatant
        To determine whether alpha-2 giardine and alpha-7.3 giardine induce p20 caspase-1 expression and play a role in activating the host NLRP3 inflammatory response, pcDNA3.1(+)-alpha-2 giardine and pcDNA3.1(+)-alpha -7.3 giardin was transfected into primary mouse peritoneal macrophages with recombinant plasmid DNA, and levels of expression, localization, and oligomerization of the key inflammatory proteins NLRP3 were determined. In this experiment, GEV was used as the positive control group, and the no treatment group (PBS only) or the pcDNA3.1(+) transfection treatment group was the negative group. The results showed that, as in the GEV group, recombinant plasmid DNA of giardin pcDNA3.1(+)-alpha-2 and giardin pcDNA3.1(+)-alpha-7.3 resulted in upregulation of NLRP3, pro-IL-1β and procaspase-1 and caspase-1 activation (Fig. 2a). In addition, both giardines induced significant IL-1β secretion (pcDNA3.1: ANOVA, F(4, 10) = 1.625, P = 0.1000; alpha-2 giardine: ANOVA, F(4, 10) = 1.625, P = 0.0007). ; alpha-7.3 giardine: ANOVA, F(4, 10) = 1.625, P<0.0001; GEV: ANOVA, F(4, 10) = 1.625, P = 0.0047) (Figure 2b). Most ASC proteins were monomeric in the no-treatment group or in the treatment group transfected with the pcDNA3.1(+) plasmid, in contrast to pcDNA3.1(+)-alpha-2 or pcDNA3.1(+)-alpha-7.3 giardine. ASC oligomerization occurred in the recombinant plasmid DNA of the GEV positive control group or group, showing an oligomeric form (Figure 2c). These preliminary data suggest that alpha-2 giardine and alpha-7,3 giardine can induce NLRP3 inflammation activation. Subsequent immunofluorescent studies of the localization of ASC and NLRP3 showed that in the negative control group, the ASC protein was scattered throughout the cytoplasm and appeared as a dot signal upon stimulation of pcDNA3.1(+)-alpha-2 with giardine or pcDNA3. 1(+)-alpha-7,3 giardine group or GEV positive control group (Figure 2d). In the negative control and plasmid-treated pcDNA 3.1 groups, the NLRP3 protein signal was not detected, while a fluorescent signal dot in response to pcDNA3.1(+)-alpha-2 giardine or pcDNA3.1(+)-alpha-7.3 was detected. . giardine are found in the cytoplasm or upon stimulation of the HEV (Fig. 2e). These data further demonstrate that G. duodenalis giardin alpha-2 and giardin alpha-7.3 activate the NLRP3 inflammasome in mouse primary peritoneal macrophages.
        pcDNA3.1(+)-alpha-2 giardin and pcDNA3.1(+)-alpha-7.3 giardin activate the NLRP3 inflammasome in mouse peritoneal macrophages. Transfect the recombinant eukaryotic expression plasmids pcDNA3.1(+)-alpha-2 giardin and pcDNA3.1(+)-alpha-7.3 giardin into primary murine peritoneal macrophages and cells, or harvest the supernatant within 24 h for analysis of expression, oligomerization, secretion. and localization of key inflammatory proteins. The PBS-only (C) group and the pcDNA3.1(+) single treatment group were used as the negative control, and the GEV treatment group was used as the positive group. a Key inflammatory proteins NLRP3, including NLRP3, pro-IL-1β, pro-caspase-1, and p20 caspase-1, were detected by Western blotting. b The levels of secretion of IL-1β in the supernatants were determined