Pregnant women infected with the pandemic influenza A(H1N1)pdm09 virus show a differential cytokine and chemokine response related to disease severity

PERIOLO NATALIA,; RUSSO MARA; AVARO MARTIN; BENEDETTI ESTEFANIA; PONTORIERO ANDREA; CAMPOS ANA; CZECH ANDREA; MARTINEZ PERALTA LILIANA; BAUMEISTER, ELSA

Cape Town, South Africa

Congreso; Options for the Control of Influenza VIII; 2013

Background: The innate immune system is the first line of defense against viruses inducing cytokine and chemokine expression. During pregnancy, immunological and hormonal alterations place women at increased risk for certain infections and associated complications. Pregnant women represent a disproportionately higher percentage of severe cases of influenza with increased risk ranging from 4- to 10-fold than that of the general population. Increased morbidity and mortality in pregnant women were reported based on data from seasonal influenza. The impact of pregnancy on the immune response is not well known. Although A(H1N1)pdm09 infection resulted in increased disease severity in pregnant women, the precise mechanisms responsible for this risk are not yet defined. The present study was aimed to investigate the host cytokine profiles in A(H1N1)pdm09 infection regarding disease severity in pregnant women. Materials and methods: This study included 41 pregnant women with confirmed A(H1N1)pdm09 infection during the 2009 pandemic: 12 died as a consequence of the infection (ID), and 29 survived the infection (IS) Samples of 17 non-infected pregnant women were included as control (NI). Respiratory samples, tracheal aspirates (TA), as well as nasopharyngeal (NP) secretions were obtained and collected from July to September 2009. Samples were sent to the National Influenza Reference Laboratory which houses the WHO National Influenza Center (NIC) for A(H1N1)pdm09 diagnosis. Specimens have been kept at -80°C since then. The A(H1N1)pdm09 infection was confirmed by real time-RT-PCR using the CDC protocol. Total RNA from cells was extracted using QIAamp® Viral RNA Mini kit QIAGEN?. cDNA was synthesized with Oligo-dT primers and Superscript III reverse transcriptase (Invitrogen) and quantified by real-time quantitative PCR analysis. The gene expression profile for cytokines (IFN-β, TNF-α, IL-6, IL-12, TGF-β, IL-17), chemokines (IL-8, RANTES, MCP-1) and viral Matrix (M1) gene was quantified and normalized using the house-keeping gene product β-actin mRNA. We used a SYBR green PCR MASTER MIX (Applied Biosystems). The M1 gene copy number was measured to evaluate viral replication in each sample. Statistical analysis was performed using the GraphPad Prism software by ANOVA. This study was approved by the Independent Ethics Committee. Results: IL-6 mRNA expression in ID resulted significantly higher than in IS or NI (5.424 ± 0.9513 vs. 1.370 ± 0.4333, p< 0. 01 and 1.578 ± 0.3566, p< 0.001, expressed as copies of IL-6 mRNA/ β- actin mRNA, respectively), while both ID and IS showed increased expression of TNF-α and IL-8 in comparison with NI. Conversely, TGF-β mRNA was lower in both groups with influenza infection than in NI. RANTES, MCP1 and IL-12 levels were not significantly different among all groups. Interestingly, the expression of INF-β in ID was significantly lower than in the other two groups (0.6000 ± 0.08528 vs. 1.275 ± 0.1058, p<0.0001 and 1.293 ± 0.1147, p= 0.000, respectively) in relation to the increased M1 gene transcription in ID compared to IS. Conclusions: The present study shows that the expression of several cytokines and chemokines may play a role in the pathogenesis of the severity of the respiratory disease in pregnant women during the A(H1N1)pdm09 virus infection. The decreased expression of TGF-β may contribute to disease progression inducing proinflammatory cytokines and chemokines in ID and IS. The highly elevated expression of IL-6 and low INF-β in ID probably allowed an increase in viral replication, indicated by the M1 gene expression. These results may serve as important biomarkers for the identification of patients at risk for severe complications during the A(H1N1)pdm09 infection, but the underlying mechanism awaits further detailed investigations. Addressing these issues will be necessary to ensure that pregnant women receive appropriate guidance and health care in the event of influenza pandemic or epidemic. The importance of a comprehensive preventive vaccination campaign in pregnant women is highlighted.