Potential role of host autophagy in Clonorchis sinensis infection

doi:10.21203/rs.3.rs-4930842/v1

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Potential role of host autophagy in Clonorchis sinensis infection

https://doi.org/10.21203/rs.3.rs-4930842/v1

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published 23 Oct, 2024

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An in vivo mouse model of Clonorchis sinensis (C. sinensis) infection with or without the administration of autophagy inhibitor chloroquine (CQ) stimulation was established to assess the possible involvement of autophagic response during C. sinensis infection. Abnormal liver function was observed at four, six, eight weeks post-infection, as indicated by elevated levels of ALT/GPT, AST/GOT, TBIL, α-SMA in the infected group. Our findings indicated C. sinensis infection activated autophagy, as shown by a decreased LC3II/I ratio and accumulated P62 expression in infected mice. Interestingly, CQ administration exhibited dual and opposing effects during the infection. In the early stage of infection, the engagement of CQ appeared to mitigates symptoms by reducing inflammation and fibrotic responses. However, in the later stage of infection, CQ might contribute to parasite survival by evading autophagic targeting, thereby exacerbating hepatic impairment and worsening liver fibrosis. Autophagy in liver was suppressed throughout the infection. These observations attested that C. sinensis infection triggered autophagy, and highlighted a complex role for CQ, with both protective and detrimental effects, in the in vivo progression of C. sinensis infection.

Clonorchis sinensis

Autophagy inhibition

Chloroquine

Infection

Liver fibrosis

Clonorchis sinensis (C. sinensis) is fish-borne parasitic trematode that has been classified as a Class I carcinogen by the International Agency for Research on Cancer under the World Health Organization (WHO) (Bian et al. 2023). Humans and other carnivorous mammals such as dogs, cats and pigs serve as definitive hosts for this parasite (Nguyen et al. 2020). Infection occurs when people consume raw or undercooked fish and shrimp, leading to C. sinensis infection (Dai et al. 2020; Keiser and Utzinger 2005). This infection causes clonorchiasis, a neglected tropical disease prevalent in several countries, including China, Vietnam, Korea, and parts of Russia (Sripa et al. 2021; Wang et al. 2004). It is estimated that 200 million people are at risk of C. sinensis infection, with approximately 15 million individuals experiencing varying degrees of clonorchiasis (Furst et al. 2012; Gao et al. 2020). Chronic infection with C. sinensis is strongly associated with inflammation, cholangitis, liver fibrosis, bile duct obstruction, and even cholangiocarcinoma (CCA) (Bouvard et al. 2009; Dai et al. 2024; Jia et al. 2013).

Autophagy is an adaptive response to exogenous stimuli such as nutrient deficiency, pathogen infection and oxidative stress, enabling cells degrade damaged cellular organelles and aberrant proteins (Khandia et al. 2019; Prakash et al. 2022). Microtubule-associated protein 1 light chain 3 (LC3), a key biomarker for autophagosomes, plays a crucial role in the autophagosome biogenesis (Su et al. 2012), with the LC3-II/LC3-I ratio reflecting the status of autophagosomes (Hong et al. 2013). A growing body of evidence demonstrate that autophagy play a crucial role in modulating the interaction between parasites and host eukaryotic cells during parasitic invasion (Priyamvada et al. 2021; Veras et al. 2019; White et al. 2024). On one hand, researches indicate that host can use autophagy to clear parasites (Ichimiya et al. 2020; Portillo et al. 2017). For instance, treatment with Angiostrongylus cantonensis L5 ESPs promotes autophagosomes formation in mouse astrocytes though Shh pathway, providing protection for the astrocytes (Chen et al. 2020). On the other hand, parasitic infection can induce or weaken host cell autophagy (Villares et al. 2024). Aberrant autophagy can aggravate the progression of various infectious and non-infectious diseases. Yu et al. demonstrate that Schistosoma japonicum infection suppresses liver autophagy, promotes hepatocyte apoptosis, and worsens hepatic fibrosis (Yu et al. 2024). Similarly, excretory/secretion products of C. sinensis (CsESPs) have been shown to activate hepatic stellate cells (HSCs) by inducing autophagy (Zheng et al. 2022). Our previous study illustrated that the total protein derived from C. sinensis (CsTP) stimulated hepatic autophagy by regulating LC3B/P62 axis in both in vitro and in vivo models. We also confirmed the role of CsTP in the activation of hepatic stellate cells and the induction of apotosis via autophagy (Shang et al. 2020). Despite these advances, uncertainties remain regarding how C. sinensis initiates hepatic autophagy, and how autophagy affects the outcome of infection.

