Roles of natural resistance-associated macrophage protein-1 in modulating bacterial distribution and immune responses during Salmonella enterica serovar Typhimurium infection in murine pregnancy

Abstract

Problem: Salmonella enterica serovar Typhimurium (S.Tm) infection in Nramp1^+/+ mice during pregnancy can lead to profound bacterial growth in the feto-placental unit and adverse pregnancy outcomes, including fetal loss, maternal illness and death. The kinetics and mechanisms by which S.Tm gains entry within individual feto-placental unit, and disseminates through tissues leading to placental resorption and fetal demise remain unclear.

Method of study: Mice were systemically infected with S.Tm. Bacterial burden within spleen and individual placentas, and placental/fetal resorptions were quantified. Flow cytometric analysis of immune cell types in the spleen and individual placentas was performed. Cytokine expression in maternal serum was determined through cytometric bead array.

Results: Systemic infection with S.Tm resulted in preferential bacterial proliferation in placentas compared to the spleen in Nramp1^+/+ mice. At 24 h post-infection, the mean infection rate of individual placentas per mouse was ∼50%, increasing to >75% by 72 h post-infection, suggesting that initial infection in few sites progresses to rapid spread of infection through the uterine milieu. This correlated with a steady increase in placental/fetal resorption rates. Placental infection was associated with local increased neutrophil percentages, whereas numbers and percentages in the spleen remained unchanged, suggesting dichotomous modulation of inflammation between the systemic compartment and the feto-maternal interface. Reduced survival rates of pregnant mice during infection correlated with decreased serum IFN-γ but increased IL-10 levels relative to non-pregnant controls.

Conclusion: Pregnancy compromises host resistance conferred by Nramp1 against S.Tm through compartment-specific regulation of maternal and placental cellular responses, and modulation of systemic cytokine expression.

Keywords: Nramp1; Salmonella; cytokines; inflammation; placenta; pregnancy.

Figure 1.. Enhanced susceptibility of pregnant Nramp1^+/+ mice to S.Tm is associated with progressive placental infection.

Pregnant (gestational day 11 and 12) and non-pregnant Nramp1^+/+ mice were infected intravenously with 10³ S.Tm CFUs. (A) Mice were monitored for signs of clinical illness and euthanized upon reaching humane endpoints. N = 11–26 mice per group. (B-F) Placentas from infected mice were collected and processed individually at various time points to determine the presence of infection in each individual placenta and their location within the uterine horns. (B) The ratios of infected to non-infected placentas were compared based on the total number of individual placentas collected per time point. (C) Bacterial CFUs in individual placentas were enumerated at various time points post-infection. Each data point represents a single placenta, and mean bacterial burden for placentas at a given time point is shown as a horizontal line. Limit of detection (LOD) for individual placentas was 1 CFU. (D) Fetal resorption rates (mean ± SEM) were calculated using the formula R/(R + V) x 100, where R is the number of resorbing/resorbed fetuses and V is the number of viable fetuses per mouse. (E) Mean ± SEM percentages of non-infected vs. infected placentas per mouse were determined. (F) The localization of individual placentas within the uterine horns are shown. Individual placentas with CFUs below the LOD were categorized as non-infected. Overall, N = 6–52 mice per group; 37–43 individual placentas per time point. PI, pregnant infected; NPI, non-pregnant infected. Statistical significance was analyzed by Log-rank (Mantel-Cox) test (A), Chi-square test (B), Kruskal-Wallis test followed by Dunn’s multiple comparisons test (C and D), and two-way ANOVA followed by Sidak’s multiple comparisons test (E). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2.. S.Tm infection promotes distinct splenic and placental distributions of innate immune cells in pregnant Nramp1^+/+ mice.

Pregnant (gestational day 11 and 12) and non-pregnant Nramp1^+/+ mice were infected intravenously with 10³ S.Tm CFUs i.v. A and B) Spleens and individual placentas were collected 72h post-infection for flow cytometric analysis of immune cell types. Total spleen cell numbers (A) and percentages (B) are shown. (C) Placental immune cell data are presented as percentages only due to overall low cell counts, which ranged from 40,000–60,000 cells per placenta. Total numbers of splenic immune cell types were determined by acquiring 100,000 events of the CD45⁺ cell population and using the formula: (percentage of immune cell type gated on CD45⁺ cells × total splenocyte count) / 100. Percentages of neutrophils, macrophages, DCs and NK cells in the spleen or placenta were determined based on the total CD45⁺ cell population acquired per sample. Data are presented as mean ± SEM. N = 6–10 mice per group; 19–47 individual placentas. Statistical significance of splenic cell numbers (A) and percentages (B) was determined using Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Placenta cell percentages (C) were analyzed by Mann Whitney test. *p < 0.05, *** p < 0.001, **** p < 0.0001.

Figure 3.. Pregnant Nramp1^+/+ mice exhibit increased systemic cytokine expression during S.Tm infection.

Pregnant (gestational day 11 and 12) and non-pregnant Nramp1^+/+ mice were infected intravenously with 10³ S.Tm CFUs i.v. Serum was collected from mice at 72h post-infection for quantification of cytokine levels. Data are presented as mean ± SEM. N = 12–14 mice per group. Statistical significance was determined by Mann Whitney test. *p < 0.05, ** p < 0.01.