Baseline T-lymphocyte and Cytokine Indices in Sheep Peripheral Blood

Overview

Journal BMC Vet Res

Publisher Biomed Central

Specialty Veterinary Medicine

Date 2022 May 5

PMID 35513847

Authors

Jihui Yang

Yongxue Lv

Yazhou Zhu

Shasha Li

Jia Tao

Liangliang Chang

Mingxing Zhu

Jiaqing Zhao

Yana Wang

Changyou Wu

Wei Zhao

Affiliations

Soon will be listed here.

Abstract

Background: Sheep are an important livestock species worldwide and an essential large-animal model for animal husbandry and veterinary research. Understanding fundamental immune indicators, especially T-lymphocyte parameters, is necessary for research on sheep diseases and vaccines, to better understand the immune response to bacteria and viruses for reducing the use of antibiotics and improving the welfare of sheep. We randomly selected 36 sheep of similar ages to analyze cell-related immune indicators in peripheral blood mononuclear cells (PBMCs). The proportions of CD4 and CD8 T cells in PBMCs were detected by flow cytometry. We used Concanavalin A (Con A) and Phorbol-12-myristate-13-acetate (PMA)/Ionomycin to stimulate PBMCs, and measured the expression of IFN-γ, IL-4, and IL-17A using enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot assay (ELISpot). Simultaneously, PMA/Ionomycin/brefeldin A (BFA) was added to PBMCs, then the expression of IFN-γ, IL-4, and IL-17A was detected by flow cytometry after 4 h of culturing. In addition, we observed the proliferation of PBMCs stimulated with Con A for 3, 4, and 5 days.

Results: The proportions of CD4 T lymphocytes (18.70 ± 4.21%) and CD8 T lymphocytes (8.70 ± 3.65%) were generally consistent among individuals, with a CD4/CD8 ratio of 2.40 ± 0.79. PBMCs produced high levels of IFN-γ, IL-4, and IL-17A after stimulation with PMA/Ionomycin and Con A. Furthermore, PMA/Ionomycin stimulation of PBMC yielded significantly higher cytokine levels than Con A stimulation. Flow cytometry showed that the level of IFN-γ (51.49 ± 11.54%) in CD8 T lymphocytes was significantly (p < 0.001) higher than that in CD4 T lymphocytes (14.29 ± 3.26%); IL-4 (16.13 ± 6.81%) in CD4 T lymphocytes was significantly (p < 0.001) higher than that in CD8 T lymphocytes (1.84 ± 1.33%), There was no difference in IL-17A between CD4 (2.83 ± 0.98%) and CD8 T lymphocytes (1.34 ± 0.67%). The proliferation of total lymphocytes, CD4 T lymphocytes, and CD8 T lymphocytes continued to increase between days 3 and 5; however, there were no significant differences in proliferation between the cell types during the stimulation period.

Conclusions: Evaluating primary sheep immune indicators, especially T lymphocytes, is significant for studying cellular immunity. This study provided valuable data and theoretical support for assessing the immune response of sheep to pathogens and improving sheep welfare.

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References

Haig D, McInnes C . Immunity and counter-immunity during infection with the parapoxvirus orf virus. Virus Res. 2002; 88(1-2):3-16. DOI: 10.1016/s0168-1702(02)00117-x. View

von Stebut E, Boehncke W, Ghoreschi K, Gori T, Kaya Z, Thaci D . IL-17A in Psoriasis and Beyond: Cardiovascular and Metabolic Implications. Front Immunol. 2020; 10:3096. PMC: 6974482. DOI: 10.3389/fimmu.2019.03096. View

van den Brom R, de Jong A, van Engelen E, Heuvelink A, Vellema P . Zoonotic risks of pathogens from sheep and their milk borne transmission. Small Rumin Res. 2020; 189:106123. PMC: 7227596. DOI: 10.1016/j.smallrumres.2020.106123. View

Rojas J, Rodriguez-Calvo T, Sevilla N . Recall T cell responses to bluetongue virus produce a narrowing of the T cell repertoire. Vet Res. 2017; 48(1):38. PMC: 5492282. DOI: 10.1186/s13567-017-0444-3. View

