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R E S EARCH ART I C L E
ASTHMA
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TH2 and TH17 inflammatory pathways are reciprocallyregulated in asthmaDavid F. Choy,1* Kevin M. Hart,2* Lee A. Borthwick,3 Aarti Shikotra,4 Deepti R. Nagarkar,1
Salman Siddiqui,4 Guiquan Jia,1 Chandra M. Ohri,4 Emma Doran,1,5 Kevin M. Vannella,2
Claire A. Butler,5 Beverley Hargadon,4 Joshua C. Sciurba,2 Richard L. Gieseck,2
Robert W. Thompson,2 Sandra White,2 Alexander R. Abbas,1 Janet Jackman,1 Lawren C. Wu,1
Jackson G. Egen,1 Liam G. Heaney,5 Thirumalai R. Ramalingam,2 Joseph R. Arron,1†‡
Thomas A. Wynn,2† Peter Bradding4†
Increasing evidence suggests that asthma is a heterogeneous disorder regulated by distinct molecular mechanisms.In a cross-sectional study of asthmatics of varying severity (n = 51), endobronchial tissue gene expression analysisrevealed three major patient clusters: TH2-high, TH17-high, and TH2/17-low. TH2-high and TH17-high patterns weremutually exclusive in individual patient samples, and their gene signatures were inversely correlated and differen-tially regulated by interleukin-13 (IL-13) and IL-17A. To understand this dichotomous pattern of T helper 2 (TH2) andTH17 signatures, we investigated the potential of type 2 cytokine suppression in promoting TH17 responses in apreclinical model of allergen-induced asthma. Neutralization of IL-4 and/or IL-13 resulted in increased TH17 cells andneutrophilic inflammation in the lung. However, neutralization of IL-13 and IL-17 protected mice from eosinophilia,mucus hyperplasia, and airway hyperreactivity and abolished the neutrophilic inflammation, suggesting that com-bination therapies targeting both pathways may maximize therapeutic efficacy across a patient population com-prising both TH2 and TH17 endotypes.
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INTRODUCTION
Asthma is a chronic disorder, characterized by episodic airway hyper-responsiveness (AHR) and remodeling with variable degrees of eosin-ophilic and neutrophilic inflammation. Asthma causes significant morbidityand mortality (1–4), and about 10% of patients have disease that is re-sistant to current therapies (1, 4). This group consumes 50 to 60% ofhealth care costs attributed to asthma, underscoring the necessity todiscover new therapies (5).
The clinical expression of asthma is heterogeneous with several dis-tinct phenotypes identified (6, 7). Identifying the molecular mechanismsdriving subtypes of asthma has the potential to reveal drug targets, bio-markers to predict treatment response, and appropriately target therapy,as evidenced by recent clinical studies of T helper 2 (TH2) cytokineantagonists (8, 9).
In addition to the TH2 pathway, attention has focused on TH17cytokines as candidate alternative drivers of severe asthma pathophys-iology (10). Interleukin-17A (IL-17A) and IL-17F can amplify selectednuclear factor kB (NF-kB)–dependent signaling pathways such as thoseinduced by tumor necrosis factor–a (TNF-a), a cytokine up-regulatedin asthmatic airways, and are further up-regulated after allergen chal-lenge and experimental rhinovirus infection (11–17). In particular,IL-17A may contribute to neutrophilic airway inflammation via up-regulation of CSF3 and CXCL chemokines (18–20), mucus gland hy-perplasia, AHR (18, 21), and corticosteroid resistance (18, 22).
1Genentech Inc., South San Francisco, CA 94080, USA. 2Program in Tissue Immunity andRepair, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases,National Institutes of Health, Bethesda, MD 20892, USA. 3Tissue Fibrosis and Repair Group,Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne NE2 4HH, UK.4Institute for Lung Health, Department of Infection, Immunity and Inflammation, Universityof Leicester, Leicester LE3 9QP, UK. 5Centre for Infection and Immunity, Health SciencesBuilding, Queens University Belfast, Lisburn Road, Belfast BT9 7AB, Northern Ireland, UK.*These authors contributed equally to this work.†These authors are co-senior authors.‡Corresponding author. E-mail: [email protected]
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Therapeutic strategies targeting TH2 and TH17 inflammatory path-ways are currently under active investigation in asthma. However, thenature and extent of the activity of these two pathways in individualpatients are unclear. TH2 cytokines can negatively regulate TH17 cy-tokine expression, and inhibiting TH2 cytokines in vitro or in vivo hasthe potential to increase IL-17A production and IL-17A–dependent air-way inflammation (23, 24). The crosstalk between TH2 and TH17 path-ways is therefore complex, and it has been proposed that targeting TH2cytokines might promote corticosteroid-resistant IL-17–dependent neu-trophilic airway inflammation (10, 24). Here, we show that TH2- andTH17-related gene expression signatures are mutually exclusive in theairways of asthma patients, but both are associated with eosinophilicinflammation. In an in vivo preclinical model, we show that therapeu-tic targeting of TH2 and TH17 cytokines can lead to amplification ofactivity of the opposing pathway.
RESULTS
IL-13 and IL-17A induce distinct gene expression patterns inbronchial epithelial cellsTo define core sets of IL-13– and IL-17–inducible transcripts for anal-ysis in bronchial tissue, we stimulated normal human bronchial ep-ithelial (NHBE) cells cultured at the air-liquid interface (ALI) with IL-13(10 ng/ml) or IL-17A (10 ng/ml) ± TNF-a (10 ng/ml), and assessed theexpression levels of transcripts associated with IL-13 and IL-17A sig-naling (19, 25, 26). We confirmed the IL-13–dependent expression ofPOSTN, CLCA1, and SERPINB2 (Fig. 1), which have been previouslydescribed as highly correlated with IL13 and IL5 expression and eosino-philic airway inflammation (25, 26) in asthmatic bronchial epithelialbrushings. IL-17A and IL-17F induce the expression of the neutrophilhematopoietic factor CSF3 and the neutrophil chemoattractants CXCL1,CXCL2, CXCL3, and IL8 (19). The expression of CSF3, CXCL1, CXCL2,
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CXCL3, and IL8 was modestly inducible by IL-17A alone or more ro-bustly in combination with TNF-a (Fig. 1). This is in keeping with exist-ing literature and relevant to asthma, where TNF-a is expressed atelevated levels (11–17). Relative to unstimulated cells, IL-13 stimulationsuppressed the expression of IL-17A–inducible transcripts. To a lesserdegree, IL-17A suppressed the expression of IL-13–inducible transcripts.
