Natalia P. Smirnova

Uterine Vein Infusion of Interferon Tau (IFNT) Extends Luteal Life Span in Ewes

Interferon tau (IFNT) from the ovine conceptus has paracrine actions on the endometrium that alter release of prostaglandin F2alpha (PGF) and protect the corpus luteum (CL). Antiviral activity in uterine vein blood and expression of interferon-stimulated genes (ISGs) in CL is greater in pregnant than in nonpregnant ewes. We hypothesized that IFNT contributes to antiviral activity in uterine vein blood and has endocrine actions on the CL. Preadsorption of IFNT with antiserum against recombinant ovine (ro) IFNT revealed that antiviral activity in uterine vein blood from pregnant ewes was mediated by IFNT. Endocrine actions of IFNT were examined after infusing either roIFNT or bovine serum albumin (BSA; 200 μg/24 h; mini-osmotic pump) into the uterine vein of nonpregnant ewes from Day 10 to Day 11 postestrus. The abundance of ISG15 mRNA and protein was greater in CL (P < 0.05) from ewes receiving 24-h roIFNT infusion compared to that from ewes receiving 24-h BSA infusion. Injection of PGF at 12 h following insertion of mini-osmotic pumps resulted in a decline in serum progesterone concentrations 6 through 12 h later in BSA-infused ewes; however, in roIFNT-infused ewes, a similar decline in progesterone concentrations at 6 h was followed by recovery to control values at 12 h. Ewes then received infusions (200 μg/day) of either roIFNT or BSA for 7 days beginning on Day 10 of the estrous cycle. All BSA-infused ewes returned to estrus by Day 19, whereas 80% of roIFNT-infused ewes maintained luteal-phase concentrations of progesterone through Day 32. In conclusion, IFNT is released from the uterus into the uterine vein and acts through an endocrine mechanism to induce ISGs in the CL and delay luteolysis.

Endocrine Delivery of Interferon Tau Protects the Corpus Luteum from Prostaglandin F2 Alpha-Induced Luteolysis in Ewes

ABSTRACT Paracrine release of ovine interferon tau (oIFNT) from the conceptus alters release of endometrial prostaglandin F2 alpha (PGF) and prevents luteolysis. Endocrine release of oIFNT into the uterine vein occurs by Day 15 of pregnancy and may impart resistance of the corpus luteum (CL) to PGF. It was hypothesized that infusion of recombinant oIFNT (roIFNT) into the uterine or jugular veins on Day 10 of the estrous cycle would protect the CL against exogenous PGF-induced luteolysis. Osmotic pumps were surgically installed in 24 ewes to deliver bovine serum albumin (BSA; n = 12) or roIFNT (200 μg/day; n = 12) for 24 h into the uterine vein. Six ewes in each treatment group received a single injection of PGF (4 mg/58 kg body weight) 12 h after pump installation. In a second experiment, BSA or roIFNT was delivered at 20 or 200 μg/day into the uterine vein or 200 μg/day into the jugular vein for 72 h in 30 ewes. One half of these ewes received an injection of PGF 24 h after pump installation. Concentrations of progesterone in serum declined in BSA-treated ewes injected with PGF, but were sustained in all ewes infused with 20 μg/day of roIFNT into the uterine vein and 200 μg of roIFNT into the jugular vein followed 24 h later with injection of PGF. All concentrations of roIFNT and modes of delivery (uterine or jugular vein) increased luteal concentrations of IFN-stimulated gene (i.e., ISG15) mRNA. Infusion of 200 μg of IFNT over 24 h induced greater mRNA concentrations for cell survival genes, such as BCL2-like 1 (BCL2L1 or Bcl-xL), serine/threonine kinase (AKT), and X-linked inhibitor of apoptosis (XIAP) and decreased prostaglandin F receptor (PTGFR) mRNA concentrations, when compared to controls. It is concluded that endocrine delivery of roIFNT, regardless of route (uterine or jugular vein), effectively protects CL from the luteolytic actions of PGF by mechanisms that involve ISGs and stabilization of cell survival genes.

