Sputum Autoantibodies in Patients with Severe Eosinophilic Asthma

Sputum autoantibodies in patients with severe eosinophilic asthma

Manali Mukherjee, PhD,a David C. Bulir, PhD,b Katherine Radford, MSc,a Melanie Kjarsgaard, RRT,a Chynna Margaret Huang, RRT,a Elizabeth A. Jacobsen, PhD,c Sergei I. Ochkur, PhD,c Ana Catuneanu, BSc,d Hanah Lamothe-Kipnes, BSc,d James Mahony, PhD,b James J. Lee, PhD,c Paige Lacy, PhD,d and Parameswaran K. Nair, MD, PhD, FRCP, FRCPCa Hamilton, Ontario, and Edmonton, Alberta, Canada, and Scottsdale, Ariz

Background: The persistence of eosinophils in sputum despite B cell–attracting chemokine 1). Immunoprecipitated sputum high doses of corticosteroids indicates disease severity in immunoglobulins from patients with increased autoantibody asthmatic patients. Chronic inflamed airways can lose tolerance levels triggered eosinophil degranulation in vitro, with release of over time to immunogenic entities released on frequent extensive histone-rich extracellular traps, an event eosinophil degranulation, which further contributes to disease unsuppressed by dexamethasone and possibly contributing to severity and necessitates an increase in maintenance the steroid-unresponsive nature of these eosinophilic patients. corticosteroids. Conclusion: This study identifies an autoimmune endotype of Objectives: We sought to investigate the possibility of a severe asthma that can be identified by the presence of sputum polyclonal autoimmune event in the airways of asthmatic autoantibodies against EPX and autologous cellular patients and to identify associated clinical and molecular components. (J Allergy Clin Immunol 2018;141:1269-79.) characteristics. Key words: Methods: The presence of autoantibodies against eosinophil Severe asthma, eosinophilia, autoantibodies, sputum, peroxidase (EPX) and anti-nuclear antibodies was investigated eosinophil degranulation, eosinophil peroxidase, autoimmunity, in patients with eosinophilic asthma maintained on high-dose anti-nuclear antibodies corticosteroids, prednisone, or both. The ability of sputum immunoglobulins to induce eosinophil degranulation in vitro was assessed. In addition, the associated inflammatory A persistence of airway eosinophilia despite daily maintenance microenvironment in patients with detectable autoantibodies oral corticosteroids (OCSs) indicates a difficult-to-control popu- was examined. lation projected to benefit most from anti-eosinophil biologics 1 Results: We report a ‘‘polyclonal’’ autoimmune event occurring currently in development. However, clinical trials targeting eo- in the airways of prednisone-dependent asthmatic patients with sinophils in prototype eosinophilic patients, although successful increased eosinophil activity, recurrent pulmonary infections, or in depleting circulating eosinophils, resulted in only an approxi- 2 both, as evident by the concomitant presence of sputum anti- mately 50% to 70% reduction in exacerbations, thereby hinting EPX and anti-nuclear antibodies of the IgG subtype. Extensive at the presence of alternative mechanisms that sustain local inflammation. Emerging hypotheses suggest the presence of cytokine profiling of sputum revealed a TH2-dominated microenvironment (eotaxin-2, IL-5, IL-18, and IL-13) and localized mechanisms that are refractory to maintenance cortico- increased signalling molecules that support the formation steroids, thereby delaying resolution of inflammation and leading of ectopic lymphoid structures (B-cell activating factor and to frequent exacerbations with characteristic clusters of free eosinophil granules (FEGs) in sputum.3,4 Increased abundance of FEGs in the airways strongly correlates with sputum eosinophil From athe Division of Respirology and bthe Department of Pathology and Molecular peroxidase (EPX) content released on luminal eosinophil degran- 5,6 Medicine, McMaster University, Hamilton; cthe Division of Pulmonary Medicine, ulation, which is indicative of ‘‘active’’ disease. Indeed, eosin- Mayo Clinic, Scottsdale; and dthe Department of Medicine, University of Alberta. ophil degranulation releasing FEGs indicative of cytolysis or Deceased. primary lysis is suggested to be pathogenic.7 Supported by the Canada Research Chair program and the Ontario Thoracic Society. Disclosure of potential conflict of interest: P. Lacy has received a grant from the Natural Increased frequency of immune cell degranulation over time Sciences and Engineering Research Council of Canada, has received a consulting fee leads to peroxidase enzyme release, which has known immuno- 8 or honorarium from MedImmune, and is employed by the University of Alberta. P. K. genic properties by virtue of its ability to cause tissue damage. Nair has received grants from AstraZeneca, Novartis, Teva, Sanofi, and GlaxoSmithK- Thyroid peroxidase and myeloperoxidase have established etio- line; has received personal fees from Roche, Teva, Novartis, Knopp, and Glax- logic roles in the pathogenesis of autoimmune disorders, such oSmithKline; and has provided consultation to a university spin-off company, 9 Cellometrics, that has a sputum filtration device. The rest of the authors declare that as Hashimoto thyroiditis and eosinophilic granulomatosis with 10 they have no relevant conflicts of interest. polyangiitis, respectively. EPX has been reported to cause Received for publication October 14, 2016; revised June 8, 2017; accepted for publica- necrotic lysis of epithelial cells in vitro.8 Therefore we speculated tion June 12, 2017. that increased eosinophil degranulation accumulates immuno- Available online July 24, 2017. Corresponding author: Parameswaran K. Nair, MD, PhD, FRCP, FRCPC, Firestone genic entities, such as EPX, in the airways and subsequent Institute for Respiratory Health, St Joseph’s Healthcare, 50 Charlton Ave E, Hamilton, markers of tissue injury, such as molecules that activate danger- Ontario L8N 4A6, Canada. E-mail: [email protected]. associated molecular pattern receptors and trigger a localized The CrossMark symbol notifies online readers when updates have been made to the adaptive immune response, leading to breach of immune toler- article such as errata or minor corrections ance per the Matzinger ‘‘danger’’ model.11 0091-6749/$36.00 Ó 2017 American Academy of Allergy, Asthma & Immunology A breach of mucosal immunity in the lungs is not unprece- http://dx.doi.org/10.1016/j.jaci.2017.06.033 dented. In the context of asthma, several studies have shown an

