Comparison of Porcine Airway and Intestinal Epithelial Cell Lines for the Susceptibility and Expression of Pattern Recognition Receptors upon Influenza Virus Infection
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Lines and Culture Conditions
2.2. Phenotyping of MK1-OSU Cells by Immunohistochemistry
2.3. Lectin Binding Assay
2.4. Culture of Swine and Human Influenza Virus and Infection of Cells
2.5. Indirect Immunoflourescence Assay for Virus Detection
2.6. Determination of Percentage of MK1-OSU Cells Infected Using Flow Cytometry
2.7. Growth Kinetics of Influenza Viruses in MK1-OSU, SD-PJEC and MDCK Cells
2.8. Estimation of Pattern Recognition Receptors (PRRs) Using Flow Cytometry
2.9. Real-Time RT-PCR Assays for Pattern-Recognition Receptors (PRRs)
2.10. Statistical Analysis
3. Results
3.1. MK1-OSU Cells are Phenotypically Epithelial Cells
3.2. Lectin Binding Profile of MK1-OSU Cells Indicated the Presence of Heterogeneous Cell Surface Sugar Moieties
3.3. MK1-OSU Cells Expressed Sial2-3Gal and Sial2-6Gal Receptors
3.4. MK1-OSU Cells Were Susceptible to SIV Infection
3.5. Influenza Infection Differentially Affected the Expression of TLRs and RLRs in MK1-OSU and SD-PJEC Cells at 24 h
4. Discussion
5. Conclusions
Author Contributions
Acknowledgments
Conflicts of Interest
References
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Lectins | Concentration (µg/mL) | Inhibitor | Concentration (mM) |
---|---|---|---|
1. Glucose/mannose group | |||
Concanavalin-A (ConA) | 10 | α-methyl mannoside, α-methyl glucoside | 200 each |
Pisum Sativum agglutinin (PSA) | 20 | α-methyl mannoside, α-methyl glucoside | 200 each |
Lens culinaris agglutinin (LCA) | 20 | α-methyl mannoside, α-methyl glucoside | 200 each |
2. N-acetylgalactosamine group | |||
Griffonia simplicifolia lectin I (GSL-1) | 10 | Galactose | 400 |
Soybean agglutinin (SBA) | 10 | N-acetyl galactosamine | 200 |
Dolichohs Biflorus agglutinin (DBA) | 10 | N-acetyl galactosamine | 200 |
Ricinus communis agglutinin (RCA-120) | 10 | Galactose | 200 |
Sophora japonica agglutinin (SJA) | 20 | No inhibitor used | |
Vicia villosa agglutinin (VVA) | 20 | N-acetyl galactosamine | 200 |
3. N-acetylglucosamine group | |||
Lycopersicon esculentum (tomato) lectin (LEL) | 20 | Chitin hydrosylate | 200 |
Solanum tuberosum(potato) lectin (STL) | 20 | Chitin hydrosylate | 200 |
Wheat germ agglutinin (WGA) | 10 | Chitin hydrosylate | 200 |
Datura stramonium lectin (DSL) | 20 | Chitin hydrosylate | 200 |
Griffornia simplicifolia lectin II (GSL-2) | 20 | No inhibitor used | |
Succinylated WGA (SWGA) | 20 | Chitin hydrosylate | 200 |
4. Galactose group | |||
Peanut agglutinin (PNA) | 20 | Galactose | 200 |
Erythrina cristagalli lectin (ECL) | 20 | Lactose | 200 |
Jacalin (JACALIN) | 20 | Galactose | 400 |
5. Fucose group | |||
Ulex europaeus agglutinin I (UEA-1) | 10 | No inhibitor used | |
6. Oligosaccharide group | |||
Phaseolus vulgaris erythroagglutinin (PHA-E) | 20 | No inhibitor used | |
Phaseolus vulgaris Leucoagglutinin (PHA-L) | 20 | No inhibitor used | |
7. Sialic acid group | |||
Sambucus nigra lectin (SNA) | 10 | No inhibitor used | |
Maackia amurensis lectin II (MAL-II) | 10 | No inhibitor used |
