Tissue levels of Alternaria allergen Alt a 1 reflect recurrence of refractory airway diseases

To the Editor,

The quantification of Alternaria allergen in the local airway tissues is quite unknown, while Alternaria alternata is a widespread fungal species in the airway discharge and known to be one source of aeroallergens which contribute to development of asthma ¹ , ² and chronic rhinosinusitis (CRS). ³ , ⁴ We have recently established the method for quantifying allergen in the local airway tissues. ⁵ Since Alt a 1 is the main sensitizing allergen component, it is useful in diagnosis for fungal allergy, and the immunotherapy with Alt a 1 could reduce the symptoms and medication consumption associated with rhinoconjunctivitis. ⁶ We have measured the levels of Alt a 1 in the local airway tissues and examined whether the quantification of Alt a 1 could reflect the allergic airway inflammation and recurrence of refractory allergic respiratory diseases.

We obtained nasal polyp tissues from 64 patients with refractory CRS with nasal polyp (CRSwNP) and homogenized them. The supernatants of nasal polyp tissue homogenates, and the tissue levels of Alt a 1, Alternaria‐specific IgE, IL‐4, IL‐5, IL‐13, IL‐33, and galectin‐10 in nasal polyp supernatant were measured using enzyme‐linked immunosorbent assay (ELISA) systems. The tissue eosinophil numbers were also counted. We searched for the existence of postoperative nasal polyp in the medical records for more than 3 years in order to examine the usefulness of Alt a 1 as a predictor of nasal polyp recurrence after surgery.

First, we measured the levels of Alt a 1 in airway tissues and ROC curve based on nasal polyp recurrence data was developed, with a cut‐off value of 1.84 ng/g of local Alt a 1 (AUC = 0.75, Figure 1A). According to the presence or absence of nasal polyp recurrence, patients were divided into two groups and the levels of Alt a 1 were compared. The tissue levels of Alt a 1 were significantly higher in the recurrence group (p < .05, Figure 1B). Kaplan–Meier curves at the cut‐off point of the local tissue Alt a 1 level shows that the recurrence‐free rate in the low‐Alt a 1 group is higher than that in the high‐Alt a 1 group (p < .05, log‐rank test, Figure 1C). Although the levels of other common aeroallergens including Japanese pollen or mite allergens were measured, we could not find any local allergen as a predictor, except Alt a 1.

FIGURE 1.

The local tissue level of Alt a 1 and the nasal polyp recurrence after surgery. The supernatants of nasal polyp tissue homogenates were subjected to duplicated measurement of Alt a 1 level using the ELISA system. (A) Based on data from Alt a 1 levels diagnostic test by a ROC curve against the recurrence group after ESS, the cut‐off level of local Alt a 1 was 1.84 ng/g. (B) The Alt a 1 group was defined as those with 1.84 or higher ng/g in NP. All others were in the low‐Alt a 1 group. p values for the comparison of local Alt a 1 level between two groups (*p < .05) (Mann–Whitney U test). (C) Kaplan–Meier curves of the recurrence‐free rate at the cut‐off point of local Alt a 1 levels (*p < .05) (log‐rank test).

We further measured Alternaria‐specific IgE level in the airway tissues in order to determine the sensitization to Alt a 1. The levels of Alt a 1 in nasal polyps had a significant positive correlation with the levels of Alternaria‐specific IgE (rs = .56, p < .0001, Figure 2A). We divided patients into two groups according to the local tissue levels of local Alt a 1, and compared the levels of Alternaria‐specific IgE antibody between two groups. As a result, the levels of Alternaria‐specific IgE antibody were significantly higher in high‐Alt a 1 group than those in the low‐Alt a 1 group (p < .0001, Figure 2B).

FIGURE 2.