1269 1270 MUKHERJEE ET AL J ALLERGY CLIN IMMUNOL APRIL 2018

Subjects’ characteristics are summarized in Table I, and information Abbreviations used collected is with respect to the date of the archived sputum sample used for

Abs600: Absorbance at 600 nm the study. Asthma diagnosis was based on either postbronchodilator revers- ANA: Anti-nuclear antibody ibility of greater than 12% and/or PC20 of less than 8 mg/mL on methacholine BAFF: B cell–activating factor of the TNF family provocation challenge, and severity was determined based on the maintenance BCA-1: B cell–attracting chemokine 1 corticosteroid dosage.15 CRS: Chronic rhinosinusitis dsDNA: Double-stranded DNA EA: Eosinophilic asthma Measurement of airway eosinophil activity (sputum EET: Eosinophil extracellular trap EPX and FEG index) 16 EPX: Eosinophil peroxidase Sputum was induced with hypertonic saline, as described previously, and FEG: Free eosinophil granule endogenous EPX content was measured by means of ELISA in the cell-free 5 HC: Healthy control supernatant. FEGs in sputum cytospin preparations were ranked by 2 inde- IP: Immunoprecipitation pendent medical technologists between 0 and 3 (0 5 none, 1 5 few, 2 5 mod- LDH: Lactose dehydrogenase erate, and 3 5 many) for the current sputum sample and 2 more samples MEA: Mixed phenotype/pleiotropic asthma preceding it (based on a sputum database maintained for all patients seen at OCS: Oral corticosteroid the Firestone Clinic). The index was computed as the sum of the ranks over OCS-EA: OCS-dependent eosinophilic asthma 3 consecutive sputum samples divided by the maximum possible outcome SEE: Standard error of estimate (ie, 3 3 3 5 9), and the mean sum of the 2 indices thus calculated was consid- TCC: Total cell count ered the FEG index. This allowed us to take into consideration both the degree of current and previous degranulation. association with autoimmunity, although these were observa- Detection of sputum autoantibodies tional in nature and without a definite causal relationship. First, To detect immunoglobulin reactivity in sputum, cell-free soluble fractions circulating anti-nuclear antibodies (ANAs) with unknown antigen of processed sputum supernatants were subjected to immunoprecipitation (IP) with Protein A/G beads to generate IP-Igs, as described recently.17 Anti-EPX specificities were seen in 55% of asthmatic patients with aspirin reactivity was assessed by using an indirect ELISA17 developed in house and sensitivity, although airways were not examined.12 Two recent expressed as absorbance at 600 nm (Abs600) after background correction. An studies reported naturally occurring circulating anti-IgE IgG in ANA Line Immunoassay strip (IMTEC-ANA-LIA-Maxx; Human Worldwide, healthy subjects, with increased titers in atopic and nonatopic Weisbaden, Germany) was adapted for detecting sputum ANAs by using the asthmatic patients,13 along with increased circulating immune manufacturer’s protocol (except for the initial step, where 100 mL of sputum complexes in the sera of asthmatic subjects with heightened dis- supernatant was incubated overnight at 48C with 700 mL of diluent). The ease severity.14 sputum reactivity against eosinophils was further validated by means of To date, an autoimmune mechanism triggered locally in the confocal microscopy (see the Methods section in this article’s Online Repos- lungs targeting eosinophil-specific antigens to explain asthma itory at www.jacionline.org). severity has not been investigated. According to our hypothesis of a degranulation-mediated increase in immunogenic entities in the Sputum cytokines airways, we aimed to investigate the presence of autoantibodies All reported cytokines and chemokines were analyzed by Eve Technolo- targeted against EPX and autologous cellular/nuclear material (ie, gies (HD65 Discovery Assay; Calgary, Alberta, Canada), except for B cell– ANAs) to further assess the possibility of a localized polyclonal activating factor of the TNF family (BAFF; Quantikine ELISA; R&D autoimmune event associated with asthma severity. Systems, Minneapolis, Minn).

