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Clinical Immunology (2014) 151, 84–99

Progression of clinical tuberculosis isassociated with a Th2 immune responsesignature in combination with elevatedlevels of SOCS3

Senait Ashenafi a,b, Getachew Aderayec, Amsalu Bekele c, Martha Zewdied,Getachew Aseffae, Anh Thu Nguyen Hoanga, Berit Carowf,Meseret Habtamud, Maria Wijkander a, Martin Rottenberg f, Abraham Aseffad,Jan Anderssona,g, Mattias Svenssona, Susanna Brighenti a,⁎

a Karolinska Institutet, Center for Infectious Medicine (CIM), F59, Department of Medicine,Karolinska University Hospital Huddinge, Stockholm, Swedenb Black Lion University Hospital and Addis Ababa University, Department of Pathology, Faculty of Medicine,Addis Ababa, Ethiopiac Black Lion University Hospital and Addis Ababa University, Department of Internal Medicine, Faculty of Medicine,Addis Ababa, Ethiopiad Armauer Hansen Research Institute (AHRI), Addis Ababa, Ethiopiae Black Lion University Hospital and Addis Ababa University, Department of Radiology, Faculty of Medicine,Addis Ababa, Ethiopiaf Karolinska Institutet, Department of Microbiology, Tumor and Cell Biology (MTC), Stockholm, Swedeng Karolinska University Hospital Huddinge, Department of Medicine, Division of Infectious Diseases, Stockholm, Sweden

Received 16 September 2013; accepted with revision 30 January 2014Available online 9 February 2014

Abbreviations: TB, tuberculosis; Mcytolytic T cell; NK, natural killerimmunoglobulin; SOCS, suppressors ofbronchoalveolar lavage; PBMC, peripheGuerin; FCS, fetal calf serum; OD, opmacrophage colony-stimulating factortuberculosis.⁎ Corresponding author at: Center fo

Hospital Huddinge, 141 86 Stockholm,E-mail address: susanna.brighenti@

http://dx.doi.org/10.1016/j.clim.2011521-6616/© 2014 The Authors. Pu

KEYWORDSTuberculosis;HIV;Human;

Abstract In this study, we explored the local cytokine/chemokine profiles in patients withactive pulmonary or pleural tuberculosis (TB) using multiplex protein analysis of bronchoalveolarlavage and pleural fluid samples. Despite increased pro-inflammation compared to theuninfected controls; there was no up-regulation of IFN-γ or the T cell chemoattractant CCL5

. tuberculosis, Mycobacterium tuberculosis; HIV, Human Immunodeficiency Virus; Th, T helper; CTL,cell; Treg, regulatory T cell; IL, interleukin; IFN, interferon; TNF, tumor necrosis factor; Ig,cytokine signaling; JAK, Janus Kinases; STAT, Signal Transducers and Activators of Transcription; BAL,ral blood mononuclear cell; TST, tuberculin skin test; LJ, Løwenstein–Jensen; BCG, Bacillus Calmettetical density; Ct, cycle threshold; M-CSF, macrophage colony-stimulating factor; GMCSF, granulocyte; IL-1RA, IL-1 receptor antagonist; IL-2R, IL-2 receptor; MOI, multiplicity of infection; PTB, pulmonary

r Infectious Medicine (CIM), F59, Department of Medicine, Karolinska Institutet, Karolinska UniversitySweden.ki.se (S. Brighenti).

4.01.010blished by Elsevier Inc. Open access under CC BY-NC-ND license.

