Sarcoidosis is a Th1/Th17 multisystem disorder

Monica Facco,1,2 Anna Cabrelle,2 Antonella Teramo,1,2 Valeria Olivieri,1,2

Marianna Gnoato,2 Sara Teolato,1 Elisa Ave,1,2 Cristina Gattazzo,1,2 Gian Paolo Fadini,3

Fiorella Calabrese,4 Gianpietro Semenzato,1,2 Carlo Agostini1,2

ABSTRACTBackground and aims Sarcoidosis is characterised bya compartmentalisation of CD4+ T helper 1 (Th1)lymphocytes and activated macrophages in involvedorgans, including the lung. Recently, Th17 effector CD4+

T cells have been claimed to be involved in thepathogenesis of granuloma formation. The objective ofthis study was to investigate the involvement of Th17cells in the pathogenesis of sarcoidosis.Methods Peripheral and pulmonary Th17 cells wereevaluated by flow cytometry, real-time PCR,immunohistochemistry analyses and functional assays inpatients with sarcoidosis in different phases of thedisease and in control subjects.Results Th17 cells were detected both in the peripheralblood (4.7262.27% of CD4+ T cells) and in thebronchoalveolar lavage (BAL) (8.8162.25% of CD4+ Tlymphocytes) of patients with sarcoidosis and T cellalveolitis. Immunohistochemical analysis of lung andlymph node specimens showed that interleukin 17(IL-17)+/CD4+ T cells infiltrate sarcoid tissuessurrounding the central core of the granuloma. IL-17 wasexpressed by macrophages infiltrating sarcoid tissueand/or forming the granuloma core (7.8862.40% ofalveolar macrophages). Analysis of some lung specimenshighlighted the persistence of IL-17+/CD4+ T cells inrelapsed patients and their absence in the recoveredcases. Migratory assays demonstrated the ability of theTh17 cell to respond to the chemotactic stimulusCCL20dthat is, the CCR6 ligand (74.868.5 vs 7.662.8migrating BAL lymphocytes/high-powered field, with andwithout CCL20, respectively).Conclusions Th17 cells participate in the alveolitic/granuloma phase and also to the progression towardsthe fibrotic phase of the disease. The recruitment of thiscell subset may be driven by CCL20 chemokine.

INTRODUCTIONSarcoidosis is a multisystemic granulomatousdisease of unknown aetiology characterised bya compartmentalisation of CD4+ T helper 1 (Th1)lymphocytes and activated monocyte/macrophagesin involved organs, including the lung, lymph nodesand skin.1 In w60% of patients the disease spon-taneously resolves, but in some subjects thepersistence of the antigenic stimulus favoursa chronic inflammatory state, granuloma lungformation and, in some cases, an evolution towardsfibrosis.2 A complex network of cytokines andchemokines orchestrates the pathogenesis ofsarcoidosis: early phases are characterised by a localoverproduction of Th1 cytokines, such as inter-leukin 2 (IL-2) and interferon g (IFNg),3 associatedwith the high expression of macrophage-derived

molecules such as IL-15,4 CXCL10,5 CXCL16,6

CCL57 and CCL20.8

Th17, a new CD4+ effector Tcell population, hasbeen recently described.9 Initially identified for itsability to produce IL-17A, IL-17F and IL-22, it isnow known that human Th17 cells develop inresponse to IL-23 and IL-1b. These cells espressCD4, CD45RO, CCR6 (actually, the unique knownreceptor of the chemokine CCL20), CCR4 and thesubunit IL-23R (the specific receptor for the p19chain of IL-23). In addition, Th17 lymphocytesexpress a specific master transcription factorknown as retinoic acid-related orphan receptor(ROR)gt10 11 and release an array of cytokines,including proinflammatory molecules such astumour necrosis factor a (TNFa) and IL-6.12 Recentstudies have highlighted the effector role of Th17cells in pathological conditions, including autoim-munity and inflammation. In particular, the Th17subset has been linked to Th1 chronic inflamma-tory diseases, such as psoriasis and inflammatorybowel diseases, and also lung fibrosis.13e15

Herein, we investigate the hypothesis that Th17cells are involved in the pathogenesis of sarcoidosis.Forty patients were stratified in different phases ofthe disease and compared with healty controls. Ourresults clearly demonstrate that Th17 cells infiltratethe sarcoid lung and that CCL20 chemokinecontributes to the recruitment of these proin-flammatory cells from the blood into the lung.