METHODS Assessing eosinophil degranulation with sputum Patient samples IP-Igs This was a retrospective study conducted with archived sputum samples Freshly prepared IP-Igs from individual patient samples with high collected from asthmatic patients during routine clinical management at the sputum autoantibody titers or low/no sputum autoantibodies and from Firestone Clinic, with approval from the local Hospital Research Ethics Board, healthy subjects were coated onto 48-well tissue-culture plates and blocked St Joseph’s Hospital, Hamilton, Ontario, Canada. Random sputum superna- with 2.5% human serum albumin for 2 hours at 378C. Eosinophils (1 3 105) tants used in this study were categorized based on the nature and history of isolated by using the MACSxpress Eosinophil Isolation Kit (Miltenyi Biotec their bronchitis: (1) patients with eosinophilic asthma (EA), with current GmbH, Bergisch Gladbach, Germany), according to the manufacturer’s pro- 2 sputum eosinophils of greater than 3%, normal total cell count (TCC) of less tocol, in serum-free RPMI 1640 medium with or without 10 6 mol/L dexa- than 9.7 3 106 cells/g, and a history of sputum eosinophilia that requires daily methasone (Sigma-Aldrich, St Louis, Mo) were added to the maintenance corticosteroid; (2) patients with neutrophilic asthma, with cur- immunoglobulin-coated wells and incubated for 12 hours at 378Cina5% 6 rent infective bronchitis with a TCC of greater than 9.7 3 10 cells/g, neutro- CO2 atmosphere. Lactose dehydrogenase (LDH) activity (LDH Assay; Ab- phils of greater than 65%, eosinophils of less than 1.5%, and no history of cam, Cambridge, United Kingdom) was measured along with double- sputum eosinophilia; and (3) patients with mixed phenotype/pleiotropic stranded DNA (dsDNA) release in the collected supernatants (Quant-iT Pi- asthma (MEA), with recurrent infective bronchitis, a TCC of greater than coGreen dsDNA Assay Kit; Molecular Probes, Eugene, Ore). To visualize 9.7 3 106 cells/g, neutrophils of greater than 65%, eosinophils of greater the event, 0.5 3 105 eosinophils were added to the IP-Ig–coated chamber than 1.5%, or a history of sputum eosinophils that requires daily maintenance slides (Lab-Tek II; Thermo Fisher, Waltham, Mass) at 3, 6, 9, and 12 hours. corticosteroids. Fifteen healthy volunteers with no known respiratory or sys- At given time points, the wells were ﬁxed with 4% paraformaldhyde, probed temic disorders, no personal/immediate family history of autoimmune dis- with 0.1 mg/mL anti-histone mAb (Millipore, Temecula, Calif), and counter- ease, and no infection within 4 weeks of sputum induction participated in stained with anti-mouse IgG Alexa Fluor 488 (Molecular Probes). Events the study with written consent. All subjects were nonsmokers (<10 per pack were visualized with a Nikon Eclipse TE2000-E (Nikon, Melville, NY) ﬂuo- year) or never smokers. rescence microscope and a Q-imaging Retiga 2000R camera (Surrey, British J ALLERGY CLIN IMMUNOL MUKHERJEE ET AL 1271 VOLUME 141, NUMBER 4

TABLE I. Demographics of subjects in the ﬁnal study cohort ICS-dependent OCS-dependent Mixed phenotype eosinophilic eosinophilic eosinophilic Neutrophilic Healthy control Patient groups asthma asthma asthma infective asthma subjects P value Abbreviation used in text ICS-EA OCS-EA MEA NIA HC Asthma severity Mild to moderate Severe Moderate to severe Mild to moderate None Bronchitis Eosinophilic Eosinophilic Pleiotropic Neutrophilic None Subjects, no. 13 20 18 13 15 Female sex, no. (%) 7 (54) 10 (50) 11 (61) 8 (61) 7 (47) .8922 Mean age (y [range]) 56 (23- 80) 53 (28-79) 60 (33-75) 55 (19-73) 34 (20-56)* <.0001 BMI (kg/m2) 26.8 6 4 33.01 6 5 30.2 6 7 30.8 6 4 24.8 6 4* .0023 Asthma onset (adult [% of no.]) 11 (84) 16 (80) 16 (88) 6 (46)* NA .031 Lung function

FEV1 (%) 77.6 6 18.9* 57.6 6 15.5 59.2 6 22 71 6 28 96.1 6 9.6* <.001 FEV1/FVC ratio 0.67 6 0.13 0.6 6 0.12 0.6 6 0.14 0.63 6 0.18 0.85 6 0.04* <.001 Daily medication ICS (mg/d), median (range) 500 (250-2000) 1200 (500-4000) 1125 (500-2000) 750 (0-3600) NA .0701 OCS (mg/d), median (range) 0 (0)* 11.25 (0-40) 6.25 (0-35) 0 (0-20)* NA .0003 Indices of airway inﬂammation Sputum TCC, mean (range) 2.3 (0.3-6.3) 7.2 (0.2-26.6) 48.6 (18.0-288)* 45.8 (14.8-150.9)* 5.19 (1.8- 9.2) <.0001 Neutrophils (% [range]) 48.0 (10.7-83.2) 43.3 (5.7-88.2) 84.4 (47-99.5)* 90.6 (67.3-99.7)* 32.0 (5.3-60.8) <.0001 Eosinophils (% [range]) 16.5 (3.7-69.3) 27.89 (3.3-87.8) 3.8 (0-14.7) 0.2 (0-0.8) 0.27 (0-1.5) <.0001 Symptoms/risk factors Atopy, no. (%) 9 (69) 10 (50) 12 (66) 6 (46) 8 (53) .6295 Sinus disease, no. (%) 9 (69) 13 (65) 13 (72) 6 (46) NA .4497 CRS with polyps, no. (%) 7 (53) 9 (45) 7 (38) 2 (15) NA .2077 Ex-smoker >10 pack-year history, no. (%) 4 (31) 2 (10) 5 (27) 8 (62) 0* .0016 Indices of systemic inﬂammation Total LKCs 8.775 6 2.5 10.68 6 4.1 11.46 6 4.1 9.892 6 3.7 NA .2782 Absolute eosinophils 0.38 6 0.27 0.5 6 0.5 0.3389 6 0.3 0.1 6 0.1* NA .005 Absolute neutrophils 5.825 6 2.1 7.767 6 3.6 8.839 6 4.2 7.523 6 3.7 NA .1385 C-reactive protein 7.914 6 15.3 6.97 6 4.9 16.37 6 23.1 22.4 6 36.2 NA .4441 Total serum IgE 260 6 319 650 6 1200 350 6 453 107 6 120 NA .2382 Autoantibodies, no. (%) 1 (7) 0 3 (16) 3 (23) 0 .1154 ANA (no.) 1 0 3 2 0 ANCA (no.) 0 0 0 1 0 Family history of autoimmunity, no. 3 1 0 1 0 Thyroid complications, no. (%) 2 (15) 1 (10) 4 (11) 1 (15) NA .3982

Data are presented as mean 6 SDs or ranges (minimums-maximums). Healthy reference range for blood LKCs: 4.0-11.0 3 109/L; eosinophils: 0.0-0.3 3 109/L; neutrophils: 2.0- 7.5 3 109/L; cutoff for C-reactive protein: 5.0 mg/L; and IgE: >165 kIU/L. ANCA, Anti-neutrophil cytoplasmic antibody; ANA, hospital panel includes antibodies against dsDNA, chromatin, ribosomal P, centromere B, Sm, smRNP, SSA, SSB, SSB (La), Scl-70, anti-topoisomerase, and Jo-1; BMI, body mass index; FVC, forced vital capacity; ICS, equivalent dose of fluticasone propionate; LKCs, leukocytes; NA, not applicable; OCS, prednisolone. *Indicates the group with significant variation. Atopy was defined as positive skin prick test response; Rh factor, erythrocyte sedimentation rate, and physician’s notes from patient charts within 3 months of sputum sample were used.