Transcriptional analysis of bronchial biopsies based on TH2and TH17 signatures identifies three distinct subgroups ofasthma patientsTo assess the relative levels and relationships between transcripts in-ducible by TH2 or TH17 cytokines, we analyzed previously reportedgene expression microarrays of endobronchial biopsies from 51 asthmapatients spanning a range of severity and corticosteroid use (27). Con-sistent with the in vitro stimulation experiments, the three IL-13–induciblegenes comprising the TH2 signature, and the five IL-17–inducible genescomprising the TH17 signature were highly intercorrelated (fig. S1). TH2and TH17 gene expression scores were calculated on the basis of averagevalues of zero-centered gene expression values. The expression levels ofcanonical TH2 and TH17 cytokines, IL13 and IL17A, measured by qPCRwere generally low, as 93% (39 of 42) and 76% (32 of 42) of availablesamples, respectively, had undetectable levels (<LLOQ, table S1). How-ever, TH2 and TH17 scores were significantly elevated (Fig. 2, A and B)in samples in which IL13 or IL17A mRNA could be detected by qPCR.
There was a significant negative correlation between TH2 and TH17gene expression scores (Fig. 2C, Spearman’s r = −0.35, P = 0.011). Inkeeping with the modest reciprocal suppression of TH2 signature tran-scripts by IL-17A + TNF-a and TH17 signature transcripts by IL-13 inNHBE cells (Fig. 1), TH2-high and TH17-high gene signatures in bron-chial biopsies were mutually exclusive: there were no patients who weresimultaneously TH2-high and TH17-high. Similarly, subjects for whomIL13 mRNA could be detected had undetectable levels of IL-17AmRNA and vice versa (table S1).
Next, we classified subjects on the basis of their TH2 and TH17scores. We used a two-step cluster analysis using the log likelihood
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method based on their factor loading score for TH2 or TH17 scores.Fifteen models were evaluated using different numbers of clusters be-tween 1 and 15. The best fitting model, and thus the number ofclusters, was determined by first calculating the Bayesian informationcriterion. On the basis of this approach, we identified three clusters asoptimally fitting the data structure that appeared to capture patientswith either TH2 score dominant (“TH2-high” asthma), TH17 scoredominant (“TH17-high” asthma), or TH2/TH17 score low (“TH2/17-low” asthma) (Fig. 2C).
TH2-high and TH17-high molecular phenotypes are bothassociated with eosinophilic inflammationTH2-high steroid-naïve asthma is associated with eosinophilic inflam-mation (25, 28), whereas TH17 signature genes include potent neutrophil
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Equal− +Fig. 1. IL-13 and IL-17A induce TH2 and TH17 signature genes in NHBEcells. NHBE cells were grown and differentiated at ALI and then stimulated
for 24 hours with IL-13 (10 ng/ml), IL-17A (10 ng/ml), TNF-a (10 ng/ml), orIL-17A (10 ng/ml) and TNF-a (10 ng/ml) (n = 3 technical replicates). Geneexpression was assessed by quantitative polymerase chain reaction (qPCR).Differential expression is represented by heatmap of averaged replicates ofuntreated control zero-centered and scaled values.●●
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Fig. 2. Three distinct asthma subgroups are defined by mutually ex-clusive TH2 and TH17 signature expression. (A and B) The expression of
endobronchial biopsy (A) TH2 and (B) TH17 signature scores is significantlyelevated (P < 0.05, Kruskal-Wallis) in samples with detectable IL13 andIL17A, respectively. (C) TH2 and TH17 scores from 51 asthmatic bronchialbiopsy microarrays are plotted by scatterplot. TH2 and TH17 signaturescores are inversely correlated (Spearman’s r = −0.35, P = 0.011). ClusteringTH2 and TH17 scores was used to classify subjects as TH2 score dominant(TH2-high, red), TH17 score dominant (TH17-high, blue), and TH2/17 scorelow (TH2/17-low, gray).eTranslationalMedicine.org 19 August 2015 Vol 7 Issue 301 301ra129 2
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chemoattractants (29, 30). We therefore hypothesized that molecularphenotypes of TH2 and TH17 inflammation would relate to phys-iologic measures of eosinophilic and neutrophilic inflammation, respec-tively. Unexpectedly, we observed evidence of eosinophilic inflammationin both TH2-high and TH17-high asthma.
Fractional exhaled nitric oxide (FeNO) and sputum eosinophilswere significantly increased in TH2-high asthma compared to TH17-highand TH2/17-low asthma (Fig. 3, A and B). Median blood eosinophilcounts were significantly elevated in TH2-high versus TH2/17-lowasthma, but were also elevated in TH17-high asthma, though not sta-tistically significant versus TH2/17-low asthma (Fig. 3C). Lamina propriaeosinophil counts in bronchial biopsies were comparable in both
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TH2-high and TH17-high asthma, and both were significantly elevatedversus TH2/17-low asthma (Fig. 3D). We assessed serum periostin, asystemic biomarker of eosinophilic airway inflammation (31), andIL-13 activity (32) in subjects for whom serum and bronchial biopsieswere collected contemporaneously. Unexpectedly, serum periostin con-centrations were highest in TH17-high asthma as compared to TH2-high and TH2/17-low asthma, but not statistically significant. Therewere no significant differences or associations between measures of neu-trophilic inflammation in peripheral or airway compartments andmolecular phenotypes of TH2 or TH17 inflammation (fig. S2).
