Colony-stimulating factor-1-dependent macrophage functions regulate the maternal decidua immune responses against Listeria monocytogenes infections during early gestation in mice

Abstract

The association between extreme-prematurity births and intrauterine infection emphasizes the importance of understanding the host immune responses against uterine-invading microbes during early pregnancy to the prevention of preterm births. Listeria monocytogenes, a clinically relevant intracellular bacterium, has a predilection for replication at the maternofetal interface during pregnancy. Here, using mice carrying the recessive null osteopetrotic mutation in the colony-stimulating factor-1 (CSF-1) gene, we show that CSF-1-dependent macrophage functions are required for the maternal decidua immune responses against L. monocytogenes infections during early gestation in mice. In the absence of CSF-1, pregnant mice were more susceptible to uterine infection by L. monocytogenes; their inability to control the expansion of colonized bacteria in the pregnant uterus led to decidual cell death, tissue disintegration, and resorption of the developing embryo. However, CSF-1-deficient mice were able to produce significant levels of both Th1 cytokines and neutrophil chemoattractants and to recruit neutrophils to the decidual tissue in response to Listeria infection. Depletion of macrophages in hormonally induced pseudopregnant mice resulted in higher uterine bacterial levels after L. monocytogenes infection. These data suggest that the anti-Listeria responses in the maternal decidual tissue are dependent on CSF-1-regulated macrophages.

FIG. 1.

CSF-1R is expressed in decidual cells. (A and C) Representative transverse sections of the GD8 uterus of a Csf1^op/op mouse immunostained using an antibody against CSF-1R. (B and D) An adjacent section immunostained using an antibody against the trophoblastic cell marker, cytokeratin. Positive cells are stained brown with the antibodies. CSF-1R-positive decidual cells do not colocalize with the cytokeratin-positive trophoblast. Bars = 100 μm.

FIG. 2.

Csf1^op/op mice are susceptible to L. monocytogenes infection during early gestation compared to Csf1^op/+ mice. (A) Pregnant mice (both Csf1^op/op and Csf1^op/+) were infected i.v. on GD5, -6, or -7 with 10⁴ CFU of L. monocytogenes. Mice were sacrificed on GD8 (at 24, 48, or 72 h p.i.), and the numbers of CFU of L. monocytogenes per gram of tissue recovered from the pregnant uteri were determined by plating serial dilutions of tissue homogenate on tryptic soy agar plates. Each data point represents one animal; data represent results from at least five independent experiments. (B) Similar to what was done for panel A, pregnant mice were infected i.v. on GD6 or -7 with 10⁴ CFU of L. monocytogenes and sacrificed on GD8 (at 24 or 48 h p.i.), and the numbers of CFU of L. monocytogenes in circulation (represented as CFU per ml of blood) were determined. Each data point represents one animal; data represent results from at least five independent experiments. (C) Ovariectomized mice (both Csf1^op/op and Csf1^op/+) were hormonally treated and injected with intrauterine oil to induce the formation of deciduomas, followed by i.v. injection of 10⁴ CFU of L. monocytogenes. Mice were sacrificed 72 h p.i., and the numbers of CFU of L. monocytogenes per gram of tissue recovered from the induced deciduomas were determined. Each data point represents one animal; data represent results from at least three independent experiments. Symbols: open circles, Csf1^op/op; filled circles, Csf1^op/+; horizontal bars, median titers (NS, not significant; ** in panel A, P = 0.002; * in panel B, P = 0.02; ** in panel C, P = 0.004; Mann-Whitney U test).

FIG. 3.

Absence of CSF-1 results in decidual tissue destruction and fetal loss after L. monocytogenes challenge during early gestation. (A) Representative photographs of GD8 pregnant uteri (U) either uninfected (iii [Csf1^op/op]) or challenged with 10⁴ CFU of L. monocytogenes i.v. on GD5 and harvested at 72 h p.i. (i [Csf1^op/op] and ii [Csf1^op/+]). * marks an example of an implantation site, which is resorbed in the Csf1^op/op uterus at 72 h p.i. (B) Pregnant mice (both Csf1^op/op and Csf1^op/+) were infected i.v. on GD5 or -6 with 10⁴ CFU of L. monocytogenes. Mice were sacrificed on GD8 (at 48 or 72 h p.i.), and cross sections at the implantation sites were stained with hematoxylin and eosin. (Top) Csf1^op/+, either uninfected (a) or 48 (b) and 72 (c) h p.i.; (bottom) Csf1^op/op, either uninfected (d) or 48 (e) and 72 (f) h p.i. Note decidual tissue necrosis and leukocyte infiltration in the Csf1^op/op animal (e) and resorption of the fetal tissue at 72 h p.i. (f). Bar = 100 μm.

