Heightened Immune Activation in Fetuses with Gastroschisis May Be Blocked by Targeting IL-5

Abstract

The development of the fetal immune system during pregnancy is a well-orchestrated process with important consequences for fetal and neonatal health, but prenatal factors that affect immune activation are poorly understood. We hypothesized that chronic fetal inflammation may lead to alterations in development of the fetal immune system. To test this hypothesis, we examined neonates with gastroschisis, a congenital abdominal wall defect that leads to exposure of the fetal intestines to amniotic fluid, with resultant intestinal inflammation. We determined that patients with gastroschisis show high systemic levels of inflammatory cytokines and chemokines such as eotaxin, as well as earlier activation of CD4(+) and CD8(+) effector and memory T cells in the cord blood compared with controls. Additionally, increased numbers of T cells and eosinophils infiltrate the serosa and mucosa of the inflamed intestines. Using a mouse model of gastroschisis, we observed higher numbers of eosinophils and both type 2 and type 3 innate lymphoid cells (ILC2 and ILC3), specifically in the portion of organs exposed to the amniotic fluid. Given the role of IL-5 produced by ILC2 in regulating eosinophil development and survival, we determined that maternal or fetal administration of the anti-IL-5 neutralizing Ab, or a depleting Ab against ILCs, can both effectively reduce intestinal eosinophilia. Thus, a congenital anomaly causing chronic inflammation can alter the composition of circulating and tissue-resident fetal immune cells. Given the high rate of prenatal and neonatal complications in these patients, such changes have clinical significance and might become targets for fetal therapy.

FIGURE 1

Elevated inflammatory markers and T cell activation in the cord blood of patients with gastroschisis. (A) Comparison of cytokine levels in unaffected controls (white, n=19) and patients with gastroschisis (black, n=21). The horizontal line represents the median for each group. *p=0.05; **p<0.01; ***p<0.001 by Mann-Whitney rank sum test. MCP-1, Monocyte Chemoattractant Protein-1; MDC, Macrophage-Derived Chemokine. (B) Supervised Principal Component Analysis (sPCA) of the cord blood biomarkers most significantly correlated with gastroschisis in healthy controls (n=19; blue), patients with gastroschisis (n=21; green) and patients with giant omphalocele (n=2; orange). Ellipsoids represent a 50% concentration of observations for controls and patients with gastroschisis. Eotaxin-1, IL-1Rα, IL-6, IL-8 and MCP-1 had the highest factor loadings on PC1. (C-E) Cord blood PBMCs were stimulated with PMA and Ionomycin and stained with antibodies to CD3, CD4, CD8, IL-17 and IFN-γ to determine the percentage of T cells producing each cytokine. (C) IFN-γ production by CD4⁺ T cells. (D) IFN-γ production by CD8⁺ T cells. (E) IL-17 production by CD4⁺ T cells. Each symbol represents a single patient; small horizontal bars indicate the mean. (F and G) On the left side, representative flow cytometric analysis of purified maternal and cord blood PBMCs from healthy controls and patients with gastroschisis stained with antibodies to CD3, CD4, CD8, CD45RA and CCR7 to detect naïve (N: CCR7⁺CD45RA⁺), central memory (CM: CCR7⁺CD45RA⁻) and effector memory (EM: CCR7⁻CD45RA⁻; EMRA: CCR7⁻CD45RA⁺) cells. On the right side, compiled analysis for cord blood (F) CD4⁺ EM T cells, (G) CD8⁺ EM T cells and CD8⁺ EMRA T cells. Each symbol represents a single patient; small horizontal bars indicate the mean. Control n=20, gastroschisis n=21. *p<0.05; **p<0.01; ***p<0.001 by Mann-Whitney test.

FIGURE 2

Infiltration of T cells and eosinophils into the intestine of patients with gastroschisis. Histological analysis of bowel specimens from healthy controls (left panels) and patients with gastroschisis (right panels). (A) Hematoxylin and eosin staining of appendix sections shows the presence of numerous vessels in the serosa in patients with gastroschisis. Lower magnification bars, 500 μm. Higher magnification bars, 100 μm. (B and C) Immunohistochemical staining of (B) appendix and (C) colon sections shows infiltration of CD3⁺ T cells (brown) in the serosa of specimens from patients with gastroschisis. Lower magnification bars, 500 μm. Higher magnification bars, 100 μm and inserts, 20 μm. (D) Enlarged lymphoid follicles and infiltration of CD3⁺ T cells (brown) in the mucosa of colon in patients with gastroschisis. Lower magnification bars, 500 μm. Higher magnification bars, 100 μm. (E) Numbers of CD3⁺ T cells infiltrating the intestine per field of view (FOV), excluding lymphoid follicles. Control n=5, gastroschisis n=6. (F) Hematoxylin and eosin staining shows the presence of eosinophils (indicated by the arrows) infiltrating the mucosa in colon of patients with gastroschisis. Control n=7, gastroschisis n=6. Lower magnification bars, 200 μm. Higher magnification bars, 50 μm and insert, 20 μm. The graph shows the numbers of eosinophils infiltrating the intestine. **p<0.01 by Mann-Whitney test.

