Inflammation-Induced Adverse Pregnancy and Neonatal Outcomes Can Be Improved by the Immunomodulatory Peptide Exendin-4

Abstract

Preterm birth is the leading cause of neonatal morbidity and mortality worldwide. Inflammation is causally linked to preterm birth; therefore, finding an intervention that dampens maternal and fetal inflammatory responses may provide a new strategy to prevent adverse pregnancy and neonatal outcomes. Using animal models of systemic maternal inflammation [intraperitoneal injection of lipopolysaccharide (LPS)] and fetal inflammation (intra-amniotic administration of LPS), we found that (1) systemic inflammation induced adverse pregnancy and neonatal outcomes by causing a severe maternal cytokine storm and a mild fetal cytokine response; (2) fetal inflammation induced adverse pregnancy and neonatal outcomes by causing a mild maternal cytokine response and a severe fetal cytokine storm; (3) exendin-4 (Ex4) treatment of dams with systemic inflammation or fetal inflammation improved adverse pregnancy outcomes by modestly reducing the rate of preterm birth; (4) Ex4 treatment of dams with systemic, but not local, inflammation considerably improved neonatal outcomes, and such neonates continued to thrive; (5) systemic inflammation facilitated the diffusion of Ex4 through the uterus and the maternal-fetal interface; (6) neonates born to Ex4-treated dams with systemic inflammation displayed a similar cytokine profile to healthy control neonates; and (7) treatment with Ex4 had immunomodulatory effects by inducing an M2 macrophage polarization and increasing anti-inflammatory neutrophils, as well as suppressing the expansion of CD8+ regulatory T cells, in neonates born to dams with systemic inflammation. Collectively, these results provide evidence that dampening maternal systemic inflammation through novel interventions, such as Ex4, can improve the quality of life for neonates born to women with this clinical condition.

Keywords: M2 macrophages; amniotic fluid; clinical chorioamnionitis; fetal inflammatory response syndrome; intra-amniotic infection/inflammation; neutrophils; preterm labor and birth; regulatory T cells.

Figure 1

Models of inflammation-induced preterm birth and adverse neonatal outcomes. (A) On 16.5 days post coitum (dpc), pregnant mice were intraperitoneally (MIR model) (10 µg/200 µL) or intra-amniotically (FIR model) (100 ng/25 µL) injected with LPS or 1× PBS (200 or 25 µL) and mice were monitored until delivery. (B,C) Rate of preterm or term birth in the MIR and FIR models. (D,E) Rate of neonatal mortality at birth in the MIR and FIR models. (F,G) Rate of neonatal mortality at one week of age in the MIR and FIR models. n = 7–10 dams with litters per group. Abbreviations: PTB, preterm birth, TB, term birth; PBS, phosphate-buffered saline; LPS, lipopolysaccharide.

Figure 2

The maternal cytokine response in the MIR and FIR models. (A) On 16.5 days post coitum (dpc), pregnant mice were intraperitoneally (MIR model) (10 µg/200 µL) or intra-amniotically (FIR model) (100 ng/25 µL) injected with lipopolysaccharide (LPS) or 1× phosphate-buffered saline (PBS) (200 or 25 µL), and on 17.5 dpc maternal serum was collected for cytokine multiplex analysis. Concentrations of (B) CCL3, (C) CCL4, (D) CCL5, (E) CXCL5, (F) CXCL10, (G) G-CSF, (H) IL-1β, (I) IL-18, (J) IL-6, (K) IFNγ, (L) TNFα, (M) CXCL2, (N) CXCL1, (O) CCL2, (P) IL-13, (Q) IL-12p70, (R) IL-10, and (S) IL-4 in the maternal serum. n = 10 dams per group.

