Rheumatoid arthtritis

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INT J TUBERC LUNG DIS 17(11):15011506

2013 The Unionhttp://dx.doi.org/10.5588/ijtld.13.0082

Indoleamine 2,3-dioxygenase in the pathogenesisof tuberculous pleurisy

Y. Suzuki,*S. Miwa,T. Akamatsu,* M. Suzuki,M. Fujie,Y. Nakamura,* N. Inui,* H. Hayakawa,

K. Chida,* T. Suda*

*Second Division, Department of Internal Medicine, Hamamatsu University School of Medicine, Hamamatsu,Respiratory Medicine, Tenryu Hospital, National Hospital Organization, Hamamatsu, Equipment Center,Hamamatsu University School of Medicine, Hamamatsu, Japan

Correspondence to: Yuzo Suzuki, 1-20-1 Hanadayama Higashi-ku, Hamamatsu, Shizuoka 431-3129, Japan. Tel: (+81)53 435 2263. Fax: (+81) 53 435 2354. e-mail: [email protected]

Article submitted 4 February 2013. Final version accepted 3 July 2013.

S U M M A R Y

THE ENZYME indoleamine 2,3-dioxygenase (IDO)catalyses tryptophan to its metabolite along the kyn-urenine pathway.16 By degradation of an essentialtryptophan amino acid in microenvironments, this en-zyme has a potent immunoregulatory effect that sup-presses T-cell function and induces regulatory T-cells,leading to immunosuppression or tolerance. Accu-mulated evidence has shown that IDO is broadly in-volved in various diseases, including cancer and infec-tion, through regulation of the immune response.715In cancer immunity, increased expression of IDO bycancer cells themselves is considered a critical immuneescape mechanism and a novel prognostic factor.14,79

During infection, the inhibition of IDO activity by1-methyl-D-tryptophan (1-MT) was reported to pro-tect mice against septic shock.12 More recently, wehave shown that serum IDO activity is a novel prog-nostic factor in patients with pulmonary tuberculosis(TB) and community-acquired pneumonia.14,15Thesendings indicate that IDO plays a crucial role in thepathogenesis of these diseases and is considered anovel therapeutic target.

Pleural uid is common in pulmonary diseases, and

may be caused by numerous pathological conditions.Of these, tuberculous pleurisy (TBP) and cancer arethe main causes.16,17TBP is thought to result from a de-layed hypersensitivity reaction to mycobacterial anti-gens in pleural disease.17,18In contrast, pleural effusioncaused by malignant diseases and parapneumonic ef-fusion are elicited by pleural and lung inammation.The diagnosis of TBP is highly dependent on biomark-ers such as adenosine deaminase (ADA) activity inpleural uid, as the Mycobacterium tuberculosis smearor culture positivity rate in pleural effusions is low.17,1922In this setting, no studies have evaluated IDO activityin pleural effusion with pulmonary diseases.

In the present study, we measured IDO activity asdetermined by the kynurenine-to-tryptophan ratio inpleural effusions and investigated the clinical signi-cance of IDO activity in pleural uid.

METHODS

Subjects

This prospective study was conducted at the Hama-matsu University School of Medicine and the Tenryu

B AC KGR O UND: Pleural fluid is a frequent manifesta-

tion in pulmonary diseases, such as lung cancer and in-

fectious diseases, including pulmonary tuberculosis (TB).

The enzyme indoleamine 2,3-dioxygenase (IDO) cataly-

ses tryptophan through the kynurenine pathway, and isconsidered a crucial immunoregulatory molecule medi-

ating immune tolerance. Recent studies have shown IDO

activity to be a novel prognostic factor not only in can-

cer patients but also in those with infectious diseases, in-

cluding pneumonia and pulmonary TB. However, no

studies have measured and determined the clinical sig-

nificance of IDO activity in pleural fluid.

ME T HO DS : We enrolled 92 patients, including 34 with

tuberculous pleurisy (TBP), 36 with malignant pleuritis

and 15 with parapneumonic effusions. IDO activity was

evaluated using liquid chromatography/electrospray

ionisation tandem mass spectrometry, and was estimated

by calculating kynurenine-to-tryptophan ratio.

