Allergen-Induced IL-6 Trans-Signalling Activates T Cells to Promote Type 2 and Type 17

Allergen-induced IL-6 trans-signalling activates  T cells to promote type 2 and type 17 airway inflammation

Md Ashik Ullah, M. Pharm, Joana A Revez, B Sci, Zhixuan Loh, B Biomed Sci, Jennifer Simpson, B Biomed Sci, Vivian Zhang, PhD, Lisa Bain, B Sci, Antiopi Varelias, PhD, Stefan Rose-John, PhD, Antje Blumenthal, PhD, Mark J Smyth, PhD, Geoffrey R Hill, MD, Maria B Sukkar, PhD, Manuel A R Ferreira, PhD,*and Simon Phipps, PhD*

METHODS

Mice

Wild-type C57Bl/6 mice (WT) were purchased from the University of Queensland Biological Resources (Brisbane, Australia). IL-17A fate-reporter mice (Il17aCreR26ReYFP on C57Bl/6 background) were generated by Prof. Brigitta Stockinger (MRC National Institute for Medical Research, London, UK) and supplied by Prof. Geoffrey R. Hill (QIMR Berghofer Medical Research Institute, Brisbane, Australia).E1 The reporter mice were generated by intercrossing R26ReYFP reporter mice with Il17aCre mice; hence, cells activated to express IL-17A also express enhanced yellow fluorescent protein (eYFP). TLR4-deficient mice (TLR4-/-) were a kind gift from Dr. Matthew J. Sweet (Institute for Molecular Bioscience, University of Queensland, Australia), TLR2-deficient mice (TLR2-/-) were provided by Dr. Antje Blumenthal (University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, Australia) and TCRδ cell-deficient mice (Tcrδ-/-) were supplied by Prof. Mark J. Smyth (QIMR Berghofer Medical Research Institute, Brisbane, Australia). Eight- to twelve-week old mice were used for experiments which were performed in accordance with the Animal Care and Ethics Committees of the University of Queensland (Brisbane, Australia).

Induction of allergic airway inflammation

Allergic airway inflammation was induced according to our previously published protocol.E2Briefly, mice were lightly anesthetized with isoflurane and sensitized intranasally with either PBS or 100 µg of HDM extract (Dermatophagoides pteronyssinus; Greer Laboratories, Lenoir, NC, USA) or 100 µg of CR extract (Blatella germanica; Greer Laboratories, Lenoir, NC, USA) at day 0. At day 14, 15, 16 and 17, mice were challenged intranasally with either PBS or 5 µg of HDM or 5 µg of CR extract. The mice were euthanized 3 hours after the last allergen challenge (Fig 1). The endotoxin concentration of HDM was 424.3 EU/100 µg of protein (according to the manufacturer certificate of analysis) and CR extract was 395 EU/100 µg of protein (determined by using Endosafe-PTS Limulus amebocyte lysate test; Charles River Laboratory, Charleston, SC, USA). To investigate the role of IL-6R signalling in the development of allergic inflammation, mice were treated via the intranasal route with 1 mg of anti-mouse IL-6R monoclonal antibody (clone MR16-1; Chugai Pharmaceutical Inc, Japan) or isotype control IgG (Sigma-Aldrich, St Louis, MO, USA) 24 hours prior to allergen challenge (i.e. day 13). Lower doses (0.50 and 0.25 mg,) of MR16-1 were also tested (not shown) but only the 1 mg dose suppressed all features of allergic airway inflammation. To assess the role of IL-6 trans-signalling, CR sensitized and challenged mice were given 250 µg of soluble gp130-Fc (generated in the laboratory of Prof. Stefan Rose-John, PhD, Christian-Albrechts-University of Kiel, Kiel, Germany)E3 24 hours prior to challenge (day 13).

Measurement of airway hyper-reactivity (AHR)

AHR was measured 3 hours after the last challenge by forced oscillation technique using Flexivent apparatus (SCIREQ, Montreal, Canada) according to the protocol described earlier.E4 Briefly, mice were anesthetized by injecting a cocktail of xylazine (0.2mg/10gm of body weight) and ketamine (0.4mg/10gm of body weight) and kept under mechanical ventilation. Airway resistance was measured at increasing doses of nebulised methacholine (0 to 30 mg/ml for 1 min) (Sigma-Aldrich, St Louis, MO, USA).

