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Co-infection of primary human adenoid epithelial cells with Influenza A and Streptococcus pneumoniae
International Congress Series 1263 (2004) 476 – 480
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Co-infection of primary human adenoid epithelial cells with Influenza A and Streptococcus pneumoniae Patricia A. McGraw, Mine R. Ikizler, Mohammed Aiyegbo, Peter F. Wright * Division of Pediatric Infectious Diseases, Department of Pediatrics, Vanderbilt University Medical Center, D7235 Medical Center North, 1161 21st Avenue South, Nashville, TN 37232-2581, USA
Abstract. Human adenoid epithelial cells (HAEC) from adenoids were used as a model for exploring potential viral/bacterial synergy. Methods: HAEC were grown on a collagen, matrix-coated tissue culture plate until the monolayer became confluent. A differentiated cell population with secretory component, mucin production, and ciliary activity was seen with histochemical staining. Cells were infected with Influenza A/Beijing/H3N2 at an MOI of 0.01 PFU for 24 h. The monolayer was washed and 107 Streptococcus pneumoniae added. After a 3-h pneumococcal infection, pneumococci were titered in the supernatant, in the cells after washing (adherent and intracellular bacteria) and in cells treated with antibiotics to destroy adherent bacteria followed by lysis (intracellular bacteria). Results: Over the 3-h infection period, neither pneumococci type 14 (encapsulated) and R6 (unencapsulated) influenced the titer of influenza released nor did influenza affect the bacterial growth. Co-localization of pneumococcal and influenza infection in individual cells was not evident. The capacity varied of pneumococcal strains to adhere and invade the epithelial surface. Encapsulated type 14 from the bloodstream was less invasive than an unencapsulated laboratory strain R6. Conclusion: The propensity for bacterial superinfection, based on increased adherence or invasion of pneumococci to influenza-infected epithelial cells, could not be explained in the HAEC model. D 2004 Elsevier B.V. All rights reserved. Keywords: Co-localization; Superinfection; Pneumococci
1. Introduction Bacterial adhesion to cells has been an area of intense interest [1]. Investigations using in vitro models have shown that virus infections can influence bacterial adherence, invasion and, hence, pathogenicity [2]. Secondary bacterial infections are clinically observed with influenza in humans though many of these reports were in the context of
* Corresponding author. Tel.: +1-615-322-2250; fax: +1-615-343-9723. E-mail address: [email protected] (P.F. Wright). 0531-5131/ D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2004.02.058
P.A. McGraw et al. / International Congress Series 1263 (2004) 476–480
477
earlier pandemics [3]. In a murine model, sequential influenza and pneumococcal infection produced a more severe infection than either agent alone [4]. In a chinchilla model of otitis media, influenza potentiated the development of pneumococcal otitis [5]. Our studies have shown that highly differentiated human adenoid epithelial cells (HAEC), derived from adenoidal tissue, efficiently supported replication of Influenza A/Beijing to titers of 106 PFU/ml [6]. The cells survived in a media in which bacterial replication could occur. HAEC are well suited for the study of pneumococcal –influenzal interactions. 2. Materials and methods 2.1. Bacteria strains Streptococcus pneumoniae unencapsulated strains R6 and CbpA-deficient R6 (kindly provided by Dr. Elaine Tuomanen, St. Jude’s Children’s Research Hospital, Memphis, TN) are derived from the capsular strain D39 [7,8]. Serotyped clinical isolates were designated as 14 and 19F (kindly provided by Dr. Kathryn Edwards, Vanderbilt University, Nashville, TN). Colonies were grown to mid-logarithmic phase in Todd – Hewitt Broth (Becton Dickinson, Sparks, MD), supplemented with 0.5% yeast extract (Becton Dickinson). 