85A Th2 immune signature and elevated SOCS3 in active TB

Immune response;Cytokines

in the lung of patients with pulmonary TB. Instead, elevated levels of IL-4 and CCL4 wereassociated with high mycobacteria-specific IgG titres as well as SOCS3 (suppressors of cytokinesignaling) mRNA and progression of moderate-to-severe disease. Contrary, IL-4, CCL4 and SOCS3

remained low in patients with extrapulmonary pleural TB, while IFN-γ, CCL5 and SOCS1 wereup-regulated. Both SOCS molecules were induced in human macrophages infected withMycobacterium tuberculosis in vitro. The Th2 immune response signature found in patientswith progressive pulmonary TB could result from inappropriate cytokine/chemokine responsesand excessive SOCS3 expression thatmay represent potential targets for clinical TBmanagement.© 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CCBY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

1. Introduction

Tuberculosis (TB) is one of the most wide-spread humanbacterial infections. The lethal synergy between TB and HIVinfections is also a major challenge to public health and hencethere is a need to understand the cellular and molecularmechanisms responsible for the progression of TB in bothHIV-negative and HIV-positive individuals [1]. Successfulcontrol of Mycobacterium tuberculosis (M. tuberculosis) aswell as HIV infection is dependent on cell mediated immunitythat is orchestrated by multiple cytokines and chemokinesinvolved in the activation and recruitment of cells to the siteof infection [2,3]. Pro-inflammatory cytokines (IL-12, TNF-α)as well as Th1 (IL-2, IFN-γ) and Th17 (IL-17, IL-22) effectorcytokines specifically promote classical activation of macro-phages (M1) and activation of CD8+ cytolytic T cells (CTLs) orNK/NKT cells that trigger target cell and bacterial killing [3].Here, HIV infection may impair cellular immunity at the site ofM. tuberculosis infection by interfering with the recruitmentand function of macrophages and CD4+ T cells [4].

We have previously reported an impairment of Th1/Th17and CD8+ CTL responses in human granulomatous TB lesions inboth the lymph nodes [5] and lung [6] that may be theconsequence of an induction of Th2 and regulatory T cell(Treg) responses [5] Consistent with these findings, growingevidence suggests that a Th1/Th2 balance is crucial to controlthe progression of active TB disease [7,8]. IL-12, IFN-γ andTNF-α contribute to the induction of Th1-mediated protectionin TB, while increased IL-4 levels promote the developmentof a Th2 response that efficiently antagonizes protectivecytokines and results in loss of TB control [9]. Th2 cytokineshave been shown to induce alternative macrophage activation(M2) that involves a less bactericidal state of the macrophage[10]. Th2 cytokines can also inhibit autophagy, which is aphysiological process known to enhance intracellular degra-dation of mycobacteria [11]. On the contrary, Th2 responsespromote antibody-mediated immunity that may fail to conferresistance in intracellular M. tuberculosis infection [12]. TheTh1/Th2 balance is likely affected by different chemokines[13] produced by various cell types. Inflammatory chemokinessuch as CCL5, CXCL9 and CXCL10, selectively attract andrecruit Th1 cells from blood to sites of infection while otherchemokines such as CCL3 and CCL4 may promote Th2immunity [13,14]. AlthoughM. tuberculosis is a potent inducerof several inflammatory chemokines [15], little is known aboutthe local chemokine profile in the chronic phase of human TB

infection. The Th1/Th2 balance could also be controlled by afamily of regulatory proteins called suppressors of cytokinesignaling (SOCS) [16]. SOCS are molecules induced bycytokines or other stimuli and function as negative feedbackinhibitors by binding either to cytokine receptors or toassociated Janus Kinases (JAK), to inhibit the activation ofSignal Transducers and Activators of Transcription (STAT) [16].SOCS1 and SOCS3 are the most studied members that inhibitSTAT1 and STAT3 signaling, respectively [16]. Accumulatingdata support a central role of SOCS proteins in the regulationof immune polarization, which may be highly relevant for theoutcome of different infectious diseases [16].

In this study, we aimed to discover adverse immuneresponse signatures relevant to the progression and severityof active TB disease in HIV-negative and HIV-positive patients.We analyzed immune mediators locally in the lung or pleura incomparison with peripheral blood, in patients with pulmonaryTB or pleural TB. While the lung is the most common site ofM.tuberculosis infection, extrapulmonary TB including pleuralTB without concomitant pulmonary infection, often repre-sents a milder clinical form of disease [17]. Low levels ofIFN-γ, IL-17 and CCL5 along with enhanced levels of IL-4, CCL4and regulatory SOCS1 and SOCS3 proteins were observed in thelungs of patients with active pulmonary TB compared to thecontrols. The Th2-like immune signature in combination withenhanced SOCS3 expression was more prominent in severeforms of TB disease and was also associated with elevatedantibody responses in patients with pulmonary TB. Futurediagnostic and therapeutic approaches of clinical TB maytherefore consider quantifying and targeting Th2 and SOCS3regulated immune response pathways.