Allergic inflammation of CRSwNP and the local tissue Alt a 1 level. The tissue level of Alternaria‐specific IgE, galectin‐10, IL‐4, IL‐5, IL‐13, and IL‐33 supernatants were measured using the ELISA systems. (A) Correlation between local Alt a 1 and Alternaria‐specific IgE levels. Spearman's rank correlation coefficient showed the relationship between Alt a 1 and Alternaria‐specific IgE levels in airway tissues (A: rs = .56, p < 0001). (B) Patients were divided into two groups according to the local tissue level of local Alt a 1 (****p < .0001) (Mann–Whitney U‐test). (C) Characteristics by the levels of local tissue Alt a 1 for chronic rhinosinusitis groups. p values for comparison between low‐and high‐Alt a 1 antigen groups (*p < .05, ****p < .0001, NP, nasal polyp; HPF, per high‐powered field).

Fungi induce alarmins, which promote the development of type 2 response via increase in the number of eosinophils, accompanied by an increase in innate lymphoid cells and effector cells such as mast cells. ⁷ , ⁸ We measured the level of type 2 cytokines (IL‐4, IL‐5, and IL‐13) in the airway tissues and analyzed between high‐ and low‐Alt a 1 group, respectively, in order to assess the contribution of type 2 inflammation to local allergic reaction to A. alternata. Patients were divided into two groups according to the tissue levels of Alt a 1. The levels of IL‐4 were significantly higher in the high‐Alt a 1 group than those in the low‐group. IL‐4 plays an essential role in IgE class switching and production. The levels of IL‐5 and IL‐13 did not show significant difference among groups (Figure 2C).

Epithelial cells, which are activated by antigens, induce proinflammatory responses due to the production of alarmins. A. alternata exposure evokes IL‐33 secretion and extracellular DNA from the airway epithelium, which functions as an alarmin to stimulate type 2 immunity in airway diseases. ⁹ The levels of IL‐33 in NPs were significantly higher in the high‐Alt a 1 group than those in the low‐group (Figure 2C). Galectin‐10 is relatively an eosinophil‐specific protein, which is released from cytolytic cells, is expected to be a biomarker for activated eosinophils in eosinophilic inflammatory diseases. ¹⁰ The levels of galectin‐10 in the airway tissues were significantly higher in the high‐Alt a 1 group than in the low group (Figure 2C).

This is the first report to have quantified the levels of Alt a 1 in airway tissues and investigated the association to the type 2 molecules of allergic reaction. We have found that the increase in the local tissue levels of Alt a 1 were associated with the recurrence of refractory CRSwNP. There is a significant difference not only in the local tissue levels of Alt a 1 but also in tissue eosinophil counts between the recurrence and non‐recurrence groups. On the other hand, there is no significant difference in the local Alt a 1 level between the asthma and non‐asthma groups, although, there is a significant difference in the tissue eosinophil counts among the groups. These results might explain that the tissue levels of Alt a 1 can be a unique predictor of recurrence of refractory respiratory airway disease.

AUTHOR CONTRIBUTIONS

Y.M. and T.Y. designed and performed the experiments and wrote the manuscript. T.A., T.Y., T.E., Y.K., and S.S. provided the clinical samples. Y.M. and T.Y. edited the manuscript. M.A. and S.U. contributed scientific advice.

FUNDING INFORMATION

This work was supported in part by and Japan Society for the Promotion of Science (JSPS) (grant numbers: JP17K11356, JP25293348, JP21H02707, JP21K19368, , 24K11593 and 24K23305)

CONFLICT OF INTEREST STATEMENT

T.Y. received honoraria from Sanofi, Mitsubishi Tanabe Pharma, and Kyorin Pharma, and grant support from Sanofi. S.U. is an advisory board member of GlaxoSmithKline K.K. (GSK), received honoraria for lectures from AstraZeneca K.K. AZ received honoraria from GSK and Sanofi, and grant support from AZ, Novartis Pharma K.K., and Maruho Co. Ltd.

INFORMED CONSENT

The approval was obtained from the Human Ethics Review Committee of Akita University (approval number 1964). Informed consent was obtained from all participants in accordance with the principles laid out in the Declaration of Helsinki.

DATA AVAILABILITY STATEMENT

This work was supported by Japan Society for the Promotion of Science (JSPS), and the data that support the findings of this study are available from the corresponding author upon reasonable request.