HuNoV Non-Structural Protein P22 Induces Maturation of IL-1β and IL-18 and N-GSDMD-Dependent Pyroptosis through Activating NLRP3 Inflammasome

Norovirus infection is the leading cause of foodborne gastroenteritis worldwide, causing more than 200,000 deaths each year. As a result of a lack of reproducible and robust in vitro culture systems and suitable animal models for human norovirus (HuNoV) infection, the pathogenesis of HuNoV is still poorly understood. In recent years, human intestinal enteroids (HIEs) have been successfully constructed and demonstrated to be able to support the replication of HuNoV. The NLRP3 inflammasome plays a key role in host innate immune responses by activating caspase1 to facilitate IL-1β and IL-18 secretion and N-GSDMD-driven apoptosis, while NLRP3 inflammasome overactivation plays an important role in the development of various inflammatory diseases. Here, we found that HuNoV activated enteric stem cell-derived human intestinal enteroids (HIEs) NLRP3 inflammasome, which was confirmed by transfection of Caco2 cells with full-length cDNA clones of HuNoV. Further, we found that HuNoV non-structural protein P22 activated the NLRP3 inflammasome and then matured IL-1β and IL-18 and processed the cleavage of gasdermin-D (GSDMD) to N-GSDMD, leading to pyroptosis. Besides, berberine (BBR) could ameliorate the pyroptosis caused by HuNoV and P22 by inhibiting NLRP3 inflammasome activation. Together, these results reveal new insights into the mechanisms of inflammation and cell death caused by HuNoV and provide potential treatments.


Introduction
Norovirus can be transmitted in various ways, including direct transmission through fecal-oral and inhalation of aerosolized vomit and indirect transmission through contaminated water, food, and surfaces. Besides, the virus is highly contagious, and it takes as little as 18 virus particles to establish infection [1]. Norovirus remains infectious on surfaces for up to 2 weeks (more than 2 months in water) and is resistant to many common disinfectants. Outbreaks of acute gastroenteritis typically occur in crowded environments, including hospitals, schools, and cruise ships, although the most common route is believed to be through infected food handlers or contact with fecal-contaminated water [2]. Because of the highly contagious and efficient transmission of norovirus, emerging virus strains have the potential to cause a global pandemic. Globally, norovirus causes approximately 0.7 billion illnesses and 0.22 million deaths per year, with direct and indirect medical costs of more than $4 billion and $60 billion, respectively [3][4][5]. The lack of reproducible and robust cell culture systems and suitable animal models represents a major challenge in studying the pathogenesis of norovirus gastroenteritis and the effects of prevention and treatment measures [6,7]. Human intestinal enteroids (HIEs) can successfully cultivate B. vulgaris [28], and it shows great antiviral and antibacterial effects with few side effects [29]. As reported, BBR inhibits the priming of the NLRP3 inflammasome by decreasing the expression of NLRP3 by suppressing the activation of the nuclear transcription factorkappa B (NF-κB) signaling pathway and inhibits the assembly of the NLRP3 inflammasome by affecting the binding between caspase1 and ASC [30,31], suggesting that BBR could suppress the activation of the NLRP3 inflammasome. Whether BBR has a potential role in suppressing the activation of the NLRP3 inflammasome caused by HuNoV has not been reported.
Here we revealed that HuNoV could activate the NLRP3 inflammasome, which further led to the maturation and release of the pro-inflammatory cytokines IL-1β and IL-18 and pyroptosis driven by N-GSDMD. In addition, we further revealed that HuNoV nonstructural protein P22 contributes to the maturation and release of the pro-inflammatory cytokines IL-1β and IL-18 and N-GSDMD-dependent pyroptosis. Moreover, the NLRP3 inflammasome activation induced by HuNoV and P22 could be inhibited by treatment with BBR, suggesting that BBR has the potential to be a therapeutic agent for relieving norovirus gastroenteritis.
After 7 days of differentiation, HIEs were released from the 3D-culture model and seeded overnight in 6-well plates for use the next day. They were then infected with HuNoV at a genome copy of 3.0 × 10 7 and incubated at 37 • C with 5% CO 2 . Forty-eight hours later, the culture supernatants were collected for later use.

