Changes in regulatory B-cell levels in bone marrow, blood, and sputum of patients with asthma following inhaled allergen challenge

• Published in: Journal of allergy and clinical immunology Vol. 141; no. 4; pp. 1495 - 1498.e9
• Main Authors: Oliveria, John-Paul, El-Gammal, Amani I., Yee, Michelle, Obminski, Caitlin D., Scime, Tara X., Watson, Richard M., Howie, Karen, O'Byrne, Paul M., Sehmi, Roma, Gauvreau, Gail M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.04.2018; Elsevier Limited
• Subjects: Adult; Allergens; Allergies; Asthma; Asthma - immunology; B-Lymphocytes, Regulatory - immunology; Bone marrow; Bone Marrow - immunology; Bronchial Provocation Tests - methods; Cytokines; Eosinophils - immunology; Female; Humans; Inflammation; Interleukin-10 - immunology; Lymphocytes B; Male; Sputum; Sputum - immunology
• ISSN: 0091-6749; 1097-6825; 1097-6825
• DOI: 10.1016/j.jaci.2017.11.013

[...]6 additional subjects (see Table E3 in this article's Online Repository at www.jacionline.org) with asthma with allergies provided blood samples to isolate CD19+ B cells and perform in vitro cultures with IL-4. [...]our data support a role of Breg cells to dampen the allergic immune response; however, a consensus on how to phenotypically define Breg cells would be helpful in delving further into the functional capacity of these cells.Methods Clinical tests Spirometry, skin prick test, methacholine challenge Spirometry was performed following American Thoracic Society guidelines.E1,E2 Skin prick tests were performed with common aeroallergen extracts, and histamine and saline were used as positive and negative controls, respectively, to confirm atopic status and determine a suitable extract for inhalation.E3-E5 A methacholine challenge test was completed using the tidal breathing method from a Wright Nebulizer to determine the provocative concentration that resulted in a 20% fall in FEV1 (PC20).E6Allergen and diluent challenges The methacholine PC20 and skin prick test were used to determine the allergen dose for inhalation, as previously described.E7 Increasing allergen concentrations were inhaled during tidal breathing for 2 minutes, and FEV1 was measured 10 minutes after each dose. The diluent control challenge consisted of three 2-minute inhalations of the diluting solution (0.9% saline).Sample collection and cell processing Sputum cells Sputum was induced by inhalation of 3%, 4%, and 5% saline and mucous plugs were selected and processed as previously described.E8,E9 Briefly, sputum was treated with Dulbecco's PBS and dithiothreitol and filtered through a nylon mesh; the cells were pelleted by centrifugation.E10 Two cytospins were prepared and stained with Diff Quik for cell differentials and the remaining cells were stored at 4°C for flow cytometry staining.Peripheral blood and bone marrow mononuclear cells Peripheral blood was collected into vacutainers containing heparin. Characteristic Controls (n = 7) Subjects with asthma with allergies (n = 11) Subjects with allergies (n = 7) Sex (M/F) 3/4 6/5 1/6 Age (y) 38 ± 5 30 ± 4 23 ± 3 FEV1 (% of predicted) 95.4 ± 1.5 94.0 ± 3.3 96.7 ± 3.4 Methacholine PC20 (mg/mL) >16 4.2 (2.1-8.4) >16 Sputum eosinophils (%) 0.06 ± 0.04 1.9 ± 0.4∗† 0.6 ± 0.2 Blood eosinophils (%) 1.3 ± 0.3 3.9 ± 1.1∗ 1.8 ± 0.4 Table E1 Subjects' demographic characteristics Sex Age (y) Predicted FEV1 (%) MCh PC20 