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The avian origin influenza A virus (IAV) H7N9 has caused a considerable number of human infections associated with high rates of death since its emergence in 2013. As a vital component of the host innate immune system, the nucleotide-binding domain leucine-rich repeat containing receptor, pyrin domain containing 3 (NLRP3) inflammasome plays a critical role against H1N1 viral infection. However, the function of NLRP3 inflammasome in host immunological responses to the lethal H7N9 virus is still obscure. Here, we demonstrated that mice deficient for NLRP3 inflammasome components, including NLRP3, caspase-1, and Apoptosis-associated speck-like protein containing a CARD (ASC), were less susceptible to H7N9 viral challenge than wild type (WT) controls. Inflammasome deficiency in these animals led to significantly milder mortality and less pulmonary inflammation compared with WT mice. Furthermore, IL-1 receptor deficient mice also exhibited a higher survival rate than WT controls. Thus, our study reveals that the NLRP3 inflammasome is deleterious for the host during H7N9 infection in mice, which is due to an overwhelming inflammatory response via caspase-1 activation and associated IL-1 signal. Therefore, fine-tuning the activity of NLRP3 inflammasome or IL-1 signaling may be beneficial for the host to control H7N9 associated lethal pathogenesis.
In March 2013, the novel avian-origin influenza A virus (IAV) H7N9 was identified in China. H7N9 exhibits low pathogenicity in birds, but has an ability to bind certain mammalian cell receptors, and replicates at temperature close to the normal body temperature of mammals1–3. The infected patients often develop severe symptoms, including pneumonia and acute respiratory distress syndrome (ARDS)4. The H7N9 infection had caused 1230 confirmed human cases, with 428 deaths by 22 February 2017 [data from FAO (Food and Agriculture Organization of the United Nations)]. The severe symptoms and high mortality associated with H7N9 infection pose a big threat to the public health. Although recent studies of H7N9 have achieved great progress, clear definition of the host immunological responses to H7N9 is still lacking1, 5, 6.
Influenza virus infection mainly induces two types of innate immune responses7. A rapid antiviral response, i.e. type I interferon production, to facilitate intracellular control of viral replication; and a proinflammatory response characterized by secretion of cytokines and chemokines, which promotes recruitment of various immune cells for viral clearance8. As a key mediator of inflammation, the proinflammatory cytokine IL-1β plays a crucial role in the pathogenesis of various diseases9. The maturation and secretion of IL-1β require a protein complex called inflammasome, which activates caspase-1 to cleave proIL-1β to its mature form10. More importantly, caspase-1 and the NLRP3 inflammasome have been found essential for protecting mice against H1N1 IAV infection11–13.
In a previous study, we have demonstrated that H7N9 influenza virus isolated from human patients could establish successful infection of mice14, 15. Employing this murine model, in the current study we demonstrate a detrimental role for the NLRP3-caspase-1 inflammasome and associated IL-1 signal in H7N9 infection of mice. Our data suggest that dampening the NLRP3 inflammasome activity or inhibiting IL-1 signaling should be beneficial for the host during a lethal H7N9 challenge.
It has been reported that H1N1 RNA plays a critical role in activating the NLRP3 inflammasome11. To assess whether the NLRP3 inflammasome could be activated during H7N9 challenge, we transfected LPS-primed BMDCs (bone marrow derived dendritic cells) with H7N9 RNA and measured the production of IL-1β. As expected, robust IL-1β secretion was detected in the H7N9 RNA-transfected samples, while the production of an inflammasome-independent cytokine TNF-α was unaltered (Fig. 1a), suggesting that H7N9 RNA-induced IL-1β production was likely dependent on inflammasome activity. To ensure the role of inflammasome in H7N9 RNA-mediated induction of IL-1β, we generated BMDCs from Nlrp3 −/−, Asc −/− and Casp1/11 −/− mice. Transfection of LPS-primed BMDCs from such mice led to significantly compromised IL-1β secretion compared to wild type controls (Fig. 1b). And western blotting revealed that caspase-1 activation and ASC oligomerization were also decreased in BMDCs from Nlrp3 −/− and Asc −/− mice (Fig. 1c), which might account for the compromised IL-1β production. Thus, our results suggest that H7N9 RNA activates caspase-1 via the NLRP3 inflammasome in mouse dendritic cells.
