Immunomodulation in the canine endometrium by uteropathogenic Escherichia coli

Abstract

This study was designed to evaluate the role of E. coli α-hemolysin (HlyA) in the pathogenesis of canine pyometra, and on the immune response of canine endometrial epithelial and stromal cells. In Experiment 1, the clinical, hematological, biochemical and uterine histological characteristics of β-hemolytic and non-hemolytic E. coli pyometra bitches were compared. More (p < 0.05) metritis cases were observed in β-hemolytic E. coli pyometra uteri than in non-hemolytic E. coli pyometra uteri. β-hemolytic E. coli pyometra endometria had higher gene transcription of IL-1β and IL-8 and lower gene transcription of IL-6 than non-hemolytic E. coli pyometra endometria (p < 0.01). In Experiment 2, the immune response of endometrial epithelial and stromal cells, to hemolytic (Pyo18) and non-hemolytic E. coli strains (Pyo18 with deleted hlya-Pyo18ΔhlyA- and Pyo14) were compared. Following 4 h of incubation, Pyo18 decreased epithelial cell numbers to 54% (p < 0.001), and induced death of all stromal cells (p < 0.0001), whereas Pyo18ΔhlyA and Pyo14 had no effect on cell numbers. Compared to Pyo18ΔhlyA and Pyo14, respectively, Pyo18 induced a lower transcription level of IL-1β (0.99 vs 152.0 vs 50.9 fold increase, p < 0.001), TNFα (3.2 vs 49.9 vs 12.9 fold increase, p < 0.05) and IL-10 (0.4 vs 3.6 vs 2.6 fold increase, p < 0.001) in stromal cells, after 1 h of incubation. This may be seen as an attempt of hemolytic E. coli to delay the activation of the immune response. In conclusion, endometrial epithelial and stromal cell damage induced by HlyA is a potential relevant step of E. coli virulence in the pathogenesis of pyometra.

Figure 1

Histology of uteri from bitches diagnosis with pyometra. A–B Histological section of uterine pyometra samples. A non-hemolytic E. coli pyometra evidencing basal glands with myeloid cells infiltration (arrow); B β-hemolytic E. coli pyometra evidencing extensive damage of basal glands (arrow) and metritis (arrow head). Staining by H&E. C–K Immunostaining of calprotectin positive cells (myeloid cells), CD79 αcy positive cells (B lymphocytes) and CD3 positive cells (T lymphocytes) in pyometra uterine sections. Myeloid cells (granulocytes/macrophages) detection: C negative control (staining with mouse isotype); D, E infiltration in apical and basal layer, respectively (cytoplasmic staining pattern). B lymphocytes detection: F negative control (PBS); G, H infiltration in apical and basal layer, respectively (membrane staining pattern). T lymphocytes detection: I negative control (staining with rabbit isotype); J, K infiltration in apical and basal layer (membrane and cytoplasmic staining pattern), respectively.

Figure 2

Transcription of TLRs signaling components in diestrous and pyometra endometria. A Representative PCR detection of transcripts of TLRs signaling components and of cytokines in diestrous and pyometra endometria. NC negative control. B Relative mRNA expression level (arbitrary units, AU) of IL-1β, IL-6, IL-8, IL-10, and TGFβ evaluated by real-time PCR, in hemolytic E. coli endometria, non-hemolytic E. coli endometria and diestrous endometria. Data are given as mean ± SEM. Columns with different superscripts differ significantly (IL-1β, IL-6, IL-8 ^{a vs b} p < 0.01^{a,b vs c}; p < 0.00001; IL-10, TGFβ ^{a vs b} p < 0.0001). MyD88 (myeloid differentiation factor 88); TRAM (TRIF related adaptor molecule); TRAF6 (TNFR-associated factor 6); TRIF (TIR domain-containing adaptor inducing interferon (IFN)-β); IRF3 (interferon regulatory factor 3).