Transplacental infection with non-cytopathic bovine viral diarrhoea virus types 1b and 2: Viral spread and molecular neuropathology

Infection with bovine viral diarrhoea virus (BVDV) represents a reproducible natural animal model in which to study mechanisms of transplacental viral infection. In the present study, BVDV-seronegative heifers were challenged intranasally with non-cytopathic BVDV of genotype 1b or 2. Fetuses were retrieved by caesarean section 7-114 days post-challenge of the dam and subjected to virological, histopathological and immunohistochemistry(IHC) studies. Gross and histopathological changes were only seen in fetuses infected at gestational age 75-85 days and retrieved at gestational age 190 days. Viral antigen could be detected in most tissues from 14 days post-infection, but the primary target organs for histopathological changes were brain, liver and spleen. In the brain, microscopical changes included leucomalacia and macrophage infiltration of meninges and neuropil. Viral antigen was detected in neurons, oligodendrocyte precursors and infiltrating macrophages. IHC revealed normal to slightly increased expression of hypoxia-inducible factor-1alpha (HIF-1alpha) in the infected fetuses, with evidence of neuronal apoptosis and induction of inducible nitric oxide synthase (iNOS) and phospho-p38alpha mitogen-activated protein kinase (MAPK). These findings suggest that hypoxia may play only a limited role in the pathogenesis of the neural lesions. By contrast, virus-induced cytokine cascades, as part of the fetal innate immune response, and apoptosis of neurons and glial precursor cells may be central to the development of lesions.

Maternal and fetal response to fetal persistent infection with bovine viral diarrhea virus

Citation Hansen TR, Smirnova NP, Van Campen H, Shoemaker ML, Ptitsyn AA, Bielefeldt‐Ohmann H. Maternal and fetal response to fetal persistent infection with bovine viral diarrhea virus. Am J Reprod Immunol 2010

Differential expression of the type I interferon pathway during persistent and transient bovine viral diarrhea virus infection.

Persistent infection with bovine viral diarrhea virus (BVDV) serves as a reservoir for the perpetuation of infection in cattle populations and causes a range of adverse effects on the health of the host. To study the interactions of the virus with the host, gene expression was compared in the blood of persistently infected (PI) and uninfected steer, and in the blood and tissues of PI fetuses, transiently infected (TI), and uninfected bovine fetuses. Microarray analysis of PI steer blood revealed differential gene expression indicative of an interferon (IFN) response including genes involved in cell cycle regulation, which may contribute to long-term adverse effects. Upregulation of IFN-stimulated genes (e.g., ISG15, PKR) and RNA helicases (RIG-I, LGP2, MDA-5) was identified in both PI fetal and steer blood in comparison to controls, indicating a continued stimulation of the innate antiviral response as a result of the persistent viremia. ISG15 was studied in further detail, implicating reticular cells as basal producers of ISG15 in the spleen, in addition to endothelial and macrophage-like cells in infected spleen. Consequences of chronic IFN pathway activation in PI cattle may include growth- and immunosuppression, the pathogenesis of which is still poorly understood. This study lends new insights into the interactions between BVDV and its host, and can serve as a model for studies of the role of the IFN system in persistent infections.

Pregnancy-associated genes contribute to antiluteolytic mechanisms in ovine corpus luteum

