Inhibition of H9N2 Virus Invasion into Dendritic Cells by the S-Layer Protein from L. acidophilus ATCC 4356

Xue Gao, Lulu Huang, Liqi Zhu, Chunxiao Mou, Qihang Hou, Qinghua Yu, Xue Gao, Lulu Huang, Liqi Zhu, Chunxiao Mou, Qihang Hou, Qinghua Yu

Abstract

Probiotics are essential for the prevention of virus invasion and the maintenance of the immune balance. However, the mechanism of competition between probiotics and virus are unknown. The objectives of this study were to isolate the surface layer (S-layer) protein from L. acidophilus ATCC 4356 as a new antiviral material, to evaluate the stimulatory effects of the S-layer protein on mouse dendritic cells (DCs) and to verify its ability to inhibit the invasion of H9N2 avian influenza virus (AIV) in DCs. We found that the S-layer protein induced DCs activation and up-regulated the IL-10 secretion. The invasion and replication of the H9N2 virus in mouse DCs was successfully demonstrated. However, the invasion of H9N2 virus into DCs could be inhibited by treatment with the S-layer protein prior to infection, which was verified by the reduced hemagglutinin (HA) and neuraminidase (NA) mRNA expression, and nucleoprotein (NP) protein expression in the DCs. Furthermore, treatment with the S-layer protein increases the Mx1, Isg15, and Ddx58 mRNA expressions, and remits the inflammatory process to inhibit H9N2 AIV infection. In conclusion, the S-layer protein stimulates the activation of mouse DCs, inhibits H9N2 virus invasion of DCs, and stimulates the IFN-I signaling pathway. Thus, the S-layer protein from Lactobacillus is a promising biological antiviral material for AIV prevention.

Keywords: L. acidophilus; S-layer protein; avian influenza virus; dendritic cells; mucosal.

