Characterization of the blastogenic response to LPS of bovine peripheral blood mononuclear cells.

Massimo Amadori,Jessica Ruggeri,Joel Fernando Soares-Filipe,Federica Riva,Nicola Lacetera,Andrea Vitali,Juan J. Loor

doi:10.1371/journal.pone.0204827

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.

Highlights

Mitogens are diverse compounds of plant and microbial origin, widely employed to test immunocompetence in animals
Liquid scintillation counting after 3H-thymidine incorporation has been the reference assay over many years, but the stepwise reduction of radioisotope usage has prompted the development and refinement of alternative assays like ELISAs for Bromodeoxyuridine (BrDU), flow-cytometry-based procedures based on Carboxyfluorescein succinimidyl ester (CFSE), DNA-intercalating fluorochromes like propidium iodide, Ki-67 nuclear antigen, as well as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based and cell counting procedures
The above hypotheses should be checked in future studies, in which the proliferative response to LPS of Peripheral Blood Mononuclear Cells (PBMC) from lactating dairy cows at different DIM should be investigated in association with the actual levels of RP105 expression, inflammatory cytokine gene expression, inflammometabolic parameters in vivo [16], and the in vitro inflammatory response to products of metabolic stress like Non-Esterified Fatty Acids (NEFA) and β-hydroxybutyrate (BHB)

Summary

Introduction

Mitogens are diverse compounds of plant and microbial origin, widely employed to test immunocompetence in animals. Non-immunocompromised hosts, they induce DNA synthesis and division of large leucocyte populations, which can be reasonably associated with immunologic competence of T or B cells. Mitogens are usually employed in diverse lymphocyte proliferation tests. Mitogens are frequently classified in terms of mitogen-reactive leukocyte population. On this basis, mitogens are classified as T cell specific, B cell specific or polyspecific. T cell mitogens, alone or in combination, include Phorbol 12-myristate 13-acetate (PMA), ionomycin, A23187, Phytohemagglutinin (PHA), Concanavalin A (Con A), anti-CD3 Ab, anti-TcR αβ Ab, anti-TcR γδ Ab, Staphylococcal toxins A, B and E. Instead, Pokeweed Mitogen (PWM) can induce proliferation of both T and B cells [1]

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