Next, we investigated the function of NLRP3 inflammasome during H7N9 infection in vivo. To this end, age and gender matched WT, Nlrp3 −/− and Casp1/11 −/− mice were intranasally (i.n.) infected with H7N9 virus as described15. We found that the Nlrp3 −/− mice survived significantly better than the WT controls (Fig. 2a). Consistently, the mortality of Casp1/11 −/− mice was also significantly lower compared to WT controls 14 days post infection (d.p.i) (Fig. 2b). In terms of body weight changes, Nlrp3 −/− mice had less weight loss compared to WT controls (P<0.01 at 3, 7, 8 d.p.i) (Fig. 2c). Moreover, WT mice lost significantly more body weights than Casp1/11 −/− mice from 3 to 7 d.p.i (P<0.01 at 3 d.p.i and P<0.05 at other time points) (Fig. 2d). However, on 10 d.p.i the survived WT mice began to recover quicker than Casp1/11 −/− mice. The gain of body weight started at 7 d.p.i for WT but 8 d.p.i. for Casp1/11 −/− mice. By 14 d.p.i, WT mice returned to the initial level of body weight but the Casp1/11 −/− mice did not (Fig. 2d). Notably, no significant difference for H7N9 viral quantity in the lungs was observed between the infected Nlrp3 −/− or Casp1/11 −/− vs. WT mice (Fig. 2e,f).
To study the pathological changes in the H7N9 infected Nlrp3 −/−, Casp1/11 −/− and WT mice, lung tissues were collected and H&E stained samples were examined by microscopy. On 3 d.p.i, infiltration of inflammatory cells was observed in the infected lungs of all strains of mice. There was an obvious thickening of alveolar septum, destruction of partial alveolar structure, as well as pulmonary septal rupture (Fig. 3a and c). However, infiltration of inflammatory cells to the lungs of WT mice was much more severe than that in Nlrp3 −/− or Casp1/11 −/− mice, especially in the bronchus. In addition, the destruction of alveolar structure in the lungs of Nlrp3 −/− and Casp1/11 −/− mice was weaker than that in WT mice (Fig. 3a and c). On 7 d.p.i, all strains of mice showed more severe infiltration of inflammatory cells to the lungs comparing to that on 3 d.p.i (Fig. 3b and c). Nonetheless, the pulmonary pathology of Nlrp3 −/− and Casp1/11 −/− mice was still milder than that in WT mice on 7 d.p.i (Fig. 3b and c). Collectively, these results suggested a role for NLRP3 and caspase-1 in promoting pathological changes in the lungs of mice infected with H7N9.
Caspase-1 is the critical executor of inflammasome activation mediating the cleavage of proIL-1β. Therefore, we further studied the impact of caspase-1 deficiency on the infiltration of proinflammatory leukocytes to the lungs after H7N9 infection. The single-cell suspensions, prepared from the bronchoalveolar lavage fluid (BALF) of H7N9 infected WT or Casp1/11 −/− mice on 0, 3 and 7 days post infection were stained with anti -CD11b, -F4/80, -CD11c, -Ly6c, -Ly6g, -CD3 and -B220 antibodies conjugated to different fluorescent conjugates, and analyzed through flow cytometry. In both WT and Casp1/11 −/− mice, the percentages of leukocytes expressing CD11b, F4/80, CD11c, Ly6c and Ly6g peaked around 3 d.p.i, then declined (Fig. 4a–e). The percentages of F4/80 (74.43%±5.027%) and CD11c (54.85%±11.50%) positive cells in the WT BALF samples were significantly higher than that in Casp1/11 −/− BALF samples (F4/80: 26.48%±11.42%; CD11c: 16.52%±6.654%) on 3 d.p.i. Meanwhile, the mean fluorescence intensities (MFIs) of CD11b (4,092±1,409), F4/80 (12,450±3,616) and CD11c (4,270±1,642) in WT BALF cells were significantly increased compared with that of Casp1/11 −/− mice (CD11b: 80.10±54.18; F4/80: 531.0±483.4; CD11c: 119.7±94.97) on 3 d.p.i (Fig. 4a–c). For Ly6c and Ly6g, albeit there was no statistical difference between the WT and Casp1/11 −/− mice, the percentage was at least more than two folds higher in the WT mice than that in Casp1/11 −/− mice; and the differences of the MFIs in those two groups were even bigger on 3 d.p.i (Fig. 4d and e). For lymphocytes, CD3 positive cells and B220 positive cells showed no significant differences between WT and Casp1/11 −/− mice on both 3 d.p.i and 7 d.p.i (Fig. 4f and g). Thus, deficiency of caspase-1 reduced the proinflammatory leukocytes recruitment to the lungs upon H7N9 IAV challenge.
