IL-6 trans-signaling mediates cytokine secretion and barrier dysfunction in hantavirus-infected cells and correlates to severity in HFRS

Abstract

Background: Hantavirus causes hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Strong inflammatory responses and vascular leakage are important hallmarks of these often fatal diseases. The mechanism behind pathogenesis is unknown and no specific treatment is available. IL-6 was recently highlighted as a biomarker for HPS/HFRS severity. IL-6 signaling is complex and context dependent: while classical signaling generally provide protective responses, trans-signaling can cause severe pathogenic responses. Here, we investigated a potential role for IL-6 trans-signaling in hantavirus pathogenesis.

Methods: Effects of IL-6 trans-signaling during in vitro hantavirus infection were assessed using primary human endothelial cells treated with recombinant soluble IL-6 receptor (sIL-6R). Plasma from Puumala orthohantavirus-infected HFRS patients (n=28) were analyzed for IL-6 trans-signaling potential and its associations to severity.

Findings: In vitro, sIL-6R treatment of infected cells enhanced IL-6 and CCL2 secretion, upregulated ICAM-1, and affected VE-cadherin leading to a disrupted cell barrier integrity. HFRS patients showed altered plasma levels of sIL-6R and soluble gp130 (sgp130) resulting in an increased sIL-6R/sgp130 ratio suggesting enhanced IL-6 trans-signaling potential. Plasma sgp130 levels negatively correlated with number of interventions and positively with albumin levels. Patients receiving oxygen treatment displayed a higher sIL-6R/sgp130 ratio compared to patients that did not.

Interpretation: IL-6 trans-signaling is linked to hantavirus pathogenesis. Targeting IL-6 trans-signaling might provide a therapeutic strategy for treatment of severe HFRS and perhaps also HPS.

Fig 1. PUUV-infected cells secrete IL-6.

HUVECs were infected with PUUV (MOI=1) and PBMCs were exposed to PUUV (MOI=3) for 24-72 h. Supernatants were assessed for IL-6, soluble gp130 (sgp130), and soluble IL-6R (sIL-6R) using ELISA. (A) Levels of IL-6 in supernatants of HUVECs (n=3) and (B) PBMCs (24-48 h, n=6, 72 h, n=2). (C) sgp130 levels in supernatants of HUVECs (n=3). (D) Levels of sgp130 (n=2) and sIL-6R (24-48 h, n=8, 72 h, n=6) in PBMC supernatants. Two-way ANOVA followed by Šídák’s multiple comparison test (factors; time after infection and condition (uninfected or PUUV-infected)). *, p<0.05, **, p<0.01.

Fig 2. IL-6 trans-signaling activates endothelial cells and drives inflammation.

HUVECs were infected with PUUV (MOI=1) for 48 h and then treated with sIL-6R at the concentrations 31.25, 62.5, 125, 250, or 500 ng/ml for 24 h or left untreated. (A) Levels of IL-6 (n=5) and (B) CCL2 (n=3) in supernatants of uninfected and PUUV-infected HUVECs. (C) Representative histogram plot and (D) graph showing median ICAM-1 expression on infected and uninfected HUVECs (n=3). Symbols depict mean and error bars indicate SD. Two-way ANOVA followed by Dunnet’s or Šídák’s multiple comparison test (factors; level of sIL-6R added and condition (PUUV-infected or uninfected)). Black asterisks indicate significance when comparing PUUV to uninfected. Red asterisks indicate significance when comparing each sIL-6R-treated condition of PUUV-infected cells with untreated PUUV-infected cells. *, p<0.05; **, p<0.01; ***, p<0.001, ****, p<0.0001.

Fig 3. IL-6 trans-signaling disrupts endothelial cell barrier functions during hantavirus-infection.

Uninfected and infected HUVECs were treated with sIL-6R at the concentrations 31.25, 62.5, 125, 250, or 500 ng/ml for 24 h or left untreated. (A) Immunofluorescence images showing expression of DAPI (blue), virus (red), and VE-cadherin (green). Representative images of three independent experiments are shown. (B) Transendothelial electrical resistance of uninfected (white symbol) and infected HUVECs (black symbol), with and without sIL-6R (n=3). Uninfected HUVECs treated with rIL-6 in addition to sIL-6R were used as control (grey symbol; n=3). Symbols depict mean and error bars indicate SD. Two-way ANOVA followed by Dunnet’s or Šídák’s multiple comparison test (factors; level of sIL-6R added and condition (uninfected, PUUV-infected, or uninfected + rIL-6)). Black asterisks indicate significance when comparing PUUV to uninfected. Red asterisks indicate significance when comparing each sIL-6R-treated condition of PUUV-infected cells with untreated PUUV-infected cells. *, p<0.05; **, p<0.01; ***, p<0.001, ****, p<0.0001.

Fig 4. The plasma sIL-6R/sgp130 ratio is increased during acute HFRS and IL-6 trans-signaling potential correlate to severity.

Plasma levels of (A) IL-6, (B) sIL-6R, (C) IL-6:sIL-6R complex, and (D) sgp130 in controls (n=20) and acute and convalescent HFRS patients (n=28). (E) Ratio of plasma sIL-6R and sgp130 in controls and HFRS patients (n=27). (F) Correlation between sgp130 levels and number of interventions during acute HFRS (n=28). (G) Correlation between sgp130 levels and serum albumin (n=13). (H) Plasma sIL-6R/sgp130 ratio in patients with or without oxygen treatment (median, interquartile range). Wilcoxon signed-rank test (acute and convalescent HFRS patients, A-E); Kruskal-Wallis test (comparisons between controls and acute and convalescent HFRS patients, A-E); red line depicts median. Spearman’s rank correlation coefficient ( F-G). Mann Whitney test ( H); bars represent median. *, p<0.05; **, p<0.01; ***, p<0.001, ****, p<0.0001.