Plasmid Constructs
The clone encoding the full-length cDNA of HuNoV was obtained as described previously [8], and then we used the clone as a template to amplify the gene of HuNoV non-structural protein P22 with a 3 × Flag tag introduced into its C-terminal, and then the PCR products were inserted into the vector pcDNA3.1(+) (Invitrogen, Waltham, MA, USA), named 3.1-P22.

Cell Transfection, siRNA Interference, and Chemical Treatment
Caco2 cells were seeded in a 6-well plate one night in advance and then transfected with plasmids expressing the full-length HuNoV cDNA or P22 or empty vector pcDNA3.1(+) (negative control (NC)) using Lipo8000 TM (Beyotime, Shanghai, China) according to the manufacturer's instructions. Briefly, for each well, 125 µL of Opti-MEM ® Medium was added into a 1.5 mL centrifuge tube, then 2 µg of plasmid was added, and 3.2 µL of lipo8000 was added after gentle blowing and mixing, and then the mixture was added into the cells and incubated at 37 • C with 5% CO 2 . Four to six hours later, the culture medium was replaced with fresh complete medium, and the incubation was continued for 48 h at 37 • C with 5% CO 2 . In the siRNA interference experiment, Caco2 cells were transfected with caspase1 or NLRP3-specific siRNA or negative control siRNA (si-NC) (Ruibo, Guangzhou, China) using Lipo8000 TM according to the manufacturer's instructions for 12 h and then transfected with plasmids expressing the full-length cDNA of HuNoV or empty vector pcDNA3.1(+) (negative control (NC)). After incubation for four to six hours at 37 • C with 5% CO 2 , the culture medium was replaced with fresh complete medium, and the incubation was continued for 48 h at 37 • C with 5% CO 2 . In BBR treatment experiment, Caco2 cells were transfected with plasmids expressing the full-length cDNA of HuNoV or plasmids expressing P22 or empty vector pcDNA3.1(+) (negative control (NC)) and incubated for 24 h at 37 • C with 5% CO 2 . Then the transfected cells were treated with BBR (100 µM, Solarbio, Guangzhou, China) for 48 h at 37 • C with 5% CO 2 . Before being treated with BBR, the medium was replaced with fresh, complete medium. Finally, the cells and supernatants were separately collected for later use.

Enzyme-Linked Immunosorbent Assay (ELISA)
We collected the cell culture supernatants and then stored them at −80 • C until use. The concentrations of IL-1β and IL-18 in the supernatants of cell culture were determined by the Human IL-1β ELISA kit (MEIMIAN, MM-0181H2, Jiangsu, China) and the Human IL-18 ELISA kit (JINMEI, JM-03294H1, Jiangsu, China) according to the manufacturer's instructions. In brief, a volume of 50 µL of gradient-diluted standards and samples was separately added to each well, and all of them were conducted in triplicate. After incubating at 37 • C for 30-60 min, the plates were washed 5 times with 1× washing buffer. Then HRPconjugated reagent in a volume of 100 µL was added to each well and incubated at 37 • C for 30-60 min. The plates were then washed 5 times again with 1 × washing buffer, and chromogen A 50 µL and chromogen B 50 µL were added, and the plates were incubated for 10 min at 37 • C. Finally, the action was stopped by adding stop solution (2 mol/L H 2 SO 4 ) in a volume of 50 µL to each well. The microplate reader (Molecular Devices, Silicon Valley, USA) was used, and the signal was quantified at 450 nm wavelength.

Protein Concentration Detection
The total protein concentration was determined by the BCA protein concentration determination kit (Beyotime, P0010, Shanghai, China). BCA reagent A and BCA reagent B were thoroughly mixed in a ratio of 50:1 to form the BCA working solution. Gradiently diluted standards and cell lysates in a volume of 20 µL was separately added to each well. After incubating at room temperature for 60 min, 200 µL of BCA working liquid was added to each well. All standards and samples were added in duplicate to the 96-well plate. The microplate reader (Molecular Devices, USA) was used, and 562 nm wavelength or wavelengths between 540 and 595 nm were set to quantify the signal. Ultimately, we calculated the total protein concentration of the sample based on the standard curve.