using enzyme-linked immunosorbent assay (ELISA). Differences between control and experimental groups were analyzed by one-way analysis of variance (ANOVA) using SPSS software version 22.0. Asterisks indicate significant differences between groups **P<0.01 and ***P<0.001. c ASC oligomerization levels in pellets were determined by DSS cross-linking analysis, while ASC levels in cell lysates were used as a loading control. d Visualization of ISC localization using immunofluorescence. e Immunofluorescence was used to visualize the localization of NLRP3. ASC, apoptotic speck-like protein; IL, interleukin; NLRP3, nucleotide-binding oligomerization-like receptor 3; ns, not significant (P > 0.05)
        Both G. duodenalis and the GEVs it secretes activate the NLRP3 inflammasome and regulate host inflammatory responses in vitro. Thus, the role of the NLRP3 inflammasome in the pathogenicity of G. duodenalis remains unclear. To investigate this issue, we designed an experiment between mice infected with G. duodenalis cyst and mice infected with G. duodenalis cyst + MCC950 inhibitor treatment and compared NLRP3 inflammasome expression when infected with G. duodenalis cyst. A detailed scheme of the experiment is shown in Fig. 3a. Changes in body weight of mice in different treatment groups were monitored for 7 days after infection with cysts, and the results are shown in Fig. 3b. Compared to the group treated with pure PBS, the results showed that (i) the body weight of mice infected with G. duodenalis cyst decreased from day 3 to day 7 after infection; (ii) treatment with the MCC950 inhibitor had no significant effect on the body weight of the mice. . Compared to the single infection group, the BW of the duodenal infection group treated with MCC950 decreased to varying degrees (Day 1: ANOVA, F(3, 24) = 1.885, P = 0.0148; Day 2: ANOVA, F( 3, 24) = 0.4602, P<0.0001; Day 3: ANOVA, F(3, 24) = 0.8360, P = 0.0010; Day 4: ANOVA, F(3, 24) = 1.683, P = 0.0052; Day 5: ANOVA, F(3, 24)=0.6497, P=0.0645; Day 6: ANOVA, F(3, 24)=5.457, P=0.0175; Day 7: ANOVA, F(3, 24) = 2.893, P = 0.0202). These data demonstrate that the NLRP3 inflammasome protects mice from significant weight loss in the early stages (2-4 days) of duodenal infection. We then aimed to detect G. duodenalis trophozoites in duodenal lavage fluid and the results are shown in Figure 3c. Compared to the G. duodenalis cyst infection group, the number of trophozoites in the duodenum significantly increased after blocking the NLRP3 inflammasome (t(12) = 2.902, P = 0.0133). Duodenal tissues stained with HE showed, compared to negative control treated with PBS and MCC950 alone: ​​(i) G. duodenalis cyst infection resulted in damage to the duodenal villi (ANOVA, F(3, 24)=0.4903, P= 0.0488) and crypt atrophy (ANOVA, F(3, 24) = 0.4716, P = 0.0089); (ii) duodenum from mice infected with G. duodenalis cysts and treated with MCC950 inhibitors. duodenal villi were damaged and dead (ANOVA, F(3, 24) = 0.4903, P = 0.0144) with atrophy and crypt branching (ANOVA, F(3, 24) = 0.4716, P = 0, 0481) (Fig. 3d-f) . These results suggest that the NLRP3 inflammasome plays a role in reducing the pathogenicity of G. duodenalis.