It is reported that the caspase-1, activated by inflammasome, plays a critical role in murine proinflammatory responses to IAV infection11, 12, 16. To investigate the role of caspase-1 in H7N9 infection related inflammation, cytokines and chemokines in the supernatants of lung homogenates were analyzed (Fig. 5). In Casp1/11 −/− lung tissues, the levels of IL-1β (118.8±30.68pg/g tissue) and IL-18 (1.168±0.3761ng/g tissue) were significantly lower than those in WT tissues (IL-1β: 309.5±28.54pg/g; IL-18: 2.856±0.3617ng/g, respectively) on 7 d.p.i (P<0.01) (Fig. 5a). Other cytokines such as IL-12/23p40, IL-1α, IFN-γ and IFN-β also exhibited decreased level in Casp1/11 −/− lung tissues compared with WT control on either 3 d.p.i or 7 d.p.i, although the differences were not significant (Fig. 5a). The level of IL-6 fluctuates and was lower in the lungs from Casp1/11 −/− mice on 3 d.p.i, but higher on 7 d.p.i compared to WT counterparts, without significant differences in both cases (Fig. 5a). Other cytokines such as IL-4, TNF-α and IL-17 did not show significant differences between tissues from Casp1/11 −/− and WT mice (Fig. 5a). As for the detected chemokines, MIP-1α was significantly lower in Casp1/11 −/− lung tissues than that in WT control on 3 d.p.i (P<0.05). The levels of other chemokines, including MIP-1β, IP-10, CXCL1, CCL2 and GM-CSF were just slightly lower in the lungs of Casp1/11 −/− mice than that in WT counterparts on either 3 d.p.i or 7 d.p.i. Therefore, caspase-1 was involved for an optimal production of IL-1β, IL-18 as well as MIP-1α in the lung in response to H7N9 challenge. To note, the production of above mentioned cytokines and chemokines from Nlrp3 −/− mice showed a similar pattern as that from Casp1/11 −/− mice (Fig. 5b), indicating that deficiency of NLRP3 inflammasome genes reduced the secretion of several critical proinflammatory mediators in the lung of mice upon H7N9 IAV challenge. In addition, the production of these cytokines and chemokines in the serum of Nlrp3 −/−, Casp1/11 −/− and WT mice showed a similar trend with that from the lung homogenates (Supplementary Fig. S1).
The adaptor protein ASC plays a vital role in the NLRP3 inflammasome formation, which is required for caspase-1 activation and maturation of IL-1β. The mature IL-1β binds to IL-1 receptor 1, the functional receptor of IL-1β, induces downstream signal transduction and executes inflammatory responses. Thus, we set out to determine whether ASC and IL-1R1 play a role similar as that of NLRP3 and caspase-1 during H7N9 IAV infection. To this end, we infected Asc −/− and Il1r1 −/− mice with H7N9 virus and monitored their weight change and mortality daily. Our results demonstrated that the mortality of Asc −/− and Il1r1 −/− mice was lower compared with that of the WT mice (Fig. 6a). The WT mice also suffered more weight loss than these knock-out animals (from 7 to 10 d.p.i) after H7N9 challenge. Of note, the difference for body weight loss between WT and Asc −/− mice was statistically significant (P<0.05) on 8 and 9 d.p.i. (Fig. 6b).
To analyze the pulmonary inflammation of Asc −/− and Il1r1 −/− mice, lung tissues of these mice were harvested for H&E staining and microscopy. On 3 d.p.i, more inflammatory cells were recruited to the alveoli and blood vessels in the lungs of WT mice compared with that of the Asc −/− or Il1r1 −/− mice, and more inflammatory cells infiltrated into the bronchus resulting in a local blockade (Fig. 7a and c). Moreover, the pulmonary exudation was even milder in the Il1r1 −/− mice compared with WT mice (Fig. 7a and c). On 7 d.p.i, all mice suffered more severe inflammation than that observed on 3 d.p.i. Furthermore, there were more inflammatory cells infiltrated in the alveoli of WT mice than in Asc −/− or Il1r1 −/− mice (Fig. 7b and c). Moreover, the damage in the lung of Asc −/− mice was milder compared with that in WT mice, and there was less inflammatory exudation in Il1r1 −/− mice than in WT controls (Fig. 7b and c). Therefore, our results suggest that signals mediated by ASC and IL-1 receptor lead to more severe disease upon H7N9 IAV challenge.