MATERIALS AND METHODSStudy populationForty patients with sarcoidosis were analysed. In allcases, the diagnosis was made from a biopsyobtained either from the lungs or from lymph nodesand showing non-caseating epithelioid granulomaswith no evidence of inorganic material known tocause granulomatous diseases. Thirty-six patientsunderwent bronchoalveolar lavage (BAL) fluidanalysis. In particular, 25 patients with sarcoidosispresenting with an episode of pulmonary involve-ment were evaluated at the onset of the disease.They were defined as having a high intensityalveolitis (ie, the active form of the disease) on thebasis of the following characteristics: lymphocyticalveolitis (>30x106 lymphocytes/L) and lung CD4to CD8 ratio >4.0. The assessment of diseaseactivity included BAL, clinical features, chest radio-graph, lung function tests, high-resolution CT androutine blood studies. BAL samples were alsoobtained from 11 patients with previously diag-nosed pulmonary sarcoidosis who repeated BALfluid analysis during their follow-up period. Thesepatients had normal lung function, normal BAL fluid

< Additional methods arepublished online only. To viewthese files please visit thejournal online (http://thorax.bmj.com).1Department of Clinical andExperimental Medicine,Hematology-ImmunologySection, Padua UniversitySchool of Medicine, Padova,Italy2Venetian Institute of MolecularMedicine, Padua UniversitySchool of Medicine, Padova,Italy3Department of Clinical andExperimental Medicine,Metabolic Diseases, PaduaUniversity School of Medicine,Padova, Italy4Department of DiagnosticMedical Sciences and SpecialTherapies, Padua UniversitySchool of Medicine, Padova,Italy

Correspondence toCarlo Agostini, Padua UniversitySchool of Medicine, Departmentof Clinical and ExperimentalMedicine, Hematology andClinical Immunology Section, ViaGiustiniani 2, 35128 Padova,Italy; carlo.agostini@unipd.it

Received 9 April 2010Accepted 29 October 2010Published Online First7 December 2010

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cell numbers and no clinical signs of acute disease. No patientreceived immunosuppressive treatment for 6 months prior to theBAL analysis.

Ten subjects were selected as controls for the BAL studies,evaluated for cough complaints without lung disease. They hadnormal physical examination, chest X rays, lung function testsand BAL cell numbers.

Written informed consent was obtained from each patient andfrom control subjects.

Preparation of cell suspensionsFollowing administration of local anaesthesia, BAL wasperformed and alveolar macrophages (AMs) and BALTcells wereenriched from the entire mononuclear cell suspensions aspreviously described.16 17 Details are reported in the onlinesupplement.

Monoclonal antibodies and cytokinesThe commercially available conjugated or unconjugated mono-clonal antibodies (mAbs) used belonged to the Becton Dickinsonand Pharmingen (San Diego, California, USA) series andincluded: CD3, CD4, CD8, CD11c, CD14, CD16, CD19,CD45R0, CD45RA, CD68 and isotype-matched controls. Anti-IL23R, anti-IL-17, anti-CCR6, anti-CCL20 and anti-IFNg mAbswere purchased from R&D Systems Inc. (Minneapolis, Minne-sota, USA).

Intracellular cytokine stainingDetection of intracellular cytokines was performed in freshlyobtained lung and peripheral blood cells and in equivalent cellsstimulated with 20 ng/ml phorbol-12-myristate 13-acetate(PMA) and 1 mM ionomycin (Sigma-Aldrich, Milan, Italy).Further details are reported in the online supplement.