2. Material and methods

2.1. Study subjects and clinical diagnosis

The study subjects were recruited at the Chest Unit,Department of Internal Medicine, Black Lion University Hospi-tal, Addis Ababa, Ethiopia, after providing signed informedconsent. The study was approved by the National EthicalCommittees in both Ethiopia and Sweden. Patients and controlswere enrolled according to the flow chart illustrated in Fig. 1.Inclusion criteria: HIV-negative and HIV-positive sputumsmear-negative individuals N18 years old with clinical symp-toms of suspected TB. Exclusion criteria: patients with miliaryTB, a history of previous TB, or N1 week of antimicrobial

86 S. Ashenafi et al.

chemotherapy, subjects who used anti-retroviral drugs or didnot consent to HIV screening. The initial clinical diagnosis of TBwas based on: 1) Typical clinical symptoms of more than4 weeks duration including persistent cough, general illness,fever, sweating, and weight loss; and 2) chest X-ray revealingeither pulmonary infiltrates/lesions or pleural effusions. Adiagnosis of pulmonary TB was later confirmed by a positiveM. tuberculosis culture of bronchoalveolar lavage (BAL) fluidsamples, while extrapulmonary pleural TB was confirmed bycyto-histopathology of pleural fluid and biopsy samples. Thus,BAL and pleura fluid samples were obtained as part of theroutine diagnosis. Patients with active TB (n = 58) receivedstandard anti-TB chemotherapy for 6 months including rifam-picin, isoniazid, pyrazinamide and ethambutol. The response totreatment was monitored at 2, 5, and 8 months and clinicalimprovement and radiologic resolution of pulmonary lesionswere used as retrospective diagnostic criteria for culture-negative pulmonary TB patients with clinical TB (n = 11).

HIV-negative individuals with a minor hemoptysis (n = 10)were used as controls for the pulmonary TB cases (n = 35).These subjects were initially screened for TB, but were finallydiagnosed as healthy individuals with a single episode ofhemoptysis caused by a minor bronchial wall irritation. Thehemoptysis did not recur and no signs of inflammatoryconditions, infection or cancer were found. HIV-negativepatients with pleural diseases other than TB (n = 5) were usedas controls for the pleural TB cases (n = 23) and includedmalignancies (n = 3), congestive heart failure (n = 1) andsystemic lupus erythematosus (n = 1). All the controls had anegative tuberculin skin test (TST).

2.2. Chest X-ray grading

Chest X-ray was used to grade disease severity amongpulmonary TB patients at the time of diagnosis into mild(grade 1), moderately severe (grade 2) or severely advanced

Screening sputum-negative patieusing clinical symptoms, chestX-ray, M. tuberculo

Lung diseases (n=56)

Pulmonary TB (n=35)TST-pos: 30/35HIV-pos: 13/35 - Malignancies (n=4)

- Pneumonia (n=3)- Bronchiectasis (n=3)TST-pos: 7/10HIV-pos: 2/10

- Haemoptysis (n=11)TST-pos: 0/11HIV-pos: 1/11

Pulmonary controls (n=10)

Other diseases (n=21)

TST-pos: 0/10HIV-pos: 0/10

Figure 1 Flow chart of participants. Sputum-negative patients wM. tuberculosis culture or cyto-histopathology of clinical specimmalignancies, pneumonia and bronchiectasis (abnormal chest X-raysingle episode of hemoptysis were finally diagnosed as healthy indivmalignancies, congestive heart failure (CHF) and systemic lupus eretiology, pleural biopsies were non-diagnostic and/or cytology waswere included as pulmonary or pleural controls. Patients and control