Western Blotting Analysis
These experiments were performed as mentioned previously [32]. In brief, the collected cells were lysed on ice for 0.5-1 h with cell lysis (Beyotime, P0013, Shanghai, China) with protease inhibitor cocktail (Roche, 11697498001, Hamburg, Germany). Then the lysate was collected into a 1.5 mL centrifuge tube and centrifuged at 10000~15000 rpm for 12 min at 4 • C to obtain the supernatant. The protein lysate was determined for protein concentration. According to the manufacturer's instructions, the total protein concentration was determined by BCA assay kit (ThermoFisher, BCA-23227, Waltham, MA, USA), and then we boiled the samples with loading buffer (50 mM Tris-HCl, 25% glycerol, 2% SDS, 1% DTT, pH 6.8) for 10 min. Further, the prepared samples were subjected to 10% SDS-PAGE and transferred onto 0.45 µm PVDF membranes (Merck Millipore, BS-00-2529, Darmstadt, Germany). We recommend soaking the PVDF membrane in methanol for at least 10 s before use. An amount of 5% non-fat milk blocked the above PVDF membranes at room temperature for 60 min, and subsequently, the primary antibodies against NLRP3, caspase1, N-GSDMD, and β-tubulin incubated them overnight at 4 • C or 60 min at room temperature. These antibodies were purchased from Proteintech, Wuhan, China. After being washed with TBST (200 mM NaCl, 50 mM Tris-HCl, 0.1% Tween-20) for 3 times, 5 min each time, the membrane was incubated with HRP-conjugated goat anti-mouse IgG (Proteintech, SA00001-1, China) or HRP-conjugated goat anti-rabbit IgG (Proteintech, B900210, China) at room temperature for 60 min. After washing with TBST for 5 times, 5 min each time, we incubated the PVDF membranes with enhanced chemiluminescence (ECL) (Biosharp, BL523B, Wuhan, China) and visualized the protein bands under a chemiluminescent imaging system. Image J was used to quantify the relative intensities of the western blots.

Cell Viability Assay
CCK-8 counting kit (Zeta life, K009) was used to measure the cell viability. CCK-8 solution contains WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt), which can be reduced by dehydrogenases in mitochondria to form a highly water-soluble orange formazan, directly proportional to cell proliferation and inversely proportional to cell toxicity. Following the manufacturer's instructions, Caco2 cells at a density of 2.5 × 10 3 cells in 100 µL of medium were plated into each well of a 96-well plate. After transfecting Caco2 cells for 48 or 72 h, CCK-8 solution was added to each well in a volume of 10 µL and incubated for 90 min at 37 • C. The microplate reader (Molecular Devices, USA) was used, and the absorbance was determined at 450 nm wavelength. Control group was assigned 100% viability, and cell viability = (treatment group/NC group) × 100%.

Statistical Analysis
All data were expressed as mean ± SD unless otherwise indicated. GraphPad Prism 8.0 (San Diego, CA, USA) was used to perform statistical analysis. Analyzing the data to determine the differences between two groups was conducted under the Student's t test. One-way ANOVA and Student-Newman-Keuls (SNK) post hoc were used to analyze the data to determine the difference among three or more groups. * p < 0.05 was considered statistically significant.

HuNoV Increases Proteolytic Maturation of IL-1β and IL-18
As a result of the lack of reproducible and robust in vitro culture systems and suitable animal models for HuNoV infection, the pathogenesis of HuNoV is still poorly understood. As described previously [8], HuNoV could infect HIEs. The HIE culture system was used to assess the effect of HuNoV on cytokines associated with inflammation, and we found that the secretion of pro-inflammatory cytokines IL-1β and IL-18 was significantly increased ( Figure 1A). We obtained the same results by transfecting Caco2 cells with plasmids encoding the full-length cDNA of HuNoV ( Figure 1B). The above results suggested that HuNoV could increase the maturation and secretion of IL-1β and IL-18. ity = (treatment group/NC group) × 100%.

Statistical Analysis
All data were expressed as mean ± SD unless otherwise indicated. GraphPad Prism 8.0 (San Diego, CA, USA) was used to perform statistical analysis. Analyzing the data to determine the differences between two groups was conducted under the Student's t test. One-way ANOVA and Student-Newman-Keuls (SNK) post hoc were used to analyze the data to determine the difference among three or more groups. * p < 0.05 was considered statistically significant.