Analysis of airway inflammation

A bronchoalveolar lavage was performed by flushing the lungs with 600 µl of ice-cold PBS. The recovered fluid was centrifuged (1600 rpm for 5 minutes) and the bronchoalveolar lavage fluid (BALF) stored at -80ºC until analysis. Following red blood cell (RBC) lysis with Gey’s lysis buffer, the total number of leukocytes from the lavage was counted, and cytospin slides prepared using a StatSpin Cytofuge 2 (Iris sample processing, Westwood, MA, USA). After air-drying, the slides were stained with May-Grunwald Giemsa solution (Sigma-Aldrich, St Louis, MO, USA). To enumerate macrophages, lymphocytes, neutrophils and eosinophils, a total of 300 cells were counted using a light microscope. For histological analysis, lung lobes were fixed in formalin and embedded in paraffin. Five micrometer thick sections were cut and stained with periodic acid-Schiff (PAS) to visualise mucus secreting cells. The total number of mucus-secreting airway epithelial cells (AECs) were counted and expressed as a percentage of total AECs. Four airways were counted for each mouse.

Splenocyte culture

Spleens from naive WT or TLR2-/- mice were excised and the cells dissociated by gentle pressure with a syringe plunger over a 70 µm cell strainer. After RBC lysis, the splenocytes were washed with ‘growth media’ (1 mM sodium pyruvate, 10% FBS, 2 mM L-gutamine, 20 mM HEPES, 100 U/ml penicillin-streptomycin and 50 µM 2-mercaptoethanol in RPMI-1640 medium), seeded into a 96-well plate at a density of 106 cells per 200 µl growth media, and stimulated with various concentrations of HDM, CR, TLR4 ligand lipopolysaccharide (LPS; Sigma-Aldrich), TLR2 ligand lipoteichoic acid (LTA; Sigma-Aldrich), dectin-1 agonist curdlan (Sigma-Aldrich), or diluent control (growth media). The culture supernatants were collected 24 hours later and stored at -80ºC prior to cytokine analysis.

Flow cytometry

Lung lobes were dissected and single cell suspensions prepared by mechanical digestion through a cell strainer. After RBC lysis, lung cells were washed twice with FACS buffer (2% FCS in PBS), seeded into a 96-well plate (106 cells/well) and incubated with Fc block for 20 minutes. Cells were stained with the following fluorescence labelled antibodies: V500 conjugated anti-CD3ε (clone 500A2), V450 conjugated NKp46 (clone 29A1.4) from BD Biosciences (San Jose, CA, USA); PerCP-cy5.5 conjugated anti-CD4 (clone RM4-5) from E-Bioscience (San Diego, CA, USA); Alexa-fluoro 647 conjugated γδ-TCR (clone GL3), and PE conjugated anti α-galactosylceramide:CD1d complex (clone L363) from Biolegend Inc, (San Diego, CA, USA). XenolightTM CF750 (Caliper Life Sciences, Massachusetts, USA) antibody labelling kit was used to conjugate CF750 fluorochrome with anti-IL-6R antibody according to the manufacturer’s protocol, and used for both flow cytometry and in-vivo imaging. Cells were enumerated using a BD LSR Fortessa cytometer (BD Biosciences, San Jose, CA, USA) and data analyzed using BD FACS DIVA software (BD Biosciences, San Jose, CA, USA).

Cytokine/chemokine analysis

The concentration of IL-6, IL-17A, IFN-γ (Biolegend Inc, San Diego, CA, USA); CCL24, CXCL1, IL-13, IL-17F, IL-6R, IL-23 (R & D systems, Minneapolis, MN, US) IL-5 (BD Biosciences, San Jose, CA, USA) expression in BALF and/or splenocyte supernatants wasanalysed by ELISA according to the manufacturer’s protocol.