McKinnon K . Flow Cytometry: An Overview. Curr Protoc Immunol. 2018; 120:5.1.1-5.1.11. PMC: 5939936. DOI: 10.1002/cpim.40. View

Matthews S, Cantrell D . New insights into the regulation and function of serine/threonine kinases in T lymphocytes. Immunol Rev. 2009; 228(1):241-52. PMC: 3520424. DOI: 10.1111/j.1600-065X.2008.00759.x. View

Chai D, Kong X, Lu S, Zhang J . CD4+/CD8+ ratio positively correlates with coronary plaque instability in unstable angina pectoris patients but fails to predict major adverse cardiovascular events. Ther Adv Chronic Dis. 2020; 11:2040622320922020. PMC: 7238310. DOI: 10.1177/2040622320922020. View

Oh-Hora M . Calcium signaling in the development and function of T-lineage cells. Immunol Rev. 2009; 231(1):210-24. DOI: 10.1111/j.1600-065X.2009.00819.x. View

Rani R, Tandon A, Wang J, Kumar S, Gandhi C . Stellate Cells Orchestrate Concanavalin A-Induced Acute Liver Damage. Am J Pathol. 2017; 187(9):2008-2019. PMC: 5682941. DOI: 10.1016/j.ajpath.2017.05.015. View

10.

Schwarzkopf S, Krawczyk A, Knop D, Klump H, Heinold A, Heinemann F . Cellular Immunity in COVID-19 Convalescents with PCR-Confirmed Infection but with Undetectable SARS-CoV-2-Specific IgG. Emerg Infect Dis. 2020; 27(1). DOI: 10.3201/2701.203772. View

11.

Rodriguez-Martin D, Louloudes-Lazaro A, Avia M, Martin V, Rojas J, Sevilla N . The Interplay between Bluetongue Virus Infections and Adaptive Immunity. Viruses. 2021; 13(8). PMC: 8402766. DOI: 10.3390/v13081511. View

12.

Quah B, Warren H, Parish C . Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester. Nat Protoc. 2007; 2(9):2049-56. DOI: 10.1038/nprot.2007.296. View

13.

Ruterbusch M, Pruner K, Shehata L, Pepper M . In Vivo CD4 T Cell Differentiation and Function: Revisiting the Th1/Th2 Paradigm. Annu Rev Immunol. 2020; 38:705-725. DOI: 10.1146/annurev-immunol-103019-085803. View

14.

Ganter M . Zoonotic risks from small ruminants. Vet Microbiol. 2015; 181(1-2):53-65. DOI: 10.1016/j.vetmic.2015.07.015. View

15.

Melzi E, Rocchi M, Entrican G, Caporale M, Palmarini M . Immunophenotyping of Sheep Paraffin-Embedded Peripheral Lymph Nodes. Front Immunol. 2019; 9:2892. PMC: 6297804. DOI: 10.3389/fimmu.2018.02892. View

16.

Matsushita M, Freigang S, Schneider C, Conrad M, Bornkamm G, Kopf M . T cell lipid peroxidation induces ferroptosis and prevents immunity to infection. J Exp Med. 2015; 212(4):555-68. PMC: 4387287. DOI: 10.1084/jem.20140857. View

17.

McRae K, Stear M, Good B, Keane O . The host immune response to gastrointestinal nematode infection in sheep. Parasite Immunol. 2015; 37(12):605-13. PMC: 4744952. DOI: 10.1111/pim.12290. View

18.

La Manna M, Agnone A, Villari S, Puleio R, Vitale M, Nicholas R . Expansion of intracellular IFN-γ positive lymphocytes during Mycoplasma agalactiae infection in sheep. Res Vet Sci. 2011; 91(3):e64-7. DOI: 10.1016/j.rvsc.2011.01.029. View

19.

Aydin S . A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA. Peptides. 2015; 72:4-15. DOI: 10.1016/j.peptides.2015.04.012. View

20.

Baron M, Hodgson S, Moffat K, Qureshi M, Graham S, Darpel K . Depletion of CD8 T cells from vaccinated goats does not affect protection from challenge with wild-type peste des petits ruminants virus. Transbound Emerg Dis. 2020; 68(6):3320-3334. PMC: 9291567. DOI: 10.1111/tbed.13936. View