This study aims to determine the effect of autophagy inhibitor CQ on liver fibrosis in mice infected with C. sinensis. Our findings will provide a theoretical basis for future research on novel therapeutic targets for parasitic infection.

Mice

Six- to eight-week-old Balb/c mice were obtained and housed in the SPF-grade animal room at Sun Yat-sen University. The animal experiment procedures were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University (Permit No. SYSU-IACUC-2022-000370).

C. sinensis and infection

Mice were intraveneously infected with alive metacercaria of C. sinensis and sacrificed at four, six and eight weeks post infection, respectively. Additionally, eight-week infected mice were divided into four groups: the eight-week infected group (Cs-8w), chloroquine (CQ, Sigma-Aldrich, USA) stimulation in the early stage of C. sinensis infection (Cs + CQ-B, each mouse intraperitoneally injected with 50 mg/kg CQ from week 1 to week 4 post-infection), CQ stimulation in the late stage of C. sinensis infection (Cs + CQ-A, each mouse intraperitoneally injected with 50 mg/kg CQ from week 5 to week 8 post-infection), and CQ stimulated group (each mouse intraperitoneally injected with 50 mg/kg CQ every other day). All mice were anesthetized and then dissected after eight weeks.

Biochemistry assay

Serum aspartate transaminase (AST), alanine transaminase (ALT) and the total bilirubin (TBil) levels were measured using a microplate diagnostic kit (Nanjing jiancheng bioengineering institute, China) according to the manufacturer’s recommended protocols.

Histopathological examination

To assess inflammatory infiltration and collagen accumulation, liver sections from C. sinensis-infected mice were fixed with 4% paraformaldehyde, embedded in paraffin, and stained with Masson’s trichrome staining. Histological features of the samples were blindly observed under light microscope (Olympus, Japan)

Realtime PCR

Total RNA was extracted from liver tissues and fully lysed using Trizol reagent (Invitrogen Life Technologies) following the manufacturer’s instructions. The concentration and quality of RNA were measured using NanoDrop2000 (ThermoFisher Scientific, USA). cDNA was synthesized using the reverse transcriptase kit (TOYOBO, Japan), according to the supplier’s recommendations. The resulting cDNA samples were tested for the expressions of ATG16L1, LC3B, TGF-β1, IFN-γ, TNF-α and MCP-1 by RT-PCR using the LightCycler 480 SYBR Green I Master Kit (Roche, CHE). GAPDH was used as an internal control. Each sample was run in triplicate on the same plate. Sequences for the forward and reverse primers used to quantify mRNA were listed as follows in Table 1. The relative CT (2^−ΔΔCt) method was used to analyze the relative expression of targeted genes.

Table 1

**Primers for Realtime PCR**
Gene Name	Primer Sequence (5'→3')
Mouse-TGF-β1	F: 5′ -CTCCCGTGGCTTCTAGTGC-3′ R: 5′ -GCCTTAGTTTGGACAGGATCTG-3′
Mouse-TNF-α	F: 5′ -GCGGTGCCTATGTCTCAGC-3′ R: 5′ -CACTTGGTGGTTTGTGAGTGT-3′
Mouse-IFN-γ	F: 5′ -ATGAACGCTACACACTGCATC-3′ R: 5′ -CCATCCTTTTGCCAGTTCCTC-3′
Mouse-MCP-1	F: 5′ -TAAAAACCTGGATCGGAACCAAA-3′ R: 5′ -GCATTAGCTTCAGATTTACGGGT-3′
Mouse-LC3B	F: 5′ -CCCACCAAGATCCCAGTGAT-3′ R: 5′ -CCAGGAACTTGGTCTTGTCCA-3′
Mouse-ATG16L1	F: 5′ -GACCTGCTAAAAGTCATCGACC-3′ R: 5′ -AGTCAGAGCCGCATTTGAATC-3′
Mouse-β-actin	F: 5′ -GTGACGTTGACATCCGTAAAGA-3′ R: 5′ -GCCGGACTCATCGTACTCC-3′

