Impaired heterologous immunity in aged ferrets during sequential influenza A H1N1 infection

The major burden of influenza morbidity resides within the elderly population. The challenge managing influenza-associated illness in the elderly is the decline of immune function, where mechanisms leading to immunological senescence have not been elucidated. To better represent the immune environment, we investigated clinical morbidity and immune function during sequential homologous and heterologous H1N1 influenza infection in an aged ferret model. Our findings demonstrated experimentally that aged ferrets had significant morbidity during monosubtypic heterologous 2° challenge with significant weight loss and respiratory symptoms. Furthermore, increased clinical morbidity was associated with slower and shorter hemagglutinin antibody generation and attenuated type 1 T-cell gene responses in peripheral blood. These results revealed dampened immune activation during sequential influenza infection in aged ferrets. With the presence of an aged model, dissecting clinical morbidity, viral dynamics and immune response during influenza infection will aid the development of future prophylactics such as age specific influenza vaccines

Inflammatory Cytokine Expression Is Associated with Chikungunya Virus Resolution and Symptom Severity

The Chikungunya virus infection zones have now quickly spread from Africa to parts of Asia, North America and Europe. Originally thought to trigger a disease of only mild symptoms, recently Chikungunya virus caused large-scale fatalities and widespread economic loss that was linked to recent virus genetic mutation and evolution. Due to the paucity of information on Chikungunya immunological progression, we investigated the serum levels of 13 cytokines/chemokines during the acute phase of Chikungunya disease and 6- and 12-month post-infection follow-up from patients of the Italian outbreak. We found that CXCL9/MIG, CCL2/MCP-1, IL-6 and CXCL10/IP-10 were significantly raised in the acute phase compared to follow-up samples. Furthermore, IL-1β, TNF-α, Il-12, IL-10, IFN-γ and IL-5 had low initial acute phase levels that significantly increased at later time points. Analysis of symptom severity showed association with CXCL9/MIG, CXCL10/IP-10 and IgG levels. These data give insight into Chikungunya disease establishment and subsequent convalescence, which is imperative to the treatment and containment of this quickly evolving and frequently re-emerging disease

Pandemic H1N1 influenza A directly induces a robust and acute inflammatory gene signature in primary human bronchial epithelial cells downstream of membrane fusion

Pandemic H1N1 influenza A (H1N1pdm) elicits stronger pulmonary inflammation than previously circulating seasonal H1N1 influenza A (sH1N1), yet mechanisms of inflammatory activation in respiratory epithelial cells during H1N1pdm infection are unclear. We investigated host responses to H1N1pdm/sH1N1 infection and virus entry mechanisms in primary human bronchial epithelial cells in vitro. H1N1pdm infection rapidly initiated a robust inflammatory gene signature (3 h post-infection) not elicited by sH1N1 infection. Protein secretion inhibition had no effect on gene induction. Infection with membrane fusion deficient H1N1pdm failed to induce robust inflammatory gene expression which was rescued with restoration of fusion ability, suggesting H1N1pdm directly triggered the inflammatory signature downstream of membrane fusion. Investigation of intra-virion components revealed H1N1pdm viral RNA (vRNA) triggered a stronger inflammatory phenotype than sH1N1 vRNA. Thus, our study is first to report H1N1pdm induces greater inflammatory gene expression than sH1N1 in vitro due to direct virus–epithelial cell interaction

Influenza Transmission in the Mother-Infant Dyad Leads to Severe Disease, Mammary Gland Infection, and Pathogenesis by Regulating Host Responses

<div><p>Seasonal influenza viruses are typically restricted to the human upper respiratory tract whereas influenza viruses with greater pathogenic potential often also target extra-pulmonary organs. Infants, pregnant women, and breastfeeding mothers are highly susceptible to severe respiratory disease following influenza virus infection but the mechanisms of disease severity in the mother-infant dyad are poorly understood. Here we investigated 2009 H1N1 influenza virus infection and transmission in breastfeeding mothers and infants utilizing our developed infant-mother ferret influenza model. Infants acquired severe disease and mortality following infection. Transmission of the virus from infants to mother ferrets led to infection in the lungs and mother mortality. Live virus was also found in mammary gland tissue and expressed milk of the mothers which eventually led to milk cessation. Histopathology showed destruction of acini glandular architecture with the absence of milk. The virus was localized in mammary epithelial cells of positive glands. To understand the molecular mechanisms of mammary gland infection, we performed global transcript analysis which showed downregulation of milk production genes such as Prolactin and increased breast involution pathways indicated by a STAT5 to STAT3 signaling shift. Genes associated with cancer development were also significantly increased including JUN, FOS and M2 macrophage markers. Immune responses within the mammary gland were characterized by decreased lymphocyte-associated genes CD3e, IL2Ra, CD4 with IL1β upregulation. Direct inoculation of H1N1 into the mammary gland led to infant respiratory infection and infant mortality suggesting the influenza virus was able to replicate in mammary tissue and transmission is possible through breastfeeding. In vitro infection studies with human breast cells showed susceptibility to H1N1 virus infection. Together, we have shown that the host-pathogen interactions of influenza virus infection in the mother-infant dyad initiate immunological and oncogenic signaling cascades within the mammary gland. These findings suggest the mammary gland may have a greater role in infection and immunity than previously thought.</p></div

STAT5 protein is decreased and STAT3 protein is nuclear localized in H1N1+ Mammary Glands.