Columbia, Canada). Care was taken not to permeabilize the cells during the When we next analyzed the nature of inflammation and protocol. bronchitis history, we identified patients within the ICS group All other reported protocols and statistical methods are provided in the with Abs600 values of greater than the cutoff who had a history Methods section in this article’s Online Repository. of recurrent infections (sputum TCC > 14 3 106 cells/g, neutrophils > 65%, eosinophils >1.5%, or a history of sputum eosinophils requiring daily prednisone). Finally, these 51 sam- RESULTS ples from patients with EA were classified into 3 distinct Identification of anti-EPX IgGs in a subset of groups based on the nature of their bronchitis, with an addi- patients with EA localized in the airways tional age- and sex-matched neutrophilic asthma (NA) group We detected anti-EPX IgG in 51 patients with EA compared added to constitute the final cohort (Table I).Thedatadistribu- with 15 healthy control (HC) subjects (Fig 1, A), the former tion for the final cohort (Fig 1, C) showed significantly comprising a subpopulation of daily OCS users with Abs600 values increased anti-EPX IgG signal in the subgroup with OCS- of greater than the cutoff. To characterize this subpopulation, we dependent eosinophilic asthma (OCS-EA) comprising segregated the 51 patients with EA based on their daily corticoste- prednisone-dependent patients with persistent sputum eosino- roid use. The proportion of anti-EPX IgG was greater in the OCS- phils and the MEA subgroup of patients with airway eosino- dependent group compared with that in both inhaled corticosteroid philia (despite corticosteroid use)/history of eosinophilia and (ICS)–dependent patients and HC subjects (Fig 1, B). recurrent infections with increased sputum TCCs. In addition, 1272 MUKHERJEE ET AL J ALLERGY CLIN IMMUNOL APRIL 2018

FIG 1. Asthmatic patients with sputum eosinophilia have anti-EPX IgG in their airways. A, Anti-EPX IgG reactivity of sputum IP-Igs from patients with EA (n 5 51; blue squares indicate patients receiving daily OCSs) compared with HC subjects (n 5 15). P 5 .0002, Mann-Whitney U test. B, Patients with ICS- dependent EA (ICS-EA) and those receiving daily maintenance OCS (OCS-EA). Purple squares indicate eosinophilic patients with recurrent bronchitis. P < .0001, Kruskal-Wallis test. C, Final cohort (n 5 79) group- ed into ICS-EA (no infection, n 5 13), OCS-EA (no infection, n 5 20), and mixed eosinophilic asthma (MEA; recurrent infection, ICS and/or OCS dependent, n 5 18) groups and an additional neutrophilic noneosino- philic asthma group (NIA;n5 13), *Significant difference between groups: Kruskal-Wallis and Dunn multiple comparison test. D, Anti-EPX IgG titers of matched sputum versus sera from 15 patients with severe asthma. Data are presented as means 6 SEMs for each titer. P < .0001, 2-way ANOVA with the Sidak multiple comparison test. E, Protocol validation for using Protein A/G eluted immunoprecipitated eluate (IP-Ig) for detection of anti-EPX IgG, IgA, IgM (n 5 10 patients with severe EA; 2-way ANOVA, Tukey multiple comparison). *P < .01, **P < .001, and ***P < .0001. F-H, Anti-EPX ELISA conducted on 3 parallel plates (n 5 20, validation cohort; see Table E1) with sputum IP-Igs probed separately for IgG, IgA, and IgM reactivity. I and J, Representative micrographs of sputum IgG reactivity shown in green, EPX (red), and nuclei visualized with 49-6-diamidino-2-phenylindole dihydrochloride. Images are presented as overlay of the 3 channels to visualize colocalization (see Fig E1 for individual channels). Fluorescence intensity for sputum IgG binding normalized to the area for all 3 subjects: P < .0001, Friedman Test (repeated measures). The calculated cutoff was based on mean absorbance values of HC subjects 1 23 SD, as represented by dotted lines. ns, Not significant. we chose 15 patients with severe EA from within our final in this article’s Online Repository at www.jacionline.org for study cohort for whom matched serum for the tested sputum patient demographics; ie, the validation cohort). Anti-EPX reac- sample was available. A direct comparison of sputum IP-Igs tivity in the sputum supernatant was significantly weaker when and serum autoantibody titers was conducted, results of which probed for IgA and IgM compared with IgG (P < .0001, 2-way suggested anti-EPX IgGs to be localized to the airways ANOVA). This further validated the use of IP-Igs as the source (Fig 1, D). for detecting anti-EPX reactivity because the signal lost in the To address the concern that Protein A/G binds to IgA and IgM wash-through fraction was minimal. We investigated the nature with a lower affinity than IgG, we tested the anti-EPX reactivity of anti-EPX immunoglobulins detected in the sputum IP-Igs for all 3 isotypes in the neat sputum supernatant, the matched of 10 eosinophilic sputa (sputum eosinophils > 3%) against non- eluted IP-Ig and non-Ig wash-through fraction, in parallel (Fig 1, eosinophilic (n 5 4, sputum eosinophils < 2%) and healthy sub- E;n5 10, severe asthma; sputum eosinophils > 3%; see Table E1 jects (n 5 6) comprising the ‘‘validation cohort’’ (n 5 20, see J ALLERGY CLIN IMMUNOL MUKHERJEE ET AL 1273 VOLUME 141, NUMBER 4