        Role of NLRP3 inflammasome in Giardia duodenum infection. Mice were gavaged (i.v.) with duodenococcal cysts and then treated with or without MCC950 (i.p.). Single treatment groups with PBS or MCC950 were used as controls. Experimental group and treatment regimen. b The body weight of mice in each of the various treatment groups was monitored for 7 days. The difference between the G. duodenalis infection group and the G. duodenalis + MCC950 infection treatment group was analyzed by t-test using SPSS software version 22.0. Asterisks indicate significant differences at *P<0.05, **P<0.01, or ***P<0.001. c Parasitic load was determined by counting the number of trophozoites in duodenal lavage fluid. The difference between the G. duodenalis infection group and the G. duodenalis + MCC950 infection treatment group was analyzed by t-test using SPSS software version 22.0. Asterisks indicate significant differences at *P < 0.05. d Hematoxylin and eosin (H&E) staining results of duodenal histopathology. Red arrows indicate damage to the villi, green arrows indicate damage to the crypts. Scale bar: 100 µm. e, f Statistical analysis of duodenal villus height and mouse crypt height. Asterisks indicate significant differences at *P<0.05 and **P<0.01. The results are taken from 7 independent biological experiments. BW, body weight; ig, intragastric delivery route; ip, intraperitoneal delivery route; ns, not significant (P > 0.05); PBS, phosphate buffered saline; WT, wild type
        The secretion of IL-1β is a hallmark of inflammation activation. To determine whether G. duodenalis alpha-2 giardine and alpha-7.3 giardine activate the NLRP3 host inflammasome in vivo, we used untreated WT mice (sham group) and NLRP3 inflammasome-blocked mice (MCC950 inhibited treatment group). A detailed scheme of the experiment is shown in Fig. 4a. Experimental groups consisted of mice treated with PBS, G. duodenalis cyst treatment by gavage, intramuscular injection of pcDNA3.1, and intramuscular injection of pcDNA3.1(+)-alpha-2 giardine or pcDNA3.1-alpha-7.3 giardine. On the 7th day after intramuscular administration of the recombinant plasmid, serum was collected and the level of IL-1β in each group was determined. As shown in Figure 4b, in the MOCK group: (i) compared to the PBS group, pcDNA3.1 treatment had no significant effect on IL-1β secretion (ANOVA, F(4.29)=4.062, P=0.9998), however, IL-β secretion was significantly elevated in the G. duodenalis cyst group (ANOVA, F(4, 29) = 4.062, P = 0.0002), (ii) pcDNA3.1-alpha-2 giardine and pcDNA3. 1- Intramuscular injection of alpha-7.3 giardine significantly increased serum IL-1β levels (ANOVA, F(4, 29) = 4.062, P<0.0001); (iii) pcDNA3.1-alpha-7,3 giardine induced high levels of IL -1β secretion in the pcDNA3.1-alpha-2 giardine intramuscular injection group (ANOVA, F(4, 29) = 4.062, P = 0.0333) . Compared with each group in the MCC950 treatment group and the MOCK group: (i) IL-1β secretion levels in the PBS control group and the pcDNA3.1 control group decreased to a certain extent after blocking the MCC950 inhibitor, but the difference was not significant (PBS: ANOVA, F (9, 58) = 3.540, P = 0.4912 pcDNA3.1: ANOVA, F(9, 58) = 3.540, P = 0.5949); (ii) after blocking MCC950. , IL-1β secretion was significantly reduced in the G. duodenalis cyst-infected group, the pcDNA3.1-alpha-2 giardine group, and the pcDNA3.1-alpha-7.3 giardine group (G. duodenalis: ANOVA, F(9, 58) = 3.540 , P = 0.0120; pcDNA3.1-alpha-2 giardine: ANOVA, F(9, 58) = 3.540, P = 0.0447; pcDNA3.1-alpha-7.3 giardine: ANOVA, F(9, 58) = 3.540, P = 0.0164). These results suggest that alpha-2 giardine and alpha-7.3 giardine mediate the activation of the NLRP3 inflammasome in vivo.
