<em>Listeria monocytogenes</em> Cytoplasmic Entry Induces Fetal Wastage by Disrupting Maternal Foxp3⁺ Regulatory T Cell-Sustained Fetal Tolerance

<div><p>Although the intracellular bacterium <em>Listeria monocytogenes</em> has an established predilection for disseminated infection during pregnancy that often results in spontaneous abortion or stillbirth, the specific host-pathogen interaction that dictates these disastrous complications remain incompletely defined. Herein, we demonstrate systemic maternal <em>Listeria</em> infection during pregnancy fractures fetal tolerance and triggers fetal wastage in a dose-dependent fashion. <em>Listeria</em> was recovered from the majority of concepti after high-dose infection illustrating the potential for <em>in utero</em> invasion. Interestingly with reduced inocula, fetal wastage occurred without direct placental or fetal invasion, and instead paralleled reductions in maternal Foxp3⁺ regulatory T cell suppressive potency with reciprocal expansion and activation of maternal fetal-specific effector T cells. Using mutants lacking virulence determinants required for <em>in utero</em> invasion, we establish <em>Listeria</em> cytoplasmic entry is essential for disrupting fetal tolerance that triggers maternal T cell-mediated fetal resorption. Thus, infection-induced reductions in maternal Foxp3⁺ regulatory T cell suppression with ensuing disruptions in fetal tolerance play critical roles in pathogenesis of immune-mediated fetal wastage.</p> </div

<i>Listeria monocytogenes</i> infection triggers dose-dependent rates of fetal wastage during allogeneic pregnancy.

<p>(A) Number of live pups born with virulent WT Lm inoculated midgestation (E10.5) at the indicated dosages for pregnant C57Bl/6 females mated with Balb/c males. (B) Percent viable and resorbed fetuses five days after WT Lm infection at midgestation. (C) Percent viable and resorbed fetuses with recoverable Lm for the mice described in B. Each data point represents results from an individual mouse combined from three independent experiments each with similar results.</p

Cytoplasmic entry is essential for <i>Listeria monocytogenes</i>-induced fetal wastage.

<p>(A) Number of live pups born with LmΔactA (10⁷ CFUs) or LmΔLLOΔPLC (10⁸ CFUs) infection at midgestation (E10.5) in C57Bl/6 females mated with Balb/c males. (B) Percent viable and resorbed fetuses five days after LmΔactA (10⁷ CFUs) or LmΔLLOΔPLC (10⁸ CFUs) infection at midgestation. Each data point represents results from an individual mouse combined from three independent experiments each with similar results.</p

<i>Listeria monocytogenes</i> cytoplasmic entry dampens maternal Foxp3⁺ regulatory CD4 T cell suppressive potency.

<p>(A) Percent GFP⁺ or Foxp3⁺ among CD4 cells in pregnant mice midgestation after infection with LmΔactA (10⁷ CFUs) or LmΔLLOΔPLC (10⁸ CFUs). (B) Representative plots demonstrating proliferation (CFSE dilution) among responder (Tresp) CD90.1⁺ CD8 cells after co-culture with each ratio of GFP⁺ Tregs isolated from LmΔactA (10⁷ CFUs) or LmΔLLOΔPLC (10⁸ CFUs) infected mice, and stimulation with anti-CD3 antibody (black line), compared with no Treg (gray filled) or no stimulation (black filled) controls (top). Relative suppression of responder cell proliferation (CFSE dilution) after co-culture with GFP⁺ Tregs for cells from mice described above normalized to suppression by GFP⁺ cells from uninfected controls at a 1∶1 Treg∶Tresp ratio (bottom). These data reflect six to eight mice per group representative of three independent experiments each with similar results.</p

<i>Listeria monocytogenes</i> infection during pregnancy does not induce quantitative changes in maternal Foxp3⁺ regulatory CD4 T cells.

<p>Absolute number (top) and percent Foxp3⁺ among CD4 T cells (bottom) three days after virulent WT Lm infection at midgestation in C57Bl/6 females mated with Balb/c males. Each data point represents results from an individual mouse combined from two independent experiments each with similar results.</p

<i>Listeria monocytogenes</i> infection during pregnancy dampens maternal Foxp3⁺ regulatory CD4 T cell suppressive potency.

