Background

Influenza is a fatal respiratory infectious disease. The excessive immune response caused by influenza infection may cause irreversible damage to the body [1, 2]. Influenza virus infection can lead to apoptosis or necrosis of tissue cells, which activates the immune system. The infected respiratory epithelial cells and vascular endothelial cells secrete cytokines and chemokines to recruit and activate various immune cells. Immune cells perform their respective functions to phagocytose pathogens and infected cells or secrete more cytokines, resulting in an expanded immune response [3]. An excessive immune response can bring serious consequences, such as immune cell infiltration and cytokine storm, which can result in pulmonary edema and may even cause death [4]. Excessive pulmonary inflammation is the main characteristic of lethal IAV infections.

Therapeutic actions for influenza are limited to vaccines and a few anti-viral drugs. However, the frequent mutation of influenza virus has limited the current treatment methods, and some studies have found that the cause of severe influenza is often an excessive immune response [5,6,7]. Therefore, antiviral therapy has been unable to meet our requirements, and a new way to resist influenza is urgently needed. Suppressing excessive immune responses to alleviate tissue damage caused by influenza virus infection could become an effective anti-influenza therapeutic strategy.

Phillyrin is one of the major bioactive components of the Chinese herbal medicine Forsythia suspensa, which has the functions of sterilization, heat clearing and detoxification [4b). These DEGs might be the important therapeutic effector molecules of phillyrin treatment on IAV-induced pneumonia.

Fig. 4
figure 4

The cytokine-cytokine receptor interaction is responsible for the suppressive effect of phillyrin on IAV-induced pneumonia. (a) Hierarchical cluster analysis of DEGs among CON group, virus group and phillyrin group. Red and blue hues indicate upregulated and downregulated expression, respectively. (b) The quantity of DEGs in virus-vs.-CON and phillyrin-vs.-virus groups, and comparison of DEGs between virus-vs.-CON and phillyrin-vs.-virus. (c) KEGG enrichment bubble diagram with common DEGs. The mRNA expression of CXCR2 (d), CCR2 (e), CCR1 (f), CCR5 (g). Mean ± SD (n = 6/group). One-way ANOVA, # p < 0.05 vs. CON, * p < 0.05 vs. IAV.

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Furthermore, pathway enrichment analysis of the 163 DEGs was performed through the KEGG database. The results showed that 15 pathways were markedly enriched for these DEGs (P < 0.05; Table 1), including the calcium signaling pathway, cytokine-cytokine receptor interaction, viral protein interaction with cytokine and cytokine receptor, chemokine signaling pathway, focal adhesion and cAMP signaling pathway (Fig. 4c). It is worth noting that there are three classic chemokine signaling pathways (ko04060, ko04061, and ko04062). These results suggest that the cytokine-cytokine receptor interaction plays a crucial role in enhancing the therapeutic effect of phillyrin on IAV-induced pneumonia.

Table 1 KEGG pathway significantly enriched
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Moreover, the levels of 5 chemokines such as CCL11/Eotaxin, CXCL1/KC, CCL5/RANTES, CCL2/MCP-1 and CCL3/MIP-1α were dramatically reduced by phillyrin in BALF of IAV-infected mice as mentioned previously (Fig. 3). These inflammatory mediators normally recruit cells of the innate immune system through selectively binding to the corresponding receptors. CCL11 is mainly involved in the recruitment of eosinophils into the lung, and its receptor is CCR3 [14]. CCL3 and CCL2 play a role in inflammatory responses through binding to the receptors CCR1, CCR2 and CCR5 [15]. As one of the natural ligands of CCR5, CCL5/CCR5 can mediate the recruitment and activation of neutrophils and monocytes during influenza, and CXCL1 can chemotactic T cells, monocytes, neutrophils and other immune cells when combined with its specific CXCR2 receptor [16,17,18]. Previous findings showed that activation of cytokine receptors at the initial stage of influenza infection could ensure proper recruitment of white blood cells and activation of antiviral pathway in epithelial cells. However, continuous or excessive activation of cytokine receptors during severe influenza infection may aggravate inflammatory reaction, leading to increased lung injury [19, 20]. One possible strategy for enhancing anti-inflammatory effects is to selectively bind and neutralize the chemokines and chemokine receptors. In view of the important role of chemokine receptors, we further detected the mRNA expression levels of CCR3, CXCR2, CCR2, CCR1 and CCR5 in the lungs. The findings demonstrated that the mRNA levels of CCR5, CCR2, CCR1 and CXCR2 genes were increased after influenza infection. Phillyrin treatment could significantly reduce the expression of CCR2 and CXCR2, among which the latter was more significantly regulated by phillyrin (Fig. 4d-g). CCR3 gene was undetermined due to its low expression in the lungs (Results not listed).