The TH17-high molecular phenotype is associated withcorticosteroid-dependent moderate to severe asthmaThe clinical characteristics of the patients separated by molecular phe-notypes of TH2/17 inflammation are shown in table S2. We did not de-tect significant differences in patient characteristics or clinical measuresincluding FEV1 (forced expiratory volume in 1 s) % predicted, FEV1/FVC(forced vital capacity) ratio, AHR, or exacerbation frequency. However,mean values of FEV1 % predicted and FEV1/FVC ratio were lowest inthe TH17-high group.
Although there was a range of doses of inhaled (ICS) or oral (OCS)corticosteroids across all three TH2/17 molecular phenotypes, the TH17signature was not observed in steroid-naïve patients (table S2). Inte-grating this observation with the associations between TH2-high andTH17-high asthma and eosinophilic inflammation (Fig. 3), we have de-lineated subsets where molecular phenotypes of TH2-high and TH17-high asthma are observed (Fig. 4). TH2-high asthma was observed onlyin subjects with evidence of eosinophilic asthma defined as blood eo-sinophils≥300/ml or sputum eosinophils≥3% or biopsy lamina propriaeosinophils ≥10/mm2. TH2-high, TH17-high, and TH2/17-low pheno-types were observed among eosinophilic moderate-severe asthma inapproximately equal proportions. The molecular phenotype of TH2/17-low asthma was observed among all subsets and was predominantamong moderate-severe asthmatics using ICS who did not display anyevidence of eosinophilia. Finally, TH17-high asthma was observed onlyin moderate to severe asthma patients, primarily in subjects who hadevidence of eosinophilia and were taking ICS or ICS + OCS.
Therapeutic blockade of TH2 cytokines during experimentalallergic asthma induces TH17 inflammationConsidering the mutually exclusive TH2 and TH17 signatures identi-fied in human asthmatic airways, and the potential for IL-13 suppressionto promote IL-17 production in vitro (23), we investigated the effects ofIL-4 and IL-13 blockade, singly or in combination, in a murine modelof allergic asthma. These studies consisted of 3 weeks of intranasalsensitization against house dust mite (HDM) extract and 4 weeks ofbiweekly intranasal HDM challenges with antibody therapy targetingIL-4, IL-13, or both cytokines on the preceding day (Fig. 5A). Anti–IL-4,anti–IL-13, and anti–IL-4/13 reduced airway inflammation (Fig. 5B).Mice receiving anti–IL-13 and anti–IL-4/13 were significantly protectedfrom arteriolar hypertrophy and fibrosis, whereas mice treated with anti–IL-4 were less protected from fibrosis and still had evidence of arteriolarhypertrophy (Fig. 5, B and C). The mucus response assessed by his-tological analysis and lung Muc5ac expression was significantly abro-gated by anti–IL-13 or anti–IL-4/13 treatment, but less so by anti–IL-4alone (Fig. 5D and fig. S3A). Anti–IL-4 and anti–IL-4/13 reduced lungIL13 expression and CD4+IL-13+ cells in lung and lymph node (Fig. 5Eand fig. S3B). All anti-TH2 interventions resulted in significantly increased
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Fig. 3. TH2-high and TH17-high asthma are both associated with ele-vated levels of physiologic T 2/eosinophilic measures of inflamma-
Htion. Pairwise comparisons (Mann-Whitney test) were made among TH2/17-low (Low), TH2-high (TH2), and TH17-high (TH17) asthmatics subjects.Red font indicates P < 0.05. (A and B) TH2-high subjects were associatedwith elevated (A) FeNO and (B) sputum eosinophil percentage as com-pared to low-inflammatory and TH17-high asthma. (C) Blood eosinophilswere elevated in TH2-high versus low-inflammatory asthma. (D) Laminapropria eosinophil counts were elevated in both TH2-high and TH17-highversus low-inflammatory asthma. (E) Median levels of serum periostin werehighest in TH17-high versus TH2-high and TH2/17-low asthma.
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tissue expression of IL17 and CD4+IL-17+ cells in lung and lymphnode, particularly anti–IL-4 (Fig. 5F and fig. S3B). Furthermore, down-stream eosinophilic and neutrophilic infiltration reflected this shift inTH2/TH17 effector cytokines. Anti–IL-4– or anti–IL-4/13-treated micehad significantly reduced eosinophil numbers in tissue, whereas anti–IL-13 alone produced a significant but less marked reduction (Fig. 5Eand fig. S3C). Meanwhile, we found marked increases in neutrophilinfiltration in lungs of all treated mice, most pronounced with anti–IL-4 (Fig. 5F). Furthermore, expression of TH2-driven genes, Clca3 (or-tholog for human CLCA1) and Ccl11, was substantially reduced withanti–IL-13 or anti–IL-4/13 treatment (Fig. 5E). Despite effectively re-ducing markers of the TH2 response, anti–IL-13 did not affect tissueexpression or CD4 staining for IL-13 and may have induced greaterIL-13 expression. These observations are consistent with previous re-ports that IL-13 serves as an important regulator of downstream TH2effector functions rather than affecting TH2 cell differentiation, andmay be explained in part by an altered balance in receptor complexavailability for IL-4 in the context of IL-13 blockade (33–36). IL-17–responsive genes Cxcl1, Cxcl3, and Csf3were increased in anti–IL-4–treatedanimals, and Csf3 was increased by both anti–IL-13 and anti–IL-4/13(Fig. 5F). Notably, the increases in neutrophils, IL-17, and IL-17–responsivegene expression were modest or nonexistent in untreated mice com-pared to saline controls, suggesting that a robust TH17 response onlyoccurred with perturbation of the TH2 pathway.
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Dexamethasone suppresses TH2 pathways but enhancesTH17 pathways in the mouse asthma modelGiven the association of corticosteroid use with patients exhibiting aTH17-high signature, we interrogated the murine HDM model to de-termine whether corticosteroid therapy alone might contribute to featuresof TH17-high asthma. Dexamethasone treatment reduced pathophys-iologic features including AHR, tissue fibrosis, eosinophil infiltration,and IL-4/13 expression. Dexamethasone treatment partially suppressedIL-17 mRNA expression but increased the frequency of neutrophils inthe lung concomitant with marked up-regulation in tissue expressionof the IL-17–inducible chemokines Cxcl3 and Cxcl1 (fig. S4). In addi-tion to recapitulating observations from published studies that corti-costeroid treatment effectively suppresses TH2-driven inflammation, butfails to significantly reduce IL-17–driven inflammation (18), these dataadd that corticosteroid use may specifically contribute to creating anenvironment permissive to an enhanced neutrophilic response.
