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Science 339 (6123): 1088-1092

Copyright © 2013 by the American Association for the Advancement of Science

Interferon-{varepsilon} Protects the Female Reproductive Tract from Viral and Bacterial Infection

Ka Yee Fung1,*, Niamh E. Mangan1,*, Helen Cumming1, Jay C. Horvat2, Jemma R. Mayall2, Sebastian A. Stifter1, Nicole De Weerd1, Laila C. Roisman1,3, Jamie Rossjohn3, Sarah A. Robertson4, John E. Schjenken4, Belinda Parker5,6, Caroline E. Gargett7,8, Hong P. T. Nguyen7, Daniel J. Carr9, Philip M. Hansbro2, and Paul J. Hertzog1,{dagger}

1 Centre for Innate Immunity and Infectious Diseases, Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australia.
2 Centre for Asthma and Respiratory Disease and Hunter Medical Research Institute, The University of Newcastle, Newcastle, New South Wales, Australia.
3 Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
4 Robinson Institute and School of Paediatrics and Reproductive Health, University of Adelaide, South Australia, Australia.
5 Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia.
6 Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Australia.
7 The Ritchie Centre, Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australia.
8 Department of Obstetrics and Gynaecology, Monash University, Clayton, Victoria, Australia.
9 Department of Ophthalmology and Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.


Figure 1
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Fig. 1. Interferon-{varepsilon} signals through the type I IFN receptor but is not induced by TLR ligands nor regulated by IRFs. (A and B) BMDMs from WT, Ifnar1/, and Ifnar2/ C57BL/6 mice were stimulated with recombinant mouse Ifn-α1, Ifn-β, or Ifn-{varepsilon} (0.1 μg/ml) for 3 hours. (A) Irf-7 and (B) 2'5'oas expression was measured by quantitative real-time fluorescence polymerase chain reaction (qRT-PCR). Data are expressed as mean ± SEM (error bars) of at least three independent experiments. (C) BMDMs from C57BL/6 WT mice were treated with a range of TLR ligands or transfected with Poly (I:C) and Poly (dA:dT) for 3 hours at 37°C. Ifn-β, Il-6, and Ifn-{varepsilon} were measured by qRT-PCR. Data are expressed as mean ± SEM of at least three independent experiments. (D) Luciferase reporter plasmids containing Ifn-α, Ifn-β, p125, or Ifn-{varepsilon} were cotransfected with empty vector or IRF-3, IRF-7, or IRF-5 expression vectors into HEK293 cells. Data are expressed as mean ± SEM. All values are means of at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (unpaired Student's t test).

 

Figure 2
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Fig. 2. Interferon-{varepsilon} is expressed in the FRT in both mice and humans. (A) Mouse organs were harvested and Ifn-{varepsilon} expression was measured by qRT-PCR, normalized to 18S RNA and presented relative to Ifn-{varepsilon} expression in kidney. Data are expressed as the mean ± SEM (error bars) of at least three individual mice. (B) Representative images showing Ifn-{varepsilon} localization in uterine tissue (at estrous stage) of WT and Ifn-{varepsilon}/ C57BL/6 mice by immunohistochemistry. Scale bars, 50 μm. These images are representative of at least five individual mice. (C and D) Ifn-{varepsilon} expression was measured by qRT-PCR in mouse uterus at different stages of (C) the estrous cycle and (D) pregnancy. Data are expressed as mean ± SEM of at least three separate experiments. (E) Ifn-{varepsilon} expression was determined by qRT-PCR in ovariectomized (OVX) mice and OVX mice treated with estrogen (E OVX). Data are expressed as mean ± SEM of at least six individual mice and are representative of at least two separate experiments. (F) A cDNA panel of human tissues was examined for IFN-{varepsilon} expression by qRT-PCR, and the results were expressed relative to IFN-{varepsilon} expression in the kidney. (G) Epithelial cells were isolated from endometrial samples of postmenopausal women or those at different stages of the menstrual cycle, and IFN-{varepsilon} expression was measured by qRT-PCR. Values are presented relative to IFN-{varepsilon} expression in the human endometrial cell line ECC-1. Data are expressed as mean ± SEM of six individual patient samples. *P < 0.05; **P < 0.01; ***P < 0.001 [(A to F) unpaired Student's t test,(G) Mann-Whitney U test].

 

Figure 3
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Fig. 3. Ifn-{varepsilon}/ mice are more susceptible to HSV-2 vaginal infection. (A) Isg15, (B) Irf-7, and (C) 2'5'oas expression between WT and Ifn-{varepsilon}/ C57BL/6 mice was determined by qRT-PCR. The values represent means ± SEM (error bars) of four individual mice. (D to F and H) Mice pretreated with medroxyprogesterone acetate (Depo-Ralovera, Pfizer) at day –5 were infected with HSV-2 (D, E, H) at a level of 2400 or (F) 24 pfu per mouse on day 0. (D) Representative images demonstrating overt genital lesions, redness, and swelling in HSV-2–infected Ifn-{varepsilon}/ mice at day 7 pi; these qualities are absent in C57BL/6 WT mice. (E)Clinical scores of WT and Ifn-{varepsilon}/ C57BL/6 mice during the 7-day course of infection. Data are means ± SEM of five individual mice and are representative of at least three separate experiments. (F and G) HSV-2 titers (pfu) from vaginal tissue of WT and Ifn-{varepsilon}/ C57BL/6 mice infected with (F) 2400 and (G) 24 pfu, respectively, at day 3 pi were determined by titration of clarified vaginal tissue samples on Vero cell monolayers by plaque assay. Data are expressed as mean ± SEM of five individual mice. (H) HSV-2 titers from homogenates of vaginal tissue, spinal cord, and brain stem of infected WT and Ifn-{varepsilon}/ C57BL/6 mice at day 7 pi were determined as in (F) and (G). Data are expressed as mean ± SEM of five individual mice. *P < 0.05; **P < 0.01 (unpaired Student's t test).

 

Figure 4
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Fig. 4. Ifn-{varepsilon}/ mice are more susceptible to Chlamydia muridarum vaginal infection. (A to D) Mice were pretreated with progesterone at day –7 and infected intravaginally with 5 x 104 inclusion-forming units (IFU) of C. muridarum. (A) Clinical scores were recorded daily for 30 days. Data are means ± SEM (error bars) of at least six individual mice. (B) Bacterial recovery from vaginal swabs of WT and Ifn-{varepsilon}/ C57BL/6 mice at different time points, as determined by qRT-PCR for bacterial major outer membrane protein. Data are means ± SEM of at least six individual mice. (C) Bacterial recovery, as measured by qRT-PCR from vaginal lavage at days 1 and 3 pi. Data are means ± SEM of at least six individual mice. (D) Bacterial 16S RNA from the uterine horns of WT and Ifn-{varepsilon}/ C57BL/6 mice at 30 days pi was examined by qRT-PCR. Data are means ± SEM of at least six individual mice. (E) WT C57BL/6 mice were pretreated with progesterone at day –7 and treated intravaginally with rIfn-{varepsilon} (2 or 4 μg) 6 hours before C. muridarum infection. Bacterial recovery from the vaginal lavage at day 3 pi was measured by qRT-PCR. PBS, phosphate-buffered saline. Data are means ± SEM of at least six individual mice. *P < 0.05; **P < 0.01; ***P < 0.001 (unpaired Student's t test).

 


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