Alternaria alternata challenge at the nasal mucosa results in eosinophilic inflammation and increased susceptibility to influenza virus infection

Abstract

Background: Eosinophils in the nasal mucosa are an elemental feature of allergic rhinitis.

Objective: Our objective was to explore eosinophilic inflammation and its impact on respiratory virus infection at the nasal mucosa.

Methods: Inflammation in the nasal mucosae of mice was evaluated in response to repetitive stimulation with strict intranasal volumes of a filtrate of Alternaria alternata. Mice were then challenged with influenza virus.

Results: Repetitive stimulation with A. alternata resulted in eosinophil recruitment to the nasal passages in association with elevated levels of IL-5, IL-13 and eotaxin-1; eosinophil recruitment was diminished in eotaxin-1^-/- mice, and abolished in Rag1^-/- mice. A. alternata also resulted in elevated levels of nasal wash IgA in both wild-type and eosinophil-deficient ∆dblGATA mice. Interestingly, A. alternata-treated mice responded to an influenza virus infection with profound weight loss and mortality compared to mice that received diluent alone (0% vs 100% survival, ***P < .001); the lethal response was blunted when A. alternata was heat-inactivated. Minimal differences in virus titre were detected, and eosinophils present in the nasal passages at the time of virus inoculation provided no protection against the lethal sequelae. Interestingly, nasal wash fluids from mice treated with A. alternata included more neutrophils and higher levels of pro-inflammatory mediators in response to virus challenge, among these, IL-6, a biomarker for disease severity in human influenza.

Conclusions and clinical relevance: Repetitive administration of A. alternata resulted in inflammation of the nasal mucosae and unanticipated morbidity and mortality in response to subsequent challenge with influenza virus. Interestingly, and in contrast to findings in the lower airways, eosinophils recruited to the nasal passages provided no protection against lethal infection. As increased susceptibility to influenza virus among individuals with rhinitis has been the subject of several clinical reports, this model may be used for further exploration of these observations.

Keywords: cytokine; eosinophil; inflammation; respiratory virus.

Fig. 1. Repetitive administration of the aeroallergen, Alternaria alternata, to the nasal passages results in eosinophilic inflammation

A. Strategy for repetitive challenge with A. alternata antigens (filtrate; 10 mg/mL) administered at strict intranasal volumes (2.5 μL per nare [19]) over a three (3) week period as described in the Methods Section. B. Eosinophils (percent of total leukocytes) are prominent in nasal wash fluid of wild-type but not eosinophil-deficient ΔdblGATA mice or lymphocyte-deficient Rag1^−/− mice in response to A. alternata administered as in Fig. 1A; inset, eosinophils from nasal wash fluid of wild-type mice stained with modified Giemsa. C. Eosinophils (total in 0.6 mL nasal wash fluid) at weeks 0, 1, 2 and 3 as in Fig. 1A. D. RNase activity, a measure of eosinophil degranulation, detected in nasal wash fluid of wild-type, but not ΔdblGATA or Rag1^−/− mice; dotted line, limit of background activity. E. IgA detected in nasal wash fluid of wild-type and ΔdblGATA mice, but not Rag1^−/− mice; n = 3 – 8 mice per time point, ***p < 0.005, **p < 0.01, *p < 0.05, ns, no statistical significance, 1-way ANOVA or Mann-Whitney U-test.

Fig. 2. Eotaxin-1 (CCL-11) promotes eosinophil recruitment to nasal passages

Detection of immunoreactive A. Interleukin-5 (IL-5) B. IL-13 and C. Eotaxin-1 (CCL-11) in nasal wash fluid after 1, 2 and 3 weeks of repetitive challenge with A. alternata as in Fig. 1A. D. Eosinophils (percent of total leukocytes) detected in the airways are diminished in eotaxin-1 gene-deleted mice; n = 3 – 5 mice per time point, **p < 0.01, *p < 0.05; 1-way ANOVA or Mann-Whitney U-test.

Fig. 3. Histology of the nasal passages of mice subjected to repetitive challenge with A. alternata antigens

A. Anterior cross section featuring the medial meatus (MM), ventral meatus (VM), and medial turbinate (MT) with areas marked B. and C. featured in enlargements on the panels to follow. B. Eosinophils (examples at arrows) are prominent in the mucosal tissue underlying the squamous epithelium and C. within the lumen of the nasal passage. D. Posterior cross section including the dorsal meatus (DM) and lateral meatus (LM); areas marked E. and F are featured in enlargements in panels to follow. E. Eosinophils are detected at the base of the ciliated columnar respiratory epithelial cells and F. within the mucosal tissue. Original magnification 4X; panels A and D and 40X; panels B, C, E and F).

