The Expression Dynamics of Key Immune-Related Genes in Response to Mannheimia Haemolytica in Sheep Alveolar Macrophages In Vitro

Abstract

Alveolar macrophages (AMs) respond to these infections as the first line of defense and trigger the lung's immune response. Knowing the expression dynamics of key immune-related genes in alveolar macrophages against M. haemolytica challenge will help deepen the understanding of disease immunopathogenesis. For this purpose, first time an in vitro obtained from bronchoalveolar lavage (BAL) fluid and treated with three doses (T1: 1800, T2: 2700, T3: 5400 CFU) of M. haemolytica inoculum, while keeping one untreated as a control. Then, total RNA was extracted, and cDNA was synthesized. The real-time quantification data indicated that the expression of IL-1β, IL-6, IL-8, IL-10, and NF-κB genes were significantly changed as compared to control. Our study revealed that the exposure to M. haemolytica stimulates the immune response in the sheep alveolar macrophages in a dose and time-dependent manner.

Keywords

• Alveolar Macrophages
• Immune related Genes
• Gene Expression
• M. haemolytica
• Pneumonia

Supporting Institution

Erciyes University Scientific Research Projects Unit

Project Number

FDK-2017-7657

Thanks

The author thanks to Prof. Dr. Kadir Semih Gümüşsoy, Department of Microbiology, Faculty of Veterinary Medicine at Erciyes University, Kayseri, Turkiye and Genome and Stem Cell Research Center (GENKÖK), Kayseri, Turkiye for the cooperation and help throughout this work.

Koyun Alveoler Makrofajlarında Mannheimia Haemolytica'ya Yanıt Olarak Bağışıklık ile İlişkili Genlerin İn Vitro mRNA Ekspresyon Dinamikleri

Abstract

Alveoler makrofajlar (AM) enfeksiyonlara ilk savunma hattı olarak yanıt verir ve akciğerin bağışıklık tepkisini tetikler. Alveoler makrofajlarda Mannheimia haemolytica mücadelesine karşı anahtar bağışıklık ile ilişkili genlerin ekspresyon dinamiklerini bilmek, hastalık immünopatogenezinin anlaşılmasını derinleştirmeye yardımcı olacaktır. Bu amaçla, ilk kez interlökin-1β, IL-6, IL-8, IL-10 ve NF-κB'nin ekspresyon desenini doz ve zamana bağlı bir şekilde araştırmak için in vitro deneysel bir enfeksiyon modeli kullanıldı. Bronkoalveoler lavajdan (BAL) sıvıdan toplam %95 saf alveoler makrofaj kültür plakaları elde edildi ve kontrol grubu ile M. haemolytica inokulumun üç dozu (T1: 1800, T2: 2700, T3: 5400 CFU) analiz edildi. Daha sonra toplam RNA ekstrakte edilerek ve cDNA sentezlendi. Gerçek zamanlı PCR verileri, IL-1β, IL-6, IL-8, IL-10 ve NF-κB genlerinin ekspresyonunun, kontrole kıyasla önemli ölçüde değiştiğini gösterdi. Çalışmamız, M. haemolytica'ya maruz kalmanın, koyun alveoler makrofajlarındaki bağışıklık tepkisini dozda ve zamana bağlı bir şekilde uyardığını ortaya koymuştur.

Keywords

• Alveolar makrofaj
• Bağışıklıkla ilişkili genler
• Gen ifadesi
• M. haemolytica
• pnömoni

Project Number

FDK-2017-7657

References

1. Ackermann, M. R., and Brogden, K. A. 2000. Response of the ruminant respiratory tract to Mannheimia (Pasteurella) haemolytica. Microbes and Infection, 2(9), 1079-1088.
2. Aksel, E. G., and Akyüz, B. 2021 Effect of LPS and LTA stimulation on the expression of TLR-pathway genes in PBMCs of Akkaraman lambs in vivo. Trop Anim Health Prod, 53(1), 65.
3. Aulik Nicole, A., Hellenbrand Katrina, M., and Czuprynski Charles, J. 2012. Mannheimia haemolytica and Its Leukotoxin Cause Macrophage Extracellular Trap Formation by Bovine Macrophages. Infection and Immunity, 80(5), 1923-1933.
4. Ayalew, S., Confer Anthony, W., and Blackwood Emily, R. 2004. Characterization of Immunodominant and Potentially Protective Epitopes of Mannheimia haemolytica Serotype 1 Outer Membrane Lipoprotein PlpE. Infection and Immunity, 72(12), 7265-7274.
5. Ayalew, S., Confer, A. W., Hartson, S. D., and Shrestha, B. 2010. Immunoproteomic analyses of outer membrane proteins of Mannheimia haemolytica and identification of potential vaccine candidates. Proteomics, 10(11), 2151-2164.
6. Ayalew, S., Shrestha, B., Montelongo, M., Wilson, A. E., and Confer, A. W. 2011. Identification and immunogenicity of Mannheimia haemolytica S1 outer membrane lipoprotein PlpF. Vaccine, 29(47), 8712-8718.
7. Bannerman, D. D., Tupper, J. C., Kelly, J. D., Winn, R. K., and Harlan, J. M. 2002. The Fas-associated death domain protein suppresses activation of NF-kappa B by LPS and IL-1 beta. J Clin Invest, 109(3), 419-425.
8. Blackwell, T. S., Hipps, A. N., Yamamoto, Y., Han, W., Barham, W. J., Ostrowski, M. C., . . . Prince, L. S. 2011. NF-κB signaling in fetal lung macrophages disrupts airway morphogenesis. J Immunol, 187(5), 2740-2747.