Aberrant and excessive inflammation during H7N9 viral infection is linked to severe morbidity and mortality in human patients17. A recent study demonstrated the histological distribution of inflammation-related genes expression in the lungs of BALB/c mice infected with H7N9 influenza A virus18. Significantly higher expression levels of NLRP3, RIP3, IL-1β and TNF-α in the lung of H7N9-infected mice were noted, indicating possible role of these molecules in driving lung pathogenesis18. For example, the involvement of TNF-α activated signaling pathway during influenza virus infection was clearly demonstrated19, 20. However, the exact roles of other molecules involved in inflammatory response during H7N9 challenged remained elusive. Specifically, the role of inflammasome signaling had not been defined before our present work.
Here, using the gene knockout mice we demonstrated that activation of the NLRP3 inflammasome was detrimental to the host upon H7N9 IAV infection. We noted that WT mice lost more body weight than Casp1/11 −/− , Asc −/− and Nlrp3 −/− mice and their overall survival rate was lower during H7N9 infection (Figs 2 and and6).6). Pathology analysis showed that there were inflammatory responses in the infected lungs of all strains of mice, both at 3 d.p.i and at 7 d.p.i; and the pulmonary inflammations at 7 d.p.i. were more severe than that at 3 d.p.i.; moreover, the pulmonary inflammations of the WT mice were stronger than Casp1/11 −/− , Asc −/− and Nlrp3 −/− mice (Figs 3 and and7).7). Flow cytometric analysis showed that inflammatory cells has started to infiltrate into the alveolar spaces in the BALF at 3 d.p.i. It seemed that CD11b+ or F4/80+ (high-expressed on microphages) or CD11c+ (high-expressed on dendritic cells) cells in the BALF of WT mice were significantly more than those in Casp1/11 −/− mice at 3 d.p.i. There were also Ly6c+ (expressed on monocytes), Ly6g+ (high-expressed on neutrophils) and CD3+ (expressed on T cells) in the BALF of WT mice exhibited higher level, though it was not significant differences (Fig. 4). However, except the CD11b+ cells were retained and B220+ (expressed on B lymphocytes) cells were increased, the percentage of the most infiltrated cells were decreased in the BALF at 7 d.p.i. (Fig. 4), which seem to be “contrast” to the severer pulmonary inflammation pathology observation at 7 d.p.i. (Figs 3 and and77).
Nevertheless, the rationale may lie in the sequential process of H7N9 virus infection in the WT mice. The H7N9 virus was intranasally inoculated and inhaled into bronchoalveolar spaces, then infected the epithelial cells. At the early stage of first few rounds of viral replication (24~72hours), the local inflammation was limited (Fig. 3a), and macrophages/dendritic cells and a few other cells started to be recruited to the infected area by chemokine MIP-1α (Figs 4a–c and and5a).5a). The production of IL-1β was still at low level at this stage (Fig. 5a). With the development of disease, at 7 d.p.i., severe inflammation occurred in the infected lung tissue (Fig. 3b). With the migration of the lymphocytes, especially B cells, to the inflammatory area (Fig. 4g), the total number of infiltrated inflammatory cells were increased but the percentage of monocytes, macrophages, dendritic cells were decreased (Supplementary Fig. S2, Fig. 4). The infiltrated inflammatory cells produced large amount of IL-1β (Fig. 5), which accelerated the pulmonary inflammation.