(grade 3) TB disease based on visual examination of thechest X-rays using the diagnostic standards and classificationof TB by the American Thoracic Society, National Tubercu-losis Association of the USA. Accordingly, mild TB disease(n = 19 pulmonary TB patients) was defined as non-confluentunilateral or bilateral infiltrates without noticeable cavita-tions. The extent did not exceed the volume of the lung onone side above the second costochondral junction or the 4thor 5th vertebrae. Moderate-to-severe TB disease (n = 16pulmonary TB patients) was defined as disseminated lesionsof slight to moderate density that extended throughout thetotal volume of one lung or equivalent volume in both lungsor dense and confluent lesion(s) that were limited to onethird of the volume of one lung lobe. Moderately severe TBinvolved cavitations with a diameter b 4 cm (n = 13 pulmo-nary TB patients), while severely advanced TB involvedcavitations with a diameter N 4 cm (n = 3 pulmonary TBpatients).

2.3. Clinical samples and HIV screening

Blood, BAL or pleural fluid samples were collected from thestudy subjects to be used for diagnostic and researchpurposes. Blood samples (20 ml) obtained were used forperipheral CD4/CD8 T cell counts (FACSCount; BD, FranklinLakes, NJ), to determine HIV status, HIV viral load (Abbottreal-time HIV-1 PCR; Abbott Molecular, Des Plaines, IL) andfor immunological analysis. HIV screening was performed atthe Black Lion University Hospital using a national algorithmin three steps: 1) HIV (1 + 2) Antibody Kit (KHB ShanghaiKehua Bio-engineering; Shanghai, China); 2) HIV 1/2 STAT-PAK Kit (CHEMBIO Diagnostic Systems; Medford, NY); and 3)Uni-Gold HIV test (Trinity Biotech; Co Wicklow, Ireland).Pre-test counseling was offered to all subjects whereaspost-test counseling was provided to HIV-positive subjectsby a trained counselor. Antiretroviral therapy was offered

nts for TB (n=90) sis culture or cyto-histopathology.

Pleural diseases (n=33)

One patient did not consent.

Pleural TB (n=23)TST-pos: 20/23HIV-pos: 7/23 -Malignancies(n=3)

- CHF (n=1)- SLE (n=1) TST-pos: 0/5HIV-pos: 0/5

- Unclear etiology (n=5)TST-pos: 4/5HIV-pos: 2/5

Other diseases (n=10)

Pleural controls (n=5)TST-pos: 0/5HIV-pos: 0/5

ere screened using clinical symptoms and chest X-ray as well asen. Individuals with other lung diseases than TB included) or a minor hemoptysis (normal chest X-ray). Subjects with aiduals. Individuals with other pleural diseases than TB includedythematosus (SLE). In patients with pleural diseases of unclearnot consistent with TB. Only TST- and HIV-negative individualss that were selected for further analysis are shown in the boxes.

87A Th2 immune signature and elevated SOCS3 in active TB

to all HIV-positive TB patients with a CD4 T count ofb350 cells/ml. Treatment for HIV was provided for free bythe government of Ethiopia (the Federal Ministry of Health).

PBMCs and plasma were isolated from whole blood usingcell preparation tubes (CPT; BD). Plasma samples werealiquoted and stored at −80 °C for cytokine/chemokineprotein analysis, while PBMCs (5 × 106) were resuspended in300 μl of RNAlater (Life Technologies, Invitrogen, Carlsbad,CA) and stored at −80 °C for mRNA analysis. Bronchoscopywas performed not only on pulmonary TB patients withabnormal chest X-ray findings (n = 35) but also on individualswith a normal chest X-ray who had a minor bleeding causedby a minor bronchial wall irritation (n = 10). In patients withpulmonary TB, a flexible fiberoptic bronchoscope (BF typeP20D bronchoscope, Olympus, Tokyo, Japan) was used tocollect BAL fluid from the affected bronchi based on theradiological findings. In the pulmonary control group, BALfluid was collected from the right middle lobe bronchususing flexible bronchoscopy. A total of 120 ml normal salinewere sequentially instilled and immediately retrieved. Inpatients with extrapulmonary pleural TB, thoracentesis wasused to collect 50 ml fluid from the pleural cavity.An aliquot of the BAL and pleural fluid was used forM. tuberculosis culture or cytology, respectively. Theremaining BAL and pleura fluid were centrifuged at1400 rpm for 25 min. Supernatants were stored at −80 °Cfor cytokine/chemokine protein analysis, while BAL andpleura cells (5 × 106) were resuspended in RNAlater andstored at −80 °C for mRNA analysis.

