Early hypercytokinemia is associated with interferon-induced transmembrane protein-3 dysfunction and predictive of fatal H7N9 infection

Abstract

A unique avian-origin A/H7N9 influenza virus has so far caused 134 cases with 44 deaths. Probing the host factors contributing to disease severity, we found that lower levels of plasma inflammatory cytokines on hospital admission correlated with faster recovery in 18 patients with A/H7N9 influenza virus, whereas high concentrations of (in particular) IL-6, IL-8, and macrophage inflammatory protein-1β were predictive of a less favorable or fatal outcome. Analysis of bronchoalveolar lavage samples showed up to 1,000-fold greater cytokine/chemokine levels relative to plasma. Furthermore, patients with the rs12252-C/C IFN-induced transmembrane protein-3 (IFITM3) genotype had more rapid disease progression and were less likely to survive. Compared with patients with the rs12252-T/T or rs12252-T/C genotype of IFITM3, patients with the C/C genotype had a shorter time from disease onset to the time point when they sought medical aid (hospital admission or antiviral therapy) and a shorter interval to development of the acute respiratory distress syndrome stage (reflected by shorter intervals between clinical onset and methylprednisolone treatments and higher rates of mechanical ventilator use), as well as experiencing elevated/prolonged lung virus titers and cytokine production and higher mortality. The present analysis provides reported data on the H7N9 influenza-induced "cytokine storm" at the site of infection in humans and identifies the rs12252-C genotype that compromises IFITM3 function as a primary genetic correlate of severe H7N9 pneumonia. Together with rs12252 sequencing, early monitoring of plasma cytokines is thus of prognostic value for the treatment and management of severe influenza pneumonia.

Keywords: avian influenza; clinical outcome.

Fig. 1.

Cytokines and chemokines associated with severe H7N9 influenza infection in plasma and BAL fluid. (A) Elevated levels of IL-10, IL-6, IL-8, and MIP-1β in the patients’ plasma correlate with fatal clinical outcomes, as specified in Table 1. Cytokine concentrations were assessed within 1–2 d after hospital admission; patients who recovered and were discharged within 35 d were assigned to group A (survival group), and those died from the infection comprise group B. (B) Kinetics of IL-6, IL-8, and MIP-1β levels in plasma during the course of H7N9 infection, represented for (i) individual patients and (ii) group A vs. group B, as outlined in Table S1. Cytokine and chemokines were measured with cytometric bead array (CBA) kits.

Fig. 2.

Cytokine levels and lung histology in fatal cases. (A) Massively increased levels of cytokines and chemokines at the site of infection (BAL) compared with plasma, measured for three individual patients (a33, a22, and a118), as outlined in Table S2. (B) Comparison of cytokine patterns between plasma and BAL within individuals. (C–F) Diffused alveolar damage with thickening of the alveolar interstitium with extensive lymphocyte infiltration and hyaline membrane formation was observed in the lung of two H7N9-infected patients (a118 and a33). (C and D) Arrows point to cellular infiltration into the alveoli. (E and F) Normal lung samples from H7N9-negative subjects are shown, with no inflammation observed. Paraffin-embedded sections were stained with H&E. (Magnification: C–F, 40×.)

Fig. 3.

Rs-12252-C association with disease severity in patients hospitalized with a H7N9 influenza virus. Due to the T→C nucleotide mutation at the SNP12252 position, human IFITM3 can have three homoforms (C/C with N-terminal 21 amino acids truncated IFITM3, T/T with WT IFITM3, and F1 T/C). (A) Comparison of allele frequencies in the normal Chinese population (from the 1,000 genomes project) and in patients with H7N9 virus from the SHAPHC. Also shown are association of the rs-12252-C group with higher influenza viral loads (quantified by real-time RT-PCR on days 8–13 after disease onset) (B), shorter time (days) from disease onset to oseltamivir treatment (C), shorter time to first methylprednisolone treatment (D), greater use of extracorporeal membrane oxygenation or mechanical respiratory ventilation (E), increased mortality rates (F), and elevated cytokines/chemokines in plasma (G). In B, dashed lines depict viral titres (log 10) of 2 and 4. In G, dashed line refers to the highest cytokines/chemokines level found for the T/T genotype.