Immunofluorescence microscopy

Liver slices were fixed in 4% paraformaldehyde for 20 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 10 min, then blocking in 5% BSA for 1h at room temperature. Samples were incubated with anti-P62 primary antibody overnight at 4°C, followed by incubation with secondary antibodies for 2 h. Images were captured using confocal microscope (Carl Zeiss Inc., Germany).

EISA

Mice serum sample from each group were collected and cytokine IL-1β concentrations were detected using commercially available ELISA kits (R&D system, USA) according to the manufacturer’s instructions.

Immunoblot analysis

Liver tissue lysates were prepared and protein concentrations were measured using the BCA protein assay (Bio-Rad Laboratories, USA). Approximately 30 µg of proteins were separated using SDS-PAGE followed by Western blot analysis. Protein bands were incubated with the following primary antibodies (1:2000 dilution) overnight at 4℃: Anti-LC3B antibody (Cell Signaling Technology, USA), Anti-P62 antibody (Proteintech, USA), Anti-α-SMA antibody (Proteintech, USA), Anti-GAPDH antibody (Cell Signaling Technology, USA). The membranes were then washed five times with wash buffer PBST and probed with HRP-linked Antibody (1:5,000 dilution, Cell Signaling Technology, USA) for 1h at room temperature. ECL detection (Merck millipore, USA) was conducted to analyze immunoreactive protein signals. Each blot was semi-quantitatively analyzed by Image J software.

Statistical analysis

Each experiment was performed with least three biological replicates. Results were expressed as mean ± SD. Statistical significance was determined using Student’s t test and one-way ANOVA with GraphPad Prism 8 software. P < 0.05 was considered statistically significant difference.

Effect of C. sinensis infection on hepatic fibrosis and liver autophagy in vivo

To explore the effect of C. sinensis infection on hepatic fibrosis and autophagy, 30 metacercariae were orally administrated to each mouse. Aspartate transaminase (AST), alanine transaminase (ALT) and the total bilirubin (TBil) levels were measured. Compared with the control group, levels of serum biochemical parameters including ALT, AST and TBIL showed a significant increase at four (4w), six (6w) and eight weeks (8w) after infection (P < 0.05) (Fig. 1). In addition, as displayed in Fig. 2A, C. sinensis infection decreased the ratio of LC3-II/LC3-I, and upregulated the expression of actin alpha 2, smooth muscle (α-SMA), a central marker of liver fibrosis. However, P62 protein levels showed a decline at four weeks post-infection, while showing an apparent increase at six and eight weeks. Furthermore, the result of P62 obtained via fluorescence microscope at different stages post-infections were consistent with the Western blot analysis (Fig. 2B). These results indicate that C. sinensis infection might induce hepatic fibrosis by inhibiting autophagy.

Influence of CQ stimulation on the autophagy in C. sinensis-infected mice

After verification of autophagy generation during liver fluke infection, we speculated that autophagy might be implicated in the pathological process of this infection. A mouse model of C. sinensis infection with or without autophagy inhibitor CQ stimulation was established. The experiments with CQ stimulation in the early and late stages of infection respectively, indicated that CQ caused a remarkable decrease in mRNA levels of autophagic markers ATG16L1 and LC3B compared to the control group at week 8 post-infection (Cs-8w), chloroquine administrated intraperitoneally from week 1 to week 4 post-infection (Cs + CQ-B), and chloroquine administration (CQ) (P < 0.01). Comparing the Cs + CQ-B group with Cs-8w revealed that this decrease of ATG16L1 mRNA expression was accentuated as autophagy was inhibited by CQ (P < 0.05). Nevertheless, for the group with CQ administrated intraperitoneally from week 5 to week 8 post-infection (Cs + CQ-A group), the expressions of ATG16L1 and LC3B remained unchanged (Fig. 3A). Meanwhile, immunofluorescence analysis showed parasitic infection and CQ involvement augmented the expression of the autophagy substrate P62 (Fig. 3B). According to these results, the further experiments were conducted to evaluate the effects of CQ on liver fibrosis, inflammation and pathogenetic changes during the infection.