HuNoV Increases Proteolytic Maturation of IL-1β and IL-18
As a result of the lack of reproducible and robust in vitro culture systems and suitable animal models for HuNoV infection, the pathogenesis of HuNoV is still poorly understood. As described previously [8], HuNoV could infect HIEs. The HIE culture system was used to assess the effect of HuNoV on cytokines associated with inflammation, and we found that the secretion of pro-inflammatory cytokines IL-1β and IL-18 was significantly increased ( Figure 1A). We obtained the same results by transfecting Caco2 cells with plasmids encoding the full-length cDNA of HuNoV ( Figure 1B). The above results suggested that HuNoV could increase the maturation and secretion of IL-1β and IL-18.

HuNoV Increases Secretion of IL-1β and IL-18 by Activating NLRP3 Inflammasome
The synthesis and release of IL-1β and IL-18 require two types of signaling, one promoting their expression and the other promoting their maturation and release by activating the NLRP3 inflammasome. Due to the scarcity of human intestinal tissues discarded clinically and the complexity of the construction process of HIEs, Caco2 cells were used as models for later research. To investigate whether HuNoV increased the secretion of IL-1β and IL-18 through activating the NLRP3 inflammasome, Caco2 cells were transfected with the plasmids encoding full-length HuNoV cDNA or empty vector (NC), and we found that the expressions of NLRP3, caspase1, and cleaved caspase1 in Caco2 cells were significantly up-regulated (Figure 2A,B). While co-transfecting Caco2 cells with specifically NLRP3 or caspase-1-targeted siRNA, the maturation of caspase-1 was obviously inhibited, and the secretion of IL-1β and IL-18 was significantly inhibited, as shown in

HuNoV Increases Secretion of IL-1β and IL-18 by Activating NLRP3 Inflammasome
The synthesis and release of IL-1β and IL-18 require two types of signaling, one promoting their expression and the other promoting their maturation and release by activating the NLRP3 inflammasome. Due to the scarcity of human intestinal tissues discarded clinically and the complexity of the construction process of HIEs, Caco2 cells were used as models for later research. To investigate whether HuNoV increased the secretion of IL-1β and IL-18 through activating the NLRP3 inflammasome, Caco2 cells were transfected with the plasmids encoding full-length HuNoV cDNA or empty vector (NC), and we found that the expressions of NLRP3, caspase1, and cleaved caspase1 in Caco2 cells were significantly up-regulated (Figure 2A,B). While co-transfecting Caco2 cells with specifically NLRP3 or caspase-1-targeted siRNA, the maturation of caspase-1 was obviously inhibited, and the secretion of IL-1β and IL-18 was significantly inhibited, as shown in Figure 2C-E. The above results indicated that NLRP3 inflammasome activation by HuNoV provoked the maturation and secretion of IL-1β and IL-18 in enterocytes.

HuNoV Induces N-GSDMD-Dependent Pyroptosis by Activating NLRP3 Inflammasome
Inflammasomes are known to provoke a lytic cell death mode termed pyroptosis, which is provoked by the proteolytic processing of GSDMD to N-GSDMD. NLRP3 inflammasome activation could activate caspase-1 and then cleave GSDMD and release N-GSDMD, leading to N-GSDMD-driven pyroptosis [25,27]. To investigate whether the activation of the NLRP3 inflammasome induced by HuNoV increased the expression of N-GSDMD, we transfected Caco2 cells with the plasmid encoding a full-length HuNoV cDNA clone and found that the level of N-GSDMD was obviously increased ( Figure 3A,B), as was the increased cell death ( Figure 3C). While the increased N-GSDMD level and pyroptosis were inhibited when co-transfected with specifically NLRP3 or caspase1 targeted siRNA ( Figure 3D-F), indicating that N-GSDMD-dependent pyroptosis caused by HuNoV is dependent on the activation of the NLRP3 inflammasome.   GSDMD, we transfected Caco2 cells with the plasmid encoding a full-length cDNA clone and found that the level of N-GSDMD was obviously increased (Figu as was the increased cell death ( Figure 3C). While the increased N-GSDMD level roptosis were inhibited when co-transfected with specifically NLRP3 or caspase1 siRNA ( Figure 3D-F), indicating that N-GSDMD-dependent pyroptosis cau HuNoV is dependent on the activation of the NLRP3 inflammasome.