Figure 3

The fetal inflammatory response in the MIR and FIR models. (A) On 16.5 days post coitum (dpc), pregnant mice were intraperitoneally (MIR model) (10 µg/200 µL) or intra-amniotically (FIR model) (100 ng/25 µL) injected with lipopolysaccharide (LPS) or 1× phosphate-buffered saline (PBS) (200 or 25 µL), and on 17.5 dpc amniotic fluid was collected for cytokine multiplex analysis. Photographs of fetuses from dams with MIR or FIR are shown. Concentration of (B) CCL3, (C) CCL4, (D) CCL5, (E) CXCL5, (F) CXCL10, (G) G-CSF, (H) IL-1β, (I) IL-18, (J) IL-6, (K) IFNγ, (L) TNFα, (M) CXCL2, (N) CXCL1, (O) CCL2, (P) IL-13, (Q) IL-12p70, (R) IL-10, and (S) IL-4 in the amniotic fluid. n = 5–14 dams per group.

Figure 4

Inflammatory gene expression in the fetal lung. (A) On 16.5 days post coitum (dpc), pregnant mice were intraperitoneally (MIR model) (10 µg/200 µL) or intra-amniotically (FIR model) (100 ng/25 µL) injected with lipopolysaccharide (LPS) or 1× phosphate-buffered saline (PBS) (200 or 25 µL), and on 17.5 dpc fetal lung was collected for gene expression analysis. Photographs of fetal lungs from dams with MIR or FIR are shown. (B) Heat map visualization of gene expression in fetal lung tissue. Expression of (C) Il1b, (D) Il6, (E) Ccl2, (F) Ccl3, (G) Ccl5, and (H) Cxcl1 in the fetal lung. n = 10–21 dams with litters per group.

Figure 5

Treatment with Ex4 improves adverse pregnancy and neonatal outcomes. (A) On 16.5 days post coitum (dpc), pregnant mice were intraperitoneally (MIR model) (10 µg/200 µL) or intra-amniotically (FIR model) (100 ng/25 µL) injected with LPS and injected intraperitoneally with 30 µg/kg Ex4. Pregnant mice were also intraperitoneally (10 µg/200 µL) or intra-amniotically (10 ng/25 µL) injected with LPS or Ex4 alone on 16.5 dpc. Mice were monitored until delivery. (B,E) Rate of preterm birth in the MIR and FIR models. (C,F) Rate of neonatal mortality at birth in the MIR and FIR models. (D,G) Rate of neonatal mortality at one week of age in the MIR and FIR models. n = 5–10 dams with litters per group. Abbreviations: PTB, preterm birth, TB, term birth; Ex4, exendin-4; dpc, days post coitum; LPS, lipopolysaccharide.

Figure 6

Exendin-4 is localized in the uterus and maternal–fetal interface. (A) On 16.5 days post coitum (dpc), pregnant mice were intraperitoneally injected with: (1) 1× phosphate-buffered saline (PBS) (200 µL); (2) lipopolysaccharide (LPS) (10 µg/200 µL); (3) Fluorescein-TRP²⁵-Exendin-4 (FLEX) (30 µg/kg) alone; and (4) LPS followed by treatment with FLEX (30 µg/kg). Imaging was performed 1 h after the second injection. (B) Representative images taken with the In Vivo Imaging System showing the fluorescence of FLEX in the uterus, decidua, placenta, fetal membranes, and fetus. n = 3 per group.

Figure 7

The cytokine profile of neonates born to dams with MIR and treated with exendin-4 (Ex4). (A) On 16.5 days post coitum (dpc), pregnant mice were intraperitoneally (10 µg/200 µL) injected with lipopolysaccharide (LPS) followed by treatment with Ex4 (30 µg/kg). Controls were injected with 1× phosphate-buffered saline (PBS, 200 µL) alone. At 15 days of age, neonatal plasma was collected for cytokine multiplex analysis. Concentrations of (B) CCL3, (C) CCL4, (D) CCL5, (E) CXCL5, (F) CXCL10, (G) G-CSF, (H) IL-1β, (I) IL-18, (J) IL-6, (K) IFNγ, (L) TNFα, (M) CXCL2, (N) CXCL1, (O) CCL2, (P) IL-13, (Q) IL-12p70, (R) IL-10, and (S) IL-4 in the neonatal plasma. n = 12–14 neonates per group.