From 7 d.p.i. to 14 d.p.i, the body weight of survived WT mice recovered completely, whereas Casp1/11 −/− mice could not returned to the initial level (0 d.p.i) (Fig. 2d). Microscopic analyses revealed that on 14 d.p.i, most tissues from WT mice exhibited complete recovery, while the Casp1/11 −/− tissues still manifested strong inflammation (data not shown). Thus, WT mice suffered more due to the lung damage and inflammation in the early phase (before 7 d.p.i) of H7N9 infection and either died of organ failure, or recovered at the later phase. In contrast, Casp1/11 −/−mice did not develop as strong inflammation and lung damage as seen in WT counterparts in the early phase, consequently fewer of them died. However, Casp1/11 −/− mice failed to recover completely at the later phase. These results might suggest a potentially dual role for caspase-1 in H7N9 infection, detrimental role to the host upon H7N9 infection before 7 d.p.i. and possibly necessary role for recovery from the lung inflammation after 7 d.p.i. phase, likely due to a contribution to tissue regeneration. But it should be kept in mind that the comparison between 20% surviving WT and 60% surviving Casp1/11 −/− is not justified, and more stringent experiments are needed for further explorations on the role of caspase-1 to host recovery.
Studies on the role of NLRP3-caspase-1 inflammasome during IAV infection produced conflicting results. Some studies have demonstrated that NLRP3-caspase-1 inflammasome is protective against H1N1 infection through viral clearance or virus induced pulmonary necrosis11, 12 . Inflammation is essential to protect host from infection, but can also cause tissue damage or dysfunction of important organs and lead to death21. Dysregulation of the proinflammatory response may result in a “cytokine storm” that contributes to a severe viral pneumonia and serious complications in the lung21. The highly pathogenic avian influenza virus H5N1 causes hypercytokinemia and severe tissue damage22. It is reported that production of proinflammatory cytokines and chemokines, including IL-6 and IP-10, is remarkably increased in H7N9 infected patients17, 23, and H7N9 infection associated inflammation in the lungs resulted in rapidly progressive pneumonia and development of ARDS in the majority of hospitalized patients24, 25. In addition, the H5N1 IAV infected patients who progressed severe lung injury have elevated levels of CCL2, IP-10, CXCL9 and IL-8 compared with the seasonal H1N1 infected patients26. Therefore, for the highly pathogenic influenza virus the hyperactivated proinflammatory responses may be detrimental for the host. Moreover, a recent study demonstrates that NLRP3 inflammasome plays both a protective and detrimental role during H1N1 and H3N2 infection of mice depending on the disease phase13. In our study, we found the inflammasome activation was deleterious in the case of H7N9 infection, possibly due to overwhelming and fatal acute inflammation in the lungs following H7N9 infection. Pathology and infiltrating cellular analyses as well as cytokine and chemokine analyses all supported this possibility (Figs 3–5). Pathology results revealed that on 3 d.p.i, there were more inflammatory cells around the blood vessels and bronchus in WT mice compared with Casp1/11 −/− mice (Fig. 3). The flow cytometric analyses further demonstrated the abundance of inflammatory cells in the BALF of WT mice compared to Casp1/11 −/− counterparts (Fig. 4), which were consistent with the observed pulmonary pathological changes.
Both Casp1/11 −/− and Nlrp3 −/− mice produced relatively less level of IFN-β in the lung compared with control mice. Although no significant difference was noted, these data indicated a possible role for the NLRP3-caspase-1 inflammasome signaling in affecting the type I interferon production. Theoretically, type I interferon response plays a role in limiting viral replication, however, we did not observe any significant difference in viral load between Casp1/11 −/− or Nlrp3 −/− versus WT mice. It is likely that the H7N9 could employ strategies to counteract the generation and function of type I interferon.
Chemokines are the driving factors promoting the recruitment of inflammatory cells. MIP-1α (CCL3) can activate granulocytes and lead to acute neutrophilic inflammation. It also induces the synthesis and release of IL-1β, IL-6 and TNF-α from fibroblasts and macrophages27. MIP-1α level is increased in H7N9 and H5N1 infected mice, but not in H9N2 infected animals28. We also noticed a clear increase of MIP-1α in WT mice upon H7N9 infection (Fig. 5). Interestingly, this elevation was abrogated in the early phase (3 d.p.i) in the absence of caspase-1 (Fig. 5). We reasoned that the MIP-1α production may be, directly or indirectly, influenced by NLRP3 or caspase-1-dependent IL-1β and/or IL-18 secretion.