P22 Induces Maturation of IL-1β and IL-18 and N-GSDMD-Dependent Pyroptosis by Activating NLRP3 Inflammasome in Caco2 Cells
P22 is a non-structural protein encoded by HuNoV [33,34], and we transfected Caco2 cells with a P22-encoding plasmid or empty vector (NC) and found that P22 significantly increased secretion of IL-1β and IL-18 ( Figure 4A). Further, we found that P22 also increased the expression of NLRP3 and cleaved-caspase1, as well as the proteolytic processing of Vaccines 2023, 11, 993 9 of 13 GSDMD to N-GSDMD ( Figure 4B,C), and we also found that P22-specific siRNA inhibited the full-length HuNoV cDNA clone that activated the NLRP3 inflammasome ( Figure 4D), suggesting that P22 plays an important role in the activation of NLRP3 inflammasome and N-GSDMD-driven pyroptosis caused by HuNoV.

P22 Induces Maturation of IL-1β and IL-18 and N-GSDMD-Dependent Pyroptosis by Activating NLRP3 Inflammasome in Caco2 Cells
P22 is a non-structural protein encoded by HuNoV [33,34], and we transfected cells with a P22-encoding plasmid or empty vector (NC) and found that P22 signifi increased secretion of IL-1β and IL-18 ( Figure 4A). Further, we found that P22 al creased the expression of NLRP3 and cleaved-caspase1, as well as the proteolyti cessing of GSDMD to N-GSDMD ( Figure 4B,C), and we also found that P22-sp siRNA inhibited the full-length HuNoV cDNA clone that activated the NLRP3 in masome ( Figure 4D), suggesting that P22 plays an important role in the activat NLRP3 inflammasome and N-GSDMD-driven pyroptosis caused by HuNoV.

BBR Inhibits the Activation of NLRP3 Inflammasome and N-GSDMD-Driven Pyroptosis Induced by HuNoV and P22
Berberine (BBR) is traditionally used to treat diarrhea and gastroenteritis and has been reported to inhibit NLRP3 inflammasome activation [30,35,36]. To explore whether BBR could inhibit HuNoV or P22-induced activation of the NLRP3 inflammasome and N-GSDMD-driven pyroptosis, we transfected Caco2 cells with plasmids encoding the full-length cDNA of HuNoV or P22, and an empty vector was used as a negative control (NC). As shown in Figure 5A, BBR could rescue the cytotoxicity caused by HuNoV and P22. Besides, HuNoV and P22 increased cleaved capase1, N-GSDMD, and secreted IL-1β and IL-18, while BBR inhibited the increase ( Figure 5B-D), indicating that BBR could inhibit the activation of the NLRP3 inflammasome and N-GSDMD-dependent pyroptosis induced by HuNoV and P22 and suggesting that BBR has the potential to treat the acute gastroenteritis caused by HuNoV infection. length cDNA of HuNoV or P22, and an empty vector was used as a negative control As shown in Figure 5A, BBR could rescue the cytotoxicity caused by HuNoV and Besides, HuNoV and P22 increased cleaved capase1, N-GSDMD, and secreted IL-1β IL-18, while BBR inhibited the increase ( Figure 5B-D), indicating that BBR could in the activation of the NLRP3 inflammasome and N-GSDMD-dependent pyroptos duced by HuNoV and P22 and suggesting that BBR has the potential to treat the gastroenteritis caused by HuNoV infection.