Immunohistochemical analysisLung samples from seven cases of sarcoidosis (three from activeforms, two from inactive forms and two obtained from nativelungs of patients requiring lung transplantation for the devel-opment of lung fibrosis), lung autoptic samples from twosubjects (death due to sarcoidosis-unrelated causes) whosesarcoidosis had recovered and lymph node samples from twopatients with active sarcoidosis were processed by immunohis-tochemistry. Details are reported in the online supplement.

RNA purification and real-time PCR for the quantification of IL-17and RORgt expression by sarcoid lung cellsTotal RNA was prepared from (1) purified T lymphocytesobtained from patients with sarcoid Tcell alveolitis or normal Tcells or (2) purified AMs from patients with active sarcoidosisand control subjects. Methods used for the real-time PCR aredetailed in the online supplement.

Statistical analysisData were analysed with the assistance of the Statistical Anal-ysis System (SAS Institute, Cary, North Carolina, USA), andstatistical significance was accepted at p<0.05. Further detailsare reported in the online supplement.

RESULTSPatient characteristicsTwenty-five subjects with high intensity alveolitis were evalu-ated at the onset of the disease (11 men and 14 women; meanage 51.569.8 years; four smokers). Twenty-two patientsrequired corticosteroid treatment; three patients spontaneouslyresolved. Eleven patients (6 men and 5 women; mean age52.567.2 years; three smokers) with previously diagnosedpulmonary sarcoidosis repeated BAL fluid analysis during theirfollow-up period (follow-up period average 50.7616.6 months,range 27e78 months). Further characteristics of patients studiedare shown in table 1. Ten subjects were selected as controls forthe BAL studies (4 men and 6 women; mean age 44.168.8 years;non-smokers).

Morphological and phenotypical analysesMorphological and phenotypical features of cells obtained fromthe BAL of 36 patients with sarcoidosis and 10 control subjectsare reported in table 2. All subjects with active sarcoidosisshowed a high intensity CD4+ lymphocytic alveolitis sustainedby CD45RO+ Tcells. The majority of these cells were equippedwith the chemokine receptor CXCR3, IL-12Rb and intra-cytoplasmatic IFNg (data not shown). CD4+ and CD8+ T cellsubsets and AMs detected in the BAL of patients with inactivesarcoidosis were superimposable on those observed in controls(table 2).

Th17 cells in the lung and the peripheral blood of patientswith sarcoidosisFirst, using a phenotypical approach, we investigated the pres-ence of IL-17+ and IL-23R+ T cells on freshly obtained pulmo-nary and peripheral blood cells of patients with active andinactive sarcoidosis and controls. The relative patterns areshown in figure 1.As shown in figure 2A, the percentage of freshly obtained

pulmonary CD4+ T lymphocytes expressing IL-17 was muchhigher in patients with the active form of the disease(8.8162.25%) as compared with inactive sarcoidosis(1.1960.83% of CD4+ T cells; p<0.001 vs active disease) and

Table 1 Disease manifestations in patients with active sarcoidosisincluded in the study

Patient no. Disease manifestation

1, 9, 11, 15, 20, 24, 27, 28, 29, 31, 32 Lung, LN

4, 6, 7, 13, 21, 22, 30, 34, 35, 37 Lung, LN, skin

10, 16, 19 Lung, LN, eyes

5 Lung, LN, eyes, salivary glands

LN, lymph node.

Table 2 Bronchoalveolar lavage features of patients with sarcoidosis and control subjects

Cell recovery Alveolar macrophages Lymphocytes CD4+ T cells CD8+ T cells

3103 3103 % 3103 % 3103 % 3103 %

Active sarcoidosis (n¼25) 333673 249658 7466 82627 2566 71625 8665 1165 1465

Inactive sarcoidosis (n¼11) 109631 102627 9464 564 462 362 5865 261 4165

Controls (n¼10) 128627 119627 9363 964 764 562 5965 462 4164

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with controls (0.4360.48% of CD4+ T lymphocytes; p<0.001 vsactive disease; analysis of variance (ANOVA) p<0.001).