Combined blockade of IL-13 and IL-17 attenuatesthe TH17 signature observed during anti–IL-13 inhibitionof the TH2 responseThe reciprocal regulation of TH2 and TH17 responses, both in vivoand in epithelial cells in vitro, provides an explanation for the mutu-ally exclusive TH2 and TH17 signatures in a large proportion of pa-tients with asthma (Figs. 2 and 4). This could be important clinicallywhen targeting these cytokine pathways, because efficacy-limiting com-pensation may occur if only a single pathway is blocked. Therefore, wetested whether combined IL-13 and IL-17 antibody blockade wouldreduce TH2-driven disease and concomitantly inhibit the TH17 response.Mice treated with either anti–IL-13 alone or anti–IL-13 + anti–IL-17had significantly reduced AHR and mucus response relative to controlantibody–treated animals or anti–IL-17 alone (Fig. 6, A and B). The in-creased IL-17 production and neutrophil infiltration associated withanti–IL-13 treatment alone were partially and completely abrogated, re-spectively, in mice receiving dual anti–IL-13/anti–IL-17 therapy (Fig.6C). Furthermore, we detected increased IL-4– and IL-13–producingCD4+ T cells in the lungs of anti–IL-17–treated animals (fig. S5), support-ing dual cross-regulation between the TH2 and TH17 cytokine networks.
DISCUSSION
Until recently, asthma was considered as a single disease entity associ-ated with eosinophilic airway inflammation driven by type 2 inflamma-tory cytokines (IL-4/5/13) (37). However, asthma can occur in the absenceof significant TH2/eosinophilic inflammation across the spectrum ofseverity (25, 38–42). There is increasing interest in the role of TH17-dependent pathways in asthma, but it is not known how TH2 and TH17pathways interact in asthmatic airways. With the potential for counter-regulation already identified, it is important to understand the conse-quences of singly inhibiting TH2 and TH17 pathways.
Here, we used TH2- and TH17-related gene signatures and individ-ual gene expression data to measure the activity of TH2 and TH17 path-ways in human asthmatic airways, and HDM-sensitized murine airways.The analysis of TH2/17-dependent gene transcription adds importantvalue by providing evidence of relevant cytokine activity. This is ex-emplified by the previously described TH2 gene signature, which isrobustly expressed in a subset of asthmatic subjects (25, 26), correlateswith airway eosinophilia, and has precipitated the development of
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Fig. 4. Proportions of TH2/17 molecular phenotypes of asthma by clin-ical evidence of eosinophilia and severity. Asthmatics were classified on
the basis of evidence of eosinophilic asthma, defined as blood eosinophilcount ≥300/ml or sputum eosinophil percentage ≥3, or biopsy lamina propriaeosinophil count ≥10/mm2 and clinical severity (British Thoracic Society/Scottish Intercollegiate Guidelines Network treatment step). Pie charts rep-resent the proportion of TH2/17-low, TH2-high (TH2), and TH17-high (TH17)asthmatic subjects per eosinophilic/severity category. The area of each piechart is proportional to the number of subjects in that category.eTranslationalMedicine.org 19 August 2015 Vol 7 Issue 301 301ra129 4
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P = 0.036P = 7.8 10−6
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P = 0.047P = 3.3 10−7
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P = 0.027P = 7.2 10−4
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Fig. 5. Therapeutic blockade of TH2 cytokines during experimental allergic asthma inducescompensatory TH17 inflammation. (A) Mice were intranasally (i.n.) sensitized against HDM ex-tract for 3 weeks at 200, 100, and 100 mg each week and subsequently challenged with biweeklyintranasal exposures for 4 weeks with or without targeted antibody therapy each day beforechallenge against IL-4, IL-13, or both cytokines [250 mg, intraperitoneally (I.P)]. (B and C) Lungpathology was assessed by scoring for gross inflammation in Giemsa-stained histological sections(B), assaying for hydroxyproline content as a surrogate for fibrosis (B), and Masson’s trichromestaining of airways (C). (D) Airway mucus production was assessed by periodic acid–Schiff
(PAS) staining. (E and F) IL-13 (E) and IL-17 (F) production was measured by gene expression and in restimulated CD4 T cells by flow cytometry. The percentof eosinophils and neutrophils was quantified from Giemsa-stained histological lung sections. (F) Whole lung tissue expression of Clca3 and Ccl11 TH2markers, and Cxcl3, Cxcl1, and Csf3 TH17 markers were assessed by qPCR (P values shown, two-tailed t tests, n = 4 to 10).www.ScienceTranslationalMedicine.org 19 August 2015 Vol 7 Issue 301 301ra129 5
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periostin as a biomarker for predicting the response to TH2-targetedtherapy (9, 31). Using this approach, we have validated a TH17-dependentgene signature (CXCL1, CXCL2, and CXCL3, IL8, and CSF3) and haveshown that the induction of these genes is further amplified by TNF-a,an important mediator associated with IL-17 inflammation, that hasbeen described to be up-regulated in the airways in many studies ofasthma (11–17, 43–46). We also confirmed in NHBE cells that IL-13and IL-17 reciprocally regulate each other’s respective signatures. Usingthis approach, we have shown that IL-17–related signaling is evidentin the airway tissue of a subset of patients with moderate-severe asthmausing corticosteroids. Intriguingly, TH2 and TH17 activity was inversely
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correlated, and clustering patients demonstrated that TH2-high andTH17-high disease were mutually exclusive. This suggests that thereis reciprocal regulation of these two pathways in vivo in human asth-ma, which is consistent with the reciprocal relationship we found forTH2 and TH17 signature genes induced by IL-13 and IL-17A in vitro,and the ability for IL-13 to attenuate IL-17A production in human TH17cells (23). However, this interpretation is limited by the inherent re-strictions of studying human subjects cross-sectionally.