Phillyrin has good binding ability to CXCR2

CXCR2 is a critical target in the efforts to suppress neutrophilic inflammation. Excessive neutrophil influx is related to greater disease severity in patients with influenza infection. A number of studies on the experimental inhibition of CXCR2 have reported its ameliorative effects on inflammatory responses and lung injury in the sublethal influenza murine models [21,22,23]. We verified the affinity of phillyrin to CXCR2 based on molecular docking. Generally, the molecular docking score of less than 0 kcal mol− 1 indicates that ligands can spontaneously bind to receptors, and the score less than − 5 kcal mol− 1 indicates that they have good binding ability [24, 25]. Our results showed that binding affinity was − 8.9 kcal mol− 1 between phillyrin and CXCR2, and the drug-target had good binding ability. In addition, their bonding types were hydrogen bonds (from residues LYS320, LYS327 and VAL72) and electrostatic force (Fig. 5.). As a special intermolecular force, hydrogen bond is a critical index to measure the affinity between protein and small molecules. Therefore, CXCR2 may be an important drug-target of phillyrin against IAV-induced pneumonia.

Fig. 5
figure 5

Phillyrin has good binding ability to CXCR2. (a) Protein structure of CXCR2. (b) Molecular structure of phillyrin. (c) 3D diagrams showed the distribution of hydrogen donors and receptors in active pockets. (d) 2D diagrams showed the bond types and amino acid residues. Green and orange dashed lines indicate hydrogen bonds and Pi-Anion, respectively

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To further confirm whether phillyrin is the direct binding to CXCR2. An SPR assay was then performed to evaluate the binding affinity of phillyrin and CXCR2. As shown in Fig. 6a, phillyrin bound to CXCR2 in a dose-dependent manner. The response units at equilibrium were plotted against phillyrin concentrations (Fig. 6b), and the binding affinity constant (KD) value was calculated by non-linear regression. The results showed that phillyrin bound to CXCR2 with a KD value of 18.58µM, indicating that phillyrin presented strong affinity for CXCR2.

Fig. 6
figure 6

The binding affinity between phillyrin and CXCR2. Interactions of CXCR2 with phillyrin measured by SPR. The CXCR2 with a his-Tag was fixed on the NTA sensor chip via capture coupling and serial dilutions of phillyrin (6.25µM, 12.5µM,25µM,50µM) were used as analytes. Changes in plasmon resonance are shown as response units. (a) Multicycle kinetics using CXCR2 as ligand and phillyrin as analyte. Different colored lines correspond to different concentrations of Phillyrin; (b)The KD value of phillyrin and CXCR2 was calculated by non-linear regression analysis

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Phillyrin inhibits overactivation of NLRP3 inflammasome