In-vivo imaging

XenolightTM CF750 (Caliper Life Sciences, Massachusetts, USA) conjugated anti-IL-6R antibody (1000 µg) was administered via the i.n. route. The mice were imaged at different time intervals to examine the bio-distribution of the antibody with the Xenogen imaging system (Xenogen IVIS 200; Caliper Life Sciences, Massachusetts, USA). After 72 hours, the mice were euthanized and the organs were dissected and images were taken. All the images were processed using Living Image Version 4 software (Caliper Life Sciences, Massachusetts, USA).

Recruitment of subjects and clinical testing

A total of 158 subjects with a doctor diagnosis of asthma were recruited from the greater Brisbane area between October 2012 and December 2013 via media appeals to participate in this study and completed the clinical protocol. Clinical testing included completing a detailed questionnaire about asthma history, symptoms, severity, medication and triggers; skin prick testing against six common allergens; spirometry (Micromedical Microlab Mk 8); measurement of fractional exhaled nitric oxide (feNO, NIOX MINO); sputum induction; and blood collection. Participants were asked to withhold anti-histamines for 72 hours, inhaled steroids for 48 hours, non-steroidal preventers or symptom controllers for 24 hours and reliever medication for 8 hours before testing. For the present study, only individuals who reported still suffering from asthma were considered for analysis (N=138). This study was approved by the QIMR Berghofer Human Research Ethics Committee and all subjects gave written informed consent.

Sputum induction and analysis

Sputum induction with hypertonic saline (4.5%) and sputum processing were performed as described previously.E5 Briefly, saline was inhaled for doubling periods (30 sec, 1 min, 2 min, 4 min) from a DeVilbiss Ultrasonic nebulizer (UltraNeb) connected to a Hans Rudolph 2700 two-way valve box with rubber mouthpiece and nose clips. Forced Expiratory Volume in 1 sec (FEV1) was measured 60 sec after each saline dose, after which participants were asked to expectorate into a sterile container. The test was stopped when either the FEV1 had fallen by more than 20% or 15.5 cumulative minutes nebulisation time had elapsed. Sputum plugs were selected, dispersed with dithiothreitol (DTT) for a minimum of 30 minutes (maximum 70 min) at room temperature and filtered through a 60 m nylon filter. A total cell count was then performed with a light microscope. The sample was then centrifuged at 400g for 10 minutes; the supernatant was stored at -80ºC and cytospin slides prepared and stained with May Grunwald and Giemsa. A differential cell count of 400 non-squamous cells was then performed. Based on the differential cell count, the sputum inflammatory subtype was classified into four groups, as proposed previouslyE6: eosinophilic (≥1% eosinophils), neutrophilic (≥61% neutrophils), mixed granulocytic (≥1% eosinophils and ≥61% neutrophils) and pauci granulocytic (<1% and <61% neutrophils).

Measurement of IL-6 and sIL-6R protein in serum and sputum supernatant

Of the 138 current asthmatics tested, 68 provided an induced sputum sample. Of these, we excluded from analysis 35 sputum samples with high saliva contamination (>40% squamous cells) or low cell viability (<40%), thus resulting in 33 samples available for analysis. A matching 10 ml blood sample was collected in a serum tube from 32 of these asthmatics immediately after sputum induction. The sample was centrifuged at 805 g for 10 min, and the serum layer extracted and stored at -80ºC until analysis. ELISA kits (R&D Systems, Minneapolis, MN, USA) were used to measure IL-6 and sIL-6R levels according to the manufacturer’s procedures. The optical density was determined using a BioTek PowerWave XS2 microplate spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA) at both 450 and 540 nm wavelengths. Results and standard curves were acquired with BioTek Gen5 2.0 Data Analysis Software.