        pcDNA3.1(+)-giardines activate the NLRP3 host inflammasome in vivo. Mice were immunized (IM) with recombinant eukaryotic expression plasmid pcDNA3.1(+)-alpha-2 giardine or pcDNA3.1(+)-alpha-7.3 giardine and then treated with MCC950 (ip; MCC950 group) or not (dummy group). The PBS or pcDNA3.1(+) plasmid treatment group was used as a negative control, the G. duodenalis cyst treatment group was used as a positive control. Experimental group and treatment regimen. b Serum levels of IL-1β in mice were measured on day 7 by ELISA assay. Differences between groups in the MOCK group were analyzed using one-way ANOVA, and differences between the MOCK group and the MCC950 group were analyzed using the t-test of SPSS software version 22.0. Asterisks indicate significant differences between treatment groups in the MOCK group, *P<0.05 and ***P<0.001; dollar signs ($) indicate significant differences between each group in the MOCK group and the MCC950 group at P<0.05. Results of seven independent biological experiments. i, intramuscular injection, ns, not significant (P > 0.05)
        To investigate the effect of alpha-2 and alpha-7.3 giardine-mediated activation of the NLRP3 host inflammasome on G. duodenalis infectivity, we used WT C57BL/6 mice and injected alpha-2 giardine and alpha-7.3 giardine. the plasmid was injected intramuscularly, after 3 days through the gastric tube of the G. duodenalis cyst, after which the mice were observed for 7 days. A detailed scheme of the experiment is shown in Fig. 5a. The body weight of each mouse was measured every day, samples of fresh duodenal tissue were collected on the 7th day after administration through a gastric tube, the number of trophozoites was measured, and histopathological changes were observed. As shown in Figure 5b, with increasing feeding time, the BW of mice in each group gradually increased. The MT of mice began to decrease on the 3rd day after intragastric administration of G. duodenalis cysts, and then gradually increased. Activation of the NLRP3 inflammasome induced by intramuscular injection of alpha-2 giardine and alpha7.3 giardine significantly attenuated weight loss in mice (Day 1: pcDNA3.1-alpha-2 giardine, ANOVA, F(4, 30) = 1.399, P = 0 .9754 Day 1: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30)=1.399, P=0.9987 Day 2: pcDNA3.1-alpha-2 giardine, ANOVA, F( 4, 30) = 0.3172, P = 0.9979; Day 2: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30) = 0.3172, P = 0.8409; Day 3 : pcDNA3.1-alpha-2 giardine, ANOVA, F( 4, 30) = 0.8222, P = 0.0262 Day 3: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4 , 30) = 0.8222, P = 0.0083; Day 4: pcDNA3.1-alpha-2 giardine, ANOVA, F(4, 30) = 0.5620, P = 0.0012, Day 4: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30) = 0.5620, P < 0.0001, Day 5: pcDNA3.1-alpha -2 giardine, ANOVA, F(4, 30) = 0.9728, P < 0.0001 Day 5: pcDNA3.1-alpha -7.3 giardine, ANOVA, F(4, 30) = 0.9728, P < 0.0001 Day 6: pcDNA3 .1 -alpha-2 giardine, ANOVA, F(4, 30) = 0.7154, P = 0.0012, Day 6: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4, 30) = 0.7154, P = 0.0006; Day 7: pcDNA3.1-alpha-2 giardine, ANOVA, F(4, 30) = 0.5369, P<0.0001 Day 7: pcDNA3.1-alpha-7.3 giardine, ANOVA, F(4 , 30) = 0.5369, P<0.0001). The parasitic load was assessed in the duodenum (Fig. 5c). Compared to the untreated positive control and the group injected with the empty pcDNA3.1 vector, the number of G. duodenalis trophozoites was significantly reduced in the groups injected with α-2 giardine and α-7,3 giardine (pcDNA3.1-alpha-2 giardine : ANOVA, F(3, 24) = 1.209, P = 0.0002, pcDNA3.1-alpha-7.3 giardine: ANOVA, F(3, 24) = 1.209, P<0.0001). In addition, giardine alfa-7.3 was more protective in mice than giardine alfa-2 (ANOVA, F(3, 24) = 1.209, P = 0.0081). The results of HE staining are shown in fig. 5d–f. Mice injected with alpha-2 giardine and alpha-7.3 giardine had fewer duodenal tissue lesions, manifested by villus damage, compared to mice injected with G. duodenalis and mice injected with G. duodenalis in combination with an empty pcDNA3 vector .1 Zoom. (pcDNA3.1-alpha-2 giardine: ANOVA, F(3, 24) = 2.466, P = 0.0035 or P = 0.0068; pcDNA3.1-alpha-7.3 giardine: ANOVA, F(3, 24) = 2.466, P = 0.0028 or P = 0.0055) and reduced crypt atrophy (pcDNA3.1-alpha-2 giardine: ANOVA, F(3, 24) = 1.470, P = 0.0264 or P = 0.0158; pcDNA3.1-alpha-7.3 giardine: ANOVA, F(3, 24) = 1.470, P = 0.0371 or P = 0.0191). These results suggest that alpha-2 giardine and alpha-7,3 giardine reduce the infectivity of G. duodenalis by activating the NLRP3 inflammasome in vivo.