RESULTS: Pleural fluid from patients with TBP had sig-

nificantly higher kynurenine concentrations and signifi-cantly lower tryptophan concentrations, resulting in sig-

nificantly higher IDO activity compared with pleural

effusion or serum from non-tuberculous pleuritis (allP


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1502 The International Journal of Tuberculosis and Lung DiseaseHospital, Hamamatsu, Japan. It was approved by theethics committees of the Hamamatsu University Schoolof Medicine and the Tenryu Hospital. Written in-formed consent was obtained from all patients in ac-cordance with institutional guidelines.

From March 2010 to February 2011, 92 consecu-tive patients (68 men and 24 women, with a meanage of 72.9 years) admitted to our institutions withpleural uid were enrolled in the study. TBP wasdiagnosed if 1) M. tuberculosis was isolated frompleural uid or pleural tissue, 2) granulomas in thepleural tissue showed staining for acid-fast bacilli(AFB), 3) granulomas in the pleural tissue did not stainfor AFB, but showed a response to anti-tuberculosistreatment, or 4) a sputum culture was positive for TB.Carcinomatous pleurisy was diagnosed either by apositive pleural uid cytological result or by identify-

ing malignant cells in a pleural biopsy specimen. Inaddition, even when both of these test results werenegative, malignant effusion was diagnosed when aprimary cancer was known to have disseminated andconcentrations of tumour marker in pleural uidwere elevated. Pleural effusions arising from pneu-monia or empyema were dened as parapneumoniceffusions. Miscellaneous pleural effusions were thosethat were not linked to either infection, including TB,or malignant disease.

Sample preparation

Serum samples were obtained at the time of admis-

sion, and pleural effusions were collected from eachsubject using a standard thoracocentesis techniquewithin 24 h of hospitalisation. Collected pleural uidand serum samples were frozen at 20C until anal-ysis, and routine laboratory examinations, such asblood cell counts and biochemical analysis, were per-formed subsequently. Pleural tissue was obtained frompatients with undiagnosed pleural effusion using tho-racoscopy under local anaesthetic.

Measurement of tryptophan and kynurenine

L-kynurenine and 3-nitro-tyrosine, used as an internal

standard, were purchased from Sigma-Aldrich (StLouis, MO, USA). L-tryptophan was purchased fromThermo Fisher Scientic (Waltham, MA, USA). Am-monium formate (HCO2NH4) and perchloric acidwere obtained from Wako Pure Chemical Industries(Osaka, Japan). The pleural uid and serum concen-trations of kynu-renine and tryptophan were deter-mined by liquid chromatography/electrospray ionisa-tion tandem mass spectrometry (LC-ESI/MS/MS, TSQ7000 LC-quadrupole mass spectrometer, Thermo-Fisher, San Jose, CA, USA), as described previously.14,15Briey, frozen samples were thawed at room tem-perature. The samples were then spiked with stan-dards and deproteinised with 0.5 N perchloric acid for10 min on ice. The samples were then centrifuged at15 000gfor 10 min, and the supernatants were vor-

texed with an equal volume of 1 M HCO2NH4. Anal-ysis and detection were performed using LC-ESI/MS/MS. Chromatographic separation of the analyteswas performed in isocratic mode using a AtrantisT3 analytical column (150 mm 2.1 mm, particle

size 5 m, Waters, Milford, MA, USA). The mobilephase of 5 mM HCO2NH4 (0.01% triuoroaceticacid)methanol (80:20, v/v) was passed at a owrate of 0.2 ml/min. Detection was performed usingsheathless electrospray tandem mass spectrometryin the multiple reaction monitoring mode. IDO activ-ity was determined by dividing the concentration ofkynurenine by tryptophan.