As far as IL-23R is concerned, a significantlyhigher percentage oflung CD4+ Tcells obtained from patients with active sarcoidosisexpressed the receptor (9.0162.10%) with respect to pulmonaryCD4+ Tcells of patients with inactive sarcoidosis (2.3861.96%;

p<0.001 vs active disease) and controls (0.6460.73%; p<0.001 vsactive disease; ANOVA p<0.001; figure 2B).The results drawn from the peripheral blood parallel the data

we obtained from the lung and confirm the multisystemicnature of the disease. In fact, the mean percentage of freshlyisolated IL-17+ Tcells in the CD4+ Tcell subset was 4.7262.27%

Figure 1 Analysis of interleukin 17 (IL-17) and IL-23 expression in CD4+ Tlymphocytes freshly obtained from thebronchoalveolar lavage (BAL) and theperipheral blood of patients affected byactive sarcoidosis (B, E, I, L), inactivesarcoidosis (C, F, J, M) and controlsubjects (D, G, K, N). On the left, thephysical parameter (forward scatter)and CD4 expression of pulmonary (A)and peripheral blood lymphocytes (H)are reported.

Figure 2 Flow cytometry analyses ofinterleukin 17 (IL-17)+ (A) and IL-23R+

(B) expression evaluated in CD4+ Tcells freshly obtained from the lung andthe blood of patients affected by activesarcoidosis (black columns), inactivesarcoidosis (black and white columns)and from control subjects (whitecolumns). (C and D) Retinoic acid-related orphan receptor (ROR)gt (C) andIL-17 (D) mRNA expression in CD4+

lung T lymphocytes of patients affectedby active sarcoidosis (black columns),in CD3+ pulmonary T cells of patientswith inactive sarcoidosis (black andwhite columns) and in lymphocytesfrom control subjects (white columns).Data are expressed as mean6SD;*p<0.001 in post hoc analyses.

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in patients with the active form of the disease, whereas thepercentages of the IL-17+/CD4+ T cell subset in patients withthe inactive disease and in controls were 0.6360.44% (p¼0.001vs active disease) and 0.6760.45% (p¼0.001 vs active disease),respectively (ANOVA p<0.001) (figure 2A). The IL-23R expres-sion was more pronounced on peripheral blood CD4+ T cells ofpatients with active sarcoidosis (3.2260.48%) than on CD4+ Tlymphocytes isolated from patients with the inactive form ofthe disease (0.8160.81%; p<0.001 vs active disease) and fromthe controls (0.7560.21%; p<0.001 vs active disease; ANOVAp<0.001; figure 2B).

Stimulation of pulmonary and peripheral blood CD4+ T cellswith PMA and ionomycin induced a strong downmodulation ofCD4 expression.18 19 Therefore, flow cytometric analysis was

restricted to CD8e/IL-17+ T cells, that we regarded as Th17cells. Stimulated lung T cells isolated from patients with activesarcoidosis showed an increased percentage of intracellular IL-17(10.3565.84%; p¼0.053 vs unstimulated cells). In parallel, themean percentage of peripheral blood stimulated IL-17+ T cellsrose to 8.4361.65% (p¼0.082 vs unstimulated cells).

Molecular analysis of RORgt and IL-17 expressionby lung T cellsAs shown in figure 2C, real-time PCR evaluation of RORgt geneexpression by highly purified BAL T lymphocytes demonstratedthat mRNA expression of RORgt was higher in lung CD4+ Tlymphocytes of patients with active sarcoidosis (48.80614.07)than in CD3+ T cells obtained from the lung of patients with

Figure 3 Analysis of interleukin 17 (IL-17) and IL-23 expression in selected cellpopulations obtained from thebronchoalveloar lavage (alveolarmacrophages) and the peripheral bloodmonocytes of patients affected byactive sarcoidosis (B, E, I, L), patientswith inactive sarcoidosis (C, F, J, M)and control subjects (D, G, K, N). On theleft, physical parameters (side andforward scatter) of alveolarmacrophages (A) and peripheral bloodmonocytes (H) are reported. (O and P)Means of IL-17+ and IL-23R+

expression evaluated in alveolarmacrophages (O) and peripheral bloodmonocytes (P) freshly obtained frompatients affected by active sarcoidosis(black columns), inactive sarcoidosis(black and white columns) and fromcontrol subjects (white columns). Dataare expressed as mean6SD; *p<0.001in post hoc analyses.