Discussion
Norovirus infection is a major cause of gastroenteritis, and outbreaks are freq resulting in a serious medical burden. People of all ages, especially infants and y children, the elderly, and immunocompromised patients, tend to be infected by Hu Several factors are currently increasing the global health challenge of norovirus infe

Discussion
Norovirus infection is a major cause of gastroenteritis, and outbreaks are frequent, resulting in a serious medical burden. People of all ages, especially infants and young children, the elderly, and immunocompromised patients, tend to be infected by HuNoV. Several factors are currently increasing the global health challenge of norovirus infection, in particular the increasing number of infected people who are immunocompromised. In addition, the rapid evolution of circulating norovirus genes and antigens complicates the development of vaccines and therapies, which urgently needs to be resolved. While the pathogenesis of HuNoV remains unclear because of the lack of reproducible and robust in vitro culture systems and suitable animal models of infection, currently, human intestinal enteroids (HIEs), which support the infection and replication of HuNoV, have been successfully constructed and have been widely used in human norovirus research. However, the lack of human intestinal tissues discarded clinically and the complexity of the construction process of HIEs limit their wider application. In this study, we constructed a 3D culture model of HIEs and found that HuNoV-infected HIEs showed increased proinflammatory cytokines IL-1β and IL-18 in the cell supernatants, and the full-length HuNoV cDNA clone transfected Caco2 cells showed the same results, which were consistent with the phenomenon that acute gastroenteritis is easily caused by HuNoV infection. When the NLRP3 inflammasome, assembled by NLRP3, ASC, and caspase1, is activated, caspase1 is self-cleaved and activated, resulting in the maturation and release of IL-1β and IL-18 [19,20]. We further investigated the activation of the NLRP3 inflammasome and found that HuNoV increased the expression of NLRP3, caspase1, and cleaved-caspase1, while down-regulating NLRP3 or caspase1 inhibited the secretion of IL-1β and IL-18, which indicated that HuNoV promoted the maturation and secretion of IL-1β and IL-18 through activating the NLRP3 inflammasome. NLRP3 inflammasome activation could also cleave GSDMD and release N-GSDMD. N-GSDMD can migrate to the cell membrane and bind to its extracellular receptors, interfering with membrane structure and forming pores, thereby releasing various cell contents, IL-1β and IL-18 included, then activating a violent inflammatory response and causing pyroptosis, which is one of the programmed cell death modes [25]. We investigated the expression of N-GSDMD and cell death, and the results showed that HuNoV increased N-GSDMD expression as well as cell death, which were inhibited by down-regulating NLRP3 or caspase1 by specific siRNA targeting NLRP3 or caspase1. The above results suggest that HuNoV could promote the maturation and release of IL-1β and IL-18, as well as N-GSDMD-driven pyroptosis, depending on the activation of the NLRP3 inflammasome. We further revealed that HuNoV non-structural protein P22 played an important role in activating the NLRP3 inflammasome and N-GSDMD-driven pyroptosis, which suggested that P22 might be a potential therapeutic target. It is worth noting that overexpression of P22 may not completely reflect what happens in HuNoV-infected cells, and whether other HuNoV proteins influence P22's regulation of NLRP3 inflammasome activation needs to be further studied.
Berberine (BBR), a natural isoquinoline alkaloid isolated from several traditional Chinese herbal plants, is traditionally used to treat diarrhea and gastroenteritis [28,37], and it was reported to inhibit the activation of the NLRP3 inflammasome [38]. Here, we intended to investigate whether BBR could inhibit NLRP3 inflammasome activation caused by HuNoV and found that BBR treatment reduced the secretion of IL-1β and IL-18 and suppressed N-GSDMD-driven pyroptosis through inhibiting the activation of NLRP3 inflammasome caused by HuNoV and P22, suggesting that BBR has the potential to treat HuNoV-induced acute gastroenteritis through inhibiting the inflammatory response and pyroptosis.

Conclusions
In conclusion, we found that HuNoV activated the NLRP3 inflammasome, leading to the maturation and release of IL-1β and IL-18, as well as N-GSDMD-driven pyroptosis. Our work will contribute to a better understanding of HuNoV-induced inflammatory and cell death pathways. Besides, we identified that BBR treatment inhibited NLRP3 inflammasome activation induced by HuNoV and HuNoV non-structural protein P22, which suggested the potential role of BBR in curing HuNoV-caused acute gastroenteritis.
Author Contributions: N.C., M.F., S.G. and L.G. conceptualized and designed the study; N.C., P.C., S.C. and Y.Z. performed the experiments; M.F., N.C. and P.C. analyzed the data and drafted the manuscript; S.G. and L.G. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement:
The primary data used to support the findings of this study are available from the corresponding author upon request.