Chronic fetal exposure to Ureaplasma parvum suppresses innate immune responses in sheep

Abstract

The chorioamnionitis associated with preterm delivery is often polymicrobial with ureaplasma being the most common isolate. To evaluate interactions between the different proinflammatory mediators, we hypothesized that ureaplasma exposure would increase fetal responsiveness to LPS. Fetal sheep were given intra-amniotic (IA) injections of media (control) or Ureaplasma parvum serovar 3 either 7 or 70 d before preterm delivery. Another group received an IA injection of Escherichia coli LPS 2 d prior to delivery. To test for interactions, IA U. parvum-exposed animals were challenged with IA LPS and delivered 2 d later. All animals were delivered at 124 ± 1-d gestation (term = 150 d). Compared with the 2-d LPS exposure group, the U. parvum 70 d + LPS group had 1) decreased lung pro- and anti-inflammatory cytokine expression and 2) fewer CD3(+) T lymphocytes, CCL2(+), myeloperoxidase(+), and PU.1(+) cells in the lung. Interestingly, exposure to U. parvum for 7 d did not change responses to a subsequent IA LPS challenge, and exposure to IA U. parvum alone induced mild lung inflammation. Exposure to U. parvum increased pulmonary TGF-β1 expression but did not change mRNA expression of either the receptor TLR4 or some of the downstream mediators in the lung. Monocytes from fetal blood and lung isolated from U. parvum 70 d + LPS but not U. parvum 7 d + LPS animals had decreased in vitro responsiveness to LPS. These results are consistent with the novel finding of downregulation of LPS responses by chronic but not acute fetal exposures to U. parvum. The findings increase our understanding of how chorioamnionitis-exposed preterm infants may respond to lung injury and postnatal nosocomial infections.

Figure 1. Lung histology after intraamniotic exposure to LPS, acute and chronic intraamniotic exposure to U. parvum

Representative photomicrographs showing hematoxylin and eosin staining of lung sections from the animals with intraamniotic exposures to (A) Controls (B) 7d U. parvum (C) 70d U. parvum (D) 2d LPS (E) 7d U. parvum +2d LPS and (F) 70d U. parvum +2d LPS. Note the similarity of histology in panels A, B, C, and F. Panels D and E demonstrate inflammatory cells in the airways and lung interstitium. (scale bar is 100μm).

Figure 2

Chronic amniotic exposure to U. parvum decreases intraamniotic LPS induced pulmonary recruitment and activation of inflammatory cells. Bronchoalveolar lavage cells were expressed relative to body weight A) Neutrophils, B) Monocytes. Quantitation of (C) CD3+ T-cells and (D) PU.1+ cells per high power field in lung tissue sections. Representative photomicrographs of of lung sections showing immunostaining with (E–H) CD3 and (I–L) PU.1. Arrows point to immunostained inflammatory cells. (scale bar is 50μm)(*p<0.05 vs. control, ^#p<0.05 vs. 2d LPS).

Figure 3. chronic amniotic exposures to U. parvum decreased intraamniotic LPS induced increases in monocyte responses to LPS in vitro

Monocytes from (A) Blood and (B) Lung were purified over percoll gradients, 10⁶ cells were cultured and challenged with media only or 100ng/mL LPS for 16h. LPS induced secretion of IL-6 in the medium was expressed as fold increase over the media value (C= fetal control, adult=monocytes/macrophages from adult ewes, *p<0.05 vs. fetal control, ^#p<0.05 vs. 2d LPS).

Figure 4. Chronic amniotic exposure to U. parvum decreased intraamniotic LPS induced pulmonary cytokine mRNA expression

Quantification using real-time PCR assays using sheep specific primers and Taqman probes (A) IL-1β (B) IL-6 (C) IL-8 (D) MCP-1 (E) Serum Amyloid A3 (F) IL-10 and (G) IL-1ra mRNAs. The values for each cytokine were normalized to 18s rRNA. The mean mRNA signals in control animals were given the value of 1 and levels at each time point were expressed relative to controls (*p<0.05 vs. control, ^#p<0.05 vs. 2d LPS).

Figure 5. chronic amniotic exposures to U. parvum decreased intraamniotic LPS induced lung cytokine proteins

Cytokine proteins in the bronchoalveolar lavage fluid (BALF) were measured by ELISA (A) IL-1β and (B) IL-8 (*p<0.05 vs. control, ^#p<0.05 vs. 2d LPS).

Figure 6. Chronic amniotic exposure to U. parvum decreased intraamniotic LPS induced MCP-1 expression

(A) Quantitation of MCP-1 positive cells per high power field. Representative pictures from (B) Controls (C) 2d LPS (D) 70d UP + 2d LPS (Scale bar = 50μm, *p<0.05 vs. control, ^#p<0.05 vs. 2d LPS).

Figure 7. Chronic amniotic exposure to U. parvum decreased intraamniotic LPS induced myeloperoxidase expression

(A) Quantitation of MPO positive cells per high power field. Representative pictures from (B) Controls (C) 2d LPS (D) 70d UP + 2d LPS (Scale bar = 50μm, *p<0.05 vs. control).

Figure 8. Chronic amniotic exposure to U. parvum did not decrease intraamniotic LPS induced increases in acute phase reactants

Quantification using real-time PCR assays using sheep specific primers and Taqman probes for (A) Liver CRP (B) Liver Serum amyloid A3 mRNA expression. The values for each cytokine were normalized to 18s rRNA. The mean mRNA signals in control animals were given the value of 1 and levels at each time point were expressed relative to controls. (C) Haptoglobin levels were measured in the plasma by ELISA (*p<0.05 vs. control).

Figure 9. Chronic amniotic exposures to U. parvum increased pulmonary TGFβ1 secretion but did not change mRNA expression of genes in the TLR4 pathway

(A) TGFβ1 was measured in BALF by ELISA after acid activation and values expressed per kg body weight. Quantification of mRNAs of select mediators of TLR4 signaling in the lung, using real-time PCR assays with sheep specific primers and Taqman probes (B) TLR4 (C) IRAK-1 (D) IRAK-4 and (E) IRAK-M. The values for each cytokine were normalized to 18s rRNA. The mean mRNA signals in control animals were given the value of 1 and levels at each time point were expressed relative to controls. (*p<0.05 vs. control).