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inactive disease and control subjects (inactive, 2.8061.26;p<0.001 vs active disease; controls, 1.4561.03; p<0.001 vsactive disease; ANOVA p<0.001). Similarly, a real-time PCRevaluation of IL-17 gene expression by pulmonary T cellsdemonstrated that IL-17 mRNA was higher in lung CD4+ Tlymphocytes of active sarcoidosis (38.53618.41) than in CD3+

T cells from the lung of patients with inactive disease andcontrol subjects (inactive, 0.7860.73; p¼0.002 vs active disease;controls, 0.4960.47; p¼0.002 vs active disease; ANOVAp¼0.001; figure 2D).

Sarcoid AMs express IL-17 and IL-23RThe flow cytometric analysis of freshly isolated AMs obtainedfrom the BAL of the lungs of patients with active sarcoidosisshowed the intracellular expression of IL-17 (7.8862.40%) andthe surface presence of IL-23R (8.1363.47%). In contrast, a verylow expression of IL-17 and IL-23R was detected on the AMsfrom patients with inactive sarcoidosis (IL-17, 0.3960.34%;p<0.001 vs active disease; IL-23R, 0.7460.80%; p<0.001 vsactive disease) and from controls (IL-17, 0.4860.46%; p<0.001vs active disease; IL-23R, 0.6460.56%; p<0.001 vs active disease;ANOVA p<0.001 for both; figure 3AeG,O).

Interestingly, peripheral monocytes of patients with activesarcoidosis showed a significantly increased expression of IL-17(10.3365.26%) and IL-23R (8.9463.35%) in comparison withmonocytes obtained from patients with inactive sarcoidosis (IL-17, 1.6860.51%; p<0.001 vs active disease; IL-23R, 1.8660.63%;p<0.001 vs active disease) and controls (IL-17, 1.0360.40%;p<0.001 vs active disease; IL-23R, 1.7860.36%; p<0.001 vsactive disease; ANOVA p<0.001 for both; figure 3HeN,P).

Immunohistochemical analysis of IL-23R and IL-17 expressionImmunohistochemical analysis confirmed the expression of IL-23R and IL-17 by sarcoid lung T cells infiltrating surgicalpulmonary biopsies obtained from four patients with activesarcoidosis (figure 4A,B). When the cell sources of IL-17 insarcoid tissue was investigated, we showed that the cytokinewas preferentially expressed by macrophage multinucleatedgiant cells and T cells localised inside the granuloma. We iden-tified IL-17+ T cells infiltrating lymph node granulomas ofpatients with active sarcoidosis (figure 4E,1e2), thus docu-menting the presence of Th17 cells at the extrapulmonary activesites of the disease. Immunohistochemical analysis of nativelungs obtained from three patients with refractory sarcoidosis

Figure 4 Immunohistochemistry forinterleukin 17 (IL-17) and IL-23Rexpression in three representativepatients with active (A, B, E) andinactive (C, D) sarcoidosis. (A and B) IL-17 and IL-23R are expressed at highintensity by macrophagic, bothepitheliod and multinucleated, cells andlymphocytes infiltrating the lung biopsyof the patient with active sarcoidosis.(a, b) Serum isotype controls withhaematoxylin counterstain. (C and D) Inthe lung specimen obtained froma patient in the inactive phase of thedisease, pulmonary T cells were mainlynon-reactive for IL-17 and IL-23R;a weak immunostaining was only seenin some epitheliod macrophagic cells.Note the negative staining inmultinucleated cells (arrows). (E) IL-17is expressed by T cells infiltrating thelymph node biopsy of the patient withactive sarcoidosis. (1, 2) Enlargement ofareas in E. Original magnification 3200.

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and pulmonary fibrosis who underwent lung allograft trans-plantation showed that in the fibrotic phase of the disease, lungT cells were mainly non-reactive for IL-23R and IL-17, and AMswere not stained with anti-IL-17 mAb (figure 4C,D).