To examine this potential counterregulation in more detail, weturned to an in vivo HDM-driven murine asthma model. This dem-onstrated that with a strong TH2 stimulus, there is also IL-17 induc-tion. However, the downstream consequences of IL-17 induction appearedto be relatively limited because there were no changes in airway neu-trophils and the IL-17–dependent cytokines Cxcl1 and Cxcl3 did notincrease significantly. However, although IL-13 and IL-4/13 blockadein combination mitigated a wide array of pathological consequences toHDM exposure, these same interventions enhanced IL-17 expression,IL-17–dependent chemokine/cytokine expression, and lung neutrophilia.This confirms that TH2 cytokines are powerful suppressors of IL-17–driven inflammation, and also raises the possibility that TH2-targetedtreatment in asthma may contribute to the emergence of an adverseTH17-permissive environment, limiting therapeutic efficacy over time.Given recent interest in IL-17 as an alternative driver of asthma, and theproposed idea that targeting TH2 cytokines might promote corticosteroid-resistant IL-17–dependent neutrophilic airway inflammation (10, 24),these observations highlight the importance of understanding the reg-ulation and interplay of TH2 and TH17 responses for developing andoptimizing therapeutic intervention strategies.
Our identification of mutually exclusive TH2 and TH17 expressionmay seem contradictory with reports of dual TH2 and TH17 cytokine–expressing CD4+ T cells (47, 48). However, the demonstration thatIL-13 strongly represses IL-17A–dependent genes in epithelial cellssuggests that in the context of coexpression, IL-17–dependent transcrip-tion will be attenuated. Our data from human bronchial biopsies sup-port this interpretation.
Because the TH17 signature genes include potent neutrophil chemo-attractants (29, 30), we hypothesized that TH17 signature expression inpatients would relate to measures of neutrophilic airway inflamma-tion, as we described previously for inflammatory dermatoses (49).As observed previously, TH2-high asthma was associated with eosino-philic inflammation (25, 28). Unexpectedly, TH17-high asthma was alsoassociated with elevated numbers of lamina propria eosinophils, andthere were no relationships in blood, sputum, or lamina propria neu-trophils among TH2 or TH17 molecular phenotypes. However, tissueneutrophil counts are reported to be similar between health and asthma(27, 39, 50–52), and their activity may be more important than theirnumber. Nevertheless, these observations have important implicationsfor the selection of patients in clinical trials of anti–IL-17 therapy. In-deed, anti–IL-17RA therapy in symptomatic moderate-severe asthmaticsusing ICS failed to demonstrate efficacy in a recent study, which maynot have been appropriately stratified to assess subsets of patients withactivity of the IL-17 pathway (53). An ongoing study of another anti–IL-17 therapy in moderate-severe asthma excludes patients with ele-vated blood eosinophils and, hence, may exclude patients with thepotential for benefit (54).
An important question arises as to the stability of the TH2 andTH17 phenotypes and whether patients can move from one to the other.Indirect evidence from the expression of periostin suggests that this is a
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Fig. 6. Dual therapeutic blockade of IL-13 and IL-17 prevents emergenceof a TH17 signature induced by antibody inhibition of the TH2 response.
Mice on the chronic HDM model of allergic asthma were treated a daybefore each challenge with anti–IL-17, anti–IL-13, or both (150 mg). (A andB) Efficacy of IL-13/17 dual blockade was assessed by airway hyperreactiv-ity [two-way analysis of variance (ANOVA), SEM shown, n = 5 to 10] (A) andmucus response gene expression (B). (C) Immune response was measuredby gene expression analysis of IL-13 and IL-17, and flow cytometric analysisof the frequency of Siglec-F+ eosinophils and Ly6G+ neutrophils in broncho-alveolar lavage (BAL) (P values shown, two-tailed t tests, n = 5 to 28).eTranslationalMedicine.org 19 August 2015 Vol 7 Issue 301 301ra129 6
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likely scenario. Periostin gene expression is directly inducible by IL-13but not IL-17A (Fig. 1) (42), elevated serum periostin levels predictclinical benefit from IL-13 inhibition (9), and serum periostin concen-trations in anti-IL-13–treated moderate-severe asthma patients fall tolevels observed in healthy nonasthmatic subjects (32). Furthermore,IL-13 induces NOS2 expression, contributing to elevated FeNO in asth-ma patients (55), which also predicts benefit from and is decreased byanti–IL-13 treatment (9, 32). We observed elevated FeNO in TH2-highbut not in TH17-high asthma, suggesting that FeNO reflects airway IL-13activity at a given point in time, whereas the turnover times for serumperiostin and tissue eosinophils may be longer. Together, the elevatedserum periostin and bronchial tissue eosinophilia but low FeNO inTH17-high asthma suggest that moderate-severe asthma may alternatebetween TH2-high, TH17-high, and TH2/17-low states depending on re-cent exposures to immunostimulatory factors. This cannot be deter-mined formally due to the cross-sectional nature of the present study,but future studies should examine these patterns over multiple longitu-dinal samples and exposures.
We observed TH17-high asthma exclusively in corticosteroid-treatedmoderate-severe asthma (Fig. 4), consistent with the demonstrationthat corticosteroids may promote IL-17 production in some patients(56, 57). In addition, dexamethasone intervention in the HDM modelpotently inhibited disease and the TH2 response, but resulted in en-hancedmarkers of TH17 inflammation despite reducing IL-17. This couldbe the result of experimental timing or potentiated signaling fromresidual IL-17 due to deregulation of the TH2 inhibitory pathway. To-gether, our data suggest that inhibition of TH2 responses and the con-comitant loss of IL-17 regulation resulting from corticosteroid exposureor selective TH2 inhibition in asthmatic airways may create a TH17-permissive environment. Subsequent exposure to frequently encounteredexogenous stimuli such as allergen, infection, pollution, and perhapscorticosteroids themselves may then enhance IL-17 expression. Thesame environment would exist in true TH2-low patients who shouldalso be susceptible to IL-17 up-regulation (summarized in fig. S6). Inour preclinical mouse model, combined treatment with anti–IL-13/17ameliorated AHR, lung pathology, and the TH2 response, and markedlyreduced the IL-17–dependent chemokine and neutrophil response ob-served with anti–IL-13 treatment alone. Thus, optimal therapeutic ben-efit in asthma may be achieved through simultaneous TH2 and TH17pathway–directed therapy. However, although therapies targeting TH2cytokines have shown efficacy in subsets of patients with moderate-severe asthma (58), formal assessments of the impact of IL-4, IL-5,and/or IL-13 inhibition on TH17 activity have not been described inthose studies and represent an area for future investigation.