        Role of pcDNA3.1(+)-giardins in G. duodenalis infection. Mice were immunized (IM) with recombinant eukaryotic expression plasmids pcDNA3.1(+)-alpha-2 giardine or pcDNA3.1(+)-alpha-7.3 giardine and then challenged with G. duodenalis cysts (ig). The PBS group and the pcDNA3.1(+) + duodenal cyst treatment group were used as negative control groups, and the duodenal cyst treatment group was used as positive control group. Experimental group and treatment regimen. b The MT of mice in each of the various treatment groups was monitored for 7 days post-challenge. Asterisks indicate significant differences between groups in the G. duodenalis group and the pcDNA3.1(+)-alpha-2 giardine group, *P < 0.05, **P < 0.01, and ***P < 0.001; the dollar sign ($) indicates a significant difference between each group of G. duodenalis and the pcDNA3.1(+)-alpha-7.3 jardine group, $$P<0.01 and $$$P<0.001. c Parasitic load was determined by counting the number of trophozoites in 1 ml of duodenal lavage from the duodenum (3 cm long) and expressed as the number of parasites per cm of duodenum. Differences between the G. duodenalis infection group, the pcDNA3.1(+)-alpha-2 giardine group, and the pcDNA3.1(+)-alpha-7.3 giardine group were analyzed by one-way ANOVA using SPSS software version 22.0. Asterisks indicate significant differences at **P<0.01 and ***P<0.001. d Histopathological changes in the duodenum. Red arrows indicate damage to the villi, green arrows indicate damage to the crypts. Scale bar: 100 µm. e, f Statistical analysis of mouse duodenal villus height (e) and crypt height (f). Differences between groups in Figure 1d were analyzed by one-way ANOVA using SPSS software version 22.0. Asterisks indicate significant differences at *P<0.05 and **P<0.01. Results of seven independent biological experiments. ns, not significant (P > 0.05)
        Giardia duodenum is a well-known intestinal parasite of humans and other mammals that causes giardiasis. In 2004, it was included in the WHO Neglected Diseases Initiative due to its high prevalence over 6 years, especially in communities of low socioeconomic status [32]. The innate immune system plays a critical role in the immune response to G. duodenalis infection. Mouse macrophages have been reported to engulf and kill G. duodenalis by releasing extracellular traps [33]. Our previous studies have shown that G. duodenalis, a non-invasive extracellular parasite, activates p38 MAPK, ERK, NF-κB p65, and NLRP3 inflammatory signaling pathways in mouse macrophages to regulate host inflammatory responses, and released GEV may enhance this process. 13], 24]. However, the exact PAMPs involved in NLRP3 inflammasome-regulated inflammation in GEV and the role of NLRP3 inflammasome in giardiasis remain to be elucidated. To shed light on these two questions, we conducted this study.