To investigate the role of Th17 cells in different phases of thedisease, we analysed surgical pulmonary biopsies obtained fromtwo patients with sarcoidosis relapse and from two subjectswhose sarcoidosis underwent resolution (figure 5). Inter-estingly, we identified IL-17+ T cells only in the relapsedpatient, suggesting the critical role of Th17 cells in sarcoidosisprogression.

Pulmonary Th17 cells expressed a functional CCR6 receptorTh17 cells are characterised by the expression of CCR6, thereceptor of the chemokine CCL20.13 By flow cytometric anal-ysis, we showed that this receptor is highly expressed by Th17cells of patients with sarcoidosis (79.33611.19% of IL-17+/CD4+ T cells) (figure 6). In a previous work,8 we demonstratedthe involvement of the CCR6/CCL20 axis in the lung micro-environment of patients with sarcoidosis. Herein, we confirmedthat sarcoid lung T lymphocytes bear CCR6 (45.667.8% ofCD4+ T cells; 52.367.1% of CCR6+CD4+ T cells were CXCR3negative) and migrate in the presence of CCL20 (74.868.5 vs7.662.8 migrating BAL lymphocytes/high-powered field, in thepresence and absence of CCL20, respectively; p<0.001). It isnoteworthy that we showed that sarcoid macrophages releaseCCL20 and that detectable levels of CCL20 protein can bedemonstrated in the BAL fluid components.8

DISCUSSIONIn this report we have provided evidence that Th17 cells infil-trate sarcoid lung, localising around and inside the granuloma, atthe sites of disease activity, not only in the early phase, but alsoin the progression towards the fibrotic phase of the disease.In patients with sarcoidosis, the pulmonary microenviron-

ment is characterised by a well-known highly polarised Th1profile.6 20 Despite the in vitro inhibition of Th17 developmentby Th1-derived IFNg,21 22 the presence in vivo of a Th1/Th17population has been extensively demonstrated in differentpathological conditions characterised by an exaggerated inflam-matory response, including psoriasis,13 rheumatoid arthritis,23

Crohn’s disease,24 hypersensitivity pneumonitis25 and tubercu-losis.26 We detected high levels of IL-17+/CD4+ T lymphocytesamong cells retrieved from the BAL of patients with sarcoidosis.In particular, patients with a marked alveolitis showed thehighest amounts of Th17 cells, both in the lung and in theperipheral blood, suggesting an overall increase of this subset,spreading from the periphery to the sites of active disease. In aprevious report, Meloni et al27 assessed the frequency ofTh1, Th2 and Th17 lymphocytes in the BAL of patients withinterstitial lung diseases, including sarcoidosis (15 cases),describing no variation of Th17 clones in the patients comparedwith controls. The different technical approaches (an IL-17Elispot and a different stimulation of Tcells) may explain thecontradictory results they obtained. In addition, the reportedabsence of any increase in the basal frequency of lung IFNg-producing cells in the patients with sarcoidosis included in thisstudy is a feature conflicting with the classical, active Th1pulmonary microenvironment.The evidence that Th17 cells are increased in the lung and the

peripheral blood of patients with active sarcoidosis supportsthe multisystem nature of the disease but raises the issue of thediminished cellular immune response in the blood of patientswith sarcoidosis. Regulatory T lymphocytes (Tregs) of patientswith sarcoidosis deeply affect the concomitant presence of anintense immune response in the affected organs and a reduced/suppressed antigen immune response in the periphery. Actually,the interactions between Tregs and Th17 cells are not completlyunderstood. Intriguingly, recent data demonstrated the plas-ticity/instability of Tregs, mainly in the presence of someproinflammatory cytokines, such as IL-6, that converted T cellreceptor (TCR)-stimulated Tregs into Th17 lymphocytes invitro.28

Sarcoid AMs produce elevated levels of IL-17. Recent studiesshowed the relevant role of monocytes/macrophages in the

Figure 5 Immunohistochemistry forlung interleukin 17 (IL-17) expression ina representative patient with sarcoidosisrelapse (A) and in lung autoptic samplesfrom two subjects (death due tosarcoidosis-unrelated causes) whosesarcoidosis had recovered (B, C, D, E).(A) IL-17 is expressed at high intensityby macrophagic cells and lymphocytesinfiltrating the lung biopsy of therelapsed patient. (B, C, D and E) In thelung autoptic specimens, pulmonary Tcells were non-reactive for IL-17antibody.