Capability of CQ stimulation on liver function and autophagy in C. sinensis-infected mice

Next, we examined whether impaired autophagy by CQ had beneficial effects on hepatic function in a parasitic infection mouse model. Remarkably, as shown in Fig. 4, the liver/body weight ratio, spleen/body weight ratio, spleen weight, serum AST, ALT and TBIL levels all increased after C. sinensis infection, while final body weight decreased post-infection (P < 0.05). Compared with the infected group (Cs-8w group), liver/body weight ratio, spleen/body weight ratio, spleen weight, serum AST, ALT, and TBIL levels were reduced in the Cs + CQ-B group while final body weight showed an increase. However, in the group with CQ administrated intraperitoneally from week 5 to week 8 post-infection (Cs + CQ-A group), serum AST, ALT and TBIL levels were higher than those in Cs-8w group (P < 0.01). These data hint that CQ might reduce symptoms in the early stage of infection, but exacerbate hepatic impairment in the later stage.

Changes in fibrotic and pathologic processes in response to CQ stimulation in C. sinensis-infected mice

To confirm the above conjecture, Masson’s trichrome staining was used to evaluate inflammatory infiltration and collagen deposition. Microscopic evaluation revealed that the arrangement of liver cells was irregular with large aggregates of inflammatory cell infiltrations in the Cs-8w and Cs + CQ-A groups. Additionally, blocking autophagy by administrating autophagy flux inhibitor CQ in the early stage of parasitic infection (Cs + CQ-B) markedly attenuated inflammation and fibrotic symptom. Disrupted autophagy in the late stage of parasitic infection (Cs + CQ-A) by CQ possibly promoted chronic inflammation and liver fibrosis in C. sinensis-infected mice (Fig. 5A).

Impact of CQ stimulation on C. sinensis-induced inflammatory signaling pathway

Studies have suggested that abnormal autophagy closely linked with inflammatory signaling pathway (Levine et al. 2011; Xiao et al. 2024). Several crucial pro-inflammation mediators, including TGF-β1, TNF-α, IFN-γ, and IL-1β, trigger innate and adaptive immune response to defend the host from pathogen invasion (Joosten et al. 2013; Yue et al. 2024). Therefore, we wonder whether blocking autophagy by CQ interfered the secretions of pro-inflammatory cytokines in the progression of liver fibrosis induced by C. sinensis infection. Liver specimens and blood samples from C. sinensis infected mice with or without CQ administration were collected and assessed by RT-PCR and ELISA, respectively. mRNA levels of TGF-β1, IFN-γ, TNF-α in Cs + CQ-A group were apparently higher than that in Cs-8w group. Compared to the control group, MCP-1 mRNA expression in experimental groups raised, and CQ significantly increased expression of TGF-β1, IFN-γ, TNF-αin the group, which autophagy being interrupted by CQ administrated intraperitoneally from week 5 to week 8 post-infection (Fig. 5B). Subsequent experiments were conducted using specific ELISA kits to detect the levels of IL-1β. Compared to the control group, significant increase in IL-1β levels were detected in experimental groups (Cs-8w, Cs + CQ-B, Cs + CQ-A groups). Notably, CQ treatment in the early stage of parasitic infection abolished IL-1β secretion, but an upregulated trend was observed in the late stage of parasitic infection (Fig. 5C).

Function of autophagy inhibition by CQ in the progression of liver fibrosis induced by C. sinensis infection

To assess the role of autophagy inhibitor CQ in the progression of hepatic fibrosis evoked by C. sinensis infection, we determined the protein expressions of autophagic biomarkers, LC3B and P62, as well as the fibrotic molecule α-SMA by Western blot. As shown in Fig. 6, we found that both the LC3II/I ratio and P62 were significantly increased in the CQ treated group, with no effect on the α-SMA expression. The result of infection with or without CQ stimulation showed that C. sinensis infection effectively upregulated the expressions of α-SMA, P62, while downregulating the LC3II/I ratio. Notably, compared with the infection group, α-SMA expression in the Cs + CQ-B group was decreased, but accumulated as expected when CQ was added in the late stage of parasite infection (Cs + CQ-A group). The treatment of CQ plus infection inhibited P62 expression compared to the infection group (Cs-8w group). However, adding CQ caused an accumulation of LC3 II versus its precursor LC3 I compared to the Cs-8w group. The LC3II/I ratio was higher in the Cs + CQ-B group than in the Cs-8w group. Taken together, these data uncover that CQ may attenuate liver fibrosis in the early stage of C. sinensis infection, but promote its progression in the late stage of infection.

