Differential cytokine mRNA expression in heterophils isolated from Salmonella-resistant and -susceptible chickens

Abstract

We recently showed that increased in vitro heterophil functional efficiency translates to increased in vivo resistance to a systemic Salmonella enteritidis (SE) infection utilizing a parental pair of broiler chickens (lines A and B) and the F1 reciprocal crosses (C and D). Heterophils produce cytokines and modulate acute protection against Salmonella in young poultry. Therefore, we hypothesize that heterophils from SE-resistant chickens (A and D) have the ability to produce an up-regulated pro-inflammatory cytokine response compared to that of heterophils from SE-susceptible chickens (B and C). In this study, heterophils were isolated from day-old chickens and treated with either RPMI-1640 (as the control), or phagocytic agonists (SE, or SE opsonized with either normal chicken serum or immune serum against SE) and cytokine mRNA expression assessed using real-time quantitative reverse transcription-polymerase chain reaction. Heterophils from SE-resistant chickens (A and D) had significantly higher levels of pro-inflammatory cytokine (interleukin (IL)-6, IL-8, and IL-18) mRNA expression upon treatment with all agonists compared to heterophils from SE-susceptible lines (B and C). Further, heterophils from SE-resistant chickens had significantly decreased mRNA expression levels of transforming growth factor-beta4, an anti-inflammatory cytokine, when compared to heterophils from SE-susceptible chickens. These data indicate cytokine gene expression in heterophils may be a useful parameter in determining resistance to Salmonella, as indicated by our previous in vivo SE studies. Therefore, heterophil functional efficiency and cytokine production may be useful biomarkers for poultry breeders to consider when developing new immunocompetent lines of birds.

Figure 1

Quantitation of IL-6 mRNA expression in heterophils isolated from day-old chickens in (a) line A (b) line B (c) line C, and (d) line D. Data are expressed as fold-change in IL-6 mRNA levels when treated samples (SE, NCS-OpSE, and IgY-OpSE) were compared to control heterophils treated with RPMI-1640. Error bars show SEM of three separate experiments including 35 chickens per experiment.

Figure 2

Quantitation of IL-8 mRNA expression in heterophils isolated from day-old chickens in (a) line A (b) line B (c) line C, and (d) line D. Data are expressed as fold-change in IL-8 mRNA levels when treated samples (SE, NCS-OpSE, and IgY-OpSE) were compared to control heterophils treated with RPMI-1640. Error bars show SEM of three separate experiments including 35 chickens per experiment.

Figure 3

Quantitation of IL-18 mRNA expression in heterophils isolated from day-old chickens in (a) line A (b) line B (c) line C, and (d) line D. Data are expressed as fold-change in IL-18 mRNA levels when treated samples (SE, NCS-OpSE, and IgY-OpSE) were compared to control heterophils treated with RPMI-1640. Error bars show SEM of three separate experiments including 35 chickens per experiment.

Figure 4

Quantitation of TGF-β4 mRNA expression in heterophils isolated from day-old chickens in (a) line A (b) line B (c) line C, and (d) line D. Data are expressed as fold-change in TGF-β4 mRNA levels when treated samples (SE, NCS-OpSE, and IgY-OpSE) were compared to control heterophils treated with RPMI-1640. Error bars show SEM of three separate experiments including 35 chickens per experiment.