FIG 2. Anti-nuclear/extractable antigen antibodies detectable in sputum of asthmatic patients. A, A representative scoring pattern is presented for estimating the ANA index score. Two blinded observers assigned a score of between 0 and 4 to each band specific for the given autoantigen. A score of 1 is given to a band showing comparable intensity to the internal cutoff control. The sum total score for all 17 autoantigens was divided by the maximum possible score (ie, 17 3 4 5 68), and the average of both the scores was used to generate an ANA index for each patient sample. B, ANA indices were validated with Hep-2 substrate slides, and representative micrographs are presented. C, ANA scores were plotted, and each symbol represents an individual subject score represented in the final cohort (n 5 79; P < .0001, Kruskal-Wallis test, Dunn multiple comparison test). The dotted line represents the cutoff value of 0.08 calculated based on the mean ANA index of 15 HCs 1 23 SDs. *P <.05 and ***P <.001. D, Significant positive correlation between sputum ANA scores and detected anti-EPX IgG levels (Spearman rank test). E, Sera ANA scores between 15 patients with severe asthma (with detectable sputum ANAs) and HC subjects (Mann-Whitney test) at 1:100 per manufacturer’s instructions. ns, Not significant.

Table E1). It was confirmed that the anti-EPX antibodies were of of possible multiple reactivities to autologous eosinophilic the IgG subtype (Fig 1, F-H). Finally, significant binding cellular components. of sputum IP-Igs to fixed permeabilized eosinophils allowed a visual confirmation of the phenomenon (Fig 1, I and J, and see Autoantibodies to nuclear and extractable nuclear Fig E1 for complete panel staining). The modest colocalization antigens in the airways of sputum IgG staining with EPX (r 5 0.23-0.71) and extensive To investigate the underlying propensity toward an autoimmune cytoplasmic staining suggested the occurrence of autoantibodies phenomenon coupled with the evidence of detectable ANAs in 1274 MUKHERJEE ET AL J ALLERGY CLIN IMMUNOL APRIL 2018

FIG 3. Sputum immunoglobulins with increased autoantibody levels cause eosinophil cytolysis with extracellular traps in vitro. Representative images of eosinophil cytolysis induced on 12 hours of incubation with immobilized IP-Igs from patients with high autoantibody (aAb) levels (n 5 8), patients with low aAb levels (n 5 7), and healthy volunteers (n 5 5). Each symbol represents sputum from an individual subject. A, Immunostaining with anti-histone mAb (green) shows visual conﬁrmation of increased histone-rich extracellular traps on ﬁxed nonpermeabilized eosinophils with the respective Ip-Igs or uncoated controls. Nuclei were stained with 49-6-diamidino-2-phenylindole dihydrochloride. Images were captured at 310

and 340 magnification. B, Respective anti-EPX IgG Abs600 values of sputum IP-Igs used for this experi- mental set are presented (Kruskal-Wallis test, followed by the Dunn post hoc multiple comparison test). C and D, dsDNA release in collected media was quantified (Fig 3, C), and LDH activity (Fig 4, D), measured as NADH conversion as a function of time, was monitored in media supernatants from 48-well plates run in parallel as a quantitative measure of the extent of cytolysis between the samples and uncoated controls and assess the suppressive effect of 1026 mol/L dexamethasone (Kruskal-Wallis test, followed by the Dunn post hoc multiple comparison test). Significant difference between groups: *P < .05, **P < .01, and ***P 5.001. Data are represented as means 6 SEMs. ns, Not significant. airways of patients with chronic obstructive pulmonary disease18 This further reiterated the presence of autoantibodies of multiple and anti-dsDNA IgG in nasal polyp extracts from patients with specificities typical of a polyclonal autoimmune response. No sig- chronic rhinosinusitis (CRS),19,20 we analyzed autoantibodies to nificant difference was seen in serum ANA profiles between 17 common autoantigens related to nuclear and extractable nuclear healthy volunteers and 15 patients with severe EA (reported in components (ANAs). The ANA scores on the immunoassay strips Fig 1, D) with detectable sputum ANAs (Fig 2, E). Taken together, (see Fig 2, A, for an example of the scoring index and the list of 17 the presence of a localized autoimmune event with in situ genera- autoantigens) were cross-validated with Hep-2 substrate slides tion of IgG autoantibodies to EPX and nuclear/cytoplasmic anti- (Immco Diagnostics, Buffalo, NY), as described recently (Fig 2, gens was evident. B).17 A significant increase in ANA scores was observed in the OCS-EA and MEA groups (Fig 2, C). The similar distribution pattern of both ANAs and anti-EPX reactivity in the final cohort Clinical relevance: Increased autoantibody levels in (Figs 1, C,and2, C) was corroborated by the significant correlation the airways of asthmatic patients contribute to value (Fig 2, D), thereby indicating a ‘‘polyclonal’’ autoimmune eosinophil activity event. The staining patterns varied from homogenous, speckled, Statistical analysis revealed significant correlation scores be- and mitochondria-like between the samples (data not shown). tween sputum autoantibody (IgG) levels and both EPX levels and J ALLERGY CLIN IMMUNOL MUKHERJEE ET AL 1275 VOLUME 141, NUMBER 4