PRR | Primary Antibody | Isotype Control | Secondary Antibody |
---|---|---|---|
TLR2 | Mouse anti-human/mouse (ebiosciences) | Mouse IgG1 (ebiosciences) | Goat anti-mouse IgG Alexa Fluor 488 (Invitrogen) |
TLR3 | Mouse anti-human (ebiosciences) | ||
TLR4 | Mouse anti-human (ebiosciences | Mouse IgG2a (ebiosciences) | |
TLR5 | Goat anti-human (Santa Cruz biotechnology) | Goat IgG (Santa Cruz biotechnology) | Chicken anti-goat IgG Alexa Fluor 488 (Invitrogen) |
TLR6 | Rat anti-human (ebiosciences) | Rat IgG2a, kappa (ebiosciences) | Mouse anti-rat IgG FITC (ebiosciences) |
TLR7 | Goat anti-human (Santa Cruz biotechnology) | Goat IgG (Santa Cruz biotechnology) | Chicken anti-goat IgG Alexa Fluor 488 (Invitrogen) |
TLR8 | Rabbit anti-human (Santa Cruz biotechnology) | Rabbit IgG (Santa Cruz biotechnology) | Goat anti-rabbit IgG Alexa Fluor 488 (Invitrogen) |
TLR9 | Rat anti-human (ebiosciences) | Rat IgG2a, kappa (ebiosciences) | Mouse anti-rat IgG FITC (ebiosciences) |
MDA5, RIG-I | Goat anti-human (Santa Cruz biotechnology) | Goat IgG (Santa Cruz biotechnology) | Chicken anti-goat IgG Alexa Fluor 488 (Invitrogen) |
Gene | Primer Sequences | Annealing Temp (°C) | Accession Number |
---|---|---|---|
TLR-7 | 5′ACA ATG ATA TCG CCA CCT CCA CCA3′ | 55 | NM_001097434 |
3′TGG CCA AGG AGA GAG TCT TCA GAT5′ | |||
RIG-I | 5′TAT CCG AGC AGC AGG CTT TGA3′ | 58 | NM_213804 |
3′TGA AGT TTA GGG TTC TCG TTG CTG GGA5′ | |||
MDA5 | 5′TGC CCT TTC CCA GTG GAT TAC TGA3′ | 58 | NM_001100194 |
3′TGT GTC CAG CTC CAA TCA GAT GGT5′ | |||
RPL4 | 5′CAA GAG TAA CTA CAA CCT TC3′ | 57 | XM_003121741 |
3′GAA CTC TAC GAT GAA TCT TC5′ |
TLRs | MK1-OSU | SD-PJEC | ||||
---|---|---|---|---|---|---|
Uninfected | MN08 | IA07 | Uninfected | MN08 | IA07 | |
TLR-2 | 65.1 ± 3.5 | 64.5 ± 12.5 | 73.9 ± 13.7 | 56.5 ± 14.0 | 52.1 ± 11.1 | 57.2 ± 14.1 |
TLR-4 | 7.3 ± 1.5 | 6.6 ± 0.7 | 3.9 ± 3.4 | 17.2 ± 6.0 | 22.3 ± 8.7 | 21.5 ± 12.5 |
TLR-5 | 23.6 ± 10.2 | 28.7 ± 12.4 | 26.9 ± 12.5 | 25.8 ± 20.2 | 21.8 ± 11.3 | 27.5 ± 26.1 |
TLR-6 | 26.7 ± 21.3 | 22.3 ± 12.4 | 26.4 ± 19.0 | 66.8 ± 7.2 | 66.8 ± 10.8 | 67.4 ± 10.9 |
TLR-8 | 17.2 ± 2.8 | 10.1 ± 2.2 | 11.5 ± 4.9 | 4.9 ± 3.5 | 8.2 ± 2.3 | 16.5 ± 4.1 |
TLR-9 | 32.7 ± 13.3 | 46.1 ± 16.6 | 35.5 ± 21.1 | 33.8 ± 22.4 | 31.8 ± 22.9 | 37.4 ± 16.1 |
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Thomas, M.; Pierson, M.; Uprety, T.; Zhu, L.; Ran, Z.; Sreenivasan, C.C.; Wang, D.; Hause, B.; Francis, D.H.; Li, F.; et al. 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, 312. https://doi.org/10.3390/v10060312
Thomas M, Pierson M, Uprety T, Zhu L, Ran Z, Sreenivasan CC, Wang D, Hause B, Francis DH, Li F, et al. 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):312. https://doi.org/10.3390/v10060312
Chicago/Turabian StyleThomas, Milton, Max Pierson, Tirth Uprety, Laihua Zhu, Zhiguang Ran, Chithra C. Sreenivasan, Dan Wang, Ben Hause, David H. Francis, Feng Li, and et al. 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, no. 6: 312. https://doi.org/10.3390/v10060312
APA StyleThomas, M., Pierson, M., Uprety, T., Zhu, L., Ran, Z., Sreenivasan, C. C., Wang, D., Hause, B., Francis, D. H., Li, F., & 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), 312. https://doi.org/10.3390/v10060312