Characterization of Eosinophil Subsets In Allergic Disease

Abstract

ABSTRACT Eosinophilia is a hallmark of allergic diseases and has long been recognized for its role in innate type 2 inflammation. There is increasing appreciation for eosinophils' multi-functional roles in the homeostatic process, tumour suppression, tissue remodelling and fibrosis, metabolism and immunoregulation. To better understand how eosinophils contribute to the pathogenesis of allergic diseases such as allergic asthma, allergic rhinitis and atopic dermatitis (AD), eosinophils can be subdivided into functional subsets based on surface marker expressions. To explore the categorization of homeostatic and inflammatory eosinophils, we evaluated eosinophil subsets identified based on the level of surface marker expression of the pre-determined markers L-selectin (CD62L) and the IL-3 receptor (CD123) (Chapter 2). We measured the frequency of eosinophil subsets referred to as resident (rEOS: CD62L+CD123-), inflammatory (iEOS: CD62L-CD123+), double-positive (CD62L+CD123+) and double-negative (CD62L-CD123-) eosinophils in patients with asthma, a lung disease associated with eosinophilia/T2 high inflammation, and compared levels in patients with COPD and chronic cough, lung diseases not related to predominantly eosinophil/T2 high inflammation, and included a control group of healthy donors. We found that patients with chronic cough consistently showed the lowest levels of total sputum eosinophils yet had the highest proportions of sputum iEOS and eosinophils expressing the immunoregulatory marker Siglec 8 or activation marker CD69 out of the total eosinophil population. Numerically, the mild allergic asthma and the COPD groups had the highest total sputum eosinophil levels yet the lowest proportions of sputum iEOS levels. Pooling blood across all groups allowed us to compare surface markers between iEOS vs rEOS. Blood iEOS have higher proportions of eosinophils expressing IL-5 receptor (CD125), CRTH2, Siglec 8, CD63 and CD69 surface expression. We conducted inhaled allergen challenges in the mild allergic asthmatics and found a reduction in the proportion of blood rEOS and a corresponding increase in the proportion of sputum iEOS 24 hours post-allergen. This increase in sputum iEOS was accompanied by an increase in the proportion of sputum eosinophils expressing Siglec 8 and CD69. Furthermore, elevated levels of eosinophil growth factors GM-CSF and IL-5, the alarmin TSLP, and the potent eosinophil chemoattractant IL-16 were detected in the sputum 24 hours post-allergen. These results suggest that iEOS increase under the influence of allergic triggers in the airways, yet the function of this eosinophil subset has yet to be determined. In patients with atopic dermatitis, we evaluated the effects of 12 weeks of treatment with the anti-IL-5Rα biologic benralizumab on skin eosinophil levels during the late cutaneous response to intradermal allergen challenge as measured by skin wheal size (Chapter 3). Additionally, we evaluated the effect of benralizumab on eosinophil subsets identified as MBP (all eosinophils) and EG2 (activated eosinophils) positive cells as well as IL-5Rα -bearing cells, including eosinophil progenitor cells (EoP) cells, basophils and mast cells in the papillary dermis of allergen challenged biopsies. We also measured the levels of eosinophils, EoPs, hematopoietic progenitor cells (HPCs), and type 2 innate lymphoid cells (ILC2s) in the blood to understand changes in eosinophil levels across compartments. Benralizumab treatment depleted blood eosinophils and significantly lowered the levels of EoPs, HPCs, ILC2s in the blood compared to placebo. Additionally, benralizumab significantly reduced the levels of EG2+ eosinophils, and IL-5Rα -bearing cells, including EG2+ IL-5Rα + eosinophils, MBP+ IL-5Rα + eosinophils, EoPs, basophils, and mast cells in the allergen-challenged skin. MBP+ eosinophils were not reduced by benralizumab treatment. Clinically, we observed a trend toward improvement of the early cutaneous response, but benralizumab had no inhibitory effect on the late cutaneous response. These results demonstrate that while targeting IL-5Ra depletes eosinophils from the blood, this treatment only lowers eosinophils in the skin by approximately 90%. We also observed that the subset of activated eosinophils recruited to skin after intradermal challenge, as measured by EG2, are responsive to benralizumab treatment versus all eosinophils, as measured by MBP, which were not significantly reduced. A lack of clinical response indicates that the early and late cutaneous responses to allergen triggers are not driven by IL-5Rα expressing cells. Finally, we evaluated the effects of 12 weeks of treatment with benralizumab on markers of chronic AD by measuring eosinophils identified as MBP and activated eosinophils identified as EG2 positive cells, as well as other IL-5Rα -bearing cells, in the papillary dermis of chronic AD lesions (Chapter 4). We also measured changes in skin histology and validated physician and patient-reported clinical scores of atopic dermatitis. Compared to placebo, benralizumab significantly reduced IL-5Rα+ cells and MBP+EG2+ eosinophils in lesions and unaffected skin. In skin lesions, benralizumab also reduced MBP+ eosinophils and basophils but had no effect on EG2+ eosinophils, EoPs, or mast cell numbers. Finally, there was no change in other skin histological assessments or improvement in clinical scores. In the chronic AD inflammation found in lesions, in contrast to the acute inflammation induced by intradermal allergen challenge, benralizumab treatment reduced the overall eosinophil population. Activated EG2+ eosinophils were not changed in AD lesions, likely due to low numbers before treatment. The lack of improvement in histological and clinical outcomes despite the significant reduction in MBP+ eosinophils in chronic AD lesions suggests that inflammatory pathways driving disease do not involve eosinophils. Taken together, the findings of this dissertation highlight the presence of various eosinophil subsets in the circulation and mucosal tissues, including the skin and lungs. A better understanding of eosinophil subsets and their functions in the context of allergic disease may aid in fine-tuning the targets for anti-eosinophil biologics.