<p>(A) Percent GFP⁺ or Foxp3⁺ among CD4 cells in virgin, pregnant mice midgestation without infection, or three days after infection with 10⁴ WT Lm. (B) Representative plots demonstrating proliferation (CFSE dilution) among responder (Tresp) CD90.1⁺ CD8 cells after co-culture with each ratio of GFP⁺ Tregs isolated from Lm-infected mice and stimulation with anti-CD3 antibody (black line), compared with no Treg (gray filled) or no stimulation (black filled) controls (top). Relative suppression of responder cell proliferation (CFSE dilution) after co-culture with GFP⁺ Tregs for the mice described above normalized to suppression by GFP⁺ cells from uninfected controls at a 1∶1 Treg∶Tresp ratio (bottom). These data reflect six to eight mice per group representative of three independent experiments each with similar results.</p

Proposed model for immune-mediated fetal wastage induced by prenatal infection that can occur with or without <i>in utero</i> pathogen invasion.

<p>After low dose infection during pregnancy, reductions in maternal regulatory T cell suppression unleash the activation of immune effectors enough to rapidly eliminate the pathogen. However, given the requirement for sustained expansion of maternal regulatory cell suppression in maintaining fetal tolerance, these reductions in suppressive potency also trigger immune-mediated fetal wastage. By comparison with higher dosage infection, blunted maternal regulatory T cell suppression that promotes immune activation does not eradicate infection as efficiently. In turn with ongoing disruption in fetal tolerance, remaining pathogen is drawn to inflammation at the uterine-placental interface that promotes invasion into the placental-fetal unit.</p

<i>Listeria monocytogenes</i> cytoplasmic entry disrupts fetal tolerance with infection during pregnancy.

<p>Representative FACS plots (top) and composite data (bottom) illustrating expansion and IFN-γ production by fetal-OVA-specific CD8 T cells among maternal splenocytes in mice impregnated by Actin-OVA males five days after LmΔactA (10⁷ CFUs) or LmΔLLOΔPLC (10⁸ CFUs) infection at midgestation. For IFN-γ production, cells were stimulated with OVA_257–264 peptide (black line) or no peptide controls (gray filled). Each data point represents results from an individual mouse combined from three independent experiments each with similar results.</p

<i>Listeria monocytogenes</i> infection during pregnancy disrupts fetal tolerance.

<p>Representative FACS plots (top) and composite data (bottom) illustrating expansion and IFN-γ production by fetal-OVA-specific CD8 T cells among maternal splenocytes in mice impregnated by Actin-OVA males five days after Lm infection at midgestation. For IFN-γ production, cells were stimulated with OVA_257–264 peptide (black line) or no peptide controls (gray filled). Each data point represents results from an individual mouse combined from three independent experiments each with similar results.</p

The primary response dominates the memory-derived secondary response during Mtb infection.

<p><b>(a)</b> Experimental strategy for adoptive co-transfer experiments. Relative proportion of naïve and memory TB10Rg3 CD8⁺ T cells and their expression of CD62L, KLRG1, and IL-7R before transfer <b>(b)</b> and in the spleen 1d after transfer into uninfected mice <b>(c)</b>. Baseline labeling with the eFluor450 proliferation dye is shown. <b>(d)</b> Concatenated histograms of eFluor450 staining of naïve and memory-derived TB10Rg3 cells in the MLN, lung, and spleen (top) and their CD62L and CD44 expression (bottom) from a representative experiment on d11 post-infection. <b>(e)</b> Proportion of adoptively-transferred memory (Thy1.1⁺) and naïve (Thy1.2⁺)-derived TB10Rg3 CD8⁺ T cells in the lung 15, 18, or 21d after Mtb infection. <b>(f)</b> The relative proportion of memory (Thy1.1) and naïve (Thy1.2)-derived TB10Rg3 CD8⁺ T cells in the MLN, lung, and spleen after infection, compared to spleens from uninfected mice 1 day after transfer (CTRL) (top). Cell numbers of memory (Thy1.1⁺) and naïve (Thy1.2⁺)-derived TB10Rg3 CD8⁺ T cells from the same mice (bottom). (<b>g</b>) KLRG1 and IL-7R expression by memory and naïve-derived TB10Rg3 cells recovered from lung at each time point. <b>(h)</b> TCR Vα2 median fluorescence intensity (MFI) (median ± SEM) in memory and naïve-derived TB10Rg3 CD8⁺ T cells from the same mice at d15 and d18 post-infection. <b>(i)</b> EdU uptake (mean ± SEM) by memory and naïve-derived TB10Rg3 cells recovered from lung. EdU uptake was compared with a student’s t-test. * p < 0.05, ** p < 0.01, <b>***</b> p < 0.001, **** p < 0.0001 n.s. not significant, n.d. < 10 cells detected. Data are representative of 2–10 independent experiments, each with 3–4 mice per group.</p