Lactobacilli-based postbiotic differentially affects chicken macrophage-like HD11 cells depending on stimulatory lipopolysaccharide dosage

Abstract

Background: This study investigated the dose-dependent effects of a lactobacilli-based postbiotic (Post) on the transcriptional reprogramming of the chicken macrophage-like HD11 cell line when exposed to Escherichia coli lipopolysaccharide (LPS). First, the HD11cells were treated with 0, 3, 30 and 300 ng/mL LPS in combination with 0, 0.2, 0.4, 0.6 and 0.8% v/v Post. Nitric oxide (NO) production was quantified at 20 h incubation and the early transcriptome reprogramming was analysed in a subset of treatments at 5 h incubation.

Results: Post increased NO production dose-dependently and an LPS-postbiotic interaction was present, with the cells eliciting a higher NO production in response to Post at 30 and 300 ng/ml LPS compared to the zero and 3 ng/ml LPS. To further understand this interaction, the early transcriptome reprogramming was investigated for treatments with 0, 3 and 300 ng/mL LPS and 0 and 0.8% v/v Post. A number of differentially expressed genes were identified and gene set enrichment analysis of KEGG pathways revealed that Post at 0 and 300 ng/mL LPS influenced similar inflammation-related pathways, until Post at 3 ng/mL LPS which had a minimal effect. Expression of transcription factors (TFs) and immune-related genes revealed differential effects induced by Post depending on LPS concentration which would have likely influenced the inflammatory response. Specifically, the only TFs affected by Post at 300 ng/ml LPS were STAT2, SMAD3 and IFR8, which all showed increased expression. The TFs consistently affected by Post at the zero and 3 ng/ml LPS increased and were JUN, ZFP36L2, SMAD1 and E2F3.

Conclusion: Our results showed that Post had a pro-inflammatory effect, which was exacerbated in the presence of a 300 but not 3 ng/ml LPS. Furthermore, the dose of LPS affected the sensitivity of the cells to Post. Dose-response studies should be performed when investigating the effects of dietary compounds on inflammation in chicken macrophages.

Keywords: Inflammation; Lactobacillus; Lipopolysaccharide; Macrophage; Postbiotic.

Fig. 1

Effect of lactobacilli postbiotic on Escherichia coli lipopolysaccharide (LPS)-stimulated avian HD11 macrophage-like cells. Effect on nitric oxide (NO) production (A), and inducible nitric oxide synthase (iNOS) and interleukin 12b (IL-12b) mRNA expression (B and C, respectively). Cells were treated with medium supplemented with LPS and postbiotic. The effect of the postbiotic were analysed per LPS dose. Nitric oxide in the supernatant was quantified at 20 h incubation and values are expressed as a percentage of the 300 ng/mL LPS condition. Genes expressions were measured at 5 h incubation, are expressed relative to the negative control and are represented on a log scale. Data are represented as means ± standard error of the mean (SEM) of 5 independent experiments for NO and of 4 for iNOS and IL-12b. Means with different superscript within LPS dose are significantly different (p < 0.05)

Fig. 2

Effect of lactobacilli postbiotic on Escherichia coli lipopolysaccharide (LPS)-stimulated avian HD11 macrophage-like cells gene expression. (A) MDS-plot with one colour per replicate. (B) Volcano plot of the differential mRNA expression for Post effect at 0, 3 and 300 ng/mL LPS. Significant threshold was set at a false discovery rate < 0.05 (horizontal dashed line). Red, black and green dots represent upregulated, downregulated, and not significantly changed mRNA expressions, respectively

Fig. 3

Effect of lactobacilli postbiotic on Escherichia coli lipopolysaccharide (LPS)-stimulated avian HD11 macrophage-like cells gene expression. (A) Upset plot showing among the differentially expressed genes for the effect of Post at 0, 3 and 300 ng/mL LPS, the number of genes with shared and different upregulated (up), downregulated (down), and not significantly different (ns) expression. (B) Correlation between log2(Fold change) for Post effect at 0, 3 and 300 ng/mL. Fold changes of non-significantly differentially expressed genes (FDR > 0.05) were set to 0 in the representation

Fig. 4

Heatmap of the mRNA gene expression profile. Escherichia coli lipopolysaccharide (LPS)-stimulated avian HD11 macrophage-like cells were treated with a lactobacilli postbiotic (Post). Cells were treated with 0, 3 or 300 ng/mL LPS and the effect of the postbiotic were analysed per LPS dose. Expression is normalised within each row. Only the 4915 genes with a false discovery rate < 0.05 for Post effect in at least one LPS concentration are represented. Per LPS dose, ns stands for non-significant effect of the postbiotic, up for upregulation and down for downregulation of the gene expression by the postbiotic. CPM: count per million

Fig. 5

Gene set enrichment analysis of KEGG pathways. Escherichia coli lipopolysaccharide (LPS)-stimulated avian HD11 macrophage-like cells were treated with a lactobacilli postbiotic (Post). Cells were treated with 0, 3 or 300 ng/mL LPS and the effect of the postbiotic were analysed per LPS dose. Only pathways with an adjusted-p < 0.1 for Post effect are presented

Fig. 6

mRNA expression of immune related transcription factors (TFs). Escherichia coli lipopolysaccharide (LPS)-stimulated avian HD11 macrophage-like cells were treated with a lactobacilli postbiotic (Post). Cells were treated with 0, 3 or 300 ng/mL LPS and the effect of the postbiotic were analysed per LPS dose. Represented TFs have a false discovery rate < 0.05 in at least one comparison. TFs were separated in seven categories depending on Post effect at the different LPS doses; A: affected at all LPS doses, B: affected at 0 ng/mL LPS only, C: affected at 3 ng/mL LPS only, D: affected at 300 ng/mL LPS only; E: affected at 0 and 3 ng/mL LPS; F: affected at 3 and 300 ng/mL LPS and G: affected at 0 and 300 ng/mL LPS

Fig. 7

mRNA expression of immune related genes. Escherichia coli lipopolysaccharide (LPS)-stimulated avian HD11 macrophage-like cells were treated with a lactobacilli postbiotic (Post). Cells were treated with 0, 3 or 300 ng/mL LPS and the effect of Post were analysed per LPS dose. Represented genes have false discovery rate < 0.05 and a|log2(fold change)| > 1 in at least one comparison. Gene were separated in seven categories depending on Post effect at the different LPS doses; A: affected at all LPS doses, B: affected at 0 ng/mL LPS only, C: affected at 3 ng/mL LPS only, D: affected at 300 ng/mL LPS only; E: affected at 0 and 3 ng/mL LPS; F: affected at 3 and 300 ng/mL LPS and G: affected at 0 and 300 ng/mL LPS. Group GO/KEGG: 0: GO:0002376 immune system process; 1: KEGG immune system 04620, 04621, 04622, 04623, 04625 and 04672; 2: KEGG membrane transport 02010; 3: KEGG signal transduction 04010, 04020, 04060, 04350 and 04370; 4: KEGG infectious disease 05132, 05164 and 05168; 5: KEGG cellular processes 04115, 04218, 04110, 04142, 04145 and 04146