Critical role of IL-17RA in immunopathology of influenza infection

Abstract

Acute lung injury due to influenza infection is associated with high mortality, an increase in neutrophils in the airspace, and increases in tissue myeloperoxidase (MPO). Because IL-17A and IL-17F, ligands for IL-17 receptor antagonist (IL-17RA), have been shown to mediate neutrophil migration into the lung in response to LPS or Gram-negative bacterial pneumonia, we hypothesized that IL-17RA signaling was critical for acute lung injury in response to pulmonary influenza infection. IL-17RA was critical for weight loss and both neutrophil migration and increases in tissue myeloperoxidase (MPO) after influenza infection. However, IL-17RA was dispensable for the recruitment of CD8(+) T cells specific for influenza hemagglutinin or nucleocapsid protein. Consistent with this, IL-17RA was not required for viral clearance. However, in the setting of influenza infection, IL-17RA(-/-) mice showed significantly reduced levels of oxidized phospholipids, which have previously been shown to be an important mediator in several models of acute lung injury, including influenza infection and gastric acid aspiration. Taken together, these data support targeting IL-17 or IL-17RA in acute lung injury due to acute viral infection.

FIGURE 1

IL-17A and IL-17F are expressed in response to influenza. Time course, in BALB/cJ mice, of IL-17A (A and C) and IL-17F (B and D) following influenza challenge. mRNA was detected by quantitative real-time PCR and is expressed relative to naive levels, with all time points having a p value of <0.05 compared with naive controls. Protein levels were detected by ELISA, and all time points had a p value of <0.05 compared with naive controls. E, The number of IL-17A-producing cells was quantified by ELISpot. *, p = 0.016; **, p = 0.003, n = 4/time point.

FIGURE 2

Production of IL-17 by γδ T cells. Intracellular cytokine staining following influenza challenge demonstrates an increasing percentage of IL-17⁺ γδ T cells (A), even as the total population of γδ T cells expands (B) Percentage of IL-17⁺ αβ T cells is unchanged compared with naive mice (C) Challenge of γδ knockout mice results in lower levels of IL-17A and IL-17F message, expressed as fold change from naive, at 6 dpi (D). *, p = 0.03; **, p = 0.02, n = 8/time point.

FIGURE 3

Weight loss and mortality following challenge with influenza A/PR/8. Weight loss (A) and mortality (B) were assessed daily following viral challenge (n = 10/group). Viral burden (C) was determined by quantitative PCR for the influenza M1 protein (n = 4/group/time point). *, p < 0.05; **, p = 0.0067; ***, p = 0.0093.

FIGURE 4

Differences in inflammation following influenza challenge. BALB/c (A) and IL-17RA^−/− (B) lungs at 6 dpi, inflated with 10% formalin, paraffin-embedded, and H&E stained. Calibration bar, 100 μm. Pathology scored (C) as in Lung fixation and histological examination. *, p = 0.01.

FIGURE 5

Cytokine response to influenza challenge. Cytokines measured at 2 and 6 dpi. BALB/c (□) vs IL-17RA^−/− (■). *, p < 0.05 (n = 4/group/time point; A).

FIGURE 6

Markers of lung injury. Total protein (A) and LDH activity (B) in the BALF. □, BALB/c mice; ■, IL-17RA^−/− mice. *, p = 0.01; **, p = 0.01 (n = 4/group/time point).

FIGURE 7

Protection is not dependent on TNF-α or IL-6. A, Weight loss among B6.129 and p55/p75 (n = 6/group) following influenza challenge. For comparison, IL-17RA^−/− on a C57BL/6 background and wild-type controls (n = 4/group) are also provided. B, Lung injury assessed by total protein in the BALF on day 6. C, BALB/c mice were treated with either anti-IL-6 (n = 4) or control IgG (n = 4) before challenge and again on 3 dpi, weight loss monitored for 6 days and lavaged on day 6 (D) to measure total protein in the BALF. *, p < 0.05.

FIGURE 8

Differences in neutrophils and oxidized phospholipids. A, IL-17RA^−/− mice (■) show decreases in the number of neutrophils in the BAL compared with BALB/c controls (□), but no differences in macrophages or lymphocytes (n = 4/group/time point). B, Pentamer staining at 10 dpi show that there is no difference in the numbers of HA-specific or NP-specific CD8⁺ T cells (n = 8/group). C, The numbers of IFN-γ positive CD4⁺ and CD8⁺ T cells are decreased in the IL-17RA^−/− mice at 10 dpi. n = 4/group; *, p = 0.02; **, p = 0.005. D, MPO activity is decreased in the lung homogenates, and trends lower in the BALF, of the IL-17RA^−/− mice compared with wild-type (WT) controls (n = 5/group/time point). *, p = 0.03; **, p = 0.01. E, Levels of oxidized phospholipids, measured by ELISA, are lower in IL-17RA^−/− mice than in BALB/c controls. n = 5/group/time point.***, p = 0.02.