Figures

Figure 1
Figure 1
Analysis of SDS-PAGE and the CCK-8 assay. (A) The S-layer protein of L. acidophilus ATCC 4356 was extracted by GuHCl. The whole proteins of L. acidophilus ATCC 4356 and the extracted S-layer protein were analyzed by SDS-PAGE. (M, molecular weight marker. 4356, the whole proteins of L. acidophilus ATCC 4356. S, S-layer protein extracted from L. acidophilus ATCC 4356.) (B) The CCK-8 assay was used to assess the effect of different S-layer protein concentrations on DCs viability.
Figure 2
Figure 2
S-layer protein activates DCs through up-regulation of maturation marker expression and cytokine production. (A–C) Flow cytometry analysis of the maturation markers CD86, CD80, and CD40 expressed on the surface of DCs treated with the S-layer protein (400 μg/ml) is shown. (D,E) The relative mRNA levels of IL-10 and TNF-α in DCs incubated with the S-layer protein (400 μg/ml) for 24 h were analyzed by qRT-PCR. CON, relative mRNA level in untreated DCs in these experiments; LPS, relative mRNA level in DCs treated with LPS (10 ng/ml); *P < 0.05; **P < 0.01. Results are from three different experiments.
Figure 3
Figure 3
H9N2 virus infects DCs. (A) DCs infected with the DyLight 488-H9N2 virus (green) for 1 h were analyzed by FACS. **P < 0.01. (Ba,b) Confocal microscopy was used to observe the invasion of DCs by the DyLight 488-H9N2 virus. (Bc–f) Three-dimensional rendering of the images obtained using the Imaris 7.2 software. DCs were stained with CD11c (red) and DAPI (blue). Bars: 50 μm (Ba); 10 μm (Bb–f).
Figure 4
Figure 4
Efficient replication of the H9N2 virus in DCs and the response of DCs treated with the H9N2 virus. DCs infected with the H9N2 virus were harvested at different time points after infection as indicated. (A,B) The relative HA and NA mRNA levels were evaluated by qRT-PCR. The expression levels of these genes in DCs treated with the H9N2 virus is presented in relation to untreated DCs. (C) The H9N2 virus was detected in DCs by confocal microscopy at 1 h and 24 h post-infection. Bars: 20 μm (D) Then, CD86, CD80, and CD40 expression on the surface of treated and untreated DCs was analyzed at 24 h post-infection by FACS. (E) The relative mRNA levels of Mx1, Isg15, and Ddx58 were evaluated by qRT-PCR. The expression levels of these genes in DCs treated with the H9N2 virus are presented in relation to untreated DCs. (F,G) The gene transcription levels of IL-10 and TNF-α in DCs were analyzed by qRT-PCR 24 h post-infection. *P < 0.05; **P < 0.01. The results are from three different experiments.
Figure 5
Figure 5
S-layer protein inhibits H9N2 virus invasion of DCs and leading to a series of changes in the DCs. (A) Flow cytometry was used to detect DyLight 488-H9N2 virus invading into DCs which incubated with different concentrations of S-layer protein before infected with H9N2 virus. The number of DCs infected with the DyLight 488-H9N2 virus is shown. (B) The mRNA relative expression levels of HA, NA, PB1, and NP 1 h after the infection of the DCs by H9N2 virus was analyzed by qRT-PCR. (C) H9N2 virus in the DCs treated with and untreated with S-layer protein was detected by confocal microscopy 24 h post-infection. Bars: 20 μm. (D) CD86, CD80, and CD40 expression on the surface of DCs under different treatments was analyzed by FACS after 24 h. (E,F) DCs infected with the H9N2 virus after incubation with the S-layer protein at different time points were collected for comparison of the mRNA expression levels of HA, NA, and some ISGs (Mx1, Isg15, and Ddx58) to DCs untreated with the S-layer protein. (G) The relative mRNA expression levels of IL-10 and TNF-α24 h post-infection were evaluated by qRT-PCR. (H) The gene transcription levels of IFN-γ in DCs treated with the S-layer protein and untreated were evaluated by qRT-PCR 1 h post-infection with the H9N2 virus. *P < 0.05; **P < 0.01. The results are from three different experiments.
Figure 6
Figure 6
The schematic diagram of the inhibitory effect of the S-layer protein against H9N2 AIV. (A) The H9N2 AIV could invade DCs through DC-SIGN. (B) The S-layer protein isolated from Lactobacillus could compete with H9N2 AIV for binding to DC-SIGN and inhibit AIV invasion into DCs.