FEGs but none with intact eosinophils (see Fig E2 in this article’s significant increase in the prevalence of both anti-EPX IgG and Online Repository at www.jacionline.org), indicating that auto- ANAs was evident in patients with increased degranulation pre- immune response was abundant in the airways of patients with senting with a history of lymphopenia. Furthermore, we observed high eosinophil luminal activity. Therefore we next investigated that all patients with high sputum TCCs with a concurrent history the clinical relevance of increased autoantibody levels in the air- of lymphopenia and airway degranulation had autoantibodies in ways to address whether their presence was a mere epiphenom- their sputum. enon or contributory to the observed airway eosinophil activity. We did not co-IP autoantibodies to determine antigen specificity (eg, anti-EPX IgG) because the aim of this experiment was to Identifying patients with an autoantibody assess the consequence of increased IgG autoantibodies in the air- signature: Clinical and molecular parameters ways and to assess their effects on naive eosinophils using an un- To identify clinical parameters associated with localized altered physiologic pool of sputum immunoglobulins. autoimmune responses, we adopted the following strategy, We determined that sputum IP-Igs with increased autoantibody considering our sample size. Patients with autoantibody signatures titers (both anti-EPX IgG and ANAs) induced eosinophil were defined as those with anti-EPX IgG or ANAs greater than degranulation in vitro. This was evident from the loss of mem- the cutoff threshold (calculated cutoff based on mean brane integrity (LDH release) and extensive extracellular release absorbance values of HCs 123 SD, as represented by dotted of histone-coated chromatin traps, which is indicative of eosino- lines in Figs 1, C,and2, C). A nonparametric test (independent- phil extracellular traps (EETs), observed with increasing time samples Mann-Whitney test) was conducted for every clinical points (see Fig E3 in this article’s Online Repository at www. parameter based on the distribution between the 2 groups (patients jacionline.org). Indeed, extensive events of EET formation were with autoantibodies vs patients without autoantibodies). We next quantified (LDH activity and dsDNA content in the collected su- performed multivariate regression analysis (method: Stepwise, pernatants) and found to be significantly induced by sputum IP- IBM SPSS version 23.0; IBM, Armonk, NY) separately for each Igs from patients with high autoantibody titers compared with autoantibody signature (anti-EPX IgG/ANA) by using the clinical those with low autoantibody titers and healthy subjects (Fig 3). parameters with a significant P value of .01 or less (see Table E2, Addition of dexamethasone at a physiologically relevant concen- A, in this article’s Online Repository at www.jacionline.org for 2 tration (10 6 mol/L) did not suppress sputum immunoglobulin- individual P values and Table E3 in this article’s Online induced degranulation (Fig 3, C and D), reflecting similar obser- Repository at www.jacionline.org for model summary and vations by Kita et al.21 excluded variables). FEG index and sputum TCCs were selected as positive predictors for anti-EPX signatures, generating a model with an R2 value of .235, a standard error of estimate (SEE) of 0.44, Mechanism of autoimmune susceptibility in and a P value of .001, whereas the FEG index was the sole patients with severe asthma predictor for the presence of ANAs (adjusted R2 5 0.119, The finding that not all patients with EA exhibit detectable SEE 5 0.47, P 5 .01; Table II). Thus the model suggested that sputum autoantibodies suggests the presence of regulatory patients with increased events of eosinophil degranulation mechanisms to support local immunogenic tolerance.22-25 In pa- (indicated by FEG index) and increased sputum TCCs, which is tients with autoimmune disorders, such as systemic lupus erythe- suggestive of susceptibility to infection, were more likely to matosus and rheumatoid arthritis, a strong association with have localized autoinflammatory responses. lymphopenia and reduced regulatory cell counts has been estab- Nonparametric analysis of all 67 inflammatory mediators and lished to be accountable for the loss of peripheral tolerance.26 correlation studies identified a unique microenvironment in Although the majority of our study subjects have low peripheral patients with an autoantibody signature (ie, anti-EPX IgG and/ lymphocyte counts, we did not document any significant correla- or ANAs; see Table E3). Eight inflammatory mediators were tion of sputum autoantibodies with either circulating or sputum computed to be significantly increased in these patients lymphocyte levels using the absolute values documented at the (P <_ .01; Fig 5), with a significant correlation among IL-13, time of sputum induction (Fig 4, A-D). Because the pathomechan- IL-5, IL-18 (TH2 environment), eotaxin-2 (eosinophil ism initiated by lymphopenia is time dependent based on the recruitment), macrophage-derived chemokine, IL-16 (TH2 gradual compensatory homeostatic expansion of autoreactive cell recruitment), and B cell–attracting chemokine 1 (BCA-1) clones, subsequently leading to loss of peripheral tolerance, we and BAFF (increased B-cell activity). To identify molecular also considered using an index that takes into account transient predictors, we used the same strategy as before. Again, only lymphopenic events over time. The history of lymphopenia was those cytokines/mediators were chosen for the regression based on the evidence of reduced blood lymphocyte counts of model whose P values in the nonparametric test were .01 or <1.5 3 109/L on >_3 occasions. Although lymphopenia has often less (refer to Table E2, B, for individual P values and Table been shown to have causality in the development of autoimmu- E4 in this article’s Online Repository at www.jacionline. nity, in our observations neither absolute lymphocyte counts (at org for model summary and excluded variables). The regression the time of sputum induction) nor the history of lymphopenia analysis suggested BAFF and IL-13 to be positive predictors for were seen to be associated with the presence of sputum autoanti- the presence of anti-EPX IgGs (R2 5 0.458, SEE 5 0.365, bodies (Fig 4, A-F). P 5.000) and eotaxin-2 and BAFF to be positive for predicting Because eosinophil degranulation was determined to be sputum ANA levels (R2 5 0.296, SEE 5 0.428, P 5 .000; associated with the presence of autoantibodies in both clinical Table II). Thus the molecular signature suggested that and murine studies, we segregated our population into patients autoimmune responses were likely to be higher in asthmatic with concurrent presence of increased airway activity (degranu- patients who retained a TH2 environment with increased lation) and a history of lymphopenia (Fig 4, G and H). Indeed, a eosinophil recruitment and B-cell activity. 1276 MUKHERJEE ET AL J ALLERGY CLIN IMMUNOL APRIL 2018