Previous studies have reported that CXCL1-CXCR2 axis could regulate NLRP3 inflammasome activation in some inflammatory scenarios [26]. IAV can induce excessive secretion of cytokines by triggering NLRP3 activation, thus resulting in lung damage and death [27, 28]. Specifically, hyperinflammatory responses, such as the secretion of NLRP3-dependent IL-1β, are characteristic features of severe IAV infection [29, 30]. In our study, phillyrin downregulated IL-1β production and inflammatory responses in BALF from H1N1-induced pneumonia mice (Fig. 3a). To further elucidate the anti-inflammatory mechanisms of phillyrin, the expression levels of NLRP3 inflammasomes and the Caspase1 p20 subunit (representing activated Caspase1)/Caspase1 were analyzed. Notably, the mRNA and protein expression levels of Caspase1, ASC and NLRP3 were all elevated in the lungs of mice with H1N1-induced pneumonia, but these trends were suppressed in the phillyrin group (Figs. 7a-c and 8a-d). Besides, in the virus group, the proportion of Caspase1 p20 to Caspase1 and the mRNA expression of IL-1β were higher than those in CON group, but were obviously reduced in phillyrin-treated mice (Figs. 7d and 8a and e). These findings suggest that phillyrin can suppress the overactivation of NLRP3 inflammasomes in mice with IAV-induced pneumonia.

Fig. 7
figure 7

Phillyrin inhibits mRNA expression of NLRP3 inflammasome. The mRNA expression of NLRP3 (a), ASC (b), Caspase 1 (c), and IL-1β (d) in the lungs on 7 dpi, as detected by qRT-PCR. Mean ± SD (n = 6/group). One-way ANOVA, # p < 0.05 vs. CON, * p < 0.05 vs. IAV.

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Fig. 8
figure 8

Phillyrin inhibits protein expression of NLRP3 inflammasome. The protein levels of NLRP3 (b), ASC (c) and Caspase 1 (d), and Caspase1 p20 subunit/Caspase1 (e) in the lungs on 7 dpi were detected by Western blotting. Mean ± SD(n = 3 independent experiments) One-way ANOVA, # p < 0.05 vs. CON, * p < 0.05 vs. IAV.

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Phillyrin has the similar effect as CXCR2 antagonist

I repeated an in vivo experiment in the context of CXCR2 antagonist SB225002 (a potent, selective and non-peptide CXCR2 antagonist, MedChemExpress). The body weights of mice in different treatment groups are demonstrated in Fig. 9. The result showed that infection of PR8 significantly reduced the body weights of virus-infected mice compared to the control mice, while treatment with phillyrin-only, CXCR2 antagonist SB225002-only, phillyrin and SB225002 synergistically significantly alleviated this loss. Oseltamivir phosphate was employed as a positive control. Compared to phillyrin-only group or SB225002-only group, we found that phillyrin and SB225002 synergistically enhanced weight regain. This data indicated that phillyrin has the similar effect as CXCR2 antagonist in improving virus-induced weight loss. However, further testing is needed to determine what changes have occurred in the CXCR2-NLRP3-inflammasome pathway.

Fig. 9
figure 9

Phillyrin and CXCR2 antagonist SB225002 treatment alleviates percentage weight loss caused by IAV. Briefly, BALB/c mice were randomized into several groups: Control group (n = 6, Saline-treated), Phillyrin group (n = 6, intraperitoneal injection (i.p.) of 15 mg/kg Phillyrin once a day from 0 day to 4 days without infection ), IAV group (n = 6, intranasally challenged with PR8), IAV + Phillyrin group (n = 6, i.p. of 15 mg/kg Phillyrin once a day from 0 day to 4 days postinfection ), IAV + SB225002 group (n = 6, i.p. of 20 μm SB225002 once a day from 0 day to 4 days postinfection ), IAV + Phillyrin + SB225002 group (n = 6, daily i.p. of 20 μm SB225002 from 1 days pre- to 4 days post-infection and i.p. of 15 mg/kg Phillyrin once a day from 0 day to 4 days postinfection), IAV + oseltamivir group (n = 6, orally administrated with 10 mg/kg oseltamivir once a day from 0 day to 4 days postinfection). Phillyrin and SB225002 was suspended in 0.4% carboxymethylcellulose sodium. The body weight change of each group were observed daily following PR8 challenge for a total of 8 days. Mean ± SD (n = 6/group). One-way ANOVA, # p < 0.05 vs. Control, * p < 0.05 vs. IAV.