References

    1. Acevedo G., Padala N. K., Ni L., Jonakait G. M. (2013). Astrocytes inhibit microglial surface expression of dendritic cell-related co-stimulatory molecules through a contact-mediated process. J. Neurochem. 125, 575–587. 10.1111/jnc.12221
    1. Alen M. M., Kaptein S. J., De Burghgraeve T., Balzarini J., Neyts J., Schols D. (2009). Antiviral activity of carbohydrate-binding agents and the role of DC-SIGN in dengue virus infection. Virology 387, 67–75. 10.1016/j.virol.2009.01.043
    1. Alexander D. J. (2007). An overview of the epidemiology of avian influenza. Vaccine 25, 5637–5644. 10.1016/j.vaccine.2006.10.051
    1. Banchereau J., Briere F., Caux C., Davoust J., Lebecque S., Liu Y. J., et al. . (2000). Immunobiology of dendritic cells. Annu. Rev. Immunol. 18, 767–811. 10.1146/annurev.immunol.18.1.767
    1. Boot H. J., Kolen C. P., van Noort J. M., Pouwels P. H. (1993). S-layer protein of Lactobacillus acidophilus ATCC 4356: purification, expression in Escherichia coli, and nucleotide sequence of the corresponding gene. J. Bacteriol. 175, 6089–6096.
    1. da Silva R. C., Segat L., Crovella S. (2011). Role of DC-SIGN and L-SIGN receptors in HIV-1 vertical transmission. Hum. Immunol. 72, 305–311. 10.1016/j.humimm.2011.01.012
    1. Drickamer K. (1992). Engineering galactose-binding activity into a C-type mannose-binding protein. Nature 360, 183–186. 10.1038/360183a0
    1. Eslami N., Kermanshahi R. K., Erfan M. (2013). Studying the stability of S-layer protein of lactobacillus acidophilus ATCC 4356 in simulated gastrointestinal fluids using SDS-PAGE and circular dichroism. Iran. J. Pharm. Res. 12, 45–54.
    1. Foligne B., Zoumpopoulou G., Dewulf J., Ben Younes A., Chareyre F., Sirard J. C., et al. . (2007). A key role of dendritic cells in probiotic functionality. PLoS ONE 2:e313. 10.1371/journal.pone.0000313
    1. Gerritsen J., Smidt H., Rijkers G. T., de Vos W. M. (2011). Intestinal microbiota in human health and disease: the impact of probiotics. Genes Nutr. 6, 209–240. 10.1007/s12263-011-0229-7
    1. Grouard G., Rissoan M. C., Filgueira L., Durand I., Banchereau J., Liu Y. J. (1997). The enigmatic plasmacytoid T cells develop into dendritic cells with interleukin (IL)-3 and CD40-ligand. J. Exp. Med. 185, 1101–1111. 10.1084/jem.185.6.1101
    1. Guinane C. M., Cotter P. D. (2013). Role of the gut microbiota in health and chronic gastrointestinal disease: understanding a hidden metabolic organ. Therap. Adv. Gastroenterol. 6, 295–308. 10.1177/1756283X13482996
    1. Hoorelbeke B., Van Damme E. J. M., Rougé P., Schols D., Van Laethem K., Fouquaert E., et al. . (2011). Differences in the mannose oligomer specificities of the closely related lectins from Galanthus nivalis and Zea mays strongly determine their eventual anti-HIV activity. Retrovirology 8:10. 10.1186/1742-4690-8-10
    1. Jankowska A., Laubitz D., Antushevich H., Zabielski R., Grzesiuk E. (2008). Competition of Lactobacillus paracasei with Salmonella enterica for adhesion to Caco-2 cells. J. Biomed. Biotechnol. 2008:357964. 10.1155/2008/357964
    1. Konstantinov S. R., Smidt H., de Vos W. M., Bruijns S. C., Singh S. K., Valence F., et al. . (2008). S layer protein A of Lactobacillus acidophilus NCFM regulates immature dendritic cell and T cell functions. Proc. Natl. Acad. Sci. U.S.A. 105, 19474–19479. 10.1073/pnas.0810305105
    1. Li P., Yin Y., Yu Q., Yang Q. (2011b). Lactobacillus acidophilus S-layer protein-mediated inhibition of Salmonella-induced apoptosis in Caco-2 cells. Biochem. Biophys. Res. Commun. 409, 142–147. 10.1016/j.bbrc.2011.04.131
    1. Li P., Yu Q., Ye X., Wang Z., Yang Q. (2011a). Lactobacillus S-layer protein inhibition of Salmonella-induced reorganization of the cytoskeleton and activation of MAPK signalling pathways in Caco-2 cells. Microbiology 157(Pt 9), 2639–2646. 10.1099/mic.0.049148-0