IL-1β showed significant reductions in Nlrp3 −/− and Casp1/11 −/− mice compared with WT on 7 d.p.i (Fig. 5). IL-1β is a critical proinflammatory cytokine that is increased during infections with H1N1 and H5N1 influenza viruses29. IL-1β also mediates the recruitment of monocytes and neutrophils into the lung30. Deficiency of IL-1β or its receptors manifests different responses to respiratory challenge with influenza virus31–33. Hence, the function of IL-1β in controlling IAV infections remains obscure. Our data showed that Il1r1 −/− mice exhibited attenuated tissue pathogenesis upon H7N9 infection (Figs 6 and and77).
During the preparation of this manuscript, we noticed that another group found the PB1-F2 protein derived from H7N9 virus also activates the NLRP3 inflammasome34. Probably, the viral particle of H7N9 contains multiple factors such as RNA and PB1-F2 that can be detected by the host inflammasome. Beside NLRP3 inflammasome, whether other inflammasomes, such as NLRP1, IPAF, AIM2, could also be involved in H7N9 infection needs to be investigated in further study.
In summary, our present work demonstrates that the NLRP3-caspase-1 inflammasome is deleterious for the mammalian host during H7N9 influenza virus infection. Therefore, fine-tuning the NLRP3-caspase-1 activity or IL-1-mediated signaling should be beneficial to control H7N9-associated lethal pathogenesis.
A/Shanghai/4664T/2013(H7N9) (GenBank No. KC853225-KC853232) strain was used in this study. Isolation of H7N9 virus was described before and the viral titer was measured via tissue culture infective dose (TCID50) assay15, 25. All experiments related to H7N9 virus were performed in biosafety level 3 laboratories in Shanghai Public Health Clinical Center (SPHCC), and followed the standard operating protocols approved by the Institutional Biosafety Committee at SPHCC, Fudan University.
All mice used in our experiments are on a C57BL/6 genetic background and all experiments were carried out with age and gender matched mice (8–12 weeks old, female). C57BL/6 mice were purchased from the B&K Universal Group Limited (Shanghai, China). Nlrp3 deficient mice (a gift from Dr. Warren Strober) and Asc deficient mice (a gift from Dr. Vishva M. Dixit) had been described35, 36. Caspase-1/11 and IL-1 receptor 1 deficient mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Herein, these mice were described as Nlrp3 −/− , Asc −/− , Casp1/11 −/− and Il1r1 −/−. All mice were housed under specific pathogen-free (SPF) condition at the animal facility at Institut Pasteur of Shanghai. Mice were transferred to the Animal Biosafety Level 3 (ABSL3) Laboratory at SPHCC three days before infection. Mice care and experimental procedures complied with national guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC) at SPHCC, Fudan University.
Bone marrow derived dendritic cells (BMDCs) were generated as described37. Briefly, bone marrow cells were harvest and lysed with RBC lysis buffer, and seeded in 10cm dish with 10ml RPMI1640 with GM-CSF (20ng/ml). The media were refreshed 3 days later and the cells were harvested for experiments on day 6.
Antibodies for immunoblotting include: rabbit anti-mouse ASC (sc-22514-R; Santa Cruz, CA, USA), mouse anti-mouse caspase-1 (AG-20B-0042; Adipogen, San Diego, CA, USA), rabbit anti-mouse mature and pro–IL-1β (sc-7884; Santa-Cruz, CA, USA) and mouse anti-mouse β-actin (sc-47778; Santa-Cruz, CA, USA).
H7N9 RNA was extracted with QIAamp Viral RNA Mini Kit(Qiagen, Hilden, Germany) according to the manufacture’s protocol. And we measured the concentration of viral RNA with Nanodrop 2000 (Thermo Scientific, Madrid, Spain). BMDCs were primed with 500ng/ml LPS (Sigma-Aldrich, St.Louis, MO, USA) for 4hours, then the different doses of H7N9 viral RNA were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacture’s protocol. Briefly, Lipofectamine 2000 (2ul/ml) and viral RNA were diluted in opti-MEM (Invitrogen, Carlsbad, CA, USA), then mixed together for 20minutes and the mixture was added into cells. 12hours later, the supernatant was harvested for ELISA or inflammasome activation assay, and the cells were collected for Western Blotting and ASC oligomerization detection.
ASC oligomer was prepared as described36. Brifely, H7N9 RNA transfected BMDCs were lysed with lysis buffer [50mmol/L Tris (pH 7.5), 150mmol/L NaCl, 1% Nonidet P-40], pelleted and washed with PBS, and cross-linked with disuccinimidylsuberate (DSS) before subjected to immunobloting.