        The NLRP3 inflammasome is located in the cytoplasm of immune cells and can be activated by various particles such as uric acid crystals, toxins, bacteria, viruses, and parasites. In bacterial studies, toxins have been identified as key PAMPs that activate inflammatory sensors, leading to inflammation and cell death [34]. Some structurally diverse toxins, such as hemolysin from Staphylococcus aureus [35] and Escherichia coli [36], hemolysin BL (HBL) from enterotoxin (NHE) [37], induce the activation of NLRP3 inflammation. Viral studies have shown that virulence proteins such as SARS-COV-2 envelope (E) protein [38] and Zika virus NS5 protein [39] are important PAMPs recognized by the NLRP3 receptor. In parasite studies, many parasites have been reported to be associated with host inflammasome activation, such as Toxoplasma gondii, Trichomonas vaginalis [40], Trypanosoma cruzi [41], and Leishmania [42]. The dense granule proteins GRA35, GRA42, and GRA43, associated with the virulence of Toxoplasma gondii, are required for the induction of pyroptosis in Lewis rat macrophages [43]. In addition, some Leishmania studies have focused on individual molecules involved in the NLRP3 inflammasome, such as parasite membrane lipophosphoglycan [44] or zinc metalloprotease [45]. Among the annexin-like alpha-giardin family of genes, alpha-1 giardin has been shown to be a potential vaccine candidate providing protection against G. duodenalis in a mouse model [18]. In our study, we selected G. duodenalis virulence factors alpha-2 and alpha-7,3 giardines, which are unique to giardia but relatively less reported. These two target genes were cloned into the pcDNA3.1(+) eukaryotic expression system vector for analysis of inflammation activation.
        In our mouse model, cleaved caspase fragments serve as markers of inflammatory activation. Upon stimulation, NLRP3 interacts with ASC, recruits procaspases, and generates active caspases that cleave pro-IL-1β and pro-IL-18 into mature IL-1β and IL-18, respectively -18. Inflammatory caspases (caspases-1, -4, -5 and -11) are a conserved family of cysteine ​​proteases that are critical for innate defense and are involved in inflammation and programmed cell death [46]. Caspase-1 is activated by canonical inflammasomes [47], while caspases-4, -5, and -11 are cleaved during the formation of atypical inflammasomes [48]. In this study, we used mouse peritoneal macrophages as a model and investigated p20 caspase-1 cleaved caspase-1 as a marker of host NLRP3 inflammation activation in studies of G. duodenalis infection. The results showed that many alpha-giardins are responsible for the typical activation of inflammation, which is consistent with the discovery of key virulence molecules involved in bacteria and viruses. However, our study is only a preliminary screen and there are other molecules that can activate non-classical inflammasomes, as our previous study found both classical and non-classical inflammasomes in G. duodenalis infection [13]. To further determine whether the generated p20 caspase-1 is associated with the NLRP3 inflammasome, we transfected alpha-2 and alpha-7.3 giardins into mouse peritoneal macrophages to determine key molecule protein expression levels and ASC oligomerization levels, confirming that both α-giardins activate inflammasome NLRP3. Our results are slightly different from those of Manko-Prykhoda et al., who reported that stimulation of Caco-2 cells with G. muris or E. coli EPEC strains alone can increase the fluorescence intensity of NLRP3, ASC, and caspase-1, although not significantly, while how costimulation of G. muris and E. coli increased the levels of three proteins [49]. This discrepancy may be due to differences in the selection of Giardia species, cell lines, and primary cells. We also performed in vivo assays using MCC950 in 5-week-old female WT C57BL/6 mice, which are more susceptible to G. duodenalis. MCC950 is a potent and selective small molecule NLRP3 inhibitor that blocks canonical and non-canonical NLRP3 activation at nanomolar concentrations. MCC950 inhibits NLRP3 activation but does not affect the activation of AIM2, NLRC4, and NLRP1 inflammatory pathways or TLR signaling pathways [27]. MCC950 blocks NLRP3 activation but does not inhibit NLRP3 initiation, K+ efflux, Ca2+ influx, or the interaction between NLRP3 and ASC; instead, it inhibits NLRP3 inflammasome activation by blocking ASC oligomerization [27]. Therefore, we used MCC950 in an in vivo study to determine the role of the NLRP3 inflammasome after giardine injection. Activated caspase-1 p10 cleaves pro-inflammatory cytokines pro-IL-1β and pro-IL-18 into mature IL-1β and IL-18 [50]. In this study, serum IL-1β levels in giardine-treated mice with or without MCC950 were used as an indicator of whether the NLRP3 inflammasome was activated. As expected, MCC950 treatment significantly reduced serum IL-1β levels. These data clearly demonstrate that G. duodenalis giardin alfa-2 and giardin alfa-7.3 are able to activate the NLRP3 mouse inflammasome.
