Pregnancy results in increased inflammation in the lungs and enhanced viral growth in lungs but not detected in placenta.

<p>A. Lungs from mock infected (a, d) and infected (b, c, e, f) pregnant and non-pregnant mice were stained with hematoxylin and eosin (H&E) 4 d.p.i. with mouse-adapted A/Brisbane/59/07 or mouse lung lysate at an equivalent dilution. (b, e) Lung sections at 10x magnification; (c, f) Inset boxes in (b, e) at 40x magnification represent lung structures. B. Viral load was quantified from lung lysates (N = 5 per group) 4 d.p.i via plaque assay in MDCK-derived cell lines and normalized per gram of tissue weight. C. Viral RNA was isolated from placental lysates (N = 4–5 per group) and probed for the presence of M gene RNA (A/Brisbane/59/07) via qPCR. Influenza-virus specific RNA was not detected below the negative control threshold (RNA isolated from mock-infected BALB/c lungs) or positive control threshold (RNA isolated from infected BALB/c lungs). Inf.: Infected mice; NP: Non-pregnant mice.</p

H1N1 influenza virus infection results in adverse pregnancy outcomes by disrupting tissue-specific hormonal regulation

<div><p>Increased susceptibility to influenza virus infection during pregnancy has been attributed to immunological changes occurring before and during gestation in order to “tolerate” the developing fetus. These systemic changes are most often characterized by a suppression of cell-mediated immunity and elevation of humoral immune responses referred to as the Th1-Th2 shift. However, the underlying mechanisms which increase pregnant mothers’ risk following influenza virus infection have not been fully elucidated. We used pregnant BALB/c mice during mid- to late gestation to determine the impact of a sub-lethal infection with A/Brisbane/59/07 H1N1 seasonal influenza virus on completion of gestation. Maternal and fetal health status was closely monitored and compared to infected non-pregnant mice. Severity of infection during pregnancy was correlated with premature rupture of amniotic membranes (PROM), fetal survival and body weight at birth, lung viral load and degree of systemic and tissue inflammation mediated by innate and adaptive immune responses. Here we report that influenza virus infection resulted in dysregulation of inflammatory responses that led to pre-term labor, impairment of fetal growth, increased fetal mortality and maternal morbidity. We observed significant compartment-specific immune responses correlated with changes in hormonal synthesis and regulation. Dysregulation of progesterone, COX-2, PGE2 and PGF2α expression in infected pregnant mice was accompanied by significant remodeling of placental architecture and upregulation of MMP-9 early after infection. Collectively these findings demonstrate the potential of a seasonal influenza virus to initiate a powerful pro-abortive mechanism with adverse outcomes in fetal health.</p></div

Improved Humoral Immunity and Protection against Influenza Virus Infection with a 3d Porous Biomaterial Vaccine.

New vaccine platforms that activate humoral immunity and generate neutralizing antibodies are required to combat emerging pathogens, including influenza virus. A slurry of antigen-loaded hydrogel microparticles that anneal&nbsp;to form a porous scaffold with high surface area for antigen uptake by infiltrating immune cells as the biomaterial degrades is demonstrated to enhance humoral immunity. Antigen-loaded-microgels elicited a robust cellular humoral immune response, with increased CD4+ T follicular helper (Tfh) cells and prolonged germinal center (GC) B cells comparable to the commonly used adjuvant, aluminum hydroxide (Alum). Increasing the weight fraction of polymer material led to increased material stiffness and&nbsp;antigen-specific antibody titers superior to Alum. Vaccinating mice with inactivated influenza virus loaded into this more highly cross-linked formulation elicited a strong antibody response and provided protection against a high dose viral challenge. By tuning physical and chemical properties, adjuvanticity can be enhanced leading to humoral immunity and protection against a pathogen, leveraging two different types of antigenic material: individual protein antigen and inactivated virus. The flexibility of the platform may enable design of new vaccines to enhance innate and adaptive immune cell programming to generate and tune high affinity antibodies, a promising approach to generate long-lasting immunity

Infection damages placental architecture and increases activation of structural protein degrading matrix metalloproteinases (MMPs).

<p>A. Placentas from uninfected [<b>a, b, c, g</b>] (N = 8) and infected [<b>d, e, f, h</b>] (N = 8) pregnant mice (E16) were embedded in paraffin, sectioned in 4 μM, H&E stained and imaged with a Zeiss brightfield microscope. Infection increases gaps within the spongiotrophoblast layer (S) between the decidual layer (D) and the placental labyrinth (L) [<b>a, d</b>; <b>10x</b>]. Gaps are marked by fetal endothelial nuclei suspended in tissue [stars] and empty space [triangles] distinct from maternal blood sinuses and fetal blood vessels [<b>d, 10x; b, e, f, 20x</b>]. Fibrinoid necrosis [squares] beneath the decidual layer was increased in placentae from infected mice [<b>h, inset of box in f</b>] compared to uninfected mice [<b>g, inset of box in c</b>]. B. Histology slides were randomized prior to imaging and scored blindly for the incidence of fetal endothelial death (FED; N = 10, p = 0.012), degradation of the spongiotrophoblast layer (Degradation; N = 10, p>0.05), and fibrinoid necrosis (FN; N = 10, p>0.05). C. Protein expression of matrix metalloproteinases (MMP-2, MMP-9; active forms denoted with *) in placentas in uninfected and infected mice at E16 quantified by Western blot. Two-way ANOVA: protein maturation, p = 0.064, ns; infection, p = 0.007, **.</p

Infection and pregnancy increase COX-2 expression in the lungs.