Mice were infected with 5×104 TCID50 of H7N9 virus in a volume of 50µl intranasally (i.n.) after sevoflurane (Hearem, Shanghai, China) inhalation anaesthesia, and monitored for clinical signs, body weight changes and physical survival during the 14 days observation period. The infected mice were sacrificed on 3, 7 or 14 days post infection for collection of samples for analysis as reported15. To monitor the body weight changes and survival rate, 14 to 22 mice were used in each group (n=14~22). For the rest analysis of murine samples, more than 3 mice were used in each group.
Lung homogenate supernatants and serum were collected and stored at −80°C before analysis. The levels of cytokines and chemokines in the collected samples were determined quantitatively using a ProcartaPlex Multiplex Immunoassay kit (eBioscience, Viennna, Australia) according to the manufacturer’s protocol.
Bronchoalveolar lavage fluid (BALF) was obtained via flushing the lungs three times with 0.5mL/time of PBS. The single-cell suspensions from BALF of H7N9 infected mice were first treated with anti-mouse CD16/CD32 antibody (eBioscience, Vienna, Australia) to block Fc receptor, then surface stained with antibodies against B220, CD3, CD11b, CD11c, Ly6c, Ly6g and F4/80, and fixed with 4% paraformaldehyde before analysis with FACS Array (BD Biosciences, Franklin Lakes, NJ, USA). The final graphical output was generated through FlowJo software (Tree Star, Inc., Ashland, OR, USA).
Total RNA was extracted from lung homogenates using a kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Relative Quantification of H7N9 viral load via RT-PCR was conducted as reported previously15. Calculation was carried out by comparing H7N9 Hemagglutinin with GAPDH, which served as an internal control GAPDH (ΔΔCT method).
Excised mouse lung tissues were fixed in 4% paraformaldehyde followed by paraffin embedding and sectioning, then subjected to hematoxylin and eosin (H&E) staining. Images were collected by Leica DMI3000 microscopy with indicated magnifications.
Four indexes were analyzed for scoring on each slide: 1) Blood vessel region inflammation, 2) Pulmonary alveoli inflammation, 3) Lung tissue inflammation, 4) Bronchial inflammation. The detailed criteria were listed in Table 1, which was referred to a previous report38 and modified by the pathologist, Dr. Y Feng, in author list of this manuscript.
All statistical analysis was conducted using GraphPad Prism Software via parametric unpaired t-test if not noted otherwise (Version 5.0; GraphPad, La Jolla, CA, USA). P<0.05 was defined as statistically significant.
This work was supported by grants from the National Major Projects for Science and Technology (2014ZX0801011B-001, 2016YFD0500208, 2015CB554302, 2014CB541905), the Strategic Priority Research Program of Chinese Academy of Sciences (XDPB0303), National Natural Science Foundation of China (31370892, 91429307, 31570895, 81471397, 81341004, 81761128012), Science and Technology Commission of Shanghai Municipality (15DZ2290200), Scientific Research Funds of SPHCC (201514) and the Opening Project of Key Laboratory of Molecular Virology & Immunology, Institute Pasteur of Shanghai. We are grateful to Dr. Warren Strober for sharing the NLRP3 deficient mice, Dr. Vishva M. Dixit for providing ASC deficient mice. We thank Dr. Maya Kozlowski for careful proof reading of the manuscript. We also wish to thank the staff of animal facility and ABSL3 in Shanghai Public Health Clinical Center at Fudan University, and pathology core facility at Institute Pasteur of Shanghai for their technical supports.
G.M. and X.Z. conceived the experiments, R.R., S.W., J.C., Y.Y., X.R., Y.F., L.C., B.Q., C.X., H.Y., Z.S. and D.T. conducted the experiments, R.R., S.W., G.M., X.Z. and Y.H. analyzed the results. All authors reviewed the manuscript.
The authors declare that they have no competing interests.
Rongrong Ren, Shuxian Wu, Jialin Cai and Yuqin Yang contributed equally to this work.
Electronic supplementary material
Supplementary information accompanies this paper at doi:10.1038/s41598-017-07384-5
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Xiaohui Zhou, Email: gro.chpahs@iuhoaixuohz.
Guangxun Meng, Email: nc.ca.spi@gnemxg.