Regulation of inflammatory mediator expression in bovine endometrial cells: effects of lipopolysaccharide, interleukin 1 beta, and tumor necrosis factor alpha

Abstract

An abnormal uterine environment can influence maternal-fetal communication, conception rate and disrupt normal embryo development, thereby affecting fertility and the reproductive performance of dairy cows. Animal variability means that development of endometrial cell lines with appropriate characteristic are required. We evaluated the effect of an infectious agent (i.e., bacterial lipopolysaccharide; LPS) and proinflammatory mediators (i.e., Interleukin 1 beta; IL-1β, and tumor necrosis factor alpha; TNFα) on inflammatory mediator gene expression and production by bovine endometrial epithelial (bEEL) and stromal (bCSC) cell lines. Expression of CXCL8/IL8, IL1A, IL1B, and IL6 cytokine genes was significantly upregulated in both epithelial and stromal cells when treated with LPS and IL-1β. LPS treatment of epithelial cells (compared with treatment by IL-1β and TNFα) exhibited greater CXCL8/IL8, IL1A, IL1B, and IL6 cytokine gene expression. Whereas, in stromal cells, IL-1β treatment (compared with LPS and TNFα) exhibited greater CXCL8/IL8, IL1A, IL1B, and IL6 cytokine gene expression. Interestingly, bEEL and bCSC cells treated with IL-1β increased IL1B gene expression, suggesting that IL-1β may act unusually in an autocrine-positive feedback loop. Cytokine production was stimulated by these agents in both cell types. We suggest that the characteristics of these two cell lines make them excellent tools for the study of intrauterine environment.

Keywords: Bovine; cytokines; endometrium; epithelial; stromal.

Figure 1

Chemokine gene expression upregulated by LPS, IL‐1β and/or TNFα in bovine endometrial epithelial cells (bEEL). The relative fold change in chemokine (A) CCL2, (B) CCL20, (C) CCL22, (D) CX3CL1, (E) CXCL3, and (F) GRO1 gene expression in bovine endometrial epithelial cells (bEEL) when treated for 6 h with 1 μg/mL LPS, 10 ng/mL IL‐1β, and 50 ng/mL TNFα compared with untreated bEEL cells (no treatment control). Results are presented as mean ± SD (n = 3 independent experiments), and the gene expression were normalized against the expression of the endogenous reference genes (GAPDH and TBP). Differences in *P < 0.05, **P < 0.01, and ***P < 0.001 were considered statistically significant.

Figure 2

Interleukins and TNF receptor superfamily members’ gene expression upregulated by LPS, IL‐1β and/or TNFα in bovine endometrial epithelial cells (bEEL). The relative fold change in interleukins (A) CXCL8/IL8, (B) IL1A, (C) IL1B, (D) IL6, and TNF receptor superfamily members (E) TNF, (F) TNFRSF11B, and (G) LTB gene expression in bovine endometrial epithelial cells (bEEL) when treated for 6 h with 1 μg/mL LPS, 10 ng/mL IL‐1β, and 50 ng/mL TNFα compared with untreated bEEL cells (no treatment control). Results are presented as mean ± SD (n = 3 independent experiments), and the gene expression were normalized against the expression of the endogenous reference genes (GAPDH and TBP). Differences in *P < 0.05, **P < 0.01, and ***P < 0.001 were considered statistically significant.

Figure 3

Inflammatory mediator gene expression downregulated by LPS, IL‐1β, and TNFα in bovine endometrial epithelial cells (bEEL). The gene expression of inflammatory mediator (A) IL21 (B) LTA, and (C) THPO when treated with 1 μg/mL LPS, 10 ng/mL IL‐1β, and 50 ng/mL TNFα for 6 h compared with untreated bEEL cells (no treatment control). Results are presented as mean ± SD (n = 3 independent experiments), and the gene expression were normalized against the expression of the endogenous reference genes (GAPDH and TBP). Differences in *P < 0.05, **P < 0.01, and ***P < 0.001 were considered statistically significant.

Figure 4

Chemokine gene expression upregulated by LPS, IL‐1β and/or TNFα in bovine endometrial stromal cells (bCSC). The relative fold change in chemokine (A) CCL2, (B) CCL20, (C) CCL5, (D) CX3CL1, (E) CXCL3, (F) CXCL5, and (G) GRO1 gene expression in bovine endometrial stromal cells (bCSC) when treated for 6 h with 1 μg/mL LPS, 10 ng/mL IL‐1β, and 50 ng/mL TNFα compared with untreated bCSC cells (no treatment control). Results are presented as mean ± SD (n = 3 independent experiments), and the gene expression were normalized against the expression of the endogenous reference genes (GAPDH and TBP). Differences in *P < 0.05, **P < 0.01, and ***P < 0.001 were considered statistically significant.

Figure 5

Interleukins and growth factors’ gene expression upregulated by LPS, IL‐1β and/or TNFα in bovine endometrial stromal cells (bCSC). The relative fold change in interleukins (A) CXCL8/IL8, (B) IL1A, (C) IL1B, and (D) IL6, and growth factors (E) CSF2, and (F) CSF3 gene expression in bovine endometrial stromal cells (bCSC) when treated for 6 h with 1 μg/mL LPS, 10 ng/mL IL‐1β, and 50 ng/mL TNFα compared with untreated bCSC cells (no treatment control). Results are presented as mean ± SD (n = 3 independent experiments), and the gene expression were normalized against the expression of the endogenous reference genes (GAPDH and TBP). Differences in *P < 0.05, **P < 0.01, and ***P < 0.001 were considered statistically significant.

Figure 6

Inflammatory mediator gene expression downregulated by LPS, IL‐1β and TNFα on bovine endometrial stromal cells (bCSC). The gene expression of inflammatory mediator (A) CXCL9, (B) CXCL10, (C) CXCL12, (D) IL1RN, and (E) SPP1 when treated with 1 μg/mL LPS, 10 ng/mL IL‐1β, and 50 ng/mL TNFα for 6 h compared with untreated bCSC cells (no treatment control). Results are presented as mean ± SD (n = 3 independent experiments), and the gene expression were normalized against the expression of the endogenous reference genes (GAPDH and TBP). Differences in *P < 0.05, **P < 0.01, and ***P < 0.001 were considered statistically significant.

Figure 7

The effect of LPS, IL‐1β, and TNFα on IL6 and IL10 production by bovine endometrial epithelial (bEEL) and stromal (bCSC) cells. Bovine endometrial epithelial (bEEL) and stromal (bCSC) cells were treated with 1 μg/mL LPS, 10 ng/mL IL‐1β, and 50 ng/mL TNFα for 24 h. IL6 and IL10 production was determined using ELISAs. (A) IL6 production by bEEL cells treated with LPS, IL‐1β, and TNFα. (B) IL6 production by bCSC cells treated with LPS, IL‐1β, and TNFα. (C) IL10 production by bEEL cells treated with LPS, IL‐1β, and TNFα. (D) IL10 production by bCSC cells treated with LPS, IL‐1β, and TNFα. Results are presented as mean ± SD (n = 3 independent experiments). Differences in *P < 0.05, **P < 0.01, and ***P < 0.001 were considered statistically significant.