preallergen (mg/mL) MCh PC20 prediluent (mg/mL) MCh PC20 postallergen (mg/mL) MCh PC20 postdiluent (mg/mL) Allergen extract inhaled Dilution of inhaled extract EAR Allergen (%) EAR Diluent (%) LAR Allergen (%) LAR Diluent (%) M 37 85.1 7.7 20.8 4.1 ND Grass 1:512 −34.8 1.0 −12.7 4.0 F 22 79.4 1.6 5.0 1.0 2.8 Ragweed 1:8 −50.0 −0.8 −31.3 −2.0 M 24 81.0 4.0 10.0 0.88 4.8 HDM 1:128 −34.1 −5.1 −19.5 −5.1 M 23 88.9 0.3 0.6 ND ND Cat 1:512 −22.0 −2.3 −22.3 −7.3 F 23 94.5 7.5 5.7 0.7 ND Horse 1:128 −47.3 −0.6 −26.5 −1.9 M 66 95.4 3.4 12.3 ND ND Cat 1:32 −48.5 −1.8 −38.8 0.0 M 53 97.6 5.4 10.3 7.4 7.3 HDM 1:64 −36.2 −3.8 −19.5 −1.8 M 28 112.4 10.5 9.6 0.1 ND HDM 1:64 −49.4 −7.2 −17.7 −4.9 F 22 94.3 4.7 5.3 0.2 ND Tree 1:32 −35.4 −2.7 −50.7 −2.7 M 51 98.4 0.3 0.4 0.8 1.7 Cat 1:64 −33.8 −4.5 −22.5 −3.1 35 ± 5 92.7 ± 3.1 2.8 (1.1- 7.0) 5.0 (2.0- 12.6) 0.8 (0.3- 2.8)∗ 4.2 (1.3- 9.9) −39.2 ± 2.9∗ −2.8 ± 0.8 −26.2 ± 3.6∗ −2.5 ± 1.0 Table E2 Response of subjects with asthma with allergies to inhalation challenge Characteristic Allergic subjects with asthma (n = 6) Sex (M/F) 2/4 Age (y) 32 ± 6 FEV1 (% of predicted) 87.9 ± 5.1 Methacholine PC20 (mg/mL) 6.4 (0.3-13.0) Sputum eosinophils (%) 1.1 ± 0.6 Table E3 Demographic characteristics of subjects with asthma donating blood for B-cell cultures Cell subset Baseline 7 h post 24 h post Allergen Diluent Allergen Diluent Allergen Diluent Bone marrow CD19+ 15.8 ± 1.5 15.3 ± 1.4 ND ND 16.8 ± 2.1 15.8 ± 1.1 CD19+FoxP3+ 10.5 ± 1.6 9.3 ± 1.6 ND ND 5.8 ± 1.2∗ 9.2 ± 1.0 Blood CD19+ 14.4 ± 1.6 13.3 ± 1.3 18.0 ± 1.9 15.3 ± 1.4 14.3 ± 1.4 13.8 ± 1.4 CD19+FoxP3+ 7.3 ± 1.4 10.7 ± 1.0 15.8 ± 4.9∗ 5.7 ± 0.6 11.3 ± 1.9 9.7 ± 0.7 Sputum CD19+ 8.6 ± 1.3 8.0 ± 1.6 16.7 ± 1.6∗ 8.5 ± 0.9 13.5 ± 2.1 9.3 ± 1.8 CD19+FoxP3+ 7.4 ± 1.2 7.8 ± 1.1 15.5 ± 8.0 7.9 ± 0.9 18.7 ± 3.0∗ 8.9 ± 1.5 Table E4 B-cell and Breg-cell frequencies after allergen inhalation challenge Baseline 7 h post 24 h post Cell subset Allergen Diluent Allergen Diluent Allergen Diluent Bone marrow—Breg-cell phenotypes CD5+ 12.1 ± 1.2 14.0 ± 1.5 ND ND 11.8 ± 1.3 13.2 ± 0.7 CD1d+CD5+ 4.6 ± 0.3 5.6 ± 0.9 ND ND 5.3 ± 0.6 6.0 ± 0.6 CD24+CD27+ 4.5 ± 0.2 5.2 ± 0.4 ND ND 6.5 ± 0.9 5.4 ± 0.3 CD24+CD38+ 38.0 ± 2.4 34.5 ± 3.4 ND ND 25.4 ± 3.1 30.0 ± 4.0 CD5+FoxP3+ 0.8 ± 0.1 1.1 ± 0.2 0.9 ± 0.2 0.8 ± 0.1 1.8 ± 0.4∗ 1.0 ± 0.1 CD1d+CD5+FoxP3+ 0.32 ± 0.05 0.42 ± 0.06 0.25 ± 0.06 0.31 ± 0.04 0.66 ± 0.19 0.31 ± 0.05 CD24+CD27+FoxP3+ 0.8 ± 0.2 0.8 ± 0.2 1.3 ± 0.4 0.5 ± 0.1 1.2 ± 0.4∗ 0.5 ± 0.1 CD24+CD38+FoxP3+ 0.8 ± 0.2 0.8 ± 0.2 1.1 ± 0.3 0.8 ± 0.2 2.2 ± 0.7∗ 0.7 ± 0.1 Blood—Breg-cell phenotypes CD5+ 11.5 ± 1.3 10.6 ± 0.9 7.2 ± 1.4∗ 12.3 ± 1.2 12.7 ± 1.2 11.6 ± 0.5 CD1d+CD5+ 3.2 ± 0.2 3.6 ± 0.3 2.1 ± 0.4∗ 4.3 ± 0.6 4.4 ± 0.4 3.9 ± 0.4 CD24+CD27+ 5.2 ± 0.9 5.9 ± 0.8 7.2 ± 1.1 5.4 ± 0.8 8.4 ± 1.5 5.4 ± 0.6 CD24+CD38+ 7.0 ± 1.1 7.9 ± 1.3 9.8 ± 1.5 8.7 ± 1.6 14.1 ± 2.2∗ 7.3 ± 1.0 CD5+FoxP3+ 0.8 ± 0.1 1.1 ± 0.2 0.9 ± 0.2 0.8 ± 0.1 1.8 ± 0.4∗ 1.0 ± 0.1 CD1d+CD5+FoxP3+ 0.32 ± 0.05 0.42 ± 0.06 0.25 ± 0.06 0.31 ± 0.04 0.66 ± 0.19 0.31 ± 0.05 CD24+CD27+FoxP3+ 0.8 ± 0.2 0.8 ± 0.2 1.3 ± 0.4 0.5 ± 0.1 1.2 ± 0.4∗ 0.5 ± 0.1 CD24+CD38+FoxP3+ 0.8 ± 0.2 0.8 ± 0.2 1.1 ± 0.3 0.8 ± 0.2 2.2 ± 0.7∗ 0.7 ± 0.1 Table E5 Frequency of Breg cells in blood and bone marrow after allergen challenge and the effects of inhaled allergen quantified with flow cytometry assessments of FoxP3+ Breg-cell frequencies