FIGURE 3

Increased eosinophils and NK cells in the exteriorized intestines and livers of ACLP^−/− mice. (A) E18.5 ACLP^−/− mouse exhibiting a defect in the anterior abdominal wall with exteriorized liver and intestine. (B) Representative flow cytometric analysis of NK cells (Nk1.1⁺) and eosinophils (Siglec-F⁺) from the intestine of E18.5 littermate controls (control) and ACLP^−/− mice after first gating on CD45.2⁺ leukocytes. (C and D) Histograms showing the percentages and absolute cell number/mg tissue of (C) eosinophils and (D) NK cells in the exposed (out) or non-exposed (in) intestine and fetal liver. Each symbol represents a single fetus; small horizontal bars indicate the mean. Control n=13, ACLP^−/− n=9. *p<0.05; **p<0.01; ***p<0.001 by one-way ANOVA with Tukey's post-tests.

FIGURE 4

Increased innate lymphoid cells (ILCs) in the exteriorized organs of ACLP^−/− mice and patients with gastroschisis. (A) Sequential gating strategy to identify ILC2 (defined as CD45⁺Lin⁻CD3⁻Thy1.2⁺IL-7Rα⁺GATA-3⁺) in the intestine of a representative ACLP^−/− mouse. (B) Representative flow cytometric plot of ILC2 and graphs showing the percentages and absolute cell number/mg tissue in the exposed (out) or non-exposed (in) intestine in littermate controls and ACLP^−/− mice. Each symbol represents a single fetus; small horizontal bars indicate the mean. Control n=18, ACLP^−/− n=12. *p<0.05; **p<0.01; ***p<0.001 by one-way ANOVA. (C and D) Gating strategy to identify ILC3 (defined as CD45⁺Lin⁻CD3⁻CD4⁺IL-7Rα⁺c-Kit⁺RORγt⁺) from E18.5 ACLP^−/− mouse (example shown) and graphs showing the percentages (on total CD45⁺ cells) and absolute cell number/mg tissue in the exposed (out) or non-exposed (in) (C) intestine and (D) fetal liver in littermate controls and ACLP^−/− mice. In this set of experiments, the entire intestine was exteriorized in the affected mice. Control n=6, ACLP^−/− n=7. *p<0.05; **p<0.01; ***p<0.001 by one-way ANOVA. (E) Flow cytometric analysis of ILC (defined as CD3⁻CD4⁻IL-7Rα⁺c-Kit⁺) and compiled analysis of ILC of lamina propria and intraepithelial lymphocytes isolated from intestine of healthy controls (cnt, n=6 samples from 4 patients) and patients with gastroschisis (GS, n=6 samples from 3 patients). Among gastroschisis samples, each symbol (square, diamond and circle) represents a different patient; small horizontal bars indicate the mean. *p<0.05 by Mann-Whitney test.

FIGURE 5

Decreased eosinophil infiltration and ILC2 in ACLP^−/− mice after treatment with anti IL-5 antibody. (A) Schematic layout of the experiment. Fetal intestine and liver are harvested 5 days after fetal liver injection of anti IL-5 or isotype control/PBS. (B and C) Histograms showing the absolute cell number/mg tissue of eosinophils in the intestine (B) and fetal liver (C). Each symbol represents a single fetus; small horizontal bars indicate the mean. Control n=43, ACLP−/− n=23. ***p<0.001 by Student's t test. (D) Schematic layout of the experiment. Maternal intravenous injection of anti IL-5 or isotype control daily between E13.5-E18.5 and harvest of fetal intestine and liver on E18.5. (E and F) Histograms showing the percentages and absolute cell number/mg tissue of eosinophils in the intestine (E) and fetal liver (F). (G) Histograms showing the percentages and absolute cell number/mg tissue of ILC2 in the intestine of littermate control and ACLP^−/− mice. Each symbol represents a single fetus; small horizontal bars indicate the mean. Control n=14, ACLP^−/− n=8. *p<0.05; **p<0.01; ***p<0.001 by Student's t test.

FIGURE 6

Decreased eosinophil infiltration in ACLP^−/− mice after fetal treatment with anti Thy1.2 antibody to deplete innate lymphoid cells (ILC). (A) Schematic layout of the experiment. Fetal mice are injected with anti Thy1.2 or isotype control antibody and harvested on E18.5. (B) Histograms showing the depletion of ILC2 (% and absolute cell number/mg tissue) in the intestine. (C) Histograms showing the reduction of eosinophils (% and absolute cell number/mg tissue) in the intestine. Each symbol represents a single fetus; small horizontal bars indicate the mean. Control n=56, ACLP^−/− n=11. *p<0.05; **p<0.01; ***p<0.001 by Student's t test.