Immunomodulator expression in trophoblasts from the feline immunodeficiency virus (FIV)-infected cat

Abstract

Background: FIV infection frequently compromises pregnancy under experimental conditions and is accompanied by aberrant expression of some placental cytokines. Trophoblasts produce numerous immunomodulators that play a role in placental development and pregnancy maintenance. We hypothesized that FIV infection may cause dysregulation of trophoblast immunomodulator expression, and aberrant expression of these molecules may potentiate inflammation and compromise pregnancy. The purpose of this project was to evaluate the expression of representative pro-(TNF-α, IFN-γ, IL-1β, IL-2, IL-6, IL-12p35, IL-12p40, IL-18, and GM-CSF) and anti-inflammatory cytokines (IL-4, IL-5, and IL-10); CD134, a secondary co-stimulatory molecule expressed on activated T cells (FIV primary receptor); the chemokine receptor CXCR4 (FIV co-receptor); SDF-1α, the chemokine ligand to CXCR4; and FIV gag in trophoblasts from early-and late-term pregnancy.

Methods: We used an anti-cytokeratin antibody in immunohistochemistry to identify trophoblasts selectively, collected these cells using laser capture microdissection, and extracted total RNA from the captured cell populations. Real time, reverse transcription-PCR was used to quantify gene expression.

Results: We detected IL-4, IL-5, IL-6, IL-1β, IL-12p35, IL-12p40, and CXCR4 in trophoblasts from early-and late-term pregnancy. Expression of cytokines increased from early to late pregnancy in normal tissues. A clear, pro-inflammatory microenvironment was not evident in trophoblasts from FIV-infected queens at either stage of pregnancy. Reproductive failure was accompanied by down-regulation of both pro-and anti-inflammatory cytokines. CD134 was not detected in trophoblasts, and FIV gag was detected in only one of ten trophoblast specimens collected from FIV-infected queens.

Conclusion: Feline trophoblasts express an array of pro-and anti-inflammatory immunomodulators whose expression increases from early to late pregnancy in normal tissues. Non-viable pregnancies were associated with decreased expression of immunomodulators which regulate trophoblast invasion in other species. The detection of FIV RNA in trophoblasts was rare, suggesting that the high rate of reproductive failure in FIV-infected queens was not a direct result of viral replication in trophoblasts. The influence of placental immune cells on trophoblast function and pregnancy maintenance in the FIV-infected cat requires additional study.

Figure 1

Laser Capture Microdissection of IHC-stained Trophoblasts from a Representative FIV-negative Cat. Trophoblasts were labeled using antibody to cytokeratin, and cytokeratin-positive trophoblasts were evident by brown staining. (A) Trophoblasts were selectively identified. (B) Areas of trophoblast-rich populations were cut and captured using the UV cutting laser and Capture IR laser, respectively. (C) Selected trophoblasts were removed from the tissue. (D) Trophoblasts were attached to the cap. All photography was performed with the 20X objective. Bars = 200 microns.

Figure 2

Relative Expression of Immunomodulators in Trophoblasts from Early-and Late-Term Feline Placentas. Real-time RT-PCR analysis of feline placental trophoblast expression of the pro-inflammatory cytokines (IL-6, IL-1β, IL-12p35, and IL-12p40), the anti-inflammatory cytokines (IL-4 and IL-5), and the chemokine receptor (CXCR4) in: (A) early-term uninfected placentas (n = 3) versus late-term uninfected placentas (n = 3); (B) early-term FIV-infected (n = 5) versus uninfected placentas; (C) late-term FIV-infected (n = 5) versus uninfected placentas. Bars represent mean Ct values substracted from a negative endpoint (60-mean Ct), bracketed by standard errors of the mean. P values obtained from single factor ANOVA and Wilcoxon rank sum test are noted. P values ≤ 0.05 were considered significant.

Figure 3

Relative Expression of Immunomodulators in Trophoblasts from Early-and Late-Term Feline Placentas from Viable and Non-viable Pregnancies. Real-time RT-PCR analysis of trophoblast expression of the pro-inflammatory cytokines (IL-6, IL-1β, IL-12p35, and IL-12p40), the anti-inflammatory cytokines (IL-4 and IL-5), and the chemokine receptor (CXCR4) in early-(A) and late-term (B) placentas from all viable (n = 4 each for early and late tissues) versus all non-viable (n = 4 each for early and late tissues) pregnancies. Bars represent mean Ct values substracted from a negative endpoint (60-mean Ct), bracketed by standard errors of the mean. P values obtained from Wilcoxon rank sum test are noted. P values ≤ 0.05 were considered significant.