There were limitations to this study that may serve as subjects offurther investigation or alternative interpretations. This post hoc anal-ysis was based on a cross-sectional study and is unable to assess thelongitudinal intrapatient variability of TH2/17 signature classification.The number of asthmatics (n = 51) and the relative paucity of subjectswho are “non-eosinophilic” limit our ability to evaluate this populationwhere current and emerging therapies are unlikely to provide mean-ingful clinical benefit. Although our designated “TH17 signature” is con-sistent with the biological activity of IL-17A and/or IL-17F, we cannotexclude the possibility that other cytokines are contributing to it in vivo.Although TH2 and TH17 signature expression was statistically signif-icantly up-regulated (Fig. 2, A and B) in subjects in whom IL13 orIL17, respectively, was expressed, caution must be made in the inter-pretation of these data due to the high percentage of subjects with un-
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detectable cytokine levels. Furthermore, assessing this gene expressionpattern in endobronchial biopsies depends on invasive procedures andprecludes routine use, highlighting the need to identify and developnoninvasive biomarkers of this TH17 pathway activity.
In summary, we have identified TH2-high, TH17-high, and TH2-17-low clusters of patients with asthma. We propose that with suppres-sion of TH2 activity by targeted therapy or corticosteroids, or absentTH2 activity (true TH2-low), a TH17-permissive environment exists. Com-bined with data from a murine model of allergic asthma, we suggest thatin a subset of patients, a direct relationship between TH17-high andTH2-high disease exists, whereby, through mutual cross-regulation,TH17-high asthma may represent a transition or switch away from TH2-mediated disease. Our studies therefore suggest that combined targetingof IL-13 and IL-17 in patients expressing either a TH2 or a TH17 signaturecould provide additional efficacy over single TH2 or TH17 inhibition.
MATERIALS AND METHODS
Study designThe overall goals of this study were to identify gene signatures asso-ciated with IL-13– and IL-17–driven inflammation and then use thesesignatures to characterize the regulation and interaction of these cyto-kines in asthma and their association with commonly studied biomarkersof asthmatic disease. Candidate signature associated genes were iden-tified via cytokine stimulation of human bronchial epithelial cells in vitro,and then a post hoc analysis was conducted in patient bronchial biop-sies. To evaluate these pathways in the context of therapeutic blockade,we used a chronic HDM model of murine asthma with therapeuticanti-cytokine antibody treatment. In the murine studies, sample sizeswere determined on the basis of previous experience and previous sta-tistical analyses of the model. In the murine studies, standard measure-ments were used to assess inflammation, gene expression, and airwayhyperreactivity. Murine histology was scored by blinded observers. Sam-ple size and replicates for all mouse studies are included in the figuresand legends.
NHBE cell culture and stimulationPrimary NHBE cells were purchased from Lonza. Transwell plates(6.5-mm diameter, 0.4-mm pore density; Corning Life Sciences) werecollagen-coated using PureCol (100 mg/ml) from Advanced BioMatrix.NHBE cells were seeded in Transwells and maintained in serum-freebronchial epithelial cell growth medium (Lonza) for 96 hours or untilconfluent. Thereafter, the apical medium was removed, and cells werefed basolaterally with PneumaCult-ALI complete medium (Stemcell)and differentiated for a period of 21 days. Differentiated NHBE cellswere cultured alone in ALI culture medium or stimulated for 24 hourswith IL-13 (10 ng/ml), IL-17A (10 ng/ml), or TNF-a (10 ng/ml), aloneor in combinations thereof (n = 3 technical replicates). Total RNA wasextracted from NHBE cells using the Qiagen RNeasy Kit.
Gene expression analysesRNA was isolated from homogenized bronchial biopsies, and real-timeqPCR was performed as described previously (28). Whole-genomeexpression microarrays from asthmatics (n = 51) (27) were analyzed.Microarray-based TH2 and TH17 score was calculated by case-wise av-eraging of zero-centered gene expression data after annotation-basedindependent filtering (59), that is, if multiple probes correspond to an
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Entrez gene, the probe with the highest interquartile range was selected.SERPINB2, CLCA1, and POSTN were used as IL-13–responsive TH2signature genes as described previously (25, 26). CXCL1, CXCL2, CXCL3,IL8, and CSF3 were used as IL-17–responsive TH17 signature genes.
Gene expression analyses from in vitro NHBE cell stimulation ex-periments and bronchial biopsy samples were conducted using TaqManGene Expression Assays that were purchased and conducted per themanufacturer’s instructions for CXCL1 (Hs00236937_m1), CXCL2(Hs00601975_m1), CXCL3 (Hs00171061_m1), IL8 (Hs00174103_m1),CSF3 (Hs00738432_g1), SERPINB2 (Hs01010736_m1), CLCA1(Hs00976287_m1), POSTN (Hs00170815_m1), IL13 (Hs99999038_m1),and IL17A (Hs99999082_m1). Target gene expression was normalized byhousekeeping genesGAPDH (4333764F) andHPRT1 (Hs02800695_m1).
Patients and assessmentsPatients were recruited from two centers, Leicester and Belfast, and theirclinical data and tissues have been used in previously published studies(27, 60). Asthma severity was defined by British Guideline on the Man-agement of Asthma treatment steps (61). Of the 26 severe patients at step4/5, 21 met the American Thoracic Society criteria for refractory asthma (1).
The study was approved by the Research Ethics Committees of bothinstitutions (04/Q2502/74, 06/NIR02/114). Written informed consentwas gained from all participants before their involvement.
Physiologic measures are reported only for those derived during thebronchoscopic study. Consequently, FeNO, sputum eosinophil per-centage, and serum periostin measurements are reported for Leicestersubjects only.