Infection with C. sinensis is mostly prevalent in many countries, especially in East Asia. It remains an enormous challenge for developing countries and regions. Cumulative evidence indicates that autophagy plays a multifaced role in various diseases, including tumor, liver diseases, diabetes, kidney injury, heart and neurodegenerative disease, even in the infection (Ichimiya et al. 2020). Our previous research demonstrated that C. sinensis adult-derived total protein affected cell autophagy, apoptosis, proliferation, and inflammatory response, resulting in worsening hepatic injury (Shang et al. 2020). However, the role of autophagy in C. sinensis infection and induced liver fibrosis remains unclear.

Autophagy is a highly conserved process implicated in the self-eradication and recycling of subcellular organelles, enduring proteins, and intercellular pathogens. Host autophagy machinery serve as a defense against pathogen invasion (Levine et al. 2011; Villares et al. 2024). Simultaneously, pathogen subvert autophagy to evade elimination by host cells for survival (Gutierrez et al. 2004; Su et al. 2012). Emerging evidence point out that autophagic processes implicate in the dynamic interactions between host and parasites infections. It has been found host cell autophagy contributes to reduce the clearance of Leishmania spp. by T cells (Crauwels et al. 2015) and facilitates Plasmodium liver development (Thieleke-Matos et al. 2016). Yuan et al. (Yuan et al. 2024) reported that inhibiting autophagy by CQ during HPC activation might diminish liver fibrosis in schistosomiasis. Parasites exploit the autophagy machinery to escape the host cell mediated extermination, but autophagy can also assist in defending against parasitic invasion (Latre de Late et al. 2017). It is speculated that modulation of autophagy could serve as a therapeutic strategy for the pathogenesis of schistosomiasis infection. Similar argument was proposed in another study about autophagy and Toxiplasma gondii infection (Cheng et al. 2022). Recent studies indicate that autophagy not only assists cells to adapt to various metabolic stress (Khandia et al. 2019), but also is involved in the activation of LX-2 cells by CsESP (Zheng et al. 2022) or CsTP (Shang et al. 2020). In this study, we confirmed the induction of autophagy during C. sinensis infection, which disrupted hepatic function in the infected mice. The secretion of ALT, AST and TBIL obviously raised in infected mice than in uninfected controls. Quantitative analysis of protein levels further supported that C. sinensis infection was accompanied by upregulated α-SMA expression, indicative of liver fibrosis. The infection triggered host autophagy, as shown by distinct differences in protein levels of LC3 and P62 between the infected and control groups. This finding agreed with previous studies (Hu et al. 2021; Priyamvada et al. 2021; White et al. 2024; Zhen et al. 2023). It was speculated that C. sinensis infection may induce host autophagy to modulate the process of liver injury.

