Ureaplasma diversum and Its Membrane-Associated Lipoproteins Activate Inflammatory Genes Through the NF-κB Pathway via Toll-Like Receptor 4

Abstract

Objectives:Ureaplasma diversum is a pathogen of cows that may cause intense inflammatory responses in the reproductive tract and interfere with bovine reproduction. The aims of this study were to evaluate the immune response of bovine blastocysts and macrophages to U. diversum infection and to evaluate the invasion capacity of this microorganism in bovine blastocysts. Methods: Viable and heat-inactivated U. diversum strains ATCC 49782 and CI-GOTA and their extracted membrane lipoproteins were inoculated in macrophages in the presence or absence of signaling blockers of Toll-Like Receptor (TLR) 4, TLR2/4, and Nuclear Factor KB (NF-κB). In addition, the same viable U. diversum strains were used to infect bovine blastocysts. RNA was extracted from infected and lipoprotein-exposed macrophages and infected blastocysts and assayed by qPCR to evaluate the expression of Interleukin 1 beta (IL-1β), Tumor Necrosis Factor Alpha (TNF-α), TLR2 and TLR4 genes. U. diversum internalization in blastocysts was followed by confocal microscopy. Results: Both Ureaplasma strains and different concentrations of extracted lipoproteins induced a higher gene expression of IL-1β, TNF-α, TLR2, and TLR4 in macrophages (p < 0.05) when compared to non-infected cells. The used blockers inhibited the expression of IL-1β and TNF-α in all treatments. Moreover, U. diversum was able to internalize within blastocysts and induce a higher gene expression of IL-1b and TNF- α when compared to non-infected blastocysts (p < 0.05). Conclusion: The obtained results strongly suggest that U. diversum and its lipoproteins interact with TLR4 in a signaling pathway acting via NF-kB signaling to stimulate the inflammatory response. This is the first study to evaluate the in vitro immunological response of macrophages and bovine blastocysts against U. diversum. These results may contribute to a better understanding of the immunomodulatory activity and pathogenicity of this infectious agent.

Keywords: LAMPs; Ureaplasma diversum; blastocysts; immunogenicity; macrophages.

FIGURE 1

Gene expression in bovine macrophages infected with viable and inactive U. diversum (ATCC 49782 – 10⁶ Ureaplasma/mL and CI-GOTA – 10⁵ Ureaplasma/mL) for 6, 12, and 24 h. (A) IL-1β Expression induced by ATCC 49782. (B) IL-1β expression induced by CI-GOTA. (C) TNF-α expression induced by ATCC 49782. (D) TNF-α expression induced by CI-GOTA. (E) TLR2 expression Induced by ATCC 49782. (F) TLR2 expression in macrophages inoculated with the CI-GOTA. (G) TLR4 expression induced by ATCC 49782. (H) TLR4 expression induced by CI-GOTA. Groups were compared using the Kruskal–Wallis non-parametric test followed by the Dunn post-test. PBS was used as negative control (CN) and 100 ng/mL of LPS was used as positive control. Statistical significance (p < 0.05) is represented by the symbols (^∗difference with the NC group, ^#difference with the LPS group, ⁺difference with the inactive group). Data are expressed as mean ± standard deviation (n = 9).

FIGURE 2

Gene expression in bovine macrophages infected with different Ureaplasma/mL of ATCC 49782 and CI-GOTA strains for 6, 12, and 24 h. (A) IL-1β expression in macrophages inoculated with the ATCC 49782 strain. (B) IL-1β expression in macrophages inoculated with the CI-GOTA strain. (C) TNF-α expression in macrophages inoculated with ATCC 49782 strains. (D) TNF-α expression in macrophages inoculated with CI-GOTA strain. (E) TLR2 expression in macrophages inoculated with ATCC 49782 strains. (F) TLR2 expression in macrophages inoculated with the CI-GOTA strain. (G) TLR4 expression in macrophages inoculated with ATCC 49782 strains. (H) TLR4 expression in Macrophages inoculated with CI-GOTA strain. Treatments were compared using the Kruskal–Wallis non-parametric test followed by the Dunn post-test. PBS - negative control (CN) and 100 ng/mL of LPS was used as positive control. Statistical significance (p < 0.05) is represented by the symbols Statistical significance (p < 0.05) is represented by the symbols [^∗difference with the NC group, ^#difference with the LPS group, ⁺difference with the 10⁵ group (ATTC) or 10⁴ group (CI-GOTA), ^&difference with the 10⁴ group (ATTC) or 10³ group (CI-GOTA)]. Data are expressed as mean ± standard deviation (n = 9).