The hypothesis that ovine luteal gene expression differs due to pregnancy status and day of estrous cycle was tested. RNA was isolated from corpora lutea (CL) on days 12 and 14 of the estrous cycle (NP) or pregnancy (P) and analyzed with the Affymetrix bovine microarray. RNA also was isolated from luteal cells on day 10 of estrous cycle that were cultured for 24 h with luteolytic hormones (OXT and PGF) and secretory products of the conceptus (IFNT and PGE2). Differential gene expression (>1.5-fold, P < 0.05) was confirmed using semiquantitative real-time PCR. Serum progesterone concentrations decreased from day 12 to day 15 in NP ewes (P < 0.05) reflecting luteolysis and remained >1.7 ng/ml in P ewes reflecting rescue of the CL. Early luteolysis (days 12-14) was associated with differential expression of 683 genes in the CL, including upregulation of SERPINE1 and THBS1. Pregnancy on day 12 (55 genes) and 14 (734 genes) also was associated with differential expression of genes in the CL, many of which were ISGs (i.e., ISG15, MX1) that were induced when culturing luteal cells with IFNT, but not PGE2. Finally, many genes, such as PTX3, IL6, VEGF, and LHR, were stabilized during pregnancy and downregulated during the estrous cycle and in response to culture of luteal cells with luteolytic hormones. In conclusion, pregnancy circumvents luteolytic pathways and activates or stabilizes genes associated with interferon, chemokine, cell adhesion, cytoskeletal, and angiogenic pathways in the CL.

Persistent fetal infection with bovine viral diarrhea virus differentially affects maternal blood cell signal transduction pathways

The consequences of viral infection during pregnancy include impact on fetal and maternal immune responses and on fetal development. Transplacental infection in cattle with noncytopathic bovine viral diarrhea virus (ncpBVDV) during early gestation results in persistently infected (PI) fetuses with life-long viremia and susceptibility to infections. Infection of the fetus during the third trimester or after birth leads to a transient infection cleared by a competent immune system. We hypothesized that ncpBVDV infection and presence of an infected fetus would alter immune response and lead to downregulation of proinflammatory processes in pregnant dams. Naïve pregnant heifers were challenged with ncpBVDV2 on day 75 (PI fetus) and day 175 [transiently infected (TI) fetus] or kept uninfected (healthy control fetus). Maternal blood samples were collected up to day 190 of gestation. Genome-wide microarray analysis of gene expression in maternal peripheral white blood cells, performed on days 160 and 190 of gestation, revealed multiple signal transduction pathways affected by ncpBVDV infection. Acute infection and presence of a TI fetus caused upregulation of the type I interferon (IFN) pathway genes, including dsRNA sensors and IFN-stimulated genes. The presence of a PI fetus caused prolonged downregulation of chemokine receptor 4 (CXCR4) and T cell receptor (TCR) signaling in maternal blood cells. We conclude that: 1) infection with ncpBVDV induces a vigorous type I IFN response, and 2) presence of a PI fetus causes downregulation of important signaling pathways in the blood of the dam, which could have deleterious consequences on fetal development and the immune response.

Temporal Release, Paracrine and Endocrine Actions of Ovine Conceptus-Derived Interferon-Tau During Early Pregnancy

ABSTRACT The antiviral activity of interferon (IFN) increases in uterine vein serum (UVS) during early pregnancy in sheep. This antiviral activity in UVS collected on Day 15 of pregnancy is blocked by anti-IFN-tau (anti-IFNT) antibodies. Conceptus-derived IFNT was hypothesized to induce IFN-stimulated gene (ISG) expression in endometrium and extrauterine tissues during pregnancy. To test this hypothesis, blood was collected from ewes on Days 12–16 of the estrous cycle or pregnancy. Serum progesterone was >1.7 ng/ml in pregnant (P) and nonpregnant (NP) ewes until Day 13, then declined to <0.6 ng/ml by Day 15 in NP ewes. A validated IFNT radioimmunoassay detected IFNT in uterine flushings (UFs) on Days 13–16 and in UVS on Days 15–16 of pregnancy. IFNT detection in UF correlated with paracrine induction of ISGs in the endometrium and occurred prior to the inhibition of estrogen receptor 1 and oxytocin receptor expression in uterine epithelia on Day 14 of pregnancy. Induction of ISG mRNAs in corpus luteum (CL) and liver tissue occurred by Day 14 and in peripheral blood mononuclear cells by Day 15 in P ewes. Expression of mRNAs for IFN signal transducers and ISGs were greater in the CL of P than that of NP ewes on Day 14. It is concluded that: 1) paracrine actions of IFNT coincide with detection of IFNT in UF; 2) endocrine action of IFNT ensues through induction of ISGs in peripheral tissues; and 3) IFNT can be detected in UVS, but not until Days 15–16 of pregnancy, which may be limited by the sensitivity of the IFNT radioimmunoassay.