Fig. 4. Eosinophilic inflammation in the upper airways does not protect against influenza virus infection

A. Strategy for influenza virus (Inf A) infection and evaluation. Inf A was administered to mice previously challenged with A. alternata or diluent control (days −19 through days −1) as previously indicated in Fig. 1A. B. Weight loss (% original weight determined for each mouse) in response to influenza virus infection. Mice were challenged with A. alternata or diluent control and inoculated on day 0 with 30 TCID₅₀ units Inf A in 5 μL (2.5 μL per nare) as in Fig. 4A; n = 5 mice per group, **p < 0.001 at time points indicated (Student’s t-test). C. Survival of influenza virus-infected mice (30 TCID₅₀ units in 5 μL on day 0 as in B.) that had been subjected to repetitive challenge with A. alternata or diluent control; n = 5 mice per group, **p < 0.005 Log-rank test. D. Survival of wild-type and eosinophil-deficient ΔdblGATA mice subjected to repetitive challenge with A. alternata or diluent control (pbs) prior to infection with Inf A (500 TCID₅₀ units in 5 μL); n = 4 – 5 mice per group, **p < 0.001 Log-rank test.

Fig. 5. Heat-inactivation of A. alternata blunts the lethal response to influenza virus

A. Filtrate of A. alternata heated to 95°C for 10 minutes retains little to no residual protease activity using casein as a substrate; ***p < 0.001. B. Heat-inactivated filtrate of A. alternata (hi-Aa) administered to upper airways as in Fig. 1A promotes eosinophil recruitment to the upper airways as does the active filtrate (Aa), n = 5 mice per group; ns, no significant difference, Mann-Whitney U-test. C. Survival of wild-type mice challenged with A. alternata (Aa), hi-Aa or diluent control as in Fig. 4A followed by Inf A (150 TCID₅₀ units in 5 μL); n = 9 – 10 mice per group, *p = 0.05, **p < 0.005, Log-rank.

Fig. 6. Influenza virus infection of mice challenged with A. alternata results in differential recruitment of proinflammatory neutrophils

A. Strategy for influenza virus (Inf A) infection and evaluation. Influenza virus was administered to mice previously challenged with A. alternata or diluent control as in Fig. 4A and evaluated on day 6. B. Neutrophils (% total leukocytes) are more prominent in nasal wash samples from wild-type mice challenged with A. alternata prior to influenza infection (150 TCID₅₀ units in 5 μL) than those from mice challenged with diluent control; n = 5 mice per group, **p < 0.01, Mann-Whitney U-test. C. Total neutrophils in nasal wash samples from mice infected with 30 or 150 TCID₅₀ units influenza A; n = 5 mice per group, **p < 0.01, Mann Whitney U-test. D. RNase activity detected in nasal wash fluid in response to influenza infection (500 TCID₅₀ units); n = 5 mice per group, **p < 0.01; ***p < 0.005., 1-way ANOVA, Student’s t-test.

Fig. 7. Eosinophilic inflammation in the upper airways has no impact on virus recovery

No significant differences were observed in comparisons of mice treated with repetitive administration of A. alternata vs. pbs diluent control, evaluated at day 6 of infection with influenza virus (inoculation of 30, 150 or 500 TCID₅₀ in 5 μL volume). In BAL fluid, A. copies per mL or B. TCID₅₀ units/mL; in lung tissue C. copies per GAPDH x 10⁵ or TCID₅₀ units/μg lung protein or D. TCID₅₀ units per μg protein; a. three points below detectable limits at dotted line; n = 4 – 5 mice per point, *p < 0.05; **p < 0.01.

Fig. 8. Eosinophilic inflammation in the nasal passages followed by influenza virus infection results in differential release of critical proinflammatory cytokines

A. Cytokine profiling reveals differential release of numerous proinflammatory cytokines in the upper airways, including elevated levels of (a) IL-6, (b) CXCL10, and (c) CCL2, cytokines that have been previously associated with poor outcomes in acute respiratory virus infection (at arrows). Direct evaluation confirmed differential detection of B. IL-6 and C. CCL2 in nasal wash fluid of Aa-treated wild-type mice, when compared to diluent-treated, Inf A-infected mice; n = 5 mice per group, **p < 0.01, *p < 0.05.

Fig. 8. Eosinophilic inflammation in the nasal passages followed by influenza virus infection results in differential release of critical proinflammatory cytokines

A. Cytokine profiling reveals differential release of numerous proinflammatory cytokines in the upper airways, including elevated levels of (a) IL-6, (b) CXCL10, and (c) CCL2, cytokines that have been previously associated with poor outcomes in acute respiratory virus infection (at arrows). Direct evaluation confirmed differential detection of B. IL-6 and C. CCL2 in nasal wash fluid of Aa-treated wild-type mice, when compared to diluent-treated, Inf A-infected mice; n = 5 mice per group, **p < 0.01, *p < 0.05.