FIGURE 3

SDS-PAGE of the UdLAMPs. First column: Novex Protein molecular weight. Second column: UdLAMPs from ATCC 49782. Third column: UdLAMPs from CI-GOTA.

FIGURE 4

Gene expression in bovine macrophages induced by different concentrations of UdLAMPs after 2, 6, and 12 h of inoculation. (A) IL-1β gene expression incubation with UdLAMPs of ATCC 49782 strain. (B) IL-1β gene expression after incubation with UdLAMPs of the CI-GOTA strain. (C) TNF-α gene expression after incubation with UdLAMPs of ATCC 49782 strain. (D) TNF-α gene expression after incubation and others with UdLAMPs of CI-GOTA strain. (E) TLR2 gene expression after incubation with UdLAMPs of ATCC 49782 strain. (F) TLR2 gene expression after incubation with UdLAMPs of CI-GOTA strain. (G) TLR4 gene expression after incubation with UdLAMPs of ATCC 49782 strain. (H) TLR4 gene expression after incubation with UdLAMPs of CI-GOTA strain. Treatments were compared using Kruskal-Wallis non-parametric test followed by the Dunn post-test. PBS was used as negative control (CN) and 100 ng/mL of LPS was used as positive control. Statistically different groups are those presenting different symbols. Statistical significance (p < 0.05) is represented by the symbols (^∗difference with the NC group, ^#difference with the 1,5 mg/ml group, ⁺difference with the 1,0 mg/ml group, ^&difference with the 0,5 mg/ml group). Data are expressed as mean ± standard deviation (n = 9).

FIGURE 5

Gene expression of IL-1β and TNF-α from bovine macrophages exposed to U. diversum and LAMPsUd and pretreated with signaling blockers for TLR4 (CLI-095), TLR2/4 (OxPAPC), and NF-κB (Celastrol). The cells were analyzed after 2 h (LAMPs) and after 12 h (U. diversum). (A) Expression of IL-1β after incubation with ATCC 49782 (10⁵ Ureaplasma/mL) and CI-GOTA (10³ Ureaplasma/mL). (B) Expression of IL-1β after incubation with LAMPsUd of ATCC 49782 and CI-GOTA (2 μg/ml). (C) Expression of TNF-α after incubation with ATCC 49782 (10⁵ Ureaplasma/mL) and CI-GOTA (10³ Ureaplasma/mL). (D) Expression of TNF-α after LAMPsUd incubation of ATCC 49782 and CI-GOTA (2 μg/ml). The treatments were compared with positive control (inoculation of U. diversum or LAMPsUd in the absence of blocker: W/B). PBS was used as negative control (CN) and 100 ng/mL of LPS and 100 ug of Pam3CysSK4 was used as positive control. Treatments were compared using the Kruskal–Wallis non-parametric test followed by the Dunn post-test. Statistically different groups are those presenting different symbols. Statistical significance (p < 0.05) is represented by the symbols (^∗difference with groups with blockers, ^#difference with the NC group, and ⁺difference among strains). Data are expressed as mean ± standard deviation (n = 9).

FIGURE 6

(A) Confocal microscopy: internalization of U. diversum in bovine blastocysts after 24 h of infection. Scale bars: 20 μm. Ureaplasmas in red (Vybrant Dil), actin in green (FITC) and DNA in blue (TOPRO-3). U. diversum was internalized as shown in the merged image. (B) Orthogonal projection of confocal microscopy showing the internalization of U. diversum ATCC49782 in bovine blastocysts after 24 h of infection. Ureaplasmas visualized by Vybrant Dil (red), actin by FITC (green) and DNA by TOPRO-3 (blue). U. diversum was internalized as shown in the merged image.

FIGURE 7

Gene expression of cytokines and TLRs in bovine blastocysts infected for 24 h with ATCC 49782 and CI-GOTA strains. (A) Cytokine IL-1β. (B) Cytokine TNF-α. (C) TLR 2. (D) TLR 4. PBS was used as negative control (CN). Statistically different groups are those presenting different symbols. Statistical significance (p < 0.05) is represented by the symbols (^∗difference with the NC group, ^#difference among strains). Data are expressed as mean ± standard deviation (n = 9).