    1. Londrigan S. L., Tate M. D., Brooks A. G., Reading P. C. (2012). Cell-surface receptors on macrophages and dendritic cells for attachment and entry of influenza virus. J. Leukoc. Biol. 92, 97–106. 10.1189/jlb.1011492
    1. Manches O., Frleta D., Bhardwaj N. (2014). Dendritic cells in progression and pathology of HIV infection. Trends Immunol. 35, 114–122. 10.1016/j.it.2013.10.003
    1. Martínez M. G., Acosta M. P., Candurra N. A., Ruzal S. M. (2012). S-layer proteins of Lactobacillus acidophilus inhibits JUNV infection. Biochem. Biophys. Res. Commun. 422, 590–595. 10.1016/j.bbrc.2012.05.031
    1. Martinez R. C., Staliano C. D., Vieira A. D., Villarreal M. L., Todorov S. D., Saad S. M., et al. . (2015). Bacteriocin production and inhibition of Listeria monocytogenes by Lactobacillus sakei subsp. sakei 2a in a potentially synbiotic cheese spread. Food Microbiol. 48, 143–152. 10.1016/j.fm.2014.12.010
    1. Mohamadzadeh M., Olson S., Kalina W. V., Ruthel G., Demmin G. L., Warfield K. L., et al. . (2005). Lactobacilli activate human dendritic cells that skew T cells toward T helper 1 polarization. Proc. Natl. Acad. Sci. U.S.A. 102, 2880–2885. 10.1073/pnas.0500098102
    1. Peiris M., Yuen K. Y., Leung C. W., Chan K. H., Ip P. L., Lai R. W., et al. . (1999). Human infection with influenza H9N2. Lancet 354, 916–917. 10.1016/S0140-6736(99)03311-5
    1. Qin T., Yin Y., Huang L., Yu Q., Yang Q. (2015). H9N2 influenza whole inactivated virus combined with polyethyleneimine strongly enhances mucosal and systemic immunity after intranasal immunization in mice. Clin. Vaccine Immunol. 22, 421–429. 10.1128/CVI.00778-14
    1. Schoggins J. W., Rice C. M. (2011). Interferon-stimulated genes and their antiviral effector functions. Curr. Opin. Virol. 1, 519–525. 10.1016/j.coviro.2011.10.008
    1. Seder R. A., Paul W. E., Davis M. M., Fazekas de St Groth B. (1992). The presence of interleukin 4 during in vitro priming determines the lymphokine-producing potential of CD4+ T cells from T cell receptor transgenic mice. J. Exp. Med. 176, 1091–1098. 10.1084/jem.176.4.1091
    1. Shen C. I., Wang C. H., Shen S. C., Lee H. C., Liao J. W., Su H. L. (2011). The infection of chicken tracheal epithelial cells with a H6N1 avian influenza virus. PLoS ONE 6:e18894. 10.1371/journal.pone.0018894
    1. Short K. R., Brooks A. G., Reading P. C., Londrigan S. L. (2012). The fate of influenza a virus after infection of human macrophages and dendritic cells. J. Gen. Virol. 93(Pt 11), 2315–2325. 10.1099/vir.0.045021-0
    1. Sutejo R., Yeo D. S., Myaing M. Z., Hui C., Xia J., Ko D., et al. . (2012). Activation of type I and III interferon signalling pathways occurs in lung epithelial cells infected with low pathogenic avian influenza viruses. PLoS ONE 7:e33732. 10.1371/journal.pone.0033732
    1. Svajger U., Anderluh M., Jeras M., Obermajer N. (2010). C-type lectin DC-SIGN: an adhesion, signalling and antigen-uptake molecule that guides dendritic cells in immunity. Cell. Signal. 22, 1397–1405. 10.1016/j.cellsig.2010.03.018
    1. Wang S. F., Huang J. C., Lee Y. M., Liu S. J., Chan Y. J., Chau Y. P., et al. . (2008). DC-SIGN mediates avian H5N1 influenza virus infection in cis and in trans. Biochem. Biophys. Res. Commun. 373, 561–566. 10.1016/j.bbrc.2008.06.078
    1. Wang Z., Loh L., Kedzierski L., Kedzierska K. (2016). Avian influenza viruses, inflammation, and CD8(+) T cell immunity. Front. Immunol. 7:60. 10.3389/fimmu.2016.00060
    1. Westenius V., Mäkelä S. M., Ziegler T., Julkunen I., Österlund P. (2014). Efficient replication and strong induction of innate immune responses by H9N2 avian influenza virus in human dendritic cells. Virology 471–473, 38–48. 10.1016/j.virol.2014.10.002
    1. Youn H. N., Lee Y. N., Lee D. H., Park J. K., Yuk S. S., Lee H. J., et al. . (2012). Effect of intranasal administration of Lactobacillus fermentum CJL-112 on horizontal transmission of influenza virus in chickens. Poult. Sci. 91, 2517–2522. 10.3382/ps.2012-02334

Source: PubMed

Sponsors and Collaborators

Medical Conditions

Drug Interventions

3
Subscribe