<p>STAT5 (right panels) and STAT3 (left panels) protein expression was visualized in Control and H1N1+MG on Day 7 post-infant-inoculation by IHC. IHC analysis of paraffin-embedded mammary sections from nursing-mothers of infected infants stained with STAT5 or STAT3 antibodies were visualized using an Aperio ScanScope XT, Leica Biosystems, Nußloch, Germany for high resolution scans. The inset picture shows a magnification of the scanned image to show cellular detail. Scale bars indicate 100 μm or 10 μm. The images show a representative of mammary glands collected from infant intranasal virus or mock inoculations (three inoculations each).</p

Influenza virus inoculation into active mammary glands of nursing-mother ferrets leads to respiratory infection, severe disease and mortality in infants.

<p>A schematic depicting the mammary gland inoculation design is shown (<b>A</b>). Temperature (left panel) and weights (right panel) of lactating mother ferrets following 2009 H1N1 (pink lines) or PBS (light grey lines) mammary inoculation (<b>B</b>). Temperature (left panel) and weights (right panel) of infants feeding from lactating mothers with 2009 H1N1 (pink lines) or PBS (light grey lines) inoculated mammary glands (<b>C</b>). Survival of mother and infant ferrets following mammary 2009 H1N1 virus inoculation (<b>D</b>). Live viral load determined from nasal wash in both infant and mothers (<b>E</b>), and live virus shedding from expressed milk (<b>F</b>) following mammary gland 2009 H1N1 inoculation. * Indicates a p-value less than 0.05 determined by ANOVA. Error bars indicate +/- SD. Results are the mean of or represent three independent mammary gland inoculation experiments (3 mammary inoculated mothers, 18 infants) and mock mammary inoculation controls (3 mammary mock inoculated mothers, 21 infants).</p

2009 H1N1 transmission from mothers to infants results in severe lower respiratory tract pathology.

<p>Harvested lungs from control infants and infants of inoculated nursing-mothers were processed for histopathological assessment. Tissue morphology was assessed by hematoxylin & eosin staining. Data was collected from three independent litter inoculations/infections (3 inoculated/infected mothers, 16 infants, and 3 mock inoculated/infected mothers) and results are a representative of the inoculations/infections. pmi = Post-Mother-Inoculation</p

Human “normal” and adenocarcinoma mammary epithelial cells are susceptible and permissive to 2009 H1N1 infection <i>in vitro</i>.

<p>Mammary epithelial cells (MCF-7, MDA-MB-231, MCF-10A cells) inoculated at an MOI of 1 with A/Cal (H1N1) were fixed at 24 hours post-inoculation and stained for filamentous actin (red), DNA (green), and influenza A virus NP protein (blue), and imaged by confocal microscopy (<b>A</b>). Mammary epithelial cells (MCF-7, MDA-MB-231, MCF-10A cells) inoculated at an MOI of 1 with A/Cal (H1N1) were collected at 3, 24, 48, and 72 h post-inoculation for quantification of viral RNA segment 7 by qRT-PCR (<b>B</b>), determination of cell viability (<b>C</b>), and live virus quantification in supernatant (<b>D</b>). Confocal pictures are representative of three independent experiments. White arrows indicate nuclear localization of influenza NP protein. Yellow arrows show virus budding along the plasma membrane. BC, Baseline Control.</p

Intranasal 2009 H1N1 infection in infant ferrets leads to severe disease and mortality in both mother and infant ferrets.

<p>Schematic of experimental design for mother-infant dyad inoculations (<b>A</b>). Infants were intranasally inoculated with the A/Cal strain of 2009 H1N1 influenza (10⁵ EID₅₀) and housed with their nursing-mothers. Temperature (<b>B</b>) and weight (<b>C</b>) were recorded for 14 days post-infant-inoculation. Control mock inoculated infants (grey lines) were used to assess natural fluctuations in growing infants. Survival of mothers and infants was determined over 14 days (<b>D</b>). Results show the mean or are representative of 3 independent litter inoculations/infections (3 mother ferrets and 19 infant ferrets) and 3 litter mock inoculations (3 mother ferrets and 11 infant ferrets). * indicates a p-value less than 0.05 determined by ANOVA comparing 2009 H1N1 inoculated/infected to mock controls. Error bars indicate +/- SE.</p