Immunohistochemistry

Pleural specimens were xed in 10% formalin, andthe tissues were embedded in parafn. Deparafnised

sections (5 m thick) were immersed in epitope re-trieval solution (Target Retrieval Solution S1700;Dako North America Inc, Carpinteria, CA, USA), andpreheated to 120C for 10 min. After blocking en-dogenous peroxidase with 3% H2O2for 15 min, theslides were incubated with the mouse anti-humanIDO monoclonal antibody (6:1000; Abnova, Taipei,Taiwan) overnight at 4C. The sections were subse-quently incubated with visualisation reagent (Histo-ne Simple Stain MAX-PO(M); Nichirei Co. Tokyo,

Japan) for 30 min, and the immunoreaction was visu-alised using a 3,3-diaminobenzidine chromogen solu-tion (DAB substrate kit; Vector Laboratories, Inc,

Burlingame, CA, USA) and counterstained withhaematoxylin.

Statistical analysis

Discrete variables are expressed as counts (percent-age), and continuous variables are described asmean standard deviation, unless otherwise speci-ed. Wilcoxon/Kruskal-Wallis tests were used forcontinuous variables, and the analysis of variationtest, followed by post-hoc analysis, was used formulti-group comparisons. Categorical data werecompared between the groups using the 2test for

independence. A receiver operating characteristic(ROC) curve was used to evaluate the ability ofIDO activity to diagnose TBP. The optimal cut-offvalue, i.e., that ensured the best combination sensitiv-ity and specicity, was obtained. Statistical analyseswere performed using JMP Start Statistics (SAS Insti-tute Inc, Cary, NC, USA). P


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IDO in tuberculous pleurisy 1503

was diagnosed as disseminated TB. Of the 58 non-tuberculous pleurisy patients, carcinomatous pleurisywas found in 36 (39.1%), including 23 lung cancerand 6 metastatic lung cancer patients. Parapneu-

monic effusions were found in 15 patients (16.3%).No sex differences were observed between TBP andnon-tuberculous pleurisy; however, patients with TBPwere signicantly older than non-tuberculous pleurisypatients. ADA is known as a useful biomarker to dis-tinguish TBP, and higher ADA levels were found inpatients with TBP (P


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1504 The International Journal of Tuberculosis and Lung Disease

IDO activity of both TBP and non-tuberculous pleu-risy patients (P


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IDO in tuberculous pleurisy 1505

peripheral immune suppression or tolerance impli-cated in various diseases. The immunosuppressive ef-fects of IDO have been shown to be critical in vari-ous pathogenic conditions, and IDO is considered atherapeutic agent. However, no studies have investi-gated serum or pleural IDO activity in pleural disease.We rst evaluated IDO activity using both serum sam-ples and pleural effusions for assessing immune con-

ditions of pleural disease, and showed signicantlyhigher IDO activity in TBP with both serum samplesand pleural effusions than non-tuberculous pleurisy.Collectively, these results suggest that the immuno-suppressive effect of IDO is affected in the pathogen-esis of TBP.

TBP occurs as a result of a TB antigen enteringthe pleural space, followed by a delayed hypersensi-tivity reaction to mycobacterial antigens mediated byCD4+cells.17,18A number of previous studies on hu-man TB reported signicant differences in immuneproles at the site of M. tuberculosis infection com-pared with peripheral blood.23 Interferon-gamma

(IFN-), known as a key cytokine in TB immunity,has been shown to be highly enriched in pleural uidand lungs compared with blood in active TB pa-tients.2426On the other hand, anti-inammatory cy-tokines, such as interleukin (IL) 10, and proportionsof Treg are also elevated in pleural uid comparedwith serum or peripheral blood.27,28These mixed Th1and immunosuppressive responses at the site ofM. tuberculosisinfection are consistent with our nd-ing of signicantly higher pleural than serum IDOactivity in TBP, as IFN-is the most potent inducerof IDO, which in turn recruits Treg due to immuno-suppressive effects. These previous reports and our re-sults support the assumption that IDO is involved inmodifying the immune status at local sites of M. tu-berculosisinfection.