Figure 3

Effect of hemolytic E. coli in epithelial and stromal cells. Morphology of endometrial epithelial (A–F) and stromal (G–L) cell cultures stained with Giemsa after 1 and 4 h of incubation: unstimulated cells (A, D, G, J); cells incubated with Pyo14 (B, E, H, K) and Pyo18 (C, F, I, L).

Figure 4

Effect of Pyo18 isogenic hlyA deletion mutant (Pyo18ΔhlyA) in epithelial and stromal cells. Morphology of endometrial stromal cells after 1 and 4 h of incubation, evaluated after Giemsa staining (A–C) or under phase-contrast (D–L): unstimulated cells (A, D, G, J); cells incubated with Pyo18 (B, E, H, K); cells incubated with Pyo18ΔhlyA (C, F, I, L).

Figure 5

Activation of TLRs signaling pathways evaluated by immunofluorescence. Nuclear detection of NFκB (A–H) and IRF3 (I–P) in endometrial epithelial and stromal cells by immunofluorescence. A–D and I–L epithelial cells [A, I rabbit isotype negative control after 4 h of incubation; B, J unstimulated cells after 4 h of incubation; C, D and K, L stimulation with Pyo18ΔhlyA strain during 1 h (C, K) and 4 h (D, L)]; E–H and M–P stromal cells [E, M rabbit isotype negative control after 1 h of incubation; F, N unstimulated cells after 1 h of incubation; G, H and O, P stimulation with Pyo18ΔhlyA strain during 1 h (G, O) and 4 h (H, P)]. White arrow—nuclear staining.

Figure 6

Pro-inflammatory interleukins genes transcription in cultured canine endometrial epithelial and stromal cells after Escherichia coli or LPS cell stimulation. Relative mRNA expression (qRT-PCR) of IL-1β (A), IL-1α (B), IL-6 (C), TNFα (D) in cultured canine endometrial epithelial and stromal cells in response to hemolytic E. coli (Pyo18), non-hemolytic E. coli (Pyo14) and the isogenic mutant of Pyo18 (Pyo18ΔhlyA). Treatment with 1 μg/mL of LPS was used as a positive control. Data are mean ± SEM (n = 3 uteri with two replicates for all stimuli). Transcription levels (fold increase) are normalized with those of non-stimulated cells ^{a vs b} p < 0.05 [(Epithelia cells: IL-1α, IL-6; stromal cells: IL-1β, IL-1α, TNFα]; ^{a vs b} p < 0.01 (stromal cells: IL-6); ^{a vs c} p < 0.05 (stromal cells: TNFα); * ^vs **p < 0.05 (IL-1β) * ^vs **p < 0.01 (IL-1α); p < 0.05 (1 vs 4 h: IL-1α, stromal cells); p < 0.01 (1 vs 4 h: IL-6, epithelia cells).

Figure 7

Chemokines, anti-inflammatory Interleukin-10 and Interferon-β genes transcription in epithelial and stromal cells after Escherichia coli or LPS cell stimulation. Relative mRNA expression (qRT-PCR) of IL-8 (A), CXCL10 (B), IL-10 (C), INFβ (D) in cultured canine endometrial epithelial and stromal cells in response to hemolytic E. coli (Pyo18), non-hemolytic E. coli (Pyo14) and the isogenic mutant of Pyo18 (Pyo18ΔhlyA). Treatment with 1 μg/mL of LPS was used as a positive control. Data are mean ± SEM (n = 3 uteri with two replicates for all stimuli). Transcription levels (fold increase) are normalized with those of non-stimulated cells. ^{a vs b} p < 0.05 (stromal cells: IL-8); ^{a vs d} p < 0.05 (epithelial cells: IFNβ); ^{c vs d} p < 0.05 (Epithelial cells: IFNβ); ^{b vs c} p < 0.05 (epithelial cells: IFNβ); ^{a vs b} p < 0.01 (stromal cells: IL-10); * ^vs **p < 0.01 (stromal cells: IL-8); p < 0.01 (1 vs 4 h: IL-8, stromal cells; CXCL10, epithelial cells); p < 0.001 (1 vs 4 h: IFNβ, epithelial cells).