Figure 8

Inflammatory gene expression in neonates born to dams with MIR and treated with exendin-4 (Ex4). On 16.5 days post coitum (dpc), pregnant mice were intraperitoneally (10 µg/200 µL) injected with lipopolysaccharide (LPS) followed by treatment with Ex4 (30 µg/kg). Controls were injected with 1× phosphate-buffered saline (PBS, 200 µL) alone. At 15 days of age the neonatal brain, lung, liver, and small intestine were collected for gene expression analysis. Expression of (A) Il1b, (B) Il6, (C) Ccl2, (D) Ccl3, (E) Ccl5, and (F) Cxcl1 in the neonatal brain, lung, liver and small intestine. n = 12–14 neonates per group.

Figure 9

Exendin-4 (Ex4) treatment induces an M1 → M2 macrophage polarization in neonates. On 16.5 days post coitum (dpc), pregnant mice were intraperitoneally (10 µg/200 µL) injected with lipopolysaccharide (LPS) followed by treatment with Ex4 (30 µg/kg). Controls were injected with 1× phosphate-buffered saline (PBS, 200 µL) alone. At 15 days of age, the neonatal lung, liver, and large intestine were collected for immunophenotyping. (A) Gating strategy for M1- and M2-like macrophages. Dead cells were excluded using a viability dye. Empty histograms represent the autofluorescence control and colored histograms represent antibody fluorescent signals. Numbers of macrophages in the neonatal lung (B), liver (E), and large intestine (H). Numbers of M2-like macrophages in the neonatal lung (C), liver (F), and large intestine (I). Numbers of M1-like macrophages in the neonatal lung (D), liver (G), and large intestine (J). n = 12–14 neonates per group.

Figure 10

Exendin-4 (Ex4) treatment induces an increase in anti-inflammatory neutrophils in neonates. On 16.5 days post coitum (dpc), pregnant mice were intraperitoneally (10 µg/200 µL) injected with lipopolysaccharide (LPS) followed by treatment with Ex4 (30 µg/kg). Controls were injected with 1× phosphate-buffered saline (PBS, 200 µL) alone. At 15 days of age, the neonatal lung, liver, and large intestine were collected for immunophenotyping. (A) Gating strategy for neutrophil polarization. Dead cells were excluded using a viability dye. Empty histograms represent the autofluorescence control and colored histograms represent antibody fluorescent signals. Numbers of neutrophils in the neonatal lung (B), liver (E), and large intestine (H). Numbers of IL-10-expressing neutrophils in the neonatal lung (C), liver (F), and large intestine (I). Numbers of iNOS-expressing neutrophils in the neonatal lung (D), liver (G), and large intestine (J). n = 12–14 neonates per group.

Figure 11

Exendin-4 (Ex4) treatment reduces neonatal CD8+ regulatory T cells (Tregs). On 16.5 days post coitum (dpc), pregnant mice were intraperitoneally (10 µg/200 µL) injected with lipopolysaccharide (LPS) followed by treatment with Ex4 (30 µg/kg). Controls were injected with 1× phosphate-buffered saline (PBS, 200 µL) alone. At 15 days of age, the neonatal spleen and thymus were collected for immunophenotyping. (A) Gating strategy for CD4+ and CD8+ T regulatory cells. Dead cells were excluded using a viability dye. Dotted histograms represent the autofluorescence control and colored histograms represent antibody fluorescent signals. CD4+ and CD8+ Tregs co-expressed CD25 and FoxP3. (B,C) Number of splenic and thymic CD4+ Tregs. (D,E) Number of splenic and thymic CD8+ Tregs. n = 12–14 neonates per group.