Figure 6 Flow cytometry analysis of the expression of CCR6+ oninterleukin 17 (IL-17)+/CD4+ T cells in the lung (A) and the blood (B) ofa representative patient with active sarcoidosis.

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promotion of the Th17 response. In fact, activated monocytesobtained from the synovial fluid of patients with rheumatoidarthritis specifically induce the Th17 response via cell contactsignals and IL-1b/TNFa cytokines.29 Our data favour thehypothesis that a Th17 response may be driven by AMs sinceIL-1b- and TNFa-producing AMs are widely detectable insarcoid lung.

IL-17+/CD4+ T cells were further characterised for theexpression of IL-23R, the master gene RORgt and the CCR6receptor, a recently recognised Th17 marker.13 IL-23R binds IL-23, a cytokine involved in human Th17 cell differentiation.30

The overexpression of this molecule on CD4+ T cells parallelsthe increase in IL-17+/CD4+ T lymphocytes, both in the lungand in the blood of patients with active sarcoidosis. RORgt andCCR6 are two strictly linked molecules: RORgt directs Th17 celldifferentiation and specifically controls the differentiation ofCD4+ T cells towards CCR6+ Th17 cells, as demonstrated byRORgt transduction experiments in CCR6e/CD4+ T cells.23

The pulmonary and peripheral Th17 cells of our patients over-expressed the RORgt mRNA and showed a strong positivity forCCR6 protein. Actually, CCL20 is the only chemokine producedand released by Th17 cells.

In a previous work,8 we demonstrated that sarcoid macro-phages and epithelioid cells, forming the central core of thesarcoid granuloma, express and release detectable amounts ofCCL20. According to the present results, we support thehypothesis that these cells are Th17 lymphocytes since theyexpress a fully functional CCR6 receptor whose interactionswith the specific ligand CCL20 regulate Th17 cell trafficking:airway epithelial cells and AMs recruit Th17 cells into the lungthrough CCL20 release, which is enhanced by proinflammatorycytokines such as IL-1b and TNFa.7 Probably, Th17 cells favourdisease progression, as our preliminary results on patients withsarcoidosis evolving towards fibrosis (figure 5) seem to point out.The involvement of IL-17A in the development of fibrosis wasrecently demonstrated in a murine model of bleomycin-inducedpulmonary fibrosis.31 IL-17A was detected both in the earlyphase of inflammation, together with IFNg (a Th1/Th17response), and at later times, with IFNg and IL-13 (a mixed Th1/Th17/Th2 response). In addition, the experiments conductedwith C57BL/6 IL-17Ae/e mice confirmed the role of IL-17 indriving the development/progress of fibrosis, facilitated by IFNgand IL-12/23p40, and in association with transforming growthfactor b (TGFb). In this context, IL-17 may represent a commondenominator in the events leading to fibrosis development ininterstitial lung diseases.

In conclusion, our data highlight the presence of Th17 cells inthe lung and the blood of patients with sarcoidosis, particularlyin those patients with the active form of the disease, suggestinga role for the Th17 subset in sarcoidosis progression. Furtherstudies are needed to better understand the role of Th17 cells inthe various stages of the disease and to validate the possibility ofa treatment based on Th17 and/or IL-17 neutralisation.

Acknowledgements The authors wish to thank Mr Luca Braghetto for technicalassistance in the preparation of lung biopsies for immunohistochemical analysis.

Funding MF has received a grant from Padua University, Progetto di AteneoCPDA063349/06. GS (PRIN 2007) and CA (PRIN 2009) have received grants from theItalian Ministry of University, Research, and Education, Rome (Italy).

Competing interests None.

Ethics approval This study was conducted with the approval of the Padova HospitalEthics Committee.

Provenance and peer review Not commissioned; externally peer reviewed.

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