FIG. 4.

Both Csf1^op/op and Csf1^op/+ mice recruit neutrophils to their decidual tissue after L. monocytogenes infection during early gestation. (A) Pregnant mice (both Csf1^op/op and Csf1^op/+) were infected i.v. on GD5 or -6 with 10⁴ CFU of L. monocytogenes. Mice were sacrificed on GD8 (at 48 or 72 h p.i.), and cross sections at the implantation sites were immunostained using a neutrophil-specific antibody, clone 7/4; positive cells (arrowheads) were stained brown with the antibody. (Top) Csf1^op/+, either uninfected (a) or 48 (b) and 72 (c) h p.i. (respective insets [boxes] are shown below); (bottom) Csf1^op/op, either uninfected (d) or 48 (e) and 72 (f) h p.i. (respective insets [boxes] are shown below). Bars = 100 μm. (B) Pregnant mice (both Csf1^op/op and Csf1^op/+) were infected i.v. on GD6 or -7 with 10⁴ CFU of L. monocytogenes. Mice were sacrificed on GD8 (at 24 or 48 h p.i.), and the deciduomas of the animals were pooled to prepare single-cell suspensions and analyzed by fluorescence-activated cell sorting. The neutrophil percentage of total hematopoietic cells for each animal was determined. Data represent mean ± standard deviation from at least five independent experiments (***, P < 0.0001; Student's t test). Compared to uninfected deciduomas, infected Csf1^op/+ deciduomas had increased neutrophil percentages with time, until peaking at 48 h p.i.; a similar trend was also seen for Csf1^op/op animals, although at every time point postinfection, the neutrophil percentage was higher than that for the Csf1^op/+ animals.

FIG. 5.

Csf1^op/op mice fail to recruit macrophages to their decidual tissue after L. monocytogenes infection during early gestation. Pregnant mice (both Csf1^op/op and Csf1^op/+) were infected i.v. on GD5 or -6 with 10⁴ CFU of L. monocytogenes. Mice were sacrificed on GD8 (at 48 or 72 h p.i.), and cross sections at the implantation sites were immunostained using an antibody against the macrophage-specific surface marker F4/80; positive cells (arrowheads) were stained brown with the antibody. (Top), Csf1^op/+, either uninfected (a) or 48 (b) and 72 (c) h p.i. (respective insets [boxes] are shown below); (bottom), Csf1^op/op, either uninfected (d) or 48 (e) and 72 (f) h p.i. (respective insets [boxes] are shown below). Note the increased number and the change into a more dendritic morphology of macrophages in the Csf1^op/op mice (b and c) after Listeria infection and the absence of such changes in the Csf1^op/op mice (e and f). Bars = 100 μm.

FIG. 6.

Macrophage depletion results in susceptibility of the decidual tissue to L. monocytogenes infection in oil-induced decidualized animals. (A) Ovariectomized FVB mice with CD11b-DTR bone marrow transplants were hormonally treated and injected with intrauterine oil to induce the formation of deciduomas. Intraperitoneal injection of DT or a mutant form of the toxin (Glu⁵²-DT) was performed 1 day prior to and on the day of i.v. injection of 10⁴ CFU of L. monocytogenes. Mice were sacrificed 60 to 72 h p.i., and the numbers of CFU of L. monocytogenes per gram of tissue recovered from the induced deciduomas were determined. Each data point represents one animal; data represent results from at least three independent experiments. Symbols: filled circles, DT-treated animals; open circles, Glu⁵²-DT-treated animals; **, P = 0.008 (Mann-Whitney U test). (B) Cross sections of the decidualized uteri of the animals in panel A were immunostained using an antibody against the macrophage-specific surface marker F4/80 (a and d) or an antibody against the L. monocytogenes-specific antigen listeriolysin O (b, c, e, and f); positive cells or bacteria (arrowheads) were stained brown with the antibodies. Note the absence of F4/80-positive cells in the DT-treated uterus (d), which also contains a higher number of listeriolysin O-positive cells (e and f). Bars = 50 μm; panels a, b, d, and e are shown at the same magnification; panels c and f are shown at the same higher magnification.