2.2. Viral strains Wild type, Influenza A/Beijing-like H3N2, isolated from a sick child in Nashville, was grown in primary rhesus monkey kidney cells in medium 199 (GIBCO). An RSV construct expressing green fluorescent protein, r-RSV GFP, was a gift from Dr. Mark Peeples, Rush University Medical Center, Chicago, IL [9]. 2.3. Epithelial cell adherence and invasion The isolation and characterization of primary epithelial cells from adenoids was previously described by our group [6]. The invasion assay was based on an endothelial cell invasion assay described previously [10]. Cells were infected with influenza (MOI=0.01) for 24 h or r-RSV-GFP 48 h prior to the bacterial infection. S. pneumoniae was grown to mid-log phase in THBY 37 jC, 5% CO2, pelleted, washed and resuspended in DMEM F12 to a final concentration of 2107 pneumococci per well. Viral supernatants of infected monolayers were collected prior to the addition of pneumococcus, and after a 3-h incubation with bacteria. 2.4. Immunofluorescent staining Epithelial cells in chamber slides were fixed with 4 jC acetone –methanol (3:2) and blocked with 5% dry milk. The pneumococcal staining was performed at either 1:50 or 1:100 with primary type-specific antisera (Statens Serum Institute, Copenhagen, Denmark) for 1 h in a humid chamber and washed 3 with PBS. A 1:1000 dilution of rabbit-specific goat serum conjugated to tetramethylrhodamine isothiocyanate TRITC (Sigma, St. Louis, MO) [11] was added, incubated for 1 h in the humid chamber, washed 3 with PBS to
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P.A. McGraw et al. / International Congress Series 1263 (2004) 476–480
allow for visualization of labeled pneumococci and stained with a 1:5000 dilution of 4V6diamidino-2-phenylindole DAPI counter stain (Sigma) for 5 min, washed with PBS and mounted using an antifade buffered glycerol reagent (Molecular Probes, Eugene OR). Images were taken at 40 on a Zeiss Axioplan microscope equipped with a digital SPOT camera and adjusted for brightness and contrast using Adobe Photoshop. 3. Results 3.1. Growth of pneumococcus The growth of pneumococcus is shown in panel A (without influenza) and panel B (with influenza) (Fig. 1). There is a sharp gradation between bacteria in the supernatant, adherent bacteria and viable bacteria that are intracellular. The bacterial titer in the supernatant had not increased appreciably over the input bacteria. Influenza did not alter the bacterial growth. The invasive property of the pneumococcal strains differed with the unencapsulated R6, proving to be more invasive than the serotype 14. Similar differences in adhesion and invasion have been described between pneumococcal strains and have confirmed unencapsulated strains and those associated with pharyngeal colonization and otitis as opposed to those encapsulated or associated with sepsis [12,13]. 3.2. Co-localization of virus and bacteria In data not shown there was no co-localization of influenza and pneumococci on individual cells. The availability of a GFP-producing RSV strain enabled us to examine co-localization of virally infected and cells with adherent pneumococci very clearly. Data for type 14 (Fig. 2) show no evidence of RSV infection predisposing to pneumococcal
Fig. 1. S. pneumoniae infection in HAEC, with and without Influenza A/Beijing H3N2.
P.A. McGraw et al. / International Congress Series 1263 (2004) 476–480
479
Fig. 2. HAEC with r-RSV-GFP (48 h) and S. pneumoniae type 14 (3 h). Stained with Type 14 antiserum + antirabbit IgG TRITC conjugate (red). DAPI cell nuclear stain (blue).