The detection of assay standards for IL-6 was affected by DTT treatment (Fig. E10Ain the Online Repository),as previously reportedE7; similar results were obtained with the sIL-6R assay (Fig. E10Bin the Online Repository). Despite this, median IL-6 levels measured in sputum with the ELISA assay used in our study have been shown to be comparable between DTT-treated and DTT-untreated samples, suggesting that the activity of DTT is neutralized during the process of mucolysis, thus reducing its effect on the IL-6 immunoassayE7. Furthermore, we found that if we used the standard curve obtained from the DTT-treated assay standards to determine IL-6 and sIL-6R concentrations in sputum samples, the resultingmedian sputum concentration levels (213 pg/mL and 788 pg/mL, respectively) were considerably higher than those reported in the literature for sputum or bronchoalveolar lavage fluid of asthmaticsE8-12, suggesting that they were overestimated. Collectively, these results are consistent with a significant effect of DTT on the detection of the assay standards but not (or less so) on the detection of the mediators in sputum samples. Therefore, we report IL-6 and sIL-6R sputum levels based on the standard curve obtained from the DTT-untreated assay standards.

Statistical analysis

Results from mice experiments are presented as mean values ± SEM. For the analysis of differences between groups, unpaired Student’s t-test (2-tailed) was used. AHR (comparing the Rn values at each dose of methacholine) data was analyzed using two-way ANOVA with Bonferroni post-hoc testing. Dose-response curves for splenocytes cultures were analyzed using one-way ANOVA with Bonferroni post-hoc testing. The software GraphPad Prism version 6.0 (La Jolla, USA) was used for all the analyses.

The association between sputum IL-6 and sIL-6R levels and clinical features of asthma was tested using linear regression, after applying a non-parametric transformation to normalise cytokine levels. There were no significant effects of age or sex on IL-6 or sIL-6R levels.

REFERENCES

E1.Hirota K, Duarte JH, Veldhoen M, Hornsby E, Li Y, Cua DJ, et al. Fate mapping of IL-17-producing T cells in inflammatory responses. Nature immunology 2011; 12:255-63.

E2.Ullah MA, Loh Z, Gan WJ, Zhang V, Yang H, Li JH, et al. Receptor for advanced glycation end products and its ligand high-mobility group box-1 mediate allergic airway sensitization and airway inflammation. J Allergy Clin Immunol 2014; 134:440-50.e3.

E3.Jostock T, Mullberg J, Ozbek S, Atreya R, Blinn G, Voltz N, et al. Soluble gp130 is the natural inhibitor of soluble interleukin-6 receptor transsignaling responses. Eur J Biochem 2001; 268:160-7.

E4.Barry J, Loh Z, Collison A, Mazzone S, Lalwani A, Zhang V, et al. Absence of Toll–IL-1 Receptor 8/Single Immunoglobulin IL-1 Receptor–Related Molecule Reduces House Dust Mite–Induced Allergic Airway Inflammation in Mice. American Journal of Respiratory Cell and Molecular Biology 2013; 49:481-90.

E5.Gibson PG, Wlodarczyk JW, Hensley MJ, Gleeson M, Henry RL, Cripps AW, et al. Epidemiological association of airway inflammation with asthma symptoms and airway hyperresponsiveness in childhood. Am J Respir Crit Care Med 1998; 158:36-41.

E6.Simpson JL, Scott R, Boyle MJ, Gibson PG. Inflammatory subtypes in asthma: assessment and identification using induced sputum. Respirology 2006; 11:54-61.

E7. Woolhouse IS, Bayley DL, Stockley RA. Effect of sputum processing with dithiothreitol on the detection of inflammatory mediators in chronic bronchitis and bronchiectasis. Thorax 2002; 57:667-71.

E8.McSharry C, Spears M, Chaudhuri R, Cameron EJ, Husi H, Thomson NC. Increased sputum endotoxin levels are associated with an impaired lung function response to oral steroids in asthmatic patients. J Allergy Clin Immunol 2014; 134:1068-75.

E9.Thunberg S, Gafvelin G, Nord M, Gronneberg R, Grunewald J, Eklund A, et al. Allergen provocation increases TH2-cytokines and FOXP3 expression in the asthmatic lung. Allergy 2010; 65:311-8.

E10.Hernandez ML, Herbst M, Lay JC, Alexis NE, Brickey WJ, Ting JP, et al. Atopic asthmatic patients have reduced airway inflammatory cell recruitment after inhaled endotoxin challenge compared with healthy volunteers. J Allergy Clin Immunol 2012; 130:869-76 e2.

