Local IL-17A potentiates early neutrophil recruitment to the respiratory tract during severe RSV infection

Abstract

Respiratory syncytial virus (RSV) bronchiolitis triggers a strong innate immune response characterized by excessive neutrophil infiltration which contributes to RSV induced pathology. The cytokine IL-17A enhances neutrophil infiltration into virus infected lungs. IL-17A is however best known as an effector of adaptive immune responses. The role of IL-17A in early immune modulation in RSV infection is unknown. We aimed to elucidate whether local IL-17A facilitates the innate neutrophil infiltration into RSV infected lungs prior to adaptive immunity. To this end, we studied IL-17A production in newborns that were hospitalized for severe RSV bronchiolitis. In tracheal aspirates we measured IL-17A concentration and neutrophil counts. We utilized cultured human epithelial cells to test if IL-17A regulates RSV infection-induced IL-8 release as mediator of neutrophil recruitment. In mice we investigated the cell types that are responsible for early innate IL-17A production during RSV infection. Using IL-17A neutralizing antibodies we tested if IL-17A is responsible for innate neutrophil infiltration in mice. Our data show that increased IL-17A production in newborn RSV patient lungs correlates with subsequent neutrophil counts recruited to the lungs. IL-17A potentiates RSV-induced production of the neutrophil-attracting chemokine IL-8 by airway epithelial cells in vitro. Various lung-resident lymphocytes produced IL-17A during early RSV infection in Balb/c mice, of which a local population of CD4 T cells stood out as the predominant RSV-induced cell type. By removing IL-17A during early RSV infection in mice we showed that IL-17A is responsible for enhanced innate neutrophil infiltration in vivo. Using patient material, in vitro studies, and an animal model of RSV infection, we thus show that early local IL-17A production in the airways during RSV bronchiolitis facilitates neutrophil recruitment with pathologic consequences to infant lungs.

Figure 1. Early tracheal aspirate (TA) IL-17A correlates to subsequent airway neutrophilia.

(A) RSV patients in this study were younger than 4 months of age and were included during the innate phase of the immune response. Gender, average age, and duration of RSV disease prior to intubation/inclusion ± SD are shown. (B,C) IL-8 and IL-17A concentrations are elevated in TA of RSV patients compared to uninfected controls. Cytokine levels were determined by multiplex immunoassay from TA that was collected at 5 and 48 hours post-intubation. Data represent 9 patients and 7 controls. (D) TA IL-17A concentration at 5 hours post intubation correlates with neutrophil infiltration at 24 hours post intubation. Neutrophils were detected by flow cytometry and are shown as a percentage of live TA cells (n=8). Each data point represents an individual RSV patient. * denote significance of p<0.05.

Figure 2. IL-17A and RSV synergistically induce IL-8 production by airway epithelial cells.

A549 airway epithelial cells were infected with RSV or UV-inactivated RSV for 24 hours at a multiplicity of infection of 3 and in the presence of IL-17A. (A) IL-8 mRNA levels measured by qPCR. Data represents 3-4 experiments. (B) Concentration of IL-8 in the supernatant of RSV-infected and IL-17A treated A549 cells. Data represents 3-5 experiments. * denote significance of p<0.05, **** denote significance of p<0.0001.

Figure 3. High dose RSV infection induces early neutrophil infiltration in BALB/c mice.

(A) Mice were infected intranasally with a high dose RSV (10⁷ pfu/mouse) or mock infected (PBS). BAL was collected at 2 and 4 days post infection. (B) High dose RSV infection induces rapid weight loss in BALB/c mice. Relative weight to the start of infection is shown. (C) RSV replicates in mice. Viral loads were determined by qPCR in the a-cellular fraction of the BAL. (D - F) High dose RSV infection causes infiltration of neutrophils into RSV infected mouse lungs. (D) Live BAL cells were counted using a hematocytometer and trypane blue staining. (E) Absolute numbers and (F) percentages of neutrophils were determined by analysis of May-Grünwald/Giemsa stained cytospins. All data represent 4 - 16 mice per group and three independent experiments. ** denote significance p<0.01, **** denote significance of p<0.0001.

Figure 4. CD4 T cells are major local producers of IL-17A during early RSV infection.

RSV induces IL-17A production in lung lymphocytes at 2 days post infection. (A) Lung single cell suspensions were harvested at 2 days post infection and stimulated with PMA/Ionomycin for 4 hours prior to intracellular cytokine staining and analysis by flow cytometry. (B) Absolute and relative numbers of IL-17A producing lung lymphocytes. (C-F) Predominantly CD4 T cells are induced to produce IL-17A by RSV infection. Separate bar graphs of the relative IL-17A production by (C) TCRβ+CD4+ T cells, (D) TCRβ+CD4- CD8- (DN) T cells, (E) CD1d-tetramer+ NKT cells, and (F) TCRγδ+ gamma/delta T cells . Absolute numbers represent n=4 control mice and n=3 RSV infected mice from one representative experiment.* denote significance of p<0.05, ** denote significance of p<0.01, *** denote significance of p<0.001.

Figure 5. Depletion of IL-17A limits neutrophil infiltration into RSV-infected lungs.

(A) Mice received IP injections of anti-IL-17A antibody or isotype control antibody upon and two days prior to RSV infection. Mice were infected with high or low dose RSV and sacrificed 2 days post infection. (B,C) Graphs representing the weight loss of anti-IL-17A treated versus isotype treated mice that were infected with a low or a high dose RSV respectively. (D) Viral loads in the a-cellular fraction of the BAL of high dose RSV infected mice. (E,F) Total BAL cell and absolute neutrophil numbers per ml BAL at day 2 post infection in both isotype and anti-IL-17A treated mice. Data represent 3 mice per group for high dose RSV (10⁷ pfu/mouse) infected mice, and at least 5 mice per group for low dose RSV (10⁶ pfu/mouse) infected mice. (G) Representative scatter plots of CD11b and GR-1 surface stained BAL cells. * denote significance of p<0.05.