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Characterization of the blastogenic response to LPS of bovine peripheral blood mononuclear cells
Massimo Amadori1, Joel Fernando Soares-Filipe2, Federica Riva2
1Laboratory of Cellular Immunology, Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia-Romagna (IZSLER), Brescia, Italy.
Insights
Bovine immune cells (PBMC) show limited replication to lipopolysaccharides (LPS), a mitogen. Differences in B and T cell responses to LPS may influence disease resistance in dairy cows.
Area of Science:
- Immunology
- Veterinary Science
Background:
- Mitogens like lipopolysaccharides (LPS) are used to assess animal immunocompetence.
- Previous studies suggest a link between LPS blastogenic response and disease resistance in periparturient dairy cows, with low responders experiencing more post-calving diseases.
Purpose of the Study:
- To investigate the blastogenic response of bovine Peripheral Blood Mononuclear Cells (PBMC) to LPS in healthy Friesian cows.
- To explore the cellular and molecular mechanisms underlying LPS-induced PBMC proliferation and its potential correlation with disease resistance.
Main Methods:
- Utilized a 72-hour Bromodeoxyuridin (BrDU) cell proliferation assay to measure bovine PBMC replication after LPS stimulation.
- Employed cell cycle and cell growth flow cytometry analyses to confirm proliferation.
- Performed magnetic separation to identify responding cell populations (B and CD4 T cells).
- Analyzed gene expression of TNF-α, TLR4, and Indoleamine-pyrrole 2,3-dioxygenase (IDO1).
Main Results:
- LPS stimulation resulted in minimal bovine PBMC replication over 72 hours, with low percentages of cells in S phase.
- Both B cells and CD4 T cells were identified as responders to LPS, differing from human and murine models.
- LPS induced early upregulation of TNF-α and TLR4 genes, with correlations between inflammatory cytokine and IDO1 gene expression.
Conclusions:
- Differences in bovine PBMC blastogenic response to LPS may stem from the heterogeneity of responding B and T lymphocytes.
- This cellular heterogeneity could impact the induction and regulation of inflammatory responses and endotoxin tolerance in cattle.
- Findings suggest potential implications for understanding and improving disease resistance in dairy cows.
Abstract:
Mitogens are diverse compounds of plant and microbial origin, widely employed to test immunocompetence in animals. The blastogenic response of bovine Peripheral Blood Mononuclear Cells (PBMC) to lypopolysaccharides (LPS) has been investigated in our laboratories for a long time. In particular, a possible correlation between blastogenic response to LPS and disease resistance of periparturient dairy cows had been observed in previous studies. Most important, low responder cows presented a higher frequency of disease cases after calving, compared with high responder animals. Owing to the above, different aspects of the blastogenic response to LPS were investigated on PBMC of healthy Friesian cows, using a 72-hour Bromodeoxyuridin (BrDU) cell proliferation assay. Stimulation with LPS induced little if any replication of bovine PBMC over 72 hours despite consistent BrDU detection in all the PBMC samples under study. Poor replication of LPS-stimulated PBMC was confirmed by cell cycle and cell growth flow cytometry analyses. In particular, LPS stimulation gave rise to very low percentages of S phase cells, sometimes lower than in control, unstimulated cells, as opposed to Concanavalin A-stimulated PBMC. Magnetic separation and analysis of BrDU-treated bovine PBMC after exposure to LPS showed that both B and CD4 T cells are involved in the blastogenic response to LPS, in contrast with current data based on human and murine models. Finally, LPS caused an early, specific up-regulation of TNF-α and TLR4 genes in bovine PBMC, and significant correlations were shown between the expression of inflammatory cytokine and Indoleamine-pyrrole 2,3-dioxygenase (IDO1) genes. On the whole, our data indicate that differences in the blastogenic response to LPS could be partly accounted for by heterogenicity of responding cells (B and T lymphocytes), which might also have an impact on induction and regulation of inflammatory responses and endotoxin tolerance.
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