CQ, as an old antimalarial drug, is known for its autophagy-inhibiting effects. Growing evidence suggest that CQ exerts anti-fibrogenesis effects in various forms of hepatic fibrosis. According to these researches, CQ protects CCl4-induced hepatic fibrosis by inhibiting autophagy, HSCs activation, and inflammation (Dai et al. 2018; Le et al. 2022; Le et al. 2023). A study from Li et al. showed that CQ pretreatment deteriorates oxidative stress, inflammation, and apoptosis induced by ammonia stress in yellow catfish (Li et al. 2024). To explicate the potential effect of CQ in the process of C. sinensis infection, our results indicated that, treatment with CQ only affected the experimental group at the early stage of infection and the uninfected group, leading to downregulated ATG16L1 and LC3B expressions and upregulated P62. Parasitic infection might induce autophagy to initiate the host-parasite interaction. A previous study indicated that during S. japonicum infection, CQ might dimmish granuloma formation and hepatic fibrosis (Yuan et al. 2024). Further experiments in our study showed that CQ treatment during the early stage of C. sinensis infection (before the metacercaria matured into adult worms), had a protective effect of improving hepatic function and alleviating liver fibrosis as evidenced by hepatic function test and histopathological staining. These results proved a protective role of autophagy inhibition during parasitic infection. Interestingly, in the late stage of C. sinensis infection, severe hepatic dysfunction and liver fibrosis occurred in mice treated with CQ. Reports indicate that autophagy inhibition or interference may impair the ability of host cell to degrade T. gondii (Subauste 2019). Different status of autophagy produces different symptomatic manifestations. Based on our results, this study provides the first evidence demonstrating that CQ has a dual-opposite role at different stages of C. sinensis infection.

To further verify our points, we analyzed the effects of CQ on inflammation. In accordance with other studies, CQ attenuates inflammations in experimental periodontitis (He et al. 2020), anti-GBM nephritis (Torigoe et al. 2023), LPS-treated RAW264.7 cells (Ota et al. 2024), and CCl4-induced acute liver injury (Dai et al. 2018). Nevertheless, a study by An et al. finds that CQ not only protects against AKI, but also exacerbates renal injury by abolishing protective autophagy and disrupting lysosomal degradation in PTEC (An et al. 2022). As mentioned earlier, we found that in the late stages of C. sinensis infection, CQ treatment aggravated inflammation signaling, evidenced by irregular liver cell arrangement, grievous inflammatory cell infiltrations, and increased secretion of inflammation cytokines such as TGF-β1, TNF-α, IFN-γ, and IL-1β. The hallmark mature myofibroblast α-SMA expression was obviously upregulated in the experimental group treated with CQ during late-stage infection. However, the opposite results were observed in the early-stage infection group treated with CQ, which showed moderate inflammatory infiltration and downregulated expressions of TGF-β1, TNF-α, IFN-γ, and IL-1β. These results suggest that CQ acts as a double-edged sword in C. sinensis induced liver injury. Before the metacercaria of C. sinensis mature into adult worm, CQ pretreatment can alleviate the symptomatic manifestations of inflammation and liver injury. However, pathological change induced by the infection become more severe at the late stage of C. sinensis infection plus CQ treatment. The research provides a basis for clarifying specific function of CQ during C. sinensis infection by inhibiting the host autophagy. However, further explorations are necessary to uncover the precise molecular mechanism by which host autophagy modulates the pathogenesis by C. sinensis infection. Our next investigation will focus on exploring these mechanisms to provide a theoretical rationale for targeting autophagy in anti-clonorchiasis drug development.

In summary, our data provide new insights into the dual role of CQ in C. sinensis infection. CQ protects against liver inflammation and fibrosis at the early stage of infection, but aggravates these conditions at the late stage. These findings suggest that modulating autophagy could be an effective therapeutic strategy for C. sinensis infection. Future efforts will focus on developing novel anti-clonorchiasis vaccines and drugs.

Ethical standards The procedure of animal experiments was approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University (Permit No. SYSU-IACUC-2022-000370).

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Funding

This research was funded by Guangdong Basic and Applied Basic Research Foundation (2020A1515110152), Third Affiliated Hospital of Sun Yat-sen University Special Cultivation Fund of the National Natural Science Foundation of China (2021GZRPYQN02), The Science and Technology Program of Guangzhou (2024A04J4787).

Author Contribution

M.S., H.L. and W.J.C. conducted data curation and project supervision. Y.G. and Y.J.W. completed data analysis and wrote the original draft of this manuscript. M.S. and Y.G. prepared figures 1-3. W.J.C. and H. L. completed figures 4-6. B.H., H.M.D. and X.R.L. reviewed and edited the manuscript, followed by finalized the manuscript. All authors reviewed the manuscript.

Acknowledgement

We thank Ph.D Sheng Liu working on helping to finalize the manuscript.

Data availability

Raw data will be made available if requested.

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Potential role of host autophagy in Clonorchis sinensis infection

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