Aberrant placental immune parameters in the feline immunodeficiency virus (FIV)-infected cat suggest virus-induced changes in T cell function.

BACKGROUND: Immune activity during pregnancy must be tightly regulated to ensure successful pregnancy. This regulation includes the suppression of inflammatory activity that could target the semi-allogeneic fetus. Tregs are immunosuppressive; Th17 cells are pro-inflammatory. A precise balance in the two cell populations is critical to pregnancy maintenance, while dysregulation in this balance accompanies compromised pregnancy in humans and mice. FIV is known to target Tregs preferentially in the infected cat. Therefore, it may be hypothesized that FIV infection alters the placental Treg/Th17 cell balance resulting in aberrant immunomodulator expression by these cells and consequent pregnancy perturbation. METHODS: RNA was purified from random sections of whole placental tissues collected from both uninfected and FIV-infected queens at early pregnancy, including tissues from viable and nonviable fetuses. Real time qPCR was performed to quantify expression of intranuclear markers of Tregs (FoxP3) and Th17 cells (RORγ); cytokine products of Tregs (IL-10 and TGF-ß), Th17 cells (IL-2, IL-6, and IL-17a), and macrophages (IL-1ß); and the FIV gag gene. Pairwise comparisons were made to evaluate coexpression patterns between the cytokines and between the cytokines and the virus. RESULTS: Both FoxP3 and RORγ were reduced in placentas of infected animals. Neither infection status nor fetal viability affected placental expression of IL-1ß. However, fetal nonviability was associated with reduced levels of all other cytokines. Infection and fetal nonviability impacted coexpression of various cytokine pairs. No obvious bias toward Treg or Th17 cells was observed. CONCLUSIONS: FIV infection coupled with fetal nonviability alters expression patterns of T cell cytokines. These data suggest that functionally altered placental T cell leukocyte populations may occur in the infected queen and possibly contribute to fetal nonviability.

Imbalance of placental regulatory T cell and Th17 cell population dynamics in the FIV-infected pregnant cat.

BACKGROUND: An appropriate balance in placental regulatory T cells (Tregs), an immunosuppressive cell population, and Th17 cells, a pro-inflammatory cell population, is essential in allowing tolerance of the semi-allogeneic fetus. TGF-ß and IL-6 are cytokines that promote differentiation of Tregs and Th17 cells from a common progenitor; aberrant expression of the cytokines may perturb the balance in the two cell populations. We previously reported a pro-inflammatory placental environment with decreased levels of FoxP3, a Treg marker, and increased levels of IL-6 in the placentas of FIV-infected cats at early pregnancy. Thus, we hypothesized that FIV infection in the pregnant cat causes altered placental Treg and Th17 cell populations, possibly resulting in placental inflammation. METHODS: We examined the effect of FIV infection on Treg and Th17 populations in placentas at early pregnancy using quantitative confocal microscopy to measure FoxP3 or RORγ, a Th17 marker, and qPCR to quantify expression of the key cytokines TGF-ß and IL-6. RESULTS: FoxP3 and RORγ were positively correlated in FIV-infected placentas at early pregnancy, but not placentas from normal cats, indicating virus-induced alteration in the balance of these cell populations. In control cats the expression of IL-6 and RORγ was positively correlated as predicted, but this relationship was disrupted in infected animals. TGF-ß was reduced in infected queens, an occurrence that could dysregulate both Treg and Th17 cell populations. Co-expression analyses revealed a highly significant positive correlation between IL-6 and TGF-ß expression in control animals that did not occur in infected animals. CONCLUSION: Collectively, these data point toward potential disruption in the balance of Treg and Th17 cell populations that may contribute to FIV-induced inflammation in the feline placenta.

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

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-1ß, 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-1ß, 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.

An immunohistochemical assay to detect trophoblasts in frozen feline placenta.

The placenta, a fetal endocrine organ, is composed of subpopulations of trophoblasts, including cytotrophoblasts, and syncytiotrophoblasts. Trophoblastic populations can be distinguished based upon their expression of cytokeratin. The purpose of the current study was to develop an immunohistochemistry (IHC) method to identify trophoblasts selectively in frozen feline placental tissue using antibodies specific for cytokeratin. The mouse monoclonal antibody anti-human pan-cytokeratin AE1/AE3 and a commercial detection system were used. Nonspecific immunoreactivity was encountered that could not be eliminated with altered blocking methods. The nonspecific reactivity was attributed to the goat anti-mouse/rabbit immunoglobulin G (IgG) peroxidase polymer included in the commercial kit. Alternatively, a polyclonal rabbit anti-cow cytokeratin wide spectrum screening antibody with goat anti-rabbit IgG polyclonal secondary antibody was used to detect cytokeratin in feline placental tissue. The IHC procedure eliminated nonspecific immunoreactivity while specifically labeling cytokeratin. This new approach provides an IHC method to identify trophoblasts specifically in feline placenta.

Cytokine dysregulation in early- and late-term placentas from feline immunodeficiency virus (FIV)-infected cats.