adherence. Fig. 1, panel C shows the lack of any effect of short-term pneumococcal infection on the release or replication of influenza. 4. Discussion An understanding of bacterial/viral interactions is critical for defining the pathogenesis of respiratory disease. In spite of an established viral infection and substantial adherence of bacteria to the cell monolayer, we are unable to explain the phenomena of superinfection by specific adherence to or invasion of pneumococci in influenza-infected cells. Although we have no direct experimental proof, bacterial superinfection with the combination of organisms studied is more likely determined by a loss of integrity of the mucosal surface that follows influenza infection than more specific viral alterations in cell membranes that predispose to passage of bacteria into intact cells. With a negative result there are always questions of whether alteration of conditions might have led to different results. Multiple preliminary experiments were done using different time periods of co-infection (up to 24 h) and additional pneumococcal strains type 19F, R6 and CbpA—with similar outcomes. We were able to demonstrate that with Detroit 562 cells expressing the polymeric Ig receptor and pneumococcus type R6 that invasion was increased by the expression of the receptor [14]. This was both pneumococcal-type and cell-type specific. Many questions remain for future research in this or other models. We explored, only in a preliminary fashion, bacterial phase variation, which has been reported to influence nasopharyngeal colonization [15] and adherence [16]. We do not know how pneumococcal invasion occurs and whether it leads to transcytosis to the basolateral side. Finally, given that invasive pneumococcal disease is a rare event with influenza infection, is it actually possible to model? References [1] E. Tuomanen, The biology of pneumococcal infection, Pediatr. Res. 42 (1997) 253 – 258. [2] J.-M. Hament, et al., Respiratory viral infection predisposing for bacterial disease: a concise review, FEMS Immunol. Med. Microbiol. 26 (1999) 189 – 195.
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P.A. McGraw et al. / International Congress Series 1263 (2004) 476–480
[3] D. Louria, et al., Studies on influenza in the pandemic of 1957 – 58, J. Clin. Invest. 38 (1959) 213 – 265. [4] J.A. McCullers, J.E. Rehg, Lethal synergism between influenza virus and Streptococcus pneumoniae: characterization of a mouse model and the role of platelet-activating factor receptor, J. Infect. Dis. 186 (2002) 341 – 350. [5] G.S. Giebink, P.F. Wright, The pathogenesis of experimental pneumococcal otitis media during respiratory virus infection in chinchillas, Recent Advances in Otitis Media with Effusion, B.C. Decker, Hamilton, Ontario, Canada, 1984, pp. 107 – 111. [6] Y. Endo, et al., Growth of influenza A virus in primary, differentiated epithelial cells derived from adenoids, J. Virol. 70 (1996) 2055 – 2058. [7] J.G. Tiraby, M.S. Fox, Marker discrimination in transformation and mutation of pneumococcus, Proc. Natl. Acad. Sci. 70 (1973) 3541 – 3545. [8] C. Rosenow, et al., Contribution of novel choline-binding proteins to adherence, colonization and immunogenicity of Streptococcus pneumoniae, Mol. Microbiol. 25 (1997) 819 – 829. [9] L.K. Hallak, et al., Iduronic acid-containing glycosaminoglycans on target cells are required for efficient syncytial virus infection, Virology 271 (2000) 264 – 275. [10] A. Ring, J.N. Weiser, E.I. Tuomanen, Pneumococcal trafficking across the blood – brain barrier, J. Clin. Invest. 102 (1998) 347 – 360. [11] K. Wicher, et al., Fluorescent antibody technic used for identification and typing of Streptococcus pneumoniae, Am. J. Clin. Pathol. 77 (1982) 72 – 77. [12] B. Andersson, et al., Adhesion of Streptococcus pneumoniae to human pharyngeal epithelial cells in vitro: differences in adhesive capacity among strains isolated from subjects with otitis media, septicemia, or meningitis or from healthy carriers, Infect. Immun. 32 (1981) 311 – 317. [13] U.M. Talbot, A.W. Paton, J.C. Paton, Uptake of Streptococcus pneumoniae by respiratory epithelial cells, Infect. Immun. 64 (1996) 3772 – 3777. [14] S.C. Brock, et al., The human polymeric immunoglobulin receptor facilitates invasion of epithelial cells by Streptococcus pneumoniae in a strain-specific and cell type-specific manner, Infect. Immun. 70 (2002) 5091 – 5095. [15] J.N. Weiser, et al., Phase variation in pneumococcal opacity: relationship between colonial morphology and nasopharyngeal colonization, Infect. Immun. 62 (1994) 2582 – 2589. [16] D.R. Cundell, et al., Relationship between colonial morphology and adherence of Streptococcus pneumoniae, Infect. Immun. 63 (1995) 757 – 761.
www.ics-elsevier.com
Co-infection of primary human adenoid epithelial cells with Influenza A and Streptococcus pneumoniae Patricia A. McGraw, Mine R. Ikizler, Mohammed Aiyegbo, Peter F. Wright * Division of Pediatric Infectious Diseases, Department of Pediatrics, Vanderbilt University Medical Center, D7235 Medical Center North, 1161 21st Avenue South, Nashville, TN 37232-2581, USA
Abstract. Human adenoid epithelial cells (HAEC) from adenoids were used as a model for exploring potential viral/bacterial synergy. Methods: HAEC were grown on a collagen, matrix-coated tissue culture plate until the monolayer became confluent. A differentiated cell population with secretory component, mucin production, and ciliary activity was seen with histochemical staining. Cells were infected with Influenza A/Beijing/H3N2 at an MOI of 0.01 PFU for 24 h. The monolayer was washed and 107 Streptococcus pneumoniae added. After a 3-h pneumococcal infection, pneumococci were titered in the supernatant, in the cells after washing (adherent and intracellular bacteria) and in cells treated with antibiotics to destroy adherent bacteria followed by lysis (intracellular bacteria). Results: Over the 3-h infection period, neither pneumococci type 14 (encapsulated) and R6 (unencapsulated) influenced the titer of influenza released nor did influenza affect the bacterial growth. Co-localization of pneumococcal and influenza infection in individual cells was not evident. The capacity varied of pneumococcal strains to adhere and invade the epithelial surface. Encapsulated type 14 from the bloodstream was less invasive than an unencapsulated laboratory strain R6. Conclusion: The propensity for bacterial superinfection, based on increased adherence or invasion of pneumococci to influenza-infected epithelial cells, could not be explained in the HAEC model. D 2004 Elsevier B.V. All rights reserved. Keywords: Co-localization; Superinfection; Pneumococci
1. Introduction Bacterial adhesion to cells has been an area of intense interest [1]. Investigations using in vitro models have shown that virus infections can influence bacterial adherence, invasion and, hence, pathogenicity [2]. Secondary bacterial infections are clinically observed with influenza in humans though many of these reports were in the context of
* Corresponding author. Tel.: +1-615-322-2250; fax: +1-615-343-9723. E-mail address: [email protected] (P.F. Wright). 0531-5131/ D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2004.02.058
P.A. McGraw et al. / International Congress Series 1263 (2004) 476–480
477
earlier pandemics [3]. In a murine model, sequential influenza and pneumococcal infection produced a more severe infection than either agent alone [4]. In a chinchilla model of otitis media, influenza potentiated the development of pneumococcal otitis [5]. Our studies have shown that highly differentiated human adenoid epithelial cells (HAEC), derived from adenoidal tissue, efficiently supported replication of Influenza A/Beijing to titers of 106 PFU/ml [6]. The cells survived in a media in which bacterial replication could occur. HAEC are well suited for the study of pneumococcal –influenzal interactions. 2. Materials and methods 2.1. Bacteria strains Streptococcus pneumoniae unencapsulated strains R6 and CbpA-deficient R6 (kindly provided by Dr. Elaine Tuomanen, St. Jude’s Children’s Research Hospital, Memphis, TN) are derived from the capsular strain D39 [7,8]. Serotyped clinical isolates were designated as 14 and 19F (kindly provided by Dr. Kathryn Edwards, Vanderbilt University, Nashville, TN). Colonies were grown to mid-logarithmic phase in Todd – Hewitt Broth (Becton Dickinson, Sparks, MD), supplemented with 0.5% yeast extract (Becton Dickinson). 2.2. Viral strains Wild type, Influenza A/Beijing-like H3N2, isolated from a sick child in Nashville, was grown in primary rhesus monkey kidney cells in medium 199 (GIBCO). An RSV construct expressing green fluorescent protein, r-RSV GFP, was a gift from Dr. Mark Peeples, Rush University Medical Center, Chicago, IL [9]. 2.3. Epithelial cell adherence and invasion The isolation and characterization of primary epithelial cells from adenoids was previously described by our group [6]. The invasion assay was based on an endothelial cell invasion assay described previously [10]. Cells were infected with influenza (MOI=0.01) for 24 h or r-RSV-GFP 48 h prior to the bacterial infection. S. pneumoniae was grown to mid-log phase in THBY 37 jC, 5% CO2, pelleted, washed and resuspended in DMEM F12 to a final concentration of 2107 pneumococci per well. Viral supernatants of infected monolayers were collected prior to the addition of pneumococcus, and after a 3-h incubation with bacteria. 2.4. Immunofluorescent staining Epithelial cells in chamber slides were fixed with 4 jC acetone –methanol (3:2) and blocked with 5% dry milk. The pneumococcal staining was performed at either 1:50 or 1:100 with primary type-specific antisera (Statens Serum Institute, Copenhagen, Denmark) for 1 h in a humid chamber and washed 3 with PBS. A 1:1000 dilution of rabbit-specific goat serum conjugated to tetramethylrhodamine isothiocyanate TRITC (Sigma, St. Louis, MO) [11] was added, incubated for 1 h in the humid chamber, washed 3 with PBS to
478
P.A. McGraw et al. / International Congress Series 1263 (2004) 476–480
allow for visualization of labeled pneumococci and stained with a 1:5000 dilution of 4V6diamidino-2-phenylindole DAPI counter stain (Sigma) for 5 min, washed with PBS and mounted using an antifade buffered glycerol reagent (Molecular Probes, Eugene OR). Images were taken at 40 on a Zeiss Axioplan microscope equipped with a digital SPOT camera and adjusted for brightness and contrast using Adobe Photoshop. 3. Results 3.1. Growth of pneumococcus The growth of pneumococcus is shown in panel A (without influenza) and panel B (with influenza) (Fig. 1). There is a sharp gradation between bacteria in the supernatant, adherent bacteria and viable bacteria that are intracellular. The bacterial titer in the supernatant had not increased appreciably over the input bacteria. Influenza did not alter the bacterial growth. The invasive property of the pneumococcal strains differed with the unencapsulated R6, proving to be more invasive than the serotype 14. Similar differences in adhesion and invasion have been described between pneumococcal strains and have confirmed unencapsulated strains and those associated with pharyngeal colonization and otitis as opposed to those encapsulated or associated with sepsis [12,13]. 3.2. Co-localization of virus and bacteria In data not shown there was no co-localization of influenza and pneumococci on individual cells. The availability of a GFP-producing RSV strain enabled us to examine co-localization of virally infected and cells with adherent pneumococci very clearly. Data for type 14 (Fig. 2) show no evidence of RSV infection predisposing to pneumococcal
Fig. 1. S. pneumoniae infection in HAEC, with and without Influenza A/Beijing H3N2.
P.A. McGraw et al. / International Congress Series 1263 (2004) 476–480
479
Fig. 2. HAEC with r-RSV-GFP (48 h) and S. pneumoniae type 14 (3 h). Stained with Type 14 antiserum + antirabbit IgG TRITC conjugate (red). DAPI cell nuclear stain (blue).