FIG 4. Sputum autoantibodies are prevalent in patients with increased airway degranulation and a history of lymphopenia. A-D, Association of sputum autoantibodies with circulating lymphocytes (absolute counts; Fig 4, A) and sputum lymphocytes (absolute counts; Fig 4, B) and distribution in patient subsets (Fig 4, C and D) in the final cohort. Dotted lines represent the reference range for circulating lymphocytes. E and F, AN- OVA with the Holm-Sidak multiple comparison test. Anti-EPX and ANA scores plotted for all subjects were divided into asthmatic patients with normal lymphocyte counts and those with a history (Hx) of lymphopenia (assessed as an blood absolute lymphocyte count <1.5 3 109/L currently and on 2 past occasions; Mann- Whitney test). G and H, Study population divided into 4 groups based on history of lymphopenia and degree of degranulation (measured by FEG index scores). Anti-EPX IgG values and ANA index scores were plotted. Significant difference between the groups: *P < .05 and **P < .01, ANOVA with Holm-Sidak multiple correction. The cutoff thresholds for both autoantibody detections are represented by the dotted line on the respective plots. ns, Not significant. Purple symbols indicate patients with sputum TCC and neutrophil per- centages indicative of infection. J ALLERGY CLIN IMMUNOL MUKHERJEE ET AL 1277 VOLUME 141, NUMBER 4

TABLE II. Clinical identiﬁcation of patients with the airway autoimmune endotype b Coefﬁcient Regression Model Predictors (standardized) R2 Adjusted R2 SE of the estimate F (df) Sig 1A. Clinical parameters for predicting Anti-EPX IgG FEG index 0.493 0.235 0.206 0.44367 7.992 .001 Sputum TCC 0.301 1B. Clinical parameters for predicting ANAs FEG index 0.345 0.119 0.099 0.47694 6.07 .018 2A. Molecular parameters for predicting anti-EPX IgG BAFF 0.465 0.458 0.443 0.36504 30.792 .000 IL-13 0.431 2B. Molecular parameters for predicting ANAs Eotaxin-2 0.467 0.296 0.274 0.42821 13.048 .000 BAFF 0.223

The dependent variable was the sputum autoantibody signature (either based on ANA index or anti-EPX IgG). Data are summarized from SPSS (version 23) output of model summary, ANOVA table, and coefﬁcients. The model summary and list of excluded variables are provided in Tables E2-E5. Sig, Signiﬁcance based on ANOVA.

FIG 5. Distribution of mediators included in the molecular signature for autoimmune endotype. Scatter

plots for sputum expression levels of positive predictors are shown. A-D, Mediators suggestive of the TH2- dominated environment: IL-5, IL-13, IL-18, and eotaxin-2. E-F, Mediators indicative of increased B-cell activ-

ity: BAFF and BCA-1. G and H, TH2 cell recruitment: IL-16 and macrophage-derived chemokine (MDC). Each symbol represents an individual patient: red indicates sputum with positive autoantibody signature (ie, detectable anti-EPX IgG and/or ANA above respective cutoffs). Signiﬁcant difference between groups: *P < .01, **P < .001, and ***P < .0001, Kruskal-Wallis test with Dunn multiple analysis.