Development of fetal and placental innate immune responses during establishment of persistent infection with bovine viral diarrhea virus

Transplacental viral infections are dependent upon complex interactions between feto-placental and maternal immune responses and the stage of fetal development at which the infection occurs. Bovine viral diarrhea virus (BVDV) has the ability to cross the placenta and infect the fetus. Infection early in gestation with non-cytopathic (ncp) BVDV leads to persistent infection. Establishment of fetal persistent infection results in life-long viremia, virus-specific immunotolerance, and may have detrimental developmental consequences. We have previously shown that heifers infected experimentally with ncp BVDV type 2 on d. 75 of gestation had transient robust up-regulation of the type I interferon (IFN) stimulated genes (ISGs) 3-15 days after viral inoculation. Blood from persistently infected (PI) fetuses, collected 115 days post maternal infection, demonstrated moderate chronic up-regulation of ISGs. This infection model was used to delineate timing of the development of innate immune responses in the fetus and placenta during establishment of persistent infection. It was hypothesized that: (i) chronic stimulation of innate immune responses occurs following infection of the fetus and (ii) placental production of the type I IFN contributes to up-regulation of ISGs in PI fetuses. PI fetuses, generated by intranasal inoculation of pregnant heifers with ncp BVDV, and control fetuses from uninfected heifers, were collected via Cesarean sections on d. 82, 89, 97, 192, and 245 of gestation. Fetal viremia was confirmed starting on d. 89. Significant up-regulation of mRNA encoding cytosolic dsRNA sensors -RIG-I and MDA5 - was detected on d. 82-192. Detection of viral dsRNA by cytosolic sensors leads to the stimulation of ISGs, which was reflected in significant up-regulation of ISG15 mRNA in fetal blood on d. 89, 97, and 192. No difference in IFN-α and IFN-β mRNA concentration was found in fetal blood or caruncular tissue, while a significant increase in both IFN-α and IFN-β mRNA was seen in cotyledons from PI fetuses on d. 192. It is concluded that fetuses respond to early gestational ncp BVDV infection by induction of the type I IFN pathway, resulting in chronic up-regulation of ISGs. Cotyledonary tissue contributes to up-regulation of ISGs by increased production of IFNs. The innate immune response might partially curtail viral replication in PI fetuses, but is not able to eliminate the virus in the absence of a virus-specific adaptive immune response.

Neuro-invasion by a ‘Trojan Horse’ strategy and vasculopathy during intrauterine flavivirus infection

The central nervous system (CNS) is a major target of several important human and animal viral pathogens causing congenital infections. However, despite the importance of neuropathological outcomes, for humans in particular, the pathogenesis, including mode of neuro‐invasion, remains unresolved for most congenital virus infections. Using a natural model of congenital infection with an RNA virus, bovine viral diarrhoea virus in pregnant cattle, we sought to delineate the timing and mode of virus neuro‐invasion of and spread within the brain of foetuses following experimental respiratory tract infection of the dams at day 75 of pregnancy, a time of maximal risk of tissue pathology without foetal death. Virus antigen was first detected in the foetal brains 14 days postinfection of dams and was initially restricted to amoeboid microglial cells in the periventricular germinal layer. The appearance of these cells was preceded by or concurrent with vasculopathy in the same region. While the affected microvessels were negative for virus antigen, they expressed high levels of the type I interferon‐stimulated protein ISG15 and eventually disappeared in parallel with the appearance of microcavitary lesions. Subsequently, the virus spread to neurons and other glial cells. Our findings suggest that the virus enters the CNS via infected microglial precursors, the amoeboid microglial cells, in a ‘Trojan horse’ mode of invasion and that the microcavitary lesions are associated with loss of periventricular microvasculature, perhaps as a consequence of high, unrestricted induction of interferon‐regulated proteins.