2.4. M. tuberculosis culture andcyto-histopathology

M. tuberculosis culture of BAL fluid was performed at theArmauer Hansen Research Institute, Addis Ababa, Ethiopia,using the established Løwenstein–Jensen (LJ) methodology.After NaOH (4%) treatment, washing and centrifugation of BALfluids, cell pellets were resuspended in 1.5 ml of 7H9 media(Sigma-Aldrich, Fluka, St. Louis, MO) from which an aliquot(20–30 μl) was inoculated in LJ-media and incubated at 37 °Cfor 8 weeks with weekly read-out of bacterial growth. Cytologyof cell samples from pleural fluid and histology of paraffin-embedded pleural tissue biopsies involved hematoxylin andeosin staining performed at the Department of Pathology, BlackLion University Hospital, Addis Ababa, Ethiopia. M. tuberculo-sis-positive specimens revealed a granulomatous reaction withmultinucleated giant cells, epithelioid cells, numerous lym-phocytes and necrotic material consistent with TB.

2.5. Multiplex luminex assay

Cytokine and chemokine responses in fluid and plasmasamples obtained from the study subjects were assessedusing a human standard cytokine 25-plex panel (LHC0009:IL-1β, IFN-α, TNF-α, IL-6, IL-12, IL-7, IL-2, IFN-γ, IL-17, IL-4,IL-5, IL-13, IL-15, IL-10, IL-1RA, IL-2R, IL-8, GMCSF, Eotaxin,CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10), a human ultra-sensitive cytokine 10-plex antibody bead kit (LHC6004:IL-1β, TNF-α, IL-6, IL-2, IFN-γ, IL-4, IL-5, IL-10, IL-8,GMCSF) and a human chemokine 10-plex antibody bead kit(LHC6001: Eotaxin, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8,

CXCL1, CXCL9, CXCL10) (Invitrogen). The multiplex luminexassay was performed according to the manufacturer'sinstructions using the Bio-Plex Luminex detection system.Data were analyzed by the Bio-Plex manager 4.0 curvefitting software using the five-parametric algorithm. Cyto-kine concentrations in BAL and plasma ranged from 0.2 to1300 pg/mg of total protein while chemokine concentrationsin BAL and plasma ranged from 1 to 30,000 pg/mg of totalprotein. Cytokine and chemokine concentrations in pleuralfluid samples ranged from 2 to 130 pg/ml and from 15 to1000 pg/ml, respectively.

2.6. BCG-specific IgG ELISA

The release of mycobacteria-specific IgG antibodies in fluidand plasma samples obtained from the study subjects wasquantified using a Bacillus Calmette Guerin (BCG)-specificELISA. The BCG ELISA detects IgG antibodies that are specificfor mycobacterial epitopes of the BCG vaccine that is derivedfrom an attenuated Mycobacterium bovis strain. Briefly, aBCG vaccine antigen (kindly provided by Japan BCG Labora-tories, Tokyo, Japan) was used to coat Maxisorb plates (Nunc,Roskilde, Denmark) overnight at 4 °C. The plates werewashedwith PBS-0.05% Tween-20 (Sigma, Sweden) and blocked withPBS-10% FCS. Plasma (diluted 1:500), BAL (diluted withrespect to total protein concentration in each sample) orpleura (diluted 1:400) fluid samples from patients and controlswere added (100 μl/well) and incubated for 2 h at 37 °Cbefore washing and addition of a rabbit anti-human IgGhorseradish peroxidase conjugate (Jackson ImmunoresearchLaboratories, West grove, PA) for 2 h at room temperