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Comparison of porcine airway and intestinal epithelial cell lines for the susceptibility and expression of pattern recognition receptors upon influenza virus infection

Viruses (2018) 10(6)
DOI: 10.3390/v10060312
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Abstract

Influenza viruses infect the epithelial cells of the swine respiratory tract. Cell lines derived from the respiratory tract of pigs could serve as an excellent in vitro model for studying the pathogenesis of influenza viruses. In this study, we examined the replication of influenza viruses in the MK1-OSU cell line, which was clonally derived from pig airway epithelium. MK1-OSU cells expressed both cytokeratin and vimentin proteins and displayed several sugar moieties on the cell membrane. These cells also expressed both Sial2-3Gal and Sial2-6Gal receptors and were susceptible to swine influenza A, but not to human B and C viruses. Interestingly, these cells were also permissive to infection by influenza D virus that utilized 9-O-acetylated glycans. To study the differences in the expression of pattern recognition receptors (PRRs) upon influenza virus infection in the respiratory and digestive tract, we compared the protein expression of various PRRs in MK1-OSU cells with that in the SD-PJEC cell line, a clonally derived cell line from the porcine jejunal epithelium. Toll-like receptor 7 (TLR-7) and melanoma differentiation-associated protein 5 (MDA5) receptors showed decreased expression in influenza A infected MK1-OSU cells, while only TLR-7 expression decreased in SD-PJEC cells. Further research is warranted to study the mechanism behind the virus-mediated suppression of these proteins. Overall, this study shows that the porcine respiratory epithelial cell line, MK1-OSU, could serve as an in-vitro model for studying the pathogenesis and innate immune responses to porcine influenza viruses.

Figures

  • Table 1. Lectins and inhibitors used for determining the lectin binding profile of MK1-OSU cells.
  • Table 2. Primary and secondary antibodies used for the estimation of PRRs in MK1-OSU and SD-PJEC cells by flow cytometry.
  • Table 3. qPCR primers for the quantification of PRRs.
  • Figure 1. Immunohistochemical staining of MK1-OSU cells for phenotypic markers: cytokeratin, vimentin, α-smooth muscle actin (ASMA), and desmin. Cytospins were prepared and stained with marker-specific monoclonal antibodies. Isotype controls for antibodies were stained concurrently. Cells were stained with cytokeratin (A), vimentin (B), ASMA (C), and desmin (D) specific monoclonal antibodies and their isotype controls. The left panel represents the isotype controls for cytokeratin, vimentin, ASMA, and desmin, respectively. As shown in the right panel, cells stained positively for epithelial cell marker, cytokeratin, and fibroblast marker vimentin. The images are representative of three independent experiments. The scale bars on the bottom left corner of each image represents 100 µm length.
  • Figure 2. Lectin binding profile of MK1-OSU cells. (A) MK1-OSU cells were incubated with biotinylated lectins and were stai ed using streptavidin-FITC. Samples were analyzed using a FACSCalibur flow cytometer. Values represent th average for three to fi independ nt exp rim nts± SE. (B) Differential expression of sialic acid receptors for influenza A virus in MK1-OSU and MDCK cells. Both MK1-OSU and MDCK cells were incubated with biotinylated lectins binding to Sial2-3Gal (MAL-II) and Sial2-6Gal (SNA) and were stained using streptavidin-FITC. Samples were analyzed using a FACSCalibur flow cytometer. Values represent the average for four experiments ± SE.
  • Figure 3. MK1-OSU cells infected with influenza A (MN08 and IA07) virus. (A) Cells were infected with MN08 or IA07 viruses at MOI of 0.01 and incubated for 24 h at 37 ◦C and were fixed with 2% paraformaldehyde in PBS. The fixed cells were incubated with mouse anti-influenza A nucleoprotein and were stained with goat anti-mouse IgG-Alexa 488 (green color) for 1 h. Nuclei were stained using DAPI (blue color) and cells were washed with PBS and examined under an inverted Olympus AX70 fluorescent microscope at 20× magnification. (B) Percentage of MK1-OSU cells infected with influenza A (MN08 and IA07) virus. MK1-OSU were infected at MOI of 0.01 and incubated for 24 h at 37 ◦C. Following incubation, cells were fixed and permeabilized using BD Cytofix/Cytoperm™. After blocking with 1% goat serum, cells were incubated with primary antibodies against the nucleoprotein of influenza A virus and were stained using goat anti-mouse IgG-Alexa 488. Percentage of cells and mean fluorescence intensity were measured using a FACSCalibur cytometer. Values represent the average for three experiments ± SE.
  • Figure 4. Growth kinetics of type A and D influenza viruses in MK1-OSU, SD-PJEC, and MDCK cells. Growth kinetics of type A and D influenza viruses during 72 h post-infection were measured in MK1OSU, SD-PJEC, and MDCK cells. Briefly, cells were infected with the influenza viruses at MOI of 0.01 and 100 µL of media was sampled at 12 h intervals until 72 h. Viral 50% infective doses were expressed as log10 TCID50/mL. Values represent the mean ± SE of three experiments for (A) pdm09/CA04, (B) MN08, (C) IA07, (D) DOK, (E) FL06, and (F) BR08.
  • Figure 5. Expressions of TLRs and RLRs in MK1-OSU and SD-PJEC cells at 6 h and 24 h post-infection determined using a flow cytometer. MK1-OSU and SD-PJEC cells were infected with MN08 and IA07 viruses at MOI of 0.01 and incubated for 6 and 24 h at 37 °C. Cells were fixed and permeabilized using BD Cytofix/Cytoperm, incubated with primary antibodies, treated with biotinylated secondary antibody, and stained with streptavidin-FITC. (A) TLR-7 expression was decreased by both MN08 and IA07 in MK1-OSU cells 24 h post-infection but only by MN08 in SD-PJEC cells. (B) Expressions of MDA5 were reduced by MN08 and IA07 infection in MK1-OSU cells at 24 h but did not affect the expressions in SD-PJEC cells. (C) RIG-I did not alter due to treatments in both cell lines. Bars represent the mean for four to six experiments ± SE p < 0.05 (*) represented a significant difference between uninfected and infected cells. ** and *** represents p < 0.005 and p < 0.0005 respectively.

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Thomas, M., Pierson, M., Uprety, T., Zhu, L., Ran, Z., Sreenivasan, C. C., … Kaushik, R. S. (2018). Comparison of porcine airway and intestinal epithelial cell lines for the susceptibility and expression of pattern recognition receptors upon influenza virus infection. Viruses, 10(6). https://doi.org/10.3390/v10060312

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