Blood Eosinophils Subtypes and Their Survivability in Asthma Patients

Andrius Januskevicius, Egle Jurkeviciute, Ieva Janulaityte, Virginija Kalinauskaite-Zukauske, Skaidrius Miliauskas, Kestutis Malakauskas, Andrius Januskevicius, Egle Jurkeviciute, Ieva Janulaityte, Virginija Kalinauskaite-Zukauske, Skaidrius Miliauskas, Kestutis Malakauskas

Abstract

Eosinophils subtypes as lung-resident (rEOS) and inflammatory (iEOS) eosinophils are different in surface protein expression, functions, response to IL-5 and localization in lungs. rEOS- and iEOS-like eosinophils are found in blood; thus, we aimed to investigate their quantity and survivability in asthma patients. A total of 40 individuals were included: 10 steroid-free non-severe allergic asthma (AA), and 18 severe non-allergic eosinophilic asthma (SNEA) patients, the control group consisted of 12 healthy non-smoking subjects (HS). A bronchial challenge with Dermatophagoidespteronysinnus allergen was performed for AA patients and HS. Blood eosinophils subtyping was completed with magnetic beads' conjugated antibodies against surface CD62L. Eosinophils adhesion to hTERT airway smooth muscle (ASM) cells was measured by evaluating their peroxidase activity and viability by annexin V and propidium iodide staining. We found that the predominant blood eosinophil subtype in AA patients was iEOS, while rEOS prevailed in SNEA patients (p < 0.05). Moreover, rEOS demonstrated higher adhesion intensity compared with iEOS in all investigated groups. Both eosinophils subtypes of SNEA patients had higher survivability over the AA group. However, iEOS survivability from AA and SNEA groups was higher compared with rEOS under standard conditions, when rEOS survivability increased after their incubation with ASM cells. Bronchial allergen challenge abolished the dominance of blood iEOS in AA patients and prolonged only iEOS survivability. Though the challenge did not affect the adhesion of any eosinophils subtypes, the direct dependence of rEOS and iEOS survivability on their interaction with ASM cells was revealed (p < 0.05). These findings provide the premise for eosinophils subtype-oriented asthma treatment.

Keywords: adhesion; allergic asthma; eosinophils subtypes; inflammatory eosinophils; lung-resident eosinophils; severe eosinophilic asthma; survivability.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Inclusion and exclusion criteria of the study population. All recruited individuals were newly selected subjects. The inclusion and exclusion were criteria were verified after the screening visit. AA—Allergic asthma; CBC—Complete blood count; HS—Healthy subjects; SNEA—Severe non-allergic eosinophilic asthma.
Figure 2
Figure 2
Experimental study design. AA—Allergic asthma; ASM—Airway smooth muscle; FeNO—Fractional exhaled nitric oxide; HS—Healthy subjects; iEOS—inflammatory eosinophils; rEOS—lung-resident eosinophils.
Figure 3
Figure 3
Confirmation of separated eosinophils subtypes by flow cytometry. (A) Total enriched eosinophils population; (B) separated rEOS population, labeled with CD62L-APC antibody; (C) rEOS population labeled with CD101-APC antibody; (D) iEOS population labeled with CD101-APC antibody parts; (B–D) a homogeneous rEOS or iEOS population was used after the final magnetic separation steps. A non-gated enriched total eosinophils population; (B–D) panels after the gating procedures, excluding cell debris (with SSC/FSC) and non-viable (with propidium iodide) cells. SSC—side scatter; FSC—forward scatter, CD62L—L Selectine, CD101—Immunoglobulin superfamily member 2; Singlec-8—sialic acid-binding Ig-like lectin 8; APC—Allophycocyanin; FITC—Fluorescein isothiocyanate.
Figure 4
Figure 4
Blood rEOS and iEOS. (A) part of rEOS and iEOS in investigated individuals’ peripheral blood. (B) part of rEOS and iEOS in investigated individuals’ peripheral blood after bronchial allergen challenge. The results were presented as mean ± S.E.M. AA—allergic asthma; iEOS—inflammatory eosinophils; rEOS—lung-resident eosinophils; SNEA—severe non-allergic eosinophilic asthma; V1—visit 1 (before bronchial allergen challenge); V2—Visit 2 (24 h after bronchial allergen challenge). Eosinophils were counted from: Part (A) AA n = 10, SNEA n = 18, HS n = 12; Part (B) AA n = 9, HS n = 8. Statistical analysis: between investigated groups—Mann–Whitney two-sided U-test (independent data); within one study group—Wilcoxon matched-pairs signed-rank two-sided test (dependent data), comparing the rEOS and iEOS of each study individual separately.
Figure 5
Figure 5
Adhesion intensity of eosinophils subtypes. (A) adhesion of blood rEOS and iEOS on ASM cells; (B) adhesion of blood rEOS and iEOS on ASM cells after bronchial allergen challenge. The results are presented as the mean ± S.E.M. AA—allergic asthma; iEOS—inflammatory eosinophils; rEOS—lung-resident eosinophils; SNEA—severe non-allergic eosinophilic asthma; V1—visit 1 (before bronchial allergen challenge); V2—Visit 2 (24 h after bronchial allergen challenge). The results from independent experiments of: Part A—AA n = 10, SNEA n = 16, HS n = 11; Part B—AA n = 9, HS n = 7. * p < 0.005 compared with the same eosinophil subtype of HS group. Statistical analysis: between investigated groups—Mann–Whitney two-sided U-test (independent data); within one study group—Wilcoxon matched-pairs signed-rank two-sided test (dependent data), comparing the rEOS and iEOS of each study individual separately.
Figure 6
Figure 6
Viability of blood eosinophils subtypes. (A) The viability of blood eosinophils subtypes at baseline; (B) the viability of blood eosinophils subtypes after bronchial allergen challenge. The results are presented as mean ± S.E.M. AA—allergic asthma; iEOS—inflammatory eosinophils; rEOS—lung-resident eosinophils; SNEA—severe non-allergic eosinophilic asthma; V1—visit 1 (before bronchial allergen challenge); V2—Visit 2 (24 h after bronchial allergen challenge). The results from independent experiments of: Part (A) AA n = 10, SNEA n = 12, HS n = 9; Part (B) AA n = 8, HS n = 8. * p < 0.05 compared with the same eosinophil subtype of SNEA group, #p < 0.05 compared with the same eosinophil subtype of the HS group. Statistical analysis: between investigated groups—Mann–Whitney two-sided U-test (independent data); within one study group—Wilcoxon matched-pairs signed-rank two-sided test (dependent data), comparing the rEOS and iEOS of each study individual separately.

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