Salmonella enterica serovar Typhimurium-induced placental inflammation and not bacterial burden correlates with pathology and fatal maternal disease

Abstract

Food-borne infections caused by Salmonella enterica species are increasing globally, and pregnancy poses a high risk. Pregnant mice rapidly succumb to S. enterica serovar Typhimurium infection. To determine the mechanisms involved, we addressed the role of inflammation and bacterial burden in causing placental and systemic disease. In vitro, choriocarcinoma cells were a highly conducive niche for intracellular S. Typhimurium proliferation. While infection of mice with S. Typhimurium wild-type (WT) and mutant (Delta aroA and Delta invA) strains led to profound pathogen proliferation and massive burden within placental cells, only the virulent WT S. Typhimurium infection evoked total fetal loss and adverse host outcome. This correlated with substantial placental expression of granulocyte colony-stimulating factor (G-CSF), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) and increased serum inflammatory cytokines/chemokines, such as G-CSF, IL-6, CCL1, and KC, evoked by WT S. Typhimurium infection. In contrast, infection with high doses of S. Typhimurium Delta aroA, despite causing massive placental infection, resulted in reduced inflammatory cellular and cytokine response. While S. Typhimurium WT bacteria were dispersed in large numbers across all regions of the placenta, including the deeper labyrinth trophoblast, S. Typhimurium Delta aroA bacteria localized primarily to the decidua. This correlated with the widespread placental necrosis accompanied by neutrophil infiltration evoked by the S. Typhimurium WT bacteria. Thus, the ability of Salmonella to localize to deeper layers of the placenta and the nature of inflammation triggered by the pathogen, rather than bacterial burden, profoundly influenced placental integrity and host survival.

FIG. 1.

S. Typhimurium infection in pregnancy. (a and b) Splenic bacterial burden in 129.B6F1 mice on day 3 after infection with 10³ CFU of WT S. Typhimurium or the ΔinvA or ΔaroA mutant (a) or 10⁶ CFU of S. Typhimurium ΔaroA (b). **, splenic bacterial burden in pregnant mice infected with WT S. Typhimurium or the ΔinvA mutant is significantly higher than that in the nonpregnant group by Mann-Whitney U test (P < 0.01). (c and d) Placental bacterial burden (c) and fetal resorption rates (d) on day 3 postinfection. Fetal resorption rates evoked by WT S. Typhimurium and S. Typhimurium ΔinvA are significantly higher than that for the noninfected pregnant group by Mann-Whitney U test; **, P < 0.001; *, P < 0.05. Each data point indicates individual mice, and the mean value for each group is indicated by a horizontal line.

FIG. 2.

Growth kinetics of S. Typhimurium (ST). (a, b, and c) Bacterial burden in organs after infection of mice with 10³ CFU of WT S. Typhimurium or 10⁶ CFU of S. Typhimurium ΔaroA. Data indicate means ± standard errors of the means (SEM) for 4 to 6 mice per time point. (d) In vitro intracellular growth of S. Typhimurium at an MOI of 10 within HeLa and human choriocarcinoma cells (JEG-3).

FIG. 3.

Host survival kinetics following S. Typhimurium (ST) infection. Nonpregnant and pregnant 129.B6F1 mice were infected with 10³ CFU of WT S. Typhimurium (a) or 10⁶ CFU of S. Typhimurium ΔaroA (b). Survival curves are based on 6 mice/group. WT S. Typhimurium-infected mice were euthanized once they showed 2 or 3 of the following clinical signs of infection: morbidity, piloerection, slow/retarded movement, or >20% weight loss.

FIG. 4.

Cytokine proteome profiling blot. Representative chemiluminescence cytokine blots carried out on serum of individual mice in the indicated 6 groups. Infections were with 10⁶ (a and b) or 10³ (c and d) CFU of S. Typhimurium ΔaroA or WT S. Typhimurium, respectively. The spot intensity is proportional to the amount of cytokine bound. Each cytokine is represented as duplicate spots, and there are 3 sets of positive control spots on each blot. The intensity of spots was quantified relative to the positive control spot based on pixel density.

FIG. 5.

Profile of serum inflammatory cytokines/chemokines. 129.B6F1 mice were infected with either WT S. Typhimurium (ST) (10³ CFU) or S. Typhimurium ΔaroA (10⁶ CFU). Fold changes in analyte levels in the serum on day 3 of infection relative to the respective noninfected control group are indicated. Data represent mean changes ± SEM. Values were obtained for analyzed serum from individual mice (n = 3 to 5/group). *, P < 0.05 in comparison with the nonpregnant WT S. Typhimurium-infected group by Mann-Whitney U test.

FIG. 6.

Placental cytokine expression. Shown is cytokine expression determined by Q-RT-PCR of placentas obtained from noninfected and WT S. Typhimurium (ST) (10³ CFU)- or S. Typhimurium ΔaroA (10⁶ CFU)-infected mice on day 3 postinfection. Relative mRNA expression is normalized to β-actin. Each data point indicates the expression level in pooled placental tissue of individual mice, and the mean expression level for each group is indicated by a horizontal line. *, expression levels for WT S. Typhimurium-infected mice are significantly higher (P < 0.05) than those for healthy noninfected placentas; ★, expression levels in the placentas of S. Typhimurium ΔaroA-infected mice are significantly lower (P < 0.05) than those in the WT S. Typhimurium-infected group. Data were analyzed by Mann-Whitney U test.

FIG. 7.

H&E and MPO staining of placental tissue. Histology of placental tissue infected with WT S. Typhimurium (ST) (10³ CFU) or S. Typhimurium ΔaroA (10⁶ CFU) was determined on day 3 postinfection. (A to C) H&E staining of noninfected (A) and WT S. Typhimurium (B)- and S. Typhimurium ΔaroA-infected (C) placentas. Scale bars, 100 μm. Insets show tissue at higher magnification. (D to F) MPO staining of noninfected (D) and WT S. Typhimurium (E)- and S. Typhimurium ΔaroA-infected (F) placentas. Scale bar, 20 μm. The inset (E) shows polymorphonuclear cell-specific MPO production. Images are representative of tissues processed from 3 mice per group.

FIG. 8.

Gr1-positive staining of placental tissue. Shown are fluorescent microscopic images of frozen placental sections from noninfected tissue (A) and tissue infected with WT S. Typhimurium (10³ CFU) (B) or S. Typhimurium ΔaroA (10⁶ CFU) (C) stained with Alexa647-conjugated anti-GR1 (red) and Hoechst 33342 (blue). Scale bar, 20 μm. Images are representative of placentas obtained from 3 mice per group.

FIG. 9.

Localization of WT S. Typhimurium (ST) and S. Typhimurium ΔaroA bacteria to distinct regions of the placenta. Shown are images of frozen placental sections from mice infected with WT S. Typhimurium (10³ CFU) or S. Typhimurium ΔaroA (10⁶ CFU) expressing GFP 72 h postinfection. (A, B, and C) Morphology of the placentas from uninfected mice and mice infected with WT S. Typhimurium or S. Typhimurium ΔaroA, respectively. D, decidual region; L, labyrinth region. (D and E) Localization of WT S. Typhimurium-GFP in the highly necrotic labyrinth trophoblast. (F and G) Localization of S. Typhimurium ΔaroA-GFP near the decidual region of the placenta. The presence of bacteria was confirmed by observing the marked areas using a 60× oil immersion objective, and the higher-magnification images of bacteria are shown in insets. The images were captured using an Olympus IX81 fluorescence microscope. Scale bars, 200 μm. Images are representative of tissues processed from 3 mice per group.