Interleukin 5-dependent inflammatory eosinophil subtype involved in allergic insect bite hypersensitivity of horses

Insect bite hypersensitivity (IBH) is an equine allergy characterized by a Type I and delayed Type IVb eosinophilic allergy. The main cell types in IBH lesions are Th2 cells and eosinophil infiltration into the dermis. An increase of eosinophil cell counts in the blood has been shown to correlate with IBH lesions and is associated with IBH severity. An active immunization targeting eosinophils was developed using a virus-like particle-based equine interleukin (eIL)-5-CuMVTT vaccine (reviewed in1). Eosinophils depend on IL-5 for survival, maturation, and proliferation.2 The vaccine induces the production of anti-self-IL-5 antibodies in horses, thereby significantly lowering eosinophil levels being released from the bone marrow into the bloodstream thereby significantly reducing IBH lesion scores.3 In humans and mice, two different subsets of eosinophils have been described. Based on distinct roles, eosinophils were subdivided into steady-state resident (rEos) and inflammatory eosinophils (iEos) in mice.4 In humans, comparable subtypes were found, that is, normodense and hypodense eosinophils.5 The objective of this study was to assess and characterize eosinophil subsets in a new cohort of healthy and allergic IBH-affected horses. These horses were either vaccinated with eIL-5-CuMVTT, treated with a placebo, or left untreated including sub-grouping into severity of IBH of mild, moderate, and severe. Methods are described in Data S1. As described earlier eosinophil cell counts in the blood were significantly reduced in vaccinated horses when compared to placebo horses over time (Figure S1).3 At a single timepoint (August), blood was collected for a detailed eosinophil characterization. Automated differential blood analysis (Figure 1A) and percentages by flow cytometry (Figure S2) showed significantly increased eosinophils in untreated or placebo-treated IBH horses, when compared to healthy and vaccinated horses (Figure 1B). Moreover, eosinophil percentages increased with severity (Figure 1C) further emphasizing their dominant role during disease progression. Healthy horses' eosinophils (Figure 1E) were significantly smaller in size when compared to IBH horses (Figure 1F), independent of treatment (Figure 1D), and severity (Figure S1). Another characteristic is the granularity of intracellular secondary granules containing secretory cytotoxic proteins causing tissue damage. Healthy horses' eosinophils (Figure 1H) contained significantly less granules when compared to IBH horses (Figure 1I), independent of treatment (Figure 1G), and severity (Figure S1). Of note, in severely affected horses' eosinophil granularity seem to split into two groups pointing towards potential sub-phenotypes of the disease. Once eosinophils leave the bone marrow, they shortly circulate in the blood, awaiting signals for extravasation.6 Survival and effector functions are directly linked with the aim to extravasate and migrate to the final tissue destination. Tissue half-life can be increased by expression of IL-5 or eotaxin-1 (reviewed in2). We stained surface-expressed integrins CD49d and CD49f on eosinophils from healthy (Figure 2A) and IBH horses either untreated (Figure 2B), or vaccinated with hardly any detectable eosinophils (Figure 2C). Single-positive CD49d+CD49f− cells were predominantly present in healthy horses, while strongly reduced in IBH horses, independent of treatment (Figure 2D) and severity (Figure 2E). Double-positive CD49d+CD49f+ cells were strongly reduced in healthy horses, while predominantly present in IBH horses, independent of treatment (Figure 2F) and severity (Figure 2G). In conclusion, in horses, two distinct eosinophil subsets have been identified. The loss of smaller, less granulated CD49d+ single-positive cells appears to be associated with the transition towards IBH pathogenic state, which is characterized by larger, more granulated, predominantly CD49d+CD49f+ double-positive cells. The latter are likely more toxic and probably have a higher migratory potential. These findings in horses may reflect the observations in mice, where a reprogramming changes morphology and expression profile from rEos towards iEos subtype. Vaccination targeting equine IL-5 depleted numbers of inflammatory eosinophils, suggesting IL-5 dependency for CD49d+CD49f+ double-positive eosinophils. This project was supported by funding of the Swiss National Fond (SNF) BRIDGE (Grant 40B2-0_194689) to AF-G and Evax AG (CH). Stanislav Pantelyushin and Tanya Rhiner planned, performed experiments (Stanislav Pantelyushin laboratory experiments, Tanya Rhiner clinical experiments), interpreted the data, and supported writing the manuscript. Fadi Jebbawi, Federica Sella, Nina Waldern, Juwela Lam, Alex Chemnitzer, Anna Fricker, and Fabia Canonica performed experiments. Angelika Schoster, Katharina Birkmann, and Daniel Widmer contributed to discussions. Antonia Fettelschoss-Gabriel planned, performed clinical studies, interpreted the data, and wrote the manuscript. We thank all horse owners who participated with their horse(s) in our clinical studies. Open access funding provided by Universitat Zurich. KB, DW, FC, and AF-G are involved in the development of therapeutic equines vaccines. TR, FJ, FS, NW, JL, AC, AF, and AS have no financial or personal conflict of interest. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Figure S1. Figure S2. Data S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.