adherence. Fig. 1, panel C shows the lack of any effect of short-term pneumococcal infection on the release or replication of influenza. 4. Discussion An understanding of bacterial/viral interactions is critical for defining the pathogenesis of respiratory disease. In spite of an established viral infection and substantial adherence of bacteria to the cell monolayer, we are unable to explain the phenomena of superinfection by specific adherence to or invasion of pneumococci in influenza-infected cells. Although we have no direct experimental proof, bacterial superinfection with the combination of organisms studied is more likely determined by a loss of integrity of the mucosal surface that follows influenza infection than more specific viral alterations in cell membranes that predispose to passage of bacteria into intact cells. With a negative result there are always questions of whether alteration of conditions might have led to different results. Multiple preliminary experiments were done using different time periods of co-infection (up to 24 h) and additional pneumococcal strains type 19F, R6 and CbpA—with similar outcomes. We were able to demonstrate that with Detroit 562 cells expressing the polymeric Ig receptor and pneumococcus type R6 that invasion was increased by the expression of the receptor [14]. This was both pneumococcal-type and cell-type specific. Many questions remain for future research in this or other models. We explored, only in a preliminary fashion, bacterial phase variation, which has been reported to influence nasopharyngeal colonization [15] and adherence [16]. We do not know how pneumococcal invasion occurs and whether it leads to transcytosis to the basolateral side. Finally, given that invasive pneumococcal disease is a rare event with influenza infection, is it actually possible to model? References [1] E. Tuomanen, The biology of pneumococcal infection, Pediatr. Res. 42 (1997) 253 – 258. [2] J.-M. Hament, et al., Respiratory viral infection predisposing for bacterial disease: a concise review, FEMS Immunol. Med. Microbiol. 26 (1999) 189 – 195.
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P.A. McGraw et al. / International Congress Series 1263 (2004) 476–480
[3] D. Louria, et al., Studies on influenza in the pandemic of 1957 – 58, J. Clin. Invest. 38 (1959) 213 – 265. [4] J.A. McCullers, J.E. Rehg, Lethal synergism between influenza virus and Streptococcus pneumoniae: characterization of a mouse model and the role of platelet-activating factor receptor, J. Infect. Dis. 186 (2002) 341 – 350. [5] G.S. Giebink, P.F. Wright, The pathogenesis of experimental pneumococcal otitis media during respiratory virus infection in chinchillas, Recent Advances in Otitis Media with Effusion, B.C. Decker, Hamilton, Ontario, Canada, 1984, pp. 107 – 111. [6] Y. Endo, et al., Growth of influenza A virus in primary, differentiated epithelial cells derived from adenoids, J. Virol. 70 (1996) 2055 – 2058. [7] J.G. Tiraby, M.S. Fox, Marker discrimination in transformation and mutation of pneumococcus, Proc. Natl. Acad. Sci. 70 (1973) 3541 – 3545. [8] C. Rosenow, et al., Contribution of novel choline-binding proteins to adherence, colonization and immunogenicity of Streptococcus pneumoniae, Mol. Microbiol. 25 (1997) 819 – 829. [9] L.K. Hallak, et al., Iduronic acid-containing glycosaminoglycans on target cells are required for efficient syncytial virus infection, Virology 271 (2000) 264 – 275. [10] A. Ring, J.N. Weiser, E.I. Tuomanen, Pneumococcal trafficking across the blood – brain barrier, J. Clin. Invest. 102 (1998) 347 – 360. [11] K. Wicher, et al., Fluorescent antibody technic used for identification and typing of Streptococcus pneumoniae, Am. J. Clin. Pathol. 77 (1982) 72 – 77. [12] B. Andersson, et al., Adhesion of Streptococcus pneumoniae to human pharyngeal epithelial cells in vitro: differences in adhesive capacity among strains isolated from subjects with otitis media, septicemia, or meningitis or from healthy carriers, Infect. Immun. 32 (1981) 311 – 317. [13] U.M. Talbot, A.W. Paton, J.C. Paton, Uptake of Streptococcus pneumoniae by respiratory epithelial cells, Infect. Immun. 64 (1996) 3772 – 3777. [14] S.C. Brock, et al., The human polymeric immunoglobulin receptor facilitates invasion of epithelial cells by Streptococcus pneumoniae in a strain-specific and cell type-specific manner, Infect. Immun. 70 (2002) 5091 – 5095. [15] J.N. Weiser, et al., Phase variation in pneumococcal opacity: relationship between colonial morphology and nasopharyngeal colonization, Infect. Immun. 62 (1994) 2582 – 2589. [16] D.R. Cundell, et al., Relationship between colonial morphology and adherence of Streptococcus pneumoniae, Infect. Immun. 63 (1995) 757 – 761.