<p>(A) Placental and (C) lung lysates collected 4 d.p.i were probed via Western blot for COX-2 expression. Protein expression is represented as isoform density normalized to loading control anti-beta actin (42 kD) for each sample. (B) COX-2 69 kD expression was the only detectable band in these placental lysates (Uninfected, N = 4; Infected, N = 6) and it was significantly lower in infected mice. COX-2 isoforms (D) 69 kD and (E) 45 kD were detected in lung lysates (N = 5 per each group except NP Infected mice; N = 4). Group annotations are as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006757#ppat.1006757.g007" target="_blank">Fig 7</a>. P-values were determined by Student’s t-test.</p

Influenza infection inhibits gestational development and offspring health.

<p>(A) Body weight (BW) fluctuations of pregnant BALB/c mice were tracked over the course of gestation. At day 12 of gestation (dashed vertical line) a cohort of pregnant mice (N = 6) was infected with 2xLD₅₀ A/Brisbane/59/07. Weight changes were compared to uninfected pregnant controls (N = 12). <i>P</i>-values were calculated by two-way ANOVA. Error bars represent Standard Error of Mean (SEM). (B) Mean gestational length for infected (N = 13) and uninfected (N = 9) groups was calculated based on the dates of successful copulation and parturition of offspring. <i>P</i>-value was calculated by Student’s t-test. (C) The body weights (BW) of pups born to infected (N = 16 to 6 mothers) and uninfected mothers (N = 53 to 12 mothers) were recorded upon delivery. (D) The BW distribution of pups was categorized based on gestational age. Pups less than 1.25g were designated SGA. Pups greater than 1.25g were considered healthy. Average BW and SEM were plotted and <i>P</i> = value was calculated by Student’s t-test for C and D. (E) Percent distribution of health status of pups born to uninfected and infected mothers.</p

Infection changes pregnancy-supportive PIBF expression in the placenta and lungs.

<p>PIBF isoforms (90 kD, 66 kD, 55 kD) were detected using Western blots of A. placental and C. lung lysates. Protein expression is represented as isoform density normalized to loading control anti-beta actin (42 kD) for each sample. The effect of infection was measured by dividing the average density of bands from tissues from infected mice by the uninfected controls for both pregnant and non-pregnant mice. (B) Relative protein normalized to actin for uninfected (N = 4) and infected (N = 6) placental lysates. (D) Relative protein normalized to actin for lung lysates (N = 5 per group. (E) Ratio of infected vs uninfected lung lysates (normalized to actin). Uninf: uninfected; NP: Non-pregnant; Inf: Infected mice. P-values were determined by Student’s t-test.</p

Viral infection disproportionately reduces cytokine and hormone expression in the sera and lungs during pregnancy.

<p>Hormone expression in sera was quantified via ELISA in uninfected and infected non-pregnant mice for A. progesterone; C. prostaglandin F2α (PGF2α); and D. prostaglandin E2 (PGE2). Cytokine and chemokine expression in sera was quantified via Bio-Rad 23-plex assay in B. uninfected pregnant (E16) (N = 5) and non-pregnant mice (N = 5); E. uninfected and infected pregnant (E16; 4 d.p.i) (N = 5 per group); and F. infected (4 d.p.i) non-pregnant and pregnant (E16) mice (N = 5 per group). Student’s t-test was performed between selected groups and significance noted above asterisk brackets. Inf.: infected; Uninf: uninfected; NP: non-pregnant; P: pregnant mice.</p

Infection increases placental expression of contraction-inducing PGF2α and reduces pregnancy-supportive progesterone.

<p>Placental lysates were isolated from uninfected and infected pregnant mice (E16). Hormone expression was quantified via ELISA for A. progesterone (N = 6 per group); B. prostaglandin F2α (PGF2α) (N = 9, uninfected, N = 7, infected) and C. prostaglandin E2 (PGE2) (N = 9 per group). Student’s t-test was performed between selected groups and significance noted above asterisk brackets.</p

Pregnancy results in increased inflammation in the lungs and enhanced viral growth in lungs but not detected in placenta.

<p>A. Lungs from mock infected (a, d) and infected (b, c, e, f) pregnant and non-pregnant mice were stained with hematoxylin and eosin (H&E) 4 d.p.i. with mouse-adapted A/Brisbane/59/07 or mouse lung lysate at an equivalent dilution. (b, e) Lung sections at 10x magnification; (c, f) Inset boxes in (b, e) at 40x magnification represent lung structures. B. Viral load was quantified from lung lysates (N = 5 per group) 4 d.p.i via plaque assay in MDCK-derived cell lines and normalized per gram of tissue weight. C. Viral RNA was isolated from placental lysates (N = 4–5 per group) and probed for the presence of M gene RNA (A/Brisbane/59/07) via qPCR. Influenza-virus specific RNA was not detected below the negative control threshold (RNA isolated from mock-infected BALB/c lungs) or positive control threshold (RNA isolated from infected BALB/c lungs). Inf.: Infected mice; NP: Non-pregnant mice.</p