Fiber-optic bronchoscopySubjects underwent bronchoscopy conducted according to the BritishThoracic Society guidelines (62). The bronchialmucosal biopsy specimenswere taken from the right middle lobe and lower lobe carinae, fixed inacetone, and embedded in glycol methacrylate (GMA), as described pre-viously (63). The biopsies were also placed immediately in RNA preserv-ative (RNAlater, Ambion) and before processing gene expression analysis.
ImmunohistochemistryThe GMA-embedded tissue was cut and immunostained as describedpreviously (63). Primary antibodies were used against the followingantigens: eosinophil major basic protein and neutrophil elastase. Isotypecontrols were also performed.
Assessment and quantification of immunohistochemical stainingEpithelial and submucosal areas in sections were identified and mea-sured using a computer analysis system (AnalySIS, Olympus). Numbersof positively stained nucleated cells in each compartment were countedblind. Cells staining in sequential sections were colocalized using com-puter analysis.
Serum periostinSerum periostin levels were measured with a proprietary sandwichenzyme-linked immunosorbent assay (ELISA) with two monoclonalantibodies capable of detecting all known splice variants of humanperiostin as previously described (31).
Murine HDM modelSix- to 8-week-old female BALB/c mice obtained from Taconic FarmsInc. were sensitized by intranasal inhalation with 200 mg of HDM ex-
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tract in 30 ml of sterile saline on day 0, and 100 mg on days 7 and 14.Challenges were repeated twice a week with 50 mg intranasally for4 weeks starting on day 21. For therapies, 250 or 150 mg each of anti–IL-4 (11b11), anti–IL-13 (262A-5-1), anti–IL-17 (16H4.4F3), or con-trol (10E7.1D2) antibody was injected intraperitoneally the day beforeintranasal challenges starting on day 20. Mice were terminally anes-thetized on day 46 with sodium pentobarbital. All animals were housedunder specific pathogen–free conditions at the National Institutes ofHealth (NIH) in an American Association for the Accreditation of Lab-oratory Animal Care–approved facility.
Ethics statementThe National Institute of Allergy and Infectious Diseases (NIAID) Di-vision of Intramural Research Animal Care and Use Program, as partof the NIH Intramural Research Program, approved all of the exper-imental procedures (protocol LPD 16E). The Program complies withall applicable provisions of the Animal Welfare Act (www.aphis.usda.gov/animal_welfare/downloads/awa/awa.pdf) and other federal statutesand regulations relating to animals.
HistopathologyMurine lung lobes were harvested and inflated/fixed with a Bouin’s-Hollande solution. Fixed tissue was embedded in paraffin for section-ing and stained with Wright’s Giemsa for airway inflammation andcellular infiltration analysis and PAS for assessing airway mucus. Inflam-mation, PAS staining, and cellular infiltrate were scored by a blindedobserver.
Fibrosis quantificationHydroxyproline was measured to determine collagen content by hy-drolyzing a weighed lung lobe in 6 N HCl at 110°C overnight beforeneutralization with 10 N NaOH. Quantification was performed by col-orization compared to a standard curve of hydroxyproline.
Cell isolation and flow cytometryAbout 75 mg of lung tissue was diced and incubated in collagenase(100 U/ml) (Sigma) at 37°C for an hour with rocking. Tissue was thenpassed through a 100-mm nylon filter to obtain a single-cell suspen-sion. Leukocytes were isolated on a 40% Percoll (Sigma) gradient andtreated with ACK buffer to remove erythrocytes. Lymph nodes werepassed over a 100-mm filter and treated with ACK. For BAL samples,the trachea of anaesthetized mice was cannulated and lungs were la-vaged with 1 ml of 5 mM EDTA sterile phosphate-buffered saline.BAL samples were centrifuged and treated with ACK before fixation.Isolated cells from lung, lymph node, and BAL were either immedi-ately fixed for cellular analysis or stimulated with phorbol 12-myristate13-acetate (10 ng/ml) and ionomycin (1 mg/ml) in the presence ofbrefeldin A (10 mg/ml) for 3 hours and fixed. Cells were permeabilized(Cytofix/Cytoperm buffer; BD Biosciences) and stained for 30 minwith antibodies for CD16/32, CD45, Siglec-F, Ly6G, Ly6C, CD11b,CD4, IL-4, IL-13, TNF-a, IFN-g (interferon-g), and IL-17.
Murine gene expression analysesLung tissue was homogenized in TRIzol Reagent (Life Technologies)with a Precellys 24 (Bertin Technologies). Total RNA was extracted withchloroform using a MagMax-96 Total RNA Isolation Kit (Qiagen) andreverse-transcribed to complementary DNA using SuperScript II Re-verse Transcriptase (Life Technologies). Gene expression was quantified
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using Power SYBR Green PCR Master Mix (Applied Biosystems) byreal-time PCR on an ABI Prism 7900HT Sequence Detection System(Applied Biosystems). Gene expression is described relative to RPLP2mRNA levels in saline-challenged lung tissue.
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Murine airway hyperreactivity measurementMice were analyzed for AHR on day 46 of the HDM exposure modelby total body plethysmography (Data Sciences International–BuxcoResearch Systems). Mice were challenged with increasing doses ofmethacholine inhalation (3 to 50 mg/ml), and measurements collectedover the following 5 min were used to calculate Penh readouts. Datawere analyzed using FinePointe software (Buxco).
Statistical analysisPrism (version 6; GraphPad), R Project software (version 2.15.1; www.R-project.org) (64), and SPSS statistics (version 20, IBM) were used forstatistical analysis and graphing. Mann-Whitney or Kruskal-Wallistest was used for testing the dependence of continuous versus categor-ical variables. Spearman’s rank correlation was used to test for depen-dence between two numeric variables. Fisher’s exact test was used totest for dependence between categorical variables. Missing data werenot imputed and treated as “missing completely at random.” Murinedata were compared with a two-tailed t-test, with Welch’s correctionwhen an F test comparing variances had a P value of <0.05, or a two-way ANOVA.
