RIG-I and TLR4 responses and adverse outcomes in pediatric influenza-related critical illness

Abstract

Background: Decreased TNF-α production in whole blood after ex vivo LPS stimulation indicates suppression of the Toll-like receptor (TLR)4 pathway. This is associated with increased mortality in pediatric influenza critical illness. Whether antiviral immune signaling pathways are also suppressed in these patients is unclear.

Objectives: We sought to evaluate suppression of the TLR4 and the antiviral retinoic acid-inducible gene-I (RIG-I) pathways with clinical outcomes in children with severe influenza infection.

Methods: In this 24-center, prospective, observational cohort study of children with confirmed influenza infection, blood was collected within 72 hours of intensive care unit admission. Ex vivo whole blood stimulations were performed with matched controls using the viral ligand polyinosinic-polycytidylic acid-low-molecular-weight/LyoVec and LPS to evaluate IFN-α and TNF-α production capacities (RIG-I and TLR4 pathways, respectively).

Results: Suppression of either IFN-α or TNF-α production capacity was associated with longer duration of mechanical ventilation and hospitalization, and increased organ dysfunction. Children with suppression of both RIG-I and TLR4 pathways (n = 33 of 103 [32%]) were more likely to have prolonged (≥7 days) multiple-organ dysfunction syndrome (30.3% vs 8.6%; P = .004) or prolonged hypoxemic respiratory failure (39.4% vs 11.4%; P = .001) compared with those with single- or no pathway suppression.

Conclusions: Suppression of both RIG-I and TLR4 signaling pathways, essential for respective antiviral and antibacterial responses, is common in previously immunocompetent children with influenza-related critical illness and is associated with bacterial coinfection and adverse outcomes. Prospective testing of both pathways may aid in risk-stratification and in immune monitoring.

Keywords: IFN-α; LPS; Polyinosinic:polycytidylic acid; TNF-α; Toll-like receptor 4; critical care; influenza; pediatric; retinoic acid–inducible gene-I; suppression.

Figure 1.. Poly(I:C) -induced IFNα production (a).

Subjects’ IFNα response was characterised into three groups based on ex vivo whole blood IFNα production after 24 hours of unstimulated control incubation (white boxes) and poly(I:C) stimulation (gray boxes). Suppressed subjects had low production of IFNα in both unstimulated controls and poly(I:C)-stimulated blood samples, with a low delta (no significance (NS) vs control), n=56. Peaked subjects had high production of IFNα in both unstimulated controls and poly(I:C) stimulated samples, with a low delta (NS vs control), n=11. Those that Responded had variable IFNα levels in unstimulated controls but were able to produce an increase in their poly(I:C) stimulated blood, with a high delta (p<0.0001 vs control), n=38 based on the Wilcoxon matched-pairs signed rank test. Data represent samples collected ≤72hrs from PICU admission. LPS-induced TNFα production (b). In the LPS stimulated samples categorized as TNFα ≥ or < 200 pg/ml, unstimulated control samples had similar values (p=0.09) while TNFα responses were significantly different (p<0.0001) based on the Mann Whitney U test. Poly(I:C)-induced IFNα response groups with LPS-induced TNFα response data (c). Gray bars represent subjects whose LPS-induced TNFα response was < 200 pg/ml while white bars represent those with a TNFα response ≥ 200 pg/ml. Subjects with Suppressed IFNα were more likely to have a low TNFα response compared to Responders or those with a Peaked IFNα response (Pearson χ² p<0.0002).

Figure 2.. Prolonged multiple organ dysfunction in subjects with and without pathway suppression (a).

Subjects who demonstrated dual suppression had the highest incidence of prolonged MODS (defined as MODS or ECMO on/after day 7 of illness or death) or mortality. White areas represent subjects who never experienced MODS which was the smallest in the IFNα and TNFα Suppressed group (p=0.003). Prolonged acute hypoxemic respiratory failure in subjects with and without pathway suppression (b). Subjects who had dual suppression also had the highest incidence of prolonged AHRF (defined as PaO2/FiO2 <200 and remaining on mechanical ventilation on/after day 7) (p=0.001). Pearson χ² p values represent comparison of MODS status or AHRF (prolonged vs never or resolved) in each stimulation group.