9. Bowen, L., Manlove, K., Roug, A., Waters, S., LaHue, N., and Wolff, P. 2022. Using transcriptomics to predict and visualize disease status in bighorn sheep (Ovis canadensis). Conservation Physiology, 10(1).
10. Chaudhry, H., Zhou, J., Zhong, Y., Ali, M. M., McGuire, F., Nagarkatti, P. S., and Nagarkatti, M. 2013. Role of cytokines as a double-edged sword in sepsis. In Vivo, 27(6), 669-684.
11. García-Alvarez, A., Fernández-Garayzábal, J. F., Chaves, F., Pinto, C., and Cid, D. 2018 Ovine Mannheimia haemolytica isolates from lungs with and without pneumonic lesions belong to similar genotypes. Veterinary Microbiology, 219, 80-86.
12. Guth, A. M., Janssen, W. J., Bosio, C. M., Crouch, E. C., Henson, P. M., and Dow, S. W. 2009. Lung environment determines unique phenotype of alveolar macrophages. American Journal of Physiology-Lung Cellular and Molecular Physiology, 296(6), L936-L946.
13. Klima, C. L., Zaheer, R., Briggs, R. E., and McAllister, T. A. 2017. A multiplex PCR assay for molecular capsular serotyping of Mannheimia haemolytica serotypes 1, 2, and 6. Journal of Microbiological Methods, 139, 155-160.
14. Lawrence, P. K., Shanthalingam, S., Dassanayake, R. P., Subramaniam, R., Herndon, C. N., Knowles, D. P., . . . Srikumaran, S. 2010. Transmission Of Mannheimia Haemolytica From Domestic Sheep (Ovis Aries) To Bighorn Sheep (Ovis Canadensis): Unequivocal Demonstration With Green Fluorescent Protein-Tagged Organisms Journal of Wildlife Diseases, 46(3), 706-717.
15. Liu, Q., Zhou, Y.-h., and Yang, Z.-q. 2016. The cytokine storm of severe influenza and development of immunomodulatory therapy. Cellular & Molecular Immunology, 13(1), 3-10.
16. Martinez, F. O., and Gordon, S. 2014. The M1 and M2 paradigm of macrophage activation: time for reassessment. F1000Prime Rep, 6, 13.
17. McClenahan, D., Hellenbrand, K., Atapattu, D., Aulik, N., Carlton, D., Kapur, A., and Czuprynski, C. 2008. Effects of Lipopolysaccharide and Mannheimia haemolytica Leukotoxin on Bovine Lung Microvascular Endothelial Cells and Alveolar Epithelial Cells. Clinical and Vaccine Immunology, 15(2), 338-347.
18. Naugler, W. E., and Karin, M. 2008. The wolf in sheep's clothing: the role of interleukin-6 in immunity, inflammation and cancer. Trends in Molecular Medicine, 14(3), 109-119.
19. N’jai, A. U., Rivera, J., Atapattu, D. N., Owusu-Ofori, K., and Czuprynski, C. J. 2013. Gene expression profiling of bovine bronchial epithelial cells exposed in vitro to bovine herpesvirus 1 and Mannheimia haemolytica. Veterinary Immunology and Immunopathology, 155(3), 182-189.
20. Oppermann, T., Busse, N., and Czermak, P. 2017. Mannheimia haemolytica growth and leukotoxin production for vaccine manufacturing — A bioprocess review. Electronic Journal of Biotechnology, 28, 95-100.
21. Ramírez Rico, G., Martínez-Castillo, M., González-Ruíz, C., Luna-Castro, S., and de la Garza, M. 2017. Mannheimia haemolytica A2 secretes different proteases into the culture medium and in outer membrane vesicles. Microbial Pathogenesis, 113, 276-281.
22. Singh, K., Confer, A. W., Hope, J. C., Rizzi, T., Wyckoff, J. H., Weng, H.-Y., and Ritchey, J. W. 2011. Cytotoxicity and cytokine production by bovine alveolar macrophages challenged with wild type and leukotoxin-deficient Mannheimia haemolytica. The Veterinary Journal, 188(2), 221-227.
23. Singh, K., Confer, A. W., Step, D. L., Rizzi, T., Wyckoff, J. H., Weng, H.-Y., and Ritchey, J. W. 2012. Cytokine expression by pulmonary leukocytes from calves challenged with wild-type and leukotoxin-deficient Mannheimia haemolytica. The Veterinary Journal, 192(1), 112-119.