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Discussion

The influenza virus causes seasonal influenza and can even lead to outbreak and epidemic worldwide [3, 31]. Viral pneumonia from IAV infection is a common complication and leading cause of death [32]. As a lack of drugs used to control viral acute lung injury continues to persist, the development of new therapeutic agents is essential. This study found that phillyrin, a monomer TCM ingredient with anti-inflammatory activity, exerted therapeutic effects on the pneumonia induced by IAV infection. The results showed that phillyrin treatment alleviated pneumonia induced by virus infection and significantly ameliorated the upregulation of multiple pro-inflammatory cytokines and chemokines (e.g., IL-1β, CXCL1, CCL3, CCL2, CCL5 and so on) in BALF. More importantly, the results of RNA sequencing analysis revealed that the cytokine-cytokine receptor interaction plays an essential role in regulating the therapeutic effect of phillyrin on influenza-induced pneumonia, and the mRNA expression level of CXCR2 was confirmed to be markedly inhibited by phillyrin. We performed computerized molecular docking of phillyrin with CXCR2 and verified good binding activity. Furthermore, SPR analysis, the gold standard method for studying protein-protein interactions, confirmed that phillyrin direct bound to CXCR2 with high affinity activity. All of these results indicate that CXCR2 is a potential therapeutic target for phillyrin. We also showed that phillyrin treatment could affect the mRNA and protein expression levels of Caspase1, ASC and NLRP3 in the lung homogenates of mice infected with H1N1. The ameliorative effect of phillyrin on influenza-induced pulmonary pathological damage may be partly related to the antagonization of CXCR2 and inhibition of NLRP3 inflammasome activation.

Influenza patients are often afflicted with severe pneumonia that is characterized by excessive infiltration of leukocytes and secretion of proinflammatory cytokines [33, 34]. Despite the inflammatory response produced by the immune host to remove the virus, the elevated production of different inflammatory cytokines typically contributes to the pathogenesis of IAV-induced acute lung injury [35,36,37]. In the present study, phillyrin (15 mg/kg) remarkably decreased the lung index, relieved the extensive degeneration and necrosis of the bronchial and bronchiolar epithelium after IAV infection and inhibited the accumulation of multiple pro-inflammatory cytokines and chemokines, revealing that phillyrin can alleviate pulmonary inflammation in mice with virus-induced pneumonia.

  • Our study also found that oseltamivir, as a positive control, led to similar reductions in pathology, suggesting that a decrease in viral replication alone can alleviate the pathological and inflammatory responses.To determine the effect of phillyrin on influenza viral replication, the relative levels of M and NP mRNA expression and virus-induced CPE at indicated time points in vitro as well as the contents of M mRNA and the levels of HA protein of lungs at 7 dpi in vivo were examined. The cell experiments verified the inhibitory effect of phillyrin on influenza viral multiplication and the results were similar to the previous report [38], however, the inhibitory trend was not significant in vivo. Unlike a single cellular environment, lung tissue is composed of a variety of cells such as respiratory epithelial cells and macrophages [39]. The higher levels of viral genomic replication, transcription, and translation in alveolar epithelial cells probably makes IAV maintain a certain amount in lung tissue to partially “counteract” the inhibition of phillyrin on viral multiplication. Therefore, in the present study, although the research in vitro have confirmed the inhibitory effect of phillyrin on influenza viral multiplication, this inhibition was not significant in vivo. The amelioration of phillyrin on pulmonary inflammation in influenza viral pneumonia mice is not mainly dependent on its antiviral activity.