        Significant data accumulated over the past decade have demonstrated that IL-17A is the master regulator of immunity against G. muris, inducing IL-17RA signaling, producing antimicrobial peptides, and regulating complement activation [51]. However, Giardia infection occurs more frequently in young adults, and it has been reported that Giardia infection in young mice does not activate the IL-17A response to exert its protective effect [52], prompting researchers to look for other immunomodulatory Giardia. mechanisms of helminth infection. The authors of a recent study reported that G. muris can activate the NLRP3 inflammasome by E. coli EPEC, which promotes the production of antimicrobial peptides and reduces its attachment capacity and the number of trophozoites in the intestinal tract, thereby reducing the severity of colon diseases caused by bacilli [49 ]. The NLRP3 inflammasome is involved in the development of various diseases. Studies have shown that Pseudomonas aeruginosa triggers autophagy in macrophages to avoid cell death, and this process depends on the activation of the NLRP3 inflammasome [53]. For N. caninum, reactive oxygen species-mediated activation of the NLRP3 inflammasome limits its replication in the host, making it a potential therapeutic target [9]. Paracoccidioides brasiliensis has been found to induce the activation of the NLRP3 inflammasome in mouse bone marrow-derived dendritic cells, resulting in the release of the inflammatory cytokine IL-1β, which plays a critical role in host defense [10]. Several Leishmania species, including L. amazonensis, L. major, L. braziliensis, and L. infantum chagasi, activate NLRP3 and ASC-dependent caspase-1 in macrophages, as well as Leishmania infection. Parasite replication is enhanced in mice deficient in the NLRP3/ASC/caspase-1 gene [11]. Zamboni et al. Leishmania infection has been reported to induce activation of the NLRP3 inflammasome in macrophages, which limits intracellular parasite replication. Thus, Leishmania may inhibit NLRP3 activation as an avoidance strategy. In in vivo studies, the NLRP3 inflammasome contributed to the elimination of Leishmania, but did not affect tissues [54]. Conversely, in helminthiasis studies, activation of the NLRP3 inflammasome suppressed the host’s protective immunity against gastrointestinal helminthiasis [12]. Shigella is one of the main bacteria causing diarrhea worldwide. These bacteria can induce IL-1β production through P2X7 receptor-mediated K+ efflux, reactive oxygen species, lysosomal acidification, and mitochondrial damage. The NLRP3 inflammasome negatively regulates phagocytosis and bactericidal activity of macrophages against Shigella [55]. Plasmodium studies have shown that AIM2, NLRP3 or caspase-1 deficient mice infected with Plasmodium produce high levels of type 1 interferon and are more resistant to Plasmodium infection [56]. However, the role of alpha-2 giardine and alpha-7.3 giardine in inducing pathogenic activation of NLRP3 inflammation in mice is unclear.
        In this study, inhibition of the NLRP3 inflammasome by MCC950 reduced BW and increased the number of trophozoites in intestinal lavage fluid in mice, resulting in more severe pathological changes in duodenal tissue. Alpha-2 giardine and alpha-7.3 giardine activate the host mouse NLRP3 inflammasome, increase mouse body weight, reduce the number of trophozoites in intestinal lavage fluid, and alleviate pathological duodenal lesions. These results suggest that G. duodenalis can activate the NLRP3 host inflammasome via alpha-2 giardine and alpha-7,3 giardine, reducing the pathogenicity of G. duodenalis in mice.
        Collectively, our results demonstrate that alpha-2 and alpha-7.3 giardines induce the activation of the NLRP3 host inflammasome and reduce the infectivity of G. duodenalis in mice. Therefore, these molecules are promising targets for the prevention of giardiasis.
       Data supporting the results of this study can be obtained from the respective author at gongpt@jlu.edu.cn.
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