Although several studies have reported increasedIDO expression in M. tuberculosis-infected macro-phages using an experimental model and also in spu-tum cells from TB patients,29,30the origin of IDO inhuman tuberculous infection remains unknown. In

this setting, we observed greatly increased IDO ex-pression using human tissue in epithelial granulomalesions, a region where various immune cells accu-mulate. Although the role of IDO for host-pathogeninteractions in tuberculous infection is still undeter-mined, the immunosuppressive effects of IDO atlocal sites might contribute to bacterial replicationby downregulating the antimycobacterial effect ofIFN-. This may be benecial for the host by protect-ing granulomas from exaggerated inammation andavoiding tissue damage, which eventually disruptsgranulomas and allows pathogens to spread. The for-mation of granuloma is a critical step in controllingtuberculous infection through the accumulation ofimmune cells, but pathogenic mycobacteria may alsoexploit early granuloma formation for rapid expan-sion.31Similarly, the role of IDO might differ depend-ing on the stage of infection.

There are several limitations to this study. Al-though a relatively large number of patients withpleural effusion was enrolled, the sample size wasstill too small to determine the clinical signicance ofpleural IDO activity. In Japan, the prevalence of TBamong elderly people is very high: more than 53% ofpatients were aged >70 years and 32% were aged

>80 years; thus, patients with TBP were potentiallyolder than non-tuberculous pleurisy patients. Weshowed increased IDO activity in pleural effusionsof TBP, but the diagnostic value of IDO in TBP wasless than that of ADA. As ADA and M. tuberculosis-specic IFN-release assays are already of provendiagnostic value in practice, the clinical usefulnessof assessing IDO in pleural effusions is currentlylimited.

In conclusion, the present study showed increasedIDO activity in pleural effusions with TBP and IDOstaining in epithelial granulomas. These results sug-gest that IDO participates in the pathogenesis of TBP

through its immune suppressive effect.

Acknowledgements

The authors thank Y Fujisaka and N Nakamura at Tenryu Hospi-

tal for samples and data collection.

Registration: UMIN000003400, http://www.umin.ac.jp/ctr/index-j.htm

Conict of interest: none declared.

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Figure 3 Expression of IDO in TBP. Pleural tissue from pa-tients with biopsy-proven TBP was stained with IDO antibody,followed by haematoxylin counterstain (original magnification200). Enhanced IDO expression was found in epitheloid granu-lomas (arrows). IDO = indoleamine 2,3-dioxygenase; TBP =tuberculous pleurisy.


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IDO in tuberculous pleurisy i

C O NT E XT E : Les panchements pleuraux sont des mani-

festations frquentes dans les maladies pulmonaires,

comme le cancer du poumon ainsi que dans les maladies

infectieuses, notamment la tuberculose (TB) pulmo-

naire. Lindolamine 2,3-dioxygnase (IDO) est lenzyme

qui catalyse le tryptophane par la voie de la kynurnine

et est considre comme une molcule immunorgulatrice

cruciale pour la mdiation de la tolrance immunitaire.

Des tudes rcentes ont montr que lactivit de lIDO

est un facteur pronostic novateur, non seulement chez les

patients atteints de cancer, mais aussi dans les maladies

infectieuses, y compris la pneumonie et la TB pulmo-

naire. Toutefois, aucune tude na mesur ni dtermin

la signification clinique de lactivit de lIDO dans les

liquides pleuraux.

M T HO DE S : Nous avons recrut 92 patients dont 34 cas

de pleursie tuberculeuse (TB), 36 cas de pleursie ma-ligne et 15 panchements parapneumoniques. Lactivit

de lIDO a t value par spectromtrie de masse tan-

dem au moyen de chromatographie liquide et dionisation

electrospray ; elle a t estime par le calcul du ratio

kynurnine/tryptophane.

RSU LTATS : Par comparaison avec les panchements

pleuraux ou le srum provenant de pleursies non-

tuberculeuses, les panchements pleuraux de patients

atteints de TBP ont des concentrations de kynurnine

significativement plus leves et des concentrations de

tryptophane significativement plus faibles, ce qui entraine

une activit de lIDO significativement plus importante

(P

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