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

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

Virus replication and pathology in mammary glands of mothers nursing 2009 H1N1-infected infants.

<p>Infant ferrets were intranasally inoculated with A/Cal and housed with mother ferrets for a 7 Day time course where mammary glands, milk, blood, and feces were collected as shown in the schematic (<b>A</b>). Live virus was quantified by MDCK titration of homogenized mammary tissue (<b>B</b>), nipples (<b>C</b>), and expressed milk (<b>D</b>) from nursing-mothers of intranasal inoculated infant ferrets. qRT-PCR was performed on expressed milk for 2009 H1N1 viral RNA (vRNA) (<b>E</b>). Viral presence was also determined by qRT-PCR in infant feces (post-direct-inoculation) (<b>F</b>) and infant, mother, and adult ferret blood (3/4 days post-inoculation) (<b>G</b>). ND = Not Detected. Samples were collected and analyzed from at least 3 independent litter inoculated/infected infants and 3 mothers per time point. Results show the mean or are representative of 3 litters per time point. Mother ferrets have variable numbers of active mammary glands per pregnancy/postpartum.</p

2009 H1N1 virus positive mammary glands have distinct genetic signatures linked to the regulation of milk production, cancer and immune responses.

<p>A clustergram of the global expression analysis of H1N1+ MGs is shown and the most prominent functional groups are indicated for each cluster (<b>A</b>). Clustergrams of H1N1+ MG gene expression for specific signaling pathways and gene networks were produced (described in methods) (<b>B</b>). Immune Responses were analyzed by comparing the genes expression profiles of 2009 H1N1 infected Adult Ferret Lungs against the H1N1+ MGs (<b>C</b>). Clustergrams for immune response analysis were generated with genes exhibiting statistically significant differential regulation in either ferret mammary glands or lungs. Gene enrichment scores for KEGG-defined signaling cascades among significantly upregulated (red) and downregulated (red) gene subsets at Days 3/4 and Days 6/7 are shown (<b>D</b>). Values above threshold (α = 0.05) indicate statistically significant enrichment among upregulated or downregulated gene subsets at a given time-point. Samples were collected and analyzed from 3 independent litter experiments of inoculated/infected infants and 3 mothers per time point.</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

Destruction of acini and accumulation of viral protein in mammary glands of mothers of infected infants.

<p>Pathological destruction was determined by H&E (left) and IHC for IAV (right) analysis of paraffin-embedded mammary sections from nursing-mothers of infected infants. Black arrows denote areas of virus expression determined by anti-IAV staining. Yellow arrows denote areas of leukocyte infiltration. Blue arrows show milk protein accumulation. Green arrows indicate active epithelial cell layers. Control = mammary glands from nursing-mothers of mock inoculated infants. Samples were collected and analyzed from at least 3 independent litter inoculated/infected infants and 3 mothers per time point. Results show are representative of 3 litters per time point. Mother ferrets have variable numbers of active mammary glands per pregnancy/postpartum.</p

Transmission of H1N1 influenza from infants to mother ferrets causes upper and lower respiratory tract infection with significant pathology.

<p>Inoculated infants (A/Cal H1N1 influenza (10⁵ EID₅₀)) were housed with their nursing-mothers. Nasal washes were collected daily from infants and mothers. Live viral loads were determined by MDCK titration assay in nasal wash (NW) (<b>A</b>) and mother trachea and mother lungs at specific time points (<b>B</b>). Lungs were harvested on Day 3/4 and 7 post-infant-inoculation for hematoxylin & eosin (H&E) histopathological assessment from nursing-mothers of inoculated infants and directly inoculated adult ferrets as control (<b>C</b>). Green arrows denote dense cell accumulation; black arrows denote diffuse immune cell infiltration. Black arrows not included on Day 7 adult tissue due to widespread infiltration. High resolution scans were performed using an Aperio ScanScope XT, Leica Biosystems, Nußloch, Germany. The left and central columns of mother images represent a low and high magnification of each lung scan. The scale bars indicate the relative 100 μm. Results of <b>A</b> are representative of 3 independent litter inoculations where nasal washes were collected from 3 mothers and 9 infants (3 per litter) daily. Results of <b>B</b> and <b>C</b> are from 6 independent litter inoculations of serial tissue collections (3 collected Day 3/4 and 3 collected Day 7). Error bars indicate +/- SD. Mock: mother ferrets nursing mock-inoculated 4-week-old infant ferrets. Results show the mean or are representative of independent litter inoculations/infections.</p

Influenza virus infection mammary glands causes milk cessation and the destruction of gland architecture.

<p>High resolution scan of Control and 2009 H1N1 inoculated mammary glands (H&E) (<b>A</b>). qRT-PCR profiles of milk production genes in 2009 H1N1 virus and vehicle inoculated mammary tissue (<b>B</b>). *** Indicates a p-value less than 0.001. Data was collected and analyzed from three independent mammary gland inoculation experiments (3 mammary inoculated mothers, 18 infants) and mock mammary inoculation controls (3 mammary mock inoculated mothers, 21 infants). Results presented show data or are representative of 3 mammary inoculations.</p

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

Intranasal 2009 H1N1 infection in mother ferrets leads to severe disease and virus transmission to breastfeeding infants.

<p>Schematic of nursing-mothers intranasally inoculated with the Cal/07 strain of 2009 H1N1 influenza (10⁵ EID₅₀) and housed with their feeding infants (<b>A</b>). Temperature (<b>B</b>) and weights (<b>C</b>) were recorded daily for mothers and infants post-inoculation. Grey lines indicate mock inoculated mothers. Survival of mothers and infants post-mother-inoculation (<b>D</b>). Live viral loads were quantified by MDCK titration of nasal wash (NW) (<b>E</b>) and infant lung (<b>F</b>) homogenates collected throughout the infection course (infants that had reached cut-off or succumbed to illness). * indicates a p-value less than 0.05 determined by ANOVA comparing H1N1 inoculated to mock controls. Error bars indicate +/- SD. Data was collected from three independent litter inoculations/infections (3 inoculated/infected mothers, 16 infants, and 3 mock inoculated/infected mothers) and results show the mean or are a representative of the inoculations/infections.</p