The pathogenesis of pneumonia caused by influenza is not only directly associated with viral cytopathology but also forms an overzealous host immune response. Neutrophils have been recognized as an important cell type that leads to deterioration in lung functions [40]. When the virus reaches the lung epithelium, neutrophils are recruited by the immune/non-immune cells, leading to the production of chemokines (e.g., CXCL1/CXCL2) in mice [1, 41]. These chemokines act through their receptor CXCR2 in different types of cells. Previous studies have shown that the chemokine receptor CXCR2 has an essential role in regulating pulmonary inflammation and damage during severe IAV infection. The downregulation of CXCR2 could prevent such aggravated response, even after sequential IAV infections, without affecting the virus-specific adaptive immune responses [22]. In the present work, DEGs and potential pathways in the lung tissue of the virus group and phillyrin group were evaluated using RNA-seq and qRT-PCR. The data suggest that the cytokine-cytokine receptor interaction plays a vital role in regulating the inhibitory effect of phillyrin on IAV-induced pneumonia. It was found that the expression of CXCL1 and CXCR2 was markedly upregulated in the BALF and lungs of mice with IAV infection. Interestingly, phillyrin treatment could remarkably decrease the expression of CXCR2 and the secretion of CXCL1. Furthermore, we performed the computerized molecular docking of phillyrin with CXCR2 and confirmed good binding activity between them, indicating that phillyrin is a potential CXCR2 inhibitor.

The CXCL1-CXCR2 axis has been reported to be involved in NLRP3 inflammasome activation and functions in some inflammatory scenarios. Tang and co-workers showed that this axis might induce inflammatory response in diabetic nephropathy by promoting NLRP3 inflammasome overactivation [26]. The activation of NLRP3 inflammasomes in LPS-primed macrophages was observed to be specific for CXCL1 and CXCL2 but not other chemokines such as CCL2 and CCL5 [42,43,44]. The inflammatory response is an integral part of IAV infection. Recently, the NLRP3 inflammasome complex has been revealed to have essential roles in both detrimental and protective immune responses during IAV infection. NLRP3 inflammasomes are oligomeric signaling platforms that induce Caspase1-associated pyroptotic cell death and mature the prototypic inflammatory cytokines IL-18 and IL-1β [45]. Recent studies have highlighted that inhibition of NLRP3 inflammasome activation can effectively treat influenza viral pulmonary inflammation. MCC950, an inhibitor specific to NLRP3 inflammasome, has been shown to improve the survival and alleviate pulmonary inflammation in IAV-infected mice by downregulating the expression of Caspase1 and NLRP3 [46]. In this study, phillyrin downregulated the expression levels of Caspase1, ASC and NLRP3 in the lungs of mice with virus-induced pneumonia and reduced the protein ratio of Caspase1p20/Caspase1, thereby inhibiting NLRP3 inflammasome overactivation. However, the previous report [42] shows that ILK and PKCµare the linkage to CXCL1 or 2/CXCR2 and NLRP3 inflammasome activation. The detection of phosphor-ILK and phosphor-PKCµ level will help to further clarify if phillyrin affect inflammasome activity upon influenza A virus infection in the furture.

Conclusion

This study demonstrates that phillyrin can ameliorate pulmonary inflammation in mice with virus-induced pneumonia, and its anti-inflammatory mechanisms may be through antagonizing CXCR2 and suppressing NLRP3 inflammasome overactivation in the lung of infected mice. Although we observed this phenomenon and the possible mechanism, lung tissue–specific CXCR2 knockout mouse models should be constructed in future experiments in order to evaluate this pathway in vivo. In addition, some key questions also remain to be resolved. For instance, is it specific for CXCL1 or CXCR2 that phillyrin inhibits NLRP3 inflammasome activation in the lungs of IAV-infected mice? If so, what is the precise molecular mechanism by which phillyrin ameliorates influenza viral pneumonia through depressing NLRP3 inflammasome activation via declining CXCR2 expression? Still, although significant work remains to be conducted, our findings provide novel insights into the treatment of IAV-induced pulmonary inflammation.