Dectin-1-Mediated Production of Pro-Inflammatory Cytokines Induced by Yeast β-Glucans in Bovine Monocytes

Abstract

Yeast-derived products containing β-glucans have long been used as feed supplements in domesticated animals in an attempt to increase immunity. β-glucans are mainly recognized by the cell surface receptor CLEC7A, also designated Dectin-1. Although the immune mechanisms elicited through Dectin-1 activation have been studied in detail in mice and humans, they are poorly understood in other species. Here, we evaluated the response of bovine monocytes to soluble and particulate purified β-glucans, and also to Zymosan. Our results show that particulate, but not soluble β-glucans, can upregulate the surface expression of costimulatory molecules CD80 and CD86 on bovine monocytes. In addition, stimulated cells increased production of IL-8 and of TNF, IL1B, and IL6 mRNA expression, in a dose-dependent manner, which correlated positively with CLEC7A gene expression. Production of IL-8 and TNF expression decreased significantly after CLEC7A knockdown using two different pairs of siRNAs. Overall, we demonstrated here that bovine monocytes respond to particulate β-glucans, through Dectin-1, by increasing the expression of pro-inflammatory cytokines. Our data support further studies in cattle on the induction of trained immunity using dietary β-glucans.

Keywords: CLEC7A; bovine; cytokines; dectin-1; monocytes; siRNA; β-glucans.

Figure 1

Cytokine production evaluated by ELISA in the supernatants of bovine monocytes cultured for 24 h with WGP Soluble (WGP-S), WGP Dispersible (WGP-D), Zymosan (Zym), LPS, and Pam3csk4 (P3C). Data are presented as Log fold change relative to medium (M) and represent means of 12 animals for IL-8 (A), 11 animals for IL-6 (B), 7 animals for TNF-α (C), and 5 animals for IL-1β (D) and IL-10 (E). Each symbol corresponds to a different animal. ^a,b,c,d Means with different superscript letters are significantly different (P<0.05).

Figure 2

Cytokine relative mRNA expression, evaluated by RT-PCR and normalized to the mRNA expression of the reference gene MARVELD1, in bovine monocytes cultured for 24 h with WGP Soluble (WGP-S), WGP Dispersible (WGP-D), Zymosan (Zym), LPS, and Pam3csk4 (P3C). Data are presented as Log fold change relative to medium (M) and represent means of ten animals for TNF (A), IL6 (B), and IL10 (D), and eight animals for IL1B (C). Each symbol corresponds to a different animal. ^a,b,c,d,e,f Means with different superscript letters are significantly different (P<0.05).

Figure 3

Correlations between CLEC7A mRNA expression and (A–C) IL-8 cytokine production, (D–F) TNF, (G–I) IL6, (J–L) IL1B, and (M–O) IL10 mRNA expression upon stimulation with 10, 50 and 100 µg/mL of WGP Dispersible, as indicated. Results are presented as Log fold changes of each cytokine relative to medium vs Log CLEC7A mRNA. Data represent simple linear regressions, with Pearson correlation coefficients (r) and P values.

Figure 4

Expression of (A, B) CD80, (C, D) CD86, and (E, F) MHC class II molecule expression on the cell surface of bovine monocytes stimulated with WGP Soluble (WGP-S), WGP Dispersible (WGP-D), Zymosan (Zym), LPS, and Pam3csk4 (P3C) for 8 h (A, C, E) or 16 h (B, D, F), as evaluated by flow cytometry. Results correspond to means of the mean fluorescence intensities for each analyzed molecule of three independent biological samples (each represented by squares, triangles or circles). ^a,b,c Means with different superscript letters are significantly different (P<0.05).

Figure 5

Bovine CLEC7A knockdown efficiency (A), calculated relative to MISSION^® siRNA Universal Negative Control #1 (UNC) treated cells. Cells were cultured with RPMI-1640 after transfection procedure with three different siRNA duplexes (#1, #2 and #3). IL-8 production (B, C) and TNF expression (D, E) of cells transfected with duplexes #1, #2 and #3 and MISSION^® siRNA Universal Negative Control #1 (UNC) and stimulated with WGP-Dispersible (B, D) or Zymosan (C, E) at 50 µg/mL, calculated in percentual change relative to UNC transfected cells. Results correspond to means from three different animals (each one represented by squares, triangles or circles). ^a,b Means with different superscript letters are significantly different (P<0.05).