E11.Hernandez ML, Lay JC, Harris B, Esther CR, Jr., Brickey WJ, Bromberg PA, et al. Atopic asthmatic subjects but not atopic subjects without asthma have enhanced inflammatory response to ozone. J Allergy Clin Immunol 2010; 126:537-44 e1.

E12.Doganci A, Eigenbrod T, Krug N, De Sanctis GT, Hausding M, Erpenbeck VJ, et al. The IL-6R α chain controls lung CD4+ CD25+ Treg development and function during allergic airway inflammation in vivo. J Clin Invest 2005; 115:313-25.

FIGURE LEGENDS

Figure E1.In vivo biodistribution of the anti-IL-6R antibody conjugated with XenoLight CF-750. Sequential observations of fluorescence images demonstrating the biodistribution of Xenolight CF750 conjugated anti-IL-6R antibody following intranasal administration in naïve WT mice.

Figure E2.Role of IL-6 signalling in HDM- and CR-induced mucus production and AHR. A, Representative photomicrographs of PAS stained airways (left panel) and quantification of percentage of mucus producing AECs (right panel) in HDM model. B, Airway resistance was assessed 3 hours after the last PBS or HDM challenge in response to methacholine challenge. C, Representative PAS stained airways (left panel) and quantification of mucus producing AECs (right panel) in CR model. D, Airway resistance was assessed 3 hours after the last PBS or CR challenge in response to methacholine challenge. n = 4-7 mice. *P< 0.05, **P<0.01 and ***P<0.001 vs PBS. §P<0.05, §§P<0.01 and §§§P<0.001 vs HDM + IgG or CR + IgG.

Figure E3.Effect of CR exposure on sIL-6R expression and airway inflammation in non-sensitised mice. A, Study design. B, BAL differential count.C, sIL-6R expression in BALF, n = 4-5 mice. §P<0.05 vs PBS.

Figure E4.Effect of selective inhibition of the IL-6 trans-signalling pathway on CR-induced mucus production and AHR. A, Percentage of mucus producing AECs. B, Airway resistance. n = 5-12 mice. §§§P<0.001 vs CR.

Figure E5.Effect of IL-6R blockade on allergen-induced lymphocyte recruitment to the lung. A, Absolute number of αδ T, γδ T, NK T and NK cell in the lungs of PBS and CR challenged (with or without anti-IL-6R antibody treatment) mice. B, Number of αδ T, γδ T, NK T and NK cell in the lungs of CR challenged with or without sgp130Fc treatment. C, Absolute number of αδ T, γδ T, NK T and NK cell in the lungs of PBS and HDM challenged (with or without anti-IL-6R antibody treatment) mice. n = 3-6 mice. *P<0.05 and **P<0.01vs PBS. §P<0.05 vs CR + IgG or CR + PBS or HDM + IgG.

Figure E6.Expression of mIL-6R by lymphocyte populations in the lung.(A) Expression of mIL-6R by different leukocytes in the lung of naive WT mice, presented as median fluorescence intensity (MFI). (B). Representative flow plot of mIL-6R expression on Ly6highCD11b+ neutrophils. Fluorescence minus one (FMO) was used as the control. n = 3 mice. ***P<0.001 vs CR.

Figure E7.HDM-induced inflammation is unaffected in TCR-δ-deficient mice (TCR- δ-/-).A, Study design B, BAL differential count (n = 5 mice).

Figure E8. Correlation between sputum IL-6 and serum IL-6 (A) and between sputum sIL-6R and serum sIL-6R (B) in human asthmatics.

Figure E9. Correlation between sputum total cell count and sputum levels of IL-6 (A) and sIL-6R (B).

Figure E10. Correlation between the estimated concentration of assay standards treated with dithiothreitol (DTT, y-axis) and control standards (x-axis).To prepare the DTT-treated standards, the lyophilised assay standard included in the kit was first reconstituted at a higher concentration to allow for the subsequent addition of four volumes of 0.1% DTT to 1 volume of assay standard (4:1). Doubling dilutions were mixed, then the 0.1% DTT was added to each dilution. For comparison, a set of standards with the same concentration was prepared without DTT. Diagonal line indicates equality between the estimated concentrations of the two sets of standards.