SUPPLEMENTARY MATERIALS
www.sciencetranslationalmedicine.org/cgi/content/full/7/301/301ra129/DC1Fig. S1. Intercorrelation between the three IL-13–inducible genes and the five IL-17–induciblegenes in endobronchial biopsies.Fig. S2. Neutrophilic inflammation in peripheral or airway compartments is not associated withmolecular phenotypes of TH2 or TH17 inflammation.Fig. S3. Differential effects of TH2blockade onmucous and inflammatory responses in allergic asthma.Fig. S4. Dexamethasone suppresses TH2 pathways but enhances TH17 pathways in the mouseasthma model.Fig. S5. Evidence of dual cross-regulation between the TH2 and TH17 cytokine networks.Fig. S6. A summary of the potential interplay between TH2 (IL-4/13)– and IL-17–dependentsignaling in asthmatic airways.Table S1. Mutual exclusivity of IL-17 and IL-13 expression in endobronchial biopsies.Table S2. Clinical characteristics of TH2/17-low, TH2-high, and TH17-high asthma.
www.Scienc
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Murine primer sequences
Gene
Forward qPCRprimer sequence
Reverse qPCRprimer sequence
Rplp2
TACGTCGCCTCTTACCTGCT GACCTTGTTGAGCCGATCATIl13
CCTCTGACCCTTAAGGAGCTTAT CGTTGCACAGGGGAGTCTCCL11
GAATCACCAACAACAGATGCAC ATCCTGGACCCACTTCTTCTTMuc5ac
CAGGACTCTCTGAAATCGTACCA AAGGCTCGTACCACAGGGAClca3
AGGAAAACCCCAAGCAGTG GCACCGACGAACTTGATTTTIl17
CAGACTACCTCAACCGTTCC AGCATCTTCTCGACCCTGAACxcl1
CTGGGATTCACCTCAAGAAC GAAGCCAGCGTTCACCAGACCxcl3
CCCCAGGCTTCAGATAATCA TCTGATTTAGAATGCAGGTCCTTCsf3
GTGCTGCTGGAGCAGTTGT TCGGGATCCCCAGAGAGTIL4
ACGAGGTCACAGGAGAAGGGA AGCCCTACAGACGAGCTCACTCeTranslationalMedicine.org 19 August 2015 Vol 7 Issue 301 301ra129 9
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Funding: Supported in part by the Intramural Research Program of the NIH, NIAID. Work inLeicester was supported by grants from the Asthma UK project grant AUK-PG-2013-208 and agrant-in-aid from Genentech Inc., and was supported by the National Institute for Health Re-search (NIHR) Leicester Respiratory Biomedical Research Unit. The views expressed are thoseof the author(s) and not necessarily those of the National Health Service (NHS), the NIHR, orthe Department of Health. Author contributions: T.A.W., K.M.H., P.B., L.C.W., D.F.C., and J.R.A.conceived the study; D.F.C., K.M.H., L.A.B., T.R.R., J.G.E., J.R.A., and P.B. designed the experiments;D.F.C., K.M.H., L.A.B., K.M.V., J.C.S., R.L.G., R.W.T., S.W., A.S., E.D., C.A.B., D.R.N., G.J., and J.J. performedthe experiments; D.F.C., K.M.H., L.A.B., T.R.R., P.B., A.R.A., J.R.A., A.S., and S.S. analyzed the data;C.M.O., P.B., and L.G.H. performed bronchoscopy; B.H. recruited patients and performed the clinicalassessments; D.F.C., K.M.H., T.A.W., P.B., J.R.A., and T.R.R. wrote the paper. All authors reviewed themanuscript for intellectual content and approved the final version. Competing interests: D.F.C.,D.R.N., G.J., E.D., A.R.A., J.J., L.C.W., J.G.E., and J.R.A. are currently or were employees of GenentechInc. during the execution of this study. P.B. and L.G.H. received funding from Genentech Inc.toward this work. The other authors declare no competing interests.
Submitted 10 April 2015Accepted 2 July 2015Published 19 August 201510.1126/scitranslmed.aab3142
Citation: D. F. Choy, K. M. Hart, L. A. Borthwick, A. Shikotra, D. R. Nagarkar, S. Siddiqui, G. Jia,C. M. Ohri, E. Doran, K. M. Vannella, C. A. Butler, B. Hargadon, J. C. Sciurba, R. L. Gieseck,R. W. Thompson, S. White, A. R. Abbas, J. Jackman, L. C. Wu, J. G. Egen, L. G. Heaney,T. R. Ramalingam, J. R. Arron, T. A. Wynn, P. Bradding, TH2 and TH17 inflammatory pathwaysare reciprocally regulated in asthma. Sci. Transl. Med. 7, 301ra129 (2015).
ranslationalMedicine.org 19 August 2015 Vol 7 Issue 301 301ra129 10
17 inflammatory pathways are reciprocally regulated in asthmaH2 and THT
Liam G. Heaney, Thirumalai R. Ramalingam, Joseph R. Arron, Thomas A. Wynn and Peter BraddingGieseck, Robert W. Thompson, Sandra White, Alexander R. Abbas, Janet Jackman, Lawren C. Wu, Jackson G. Egen,Chandra M. Ohri, Emma Doran, Kevin M. Vannella, Claire A. Butler, Beverley Hargadon, Joshua C. Sciurba, Richard L. David F. Choy, Kevin M. Hart, Lee A. Borthwick, Aarti Shikotra, Deepti R. Nagarkar, Salman Siddiqui, Guiquan Jia,
DOI: 10.1126/scitranslmed.aab3142, 301ra129301ra129.7Sci Transl Med
pathways may better treat asthmatic individuals.promoted the other in a mouse model of asthma. These data suggest that combination therapies targeting both
17-high clusters were inversely correlated in patients. Moreover, neutralizing one signatureH2-high and THThe T2/17-low.H17-high, and TH2-high, THdemonstrate that asthma can be divided into three immunological clusters: T
et al.have shown us that sets of symptoms can be caused by different pathogenic mechanisms. Now, Choy Classifying diseases according to symptoms is rapidly becoming a thing of the past. Targeted therapeutics
A tale of two asthmas
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