DISCUSSION In our study cohort of 79 subjects, levels of both anti-EPX IgG We report a novel observation of pathogenic autoantibodies in and ANAs were greater in patients with prednisone-dependent the sputum of patients with persistent eosinophilia and severe EA and in those with severe asthma and pleiotropic bronchitis asthma. Sputum autoantibodies against EPX and several anti- compared with patients with mild-to-moderate EA and those with nuclear antigens absent from the circulation suggest that a neutrophilic asthma. Atopy, smoking status, and body mass index polyclonal autoimmune event localized to the airways has were not determinants of autoantibody levels (see Tables E2 and occurred in these patients, the presence of which directly E3). Because the prevalence of the detected autoantibodies was correlates with asthma severity. Patients with the proposed significantly less in the mild-to-moderate eosinophilic group autoimmune endotype require high doses of corticosteroids to (ICS-EA; Figs 1, C, and 2, C), it is reasonable to assume that control their asthma, which is characterized by increased airway the loss of tolerance demonstrated in our findings occurs with dis- eosinophilic degranulation coupled with sputum cellularity sug- ease progression. However, other unknown underlying events gestive of infection and systemic lymphopenia. These patients cannot be discounted at this stage that could simultaneously be presented with a unique sputum cytokine signature indicative of responsible for both the loss of tolerance and disease progression. increased eosinophil recruitment, chronic TH2 inflammation, and Finally, as a limitation of our cohort, although the healthy subjects B-cell activity. were not age matched to the asthmatic patients, there was no 1278 MUKHERJEE ET AL J ALLERGY CLIN IMMUNOL APRIL 2018 statistical difference in the ages of the 4 groups of asthmatic pa- eotaxin-2 and IL-13 levels would enhance eosinophil recruitment tients (Table I). into the lungs. It is unlikely that these observations are secondary to Recently, IgG-mediated eosinophil degranulation with for- nonspecific binding from an increased IgG level (HC subjects mation of extensive extracellular DNA traps, termed EETosis, vs 51 patients with EA; P <.001, Mann-Whitney test). When total was reported in vitro35 and in vivo36 in CRS tissue. Weiler et al37 IgG levels were analyzed in the final cohort (see Fig E4), a signif- demonstrated in 1996 that IgG caused eosinophils to degranulate icant increase was charted only for the mixed pleiotropic group (undergo exocytosis) to release EPX and potentially free gran- (MEA). In addition, staining intensities on Hep-2 slides (Fig 2, ules. Therefore an increase in IgG levels in asthmatic airways B) were not influenced by total IgG content in sputum. The pres- (caused by increased autoantibody levels) coupled to active ence of autoreactive IgE subtypes in these patients is considered eosinophil recruitment is likely to trigger eosinophil degranula- unlikely because of the unremarkable total IgE content in the final tion and EETs. However, from our current data set, it cannot be cohort (see Fig E4, D) and the nonsignificant correlation of IgE concluded whether EET formation is induced specifically by levels with sputum autoantibody levels (anti-EPX: r 520.04, downstream signaling of autoantibody-target antigen interaction P 5 .72; ANAs: r 5 0.136, P 5 .33). or IgG:Fcg receptor cross-linking. There is early clinical evi- It is interesting to note that, similar to our observation of dence that ragweed allergen–specific IgG from the sera of asth- localized autoreactive IgGs in an eosinophilic microenvironment, matic patients induced eosinophil degranulation in vitro,which there have been recent reports of anti-dsDNA IgGs in nasal polyp was abolished in the presence of anti-IgG and anti-FcgRII anti- extracts from patients with CRS,19 with no evidence of IgE autor- bodies but not with anti-IgE or anti-FcεRII.38 In addition, 20 eactivity. Despite the emerging evidence of a TH17 axis in auto- increased IgG-autoantigen complexes in the airways as a result immunity,27 we could not determine any correlation between of an autoimmune response would lead to ligation of FcgRII, detected autoantibodies and airway levels of IL-17 or IL-23 (cor- which has been shown to inhibit glucocorticoid-induced relation values are shown in Table E4). In addition, one of the eosinophil apoptosis.39 This, coupled with our observation that most commonly observed sputum reactivity bands on the ANA dexamethasone was unable to suppress the immunoglobulin- Line Immunoassay was against dsDNA similar to the observa- induced extracellular trap formation (Fig 3, C and D) possibly tions in patients with CRS.19,20 Even though a direct causality be- underlies the steroid insensitivity observed in the putative auto- tween chronic eosinophilic inflammation and development of immune endotype. local autoimmunity could not be established, observations re- In our current set of patients, we failed to demonstrate a ported in patients with CRS and our recent findings in severe significant association between the presence of lymphopenia and eosinophilic airways suggest a strong association. It is also impor- years of prednisone use (r 5 0.25, P 5.08; see Table E2). Further- tant to mention that the potential of a polyclonal autoantibody more, ‘‘prednisone dose’’ in our multivariate regression model response, as seen in these patients, might simply be the nonspe- was not computed to be a ‘‘predictor’’ (refer to excluded variable cific consequence of unresolved inflammation. OCS in Table E3). In addition, a recent study shows that variations Chronic unresolved inflammation is considered a critical in the immunome with corticosteroid treatment do not affect reg- trigger for inducible bronchus-associated lymphoid tissue forma- ulatory T-cell populations, the suppression of which is implicated tion,28,29 with a typical chemokine signature comprising BCA-1, in the development of autoimmunity.40 Finally, the l5/hE2 murine CCL21, and BAFF, as seen in patients with Sjogren€ disease30 and model (see the Methods and Results sections and Fig E5), which rheumatoid arthritis31,32 presenting with pulmonary complica- resembles our asthmatic population with respect to intense airway tions. Increased sputum BAFF and BCA-1 levels (Fig 5) coupled eosinophil degranulation, was found to develop anti-EPX IgG with BAFF as a positive predictor for the presence of lung auto- without being subjected to any steroid treatment. Nevertheless, antibodies (Table II; and see Table E5 in this article’s Online Re- we acknowledge that without a longitudinal study with a justified pository at www.jacionline.org) support the likelihood of local sample size, the role of long-term corticosteroid use in the devel- B-cell clusters as the seat of autoantibody generation. We were opment of observed autoimmunity cannot be ruled out unable to confirm the presence of B-cell clusters in our patients definitively. because we did not have access to resected lung tissue (broncho- In conclusion, these findings suggest that some patients with scopic mucosal tissue might not suffice). However, a recent severe asthma might have autoantibodies against autologous description of ‘‘asthma granulomatosis’’ was based on the immu- eosinophilic cellular components that can be detected in nohistologic evidence of granulomas in lung biopsy specimens sputum. These patients, which are crudely estimated to be from prednisone-dependent patients with severe disease.33 Again, approximately a third of patients with severe EA, can be double-transgenic l5/hE2 mice with severe respiratory inflamma- identified as those with luminal eosinophil degranulation, tion and airway eosinophil degranulation (resembling our patient lymphopenia, and recurrent infective bronchitis and a charac- population) were seen to have localized anti-EPX IgG in their teristic lung TH2 microenvironment along with increased bronchoalveolar lavage fluid in addition to positive immunohisto- B-cell activity, features that are often overlooked in routine logic staining for B-cell clusters (see the Methods and Results clinical practice. Comprehensive understanding of the molecu- sections and Fig E5 in this article’s Online Repository at www. lar events that lead to the described autoimmune endotype will jacionline.org). The presence of granulomas or ectopic lymphoid pave the way for better and novel therapeutic options and structures with higher organization (inducible bronchus- improve patient management. associated lymphoid tissue) in our autoantibody-positive patients is possible. Furthermore, localized expression of BAFF, BCA-1, We thank Brenda Helpard and Nicola LaVigne, MLT, Hargreave Sputum IL-16, and IL-15 (see Table E2) would support both T cell–depen- Laboratory, St Joseph’s Healthcare, Hamilton, Ontario, Canada, for their dent and independent B-cell activation and differentiation to expertise with sputum processing. We dedicate this manuscript to our dear antibody-secreting plasma cells in situ,28,34 whereas increased colleague Professor Jamie Lee. We sorely miss his wit and wisdom. J ALLERGY CLIN IMMUNOL MUKHERJEE ET AL 1279 VOLUME 141, NUMBER 4

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