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Table E1. Clinical and sputum characteristics of 33 mild-to-moderate asthmatics who participated in this study.

Eosinophilic / Mixed granulocytic / Neutrophilic / Pauci granulocytic
N / 14 / 10 / 4 / 5
Female % / 50 / 30 / 75 / 60
Mean age (range) / 39 (21-59) / 45 (21-58) / 39 (29-52) / 38 (31-45)
Median sputum total cell count, x106/mL (IQR) / 1.2 (0.9-1.5) / 1.1 (0.8-1.5) / 0.6 (0.4-0.9) / 0.7 (0.6-0.8)
Mean sputum cell viability (range) / 0.69 (0.42-0.93) / 0.69 (0.54-0.82) / 0.74 (0.68-0.84) / 0.66 (0.58-0.82)
Mean differential cell counts, % (range)
Squamous epithelial cells / 10 (0-32) / 10 (1-28) / 6 (2-9) / 16 (9-22)
Columnar epithelial cells / 0.2 (0-0.7) / 0.2 (0-1) / 0 (0-0) / 0.1 (0-0.7)
Neutrophils / 42.4 (26-60) / 78.5 (68-95) / 76.7 (64-90) / 33.1 (7-59)
Eosinophils / 12.2 (1-35) / 2.8 (1-7) / 0.3 (0-1) / 0.5 (0-1)
Macrophages / 41.3 (12-65) / 15.6 (4-27) / 19.6 (8-30) / 61.8 (39-89)
Lymphocytes / 3.9 (0.7-21) / 2.8 (0.2-5) / 3.4 (1-5) / 4.5 (2-7)
Mean absolute cell counts, x104/mL (range)
Neutrophils / 57.9 (19.2-116.4) / 112.7 (46.4-267.6) / 61.6 (31.7-137.2) / 29 (4.5-74)
Eosinophils / 15.2 (1.3-40.5) / 5.2 (0.9-26) / 0.3 (0-0.5) / 0.5 (0-1.2)
Macrophages / 54.8 (12.9-114.8) / 23.1 (3.1-56.4) / 12.5 (8.6-20.2) / 47.2 (24.7-61.9)
Lymphocytes / 4.1 (0.8-18.1) / 4.6 (0.2-14) / 2.1 (0.7-3.6) / 3.3 (2.1-4.7)
Mean baseline FEV1 (range) / 2.8 (1.2-4.8) / 2.8 (0.8-5.4) / 2.7 (1.8-3.3) / 2.9 (1.8-3.5)
Mean percent predicted FEV1 (range) / 75.7 (43.9-94.4) / 69.6 (34.2-113.2) / 81.5 (65.9-107.9) / 79 (56.7-98.1)
Mean FEV1/FVC ratio (range) / 0.71 (0.43-0.93) / 0.64 (0.45-0.79) / 0.74 (0.6-0.86) / 0.72 (0.62-0.84)
Mean Asthma Control Questionnaire score (range) / 1.73 (0.71-2.86) / 1.67 (0.71-3.14) / 1.96 (1.14-3.57) / 1.8 (0.71-2.71)
Median FeNO (IQR) / 34 (29-80) / 44 (24-81) / 12 (9-16) / 27 (22-49)
Asthma severity (N in GINA steps 1:2:3:4:5) / 6:0:6:2:0 / 2:1:5:1:1 / 1:0:1:2:0 / 2:0:0:3:0
Median sputum IL-6, pg/mL (IQR) / 14.2 (9.2-18.8) / 26.5 (22.4-29.3) / 27.2 (18.9-34.3) / 8.3 (1.7-32.0)
Median sputum sIL-6R, pg/mL (IQR) / 532 (249-771) / 379 (183-876) / 269 (94-510) / 305 (175-336)

Table E2. Association between normalised sputum IL-6 and sIL-6R levels and sputum immune cell counts.