PROBLEM: Experimental infection of cats with FIV-B-2542 produces high rates of fetal infection and reproductive failure. We hypothesized that dysregulation of placental cytokine expression occurs in FIV-infected queens, and aberrant expression potentiates inflammation and impacts pregnancy outcome. Our purpose was to quantify expression of representative pro-inflammatory cytokines (IL-6, IL-12p35, and IL-1ß), IL-10 (anti-inflammatory), and the chemokine SDF-1α in early- and late-term placental tissues. METHOD OF STUDY: Real-time reverse transcriptase PCR was used to measure gene expression in placental tissues. RESULTS: Increased expression of IL-6 and IL-12p35 and decreased expression of IL-10 occurred in FIV-infected tissues at early pregnancy; at late gestation, IL-6 expression increased and IL-1ß and SDF-1α decreased. At late pregnancy, IL-6 expression positively correlated with FIV load. IL-12:IL-10 ratios were higher in infected tissues at early, but not late pregnancy. Fetal non-viability accompanied decreased IL-12p35 and SDF-1α expression at both stages and decreased IL-12:IL-10 ratio at late pregnancy. CONCLUSION: FIV infection caused a pro-inflammatory placental microenvironment at early, but not late pregnancy.

Placental immunopathology in the FIV-infected cat: a role for inflammation in compromised pregnancy?

In utero transmission of feline immunodeficiency virus (FIV) occurs frequently in queens experimentally infected with FIV-B-2542 and other FIV isolates. Fetal infection has been detected as early as 3-4 weeks gestation, and the incidence of fetal infection increases with progressing gestation. Reproductive failure occurs commonly, including fetal resorptions and developmentally-arrested fetuses, demonstrating that fetal demise occurs early in gestation. Precise, temporal immunomodulation within the placenta is essential for successful pregnancy. Placental Th1 and Th2 cytokines must be appropriately balanced, typically favoring Th2 cytokines at the maternal-fetal interface. Abnormal inflammatory cytokine expression often accompanies miscarriage. Regulatory T cells (Tregs) play an essential role in maternal tolerance of the semi-allogeneic fetus by suppressing inflammation. We are using the FIV-infected cat to examine the relationship between lentivirus-induced placental immunopathology and reproductive outcome. Using TaqMan real time reverse transcriptase (RT)-PCR, we measured relative expression of key immunomodulators in the placentas of FIV-B-2542-infected and control cats, including placentas from both viable and nonviable pregnancies. Our data associate significantly-increased expression of inflammatory cytokines with failed pregnancies, identify Treg markers in the placentas, and provide preliminary evidence that Tregs or other cells bearing similar activation markers may be involved in pregnancy maintenance. Our data suggest that placental inflammation in the FIV-infected cat may compromise pregnancy.

Maternal hematological and virological characteristics during early feline immunodeficiency virus (FIV) infection of cats as predictors of fetal infection and reproductive outcome at early gestation.

The FIV-infected cat is a small animal model for HIV mother-to-child transmission (MTCT) because the two lentiviruses are biologically related and produce similar clinical syndromes. Both viruses are vertically transmissible and may negatively impact reproductive outcome. Maternal hematological and virological parameters are predictors of MTCT in HIV-infected women. Our purpose was to determine whether similar maternal characteristics during early pregnancy in FIV-infected cats influence pregnancy outcome. We inoculated 10 cats with FIV-B-2542; 10 cats were uninoculated. We quantified longitudinal CD4:CD8 T cell ratios, proviral load, and plasma viremia, monitored longitudinal serostatus, and documented clinical and reproductive outcome during early pregnancy. Inoculated queens were seropositive and provirus positive by week 4 post-infection (p.i.). CD4:CD8 ratios were depressed in the infected group by month 3.5 p.i. Proviral load was variable in the animals throughout the course of infection; plasma viremia was below the level of detection in all animals. Reduced litter sizes and increased fetal demise occurred in infected queens. Viral RNA, but not proviral DNA, was detected in representative placentas (14 of 14; 100%) and fetuses (12 of 14; 86%) collected from infected queens. However, maternal virological and hematological characteristics did not correlate either positively or negatively with reproductive outcome.

Expression of CD134 and CXCR4 mRNA in term placentas from FIV-infected and control cats.

Feline immunodeficiency virus (FIV) causes a natural infection of domestic cats that resembles HIV-1 in pathogenesis and disease progression. Feline AIDS is characterized by depression of the CD4+ T cell population and fatal opportunistic infections. Maternal-fetal transmission of FIV readily occurs under experimental conditions, resulting in infected viable kittens and resorbed or arrested fetal tissues. Although both FIV and HIV use the chemokine receptor CXCR4 as a co-receptor, FIV does not utilize CD4 as the primary receptor. Rather, CD134 (OX40), a T cell activation antigen and co-stimulatory molecule, is the primary receptor for FIV. We hypothesized that placental expression of CD134 and CXCR4 may render the placenta vulnerable to FIV infection, possibly facilitating efficient vertical transmission of FIV, and impact pregnancy outcome. The purpose of this project was to quantify the relative expression of CD134 and CXCR4 mRNA from the term placentas of three groups of cats: uninfected queens producing viable offspring, experimentally-infected queens producing only viable offspring, and experimentally-infected queens producing viable offspring among mostly non-viable fetuses. Total RNA was extracted from term placental tissues from all groups of cats. Real-time one-step reverse transcriptase-PCR was used to measure gene expression. The FIV receptors CD134 and CXCR4 were expressed in all late term feline placental tissues. Placentas from FIV-infected queens producing litters of only viable offspring expressed more CD134 and CXCR4 mRNA than those from uninfected queens, suggesting that infection may cause upregulation of the receptors. On the other hand, placentas from FIV-infected cats with non-successful pregnancies expressed similar levels of CD134 mRNA and slightly less CXCR4 mRNA than those from uninfected queens. Thus, it appears that cells expressing these receptors may play a role in pregnancy maintenance.

The feline immunodeficiency virus-infected cat: a model for lentivirus-induced placental immunopathology and reproductive failure (mini-review).

PROBLEM: Pediatric human immunodeficiency virus (HIV) infection is largely a result of transplacental transmission, and pregnancy perturbation is more frequent in HIV-infected women. Dysregulation of placental immunology may occur during HIV infection, possibly facilitating HIV vertical transfer and miscarriage. The (FIV)-infected cat is a useful small-animal model for HIV pathogenesis because the viruses share common biological and clinical features. Transplacental transmission is readily achieved experimentally, resulting in a high proportion of infected offspring and frequent reproductive failure. METHOD OF STUDY: We are using this model to examine lentivirus-induced placental immunopathology to determine the role aberrant immunology plays in intrauterine transmission and pregnancy perturbation. RESULTS: Kittens were cesarean delivered from FIV-B-2542-infected and control queens at week 8 gestation (1 week short of term), and placental and fetal specimens were collected. On average, control queens delivered 3.8 kittens/litter, and 1 of 31 kittens (3.2%) was non-viable. FIV-infected queens produced 2.7 kittens/litter with 15 of 25 fetuses (60%) non-viable. The virus was detected in 14 of 15 placentas (93%) and 21 of 22 fetuses (95%) using polymerase chain reaction (PCR). Using a one-step, real time reverse transcriptase (RT)-PCR, we measured expression of representative placental T helper 1 (Th1) cytokines, interleukin (IL)-1beta and interferon (IFN)-gamma, a Th2 cytokine, IL-10, and chemokine receptor CXCR4. A comparison of placental cytokine expression between infected and control queens did not reveal differences between the two groups. However, elevated expression of Th1 cytokines and increased Th1/Th2 ratios (IL-1beta/IL-10) occurred in placentas from resorptions, indicating that increased placental Th1 cytokine expression was associated with pregnancy failure in the FIV-infected cat. CONCLUSION: The potential to establish efficient FIV in utero transmission, coupled with the parallels in immunopathology between FIV-infected cats and HIV-infected humans, suggests the usefulness of the FIV-infected cat as a cost-effective, small-animal model to study lentivirus-induced immunopathology, transplacental infection, and reproductive failure.

National Institute on Drug Abuse Conference report on placental proteins, drug transport, and fetal development.

The use of illicit and licit drugs during pregnancy is a major public health concern because of potential adverse effects on the fetus and the risk to maternal health. Because the placenta is the primary link between the mother and the conceptus and is essential for the growth and survival of the fetus, abnormalities in placental formation and function resulting from drug use could have a major influence on pregnancy outcome. At present, little information is available on the impact of abused drugs on placental biology alone or in combination with other "host" factors (eg, stress, infections). This prompted the National Institute on Drug Abuse (NIDA) to convene a meeting of experts in placental biology to review cutting-edge research with the mission to translate existing information to new clinical and research initiatives in the drug abuse field. This report summarizes the presentations and research recommendations resulting from the workshop discussions.

Confirmation of vertical transmission of bovine immunodeficiency virus in naturally infected dairy cattle using the polymerase chain reaction.

The purpose of this study was to determine whether bovine immunodeficiency virus (BIV) is vertically transmitted in naturally infected dairy cattle. Twenty-two dam/calf pairs from a Mississippi Agriculture and Forestry Experiment Station dairy were the study group. Blood samples were collected following delivery of calves, the peripheral blood leukocytes were purified from these samples, and the leukocyte DNA was used in polymerase chain reactions targeting the pol gene region of the BIV provirus. Southern blotting and hybridization were used to confirm the BIV specificity of the amplified fragments. BIV provirus was detected in 14 of 22 calves (64%), demonstrating vertical transmission. Eight of the calves were disqualified from the final interpretation of transplacental transfer because they may have nursed their mothers prior to blood collection, allowing the possibility of lactogenic transfer of the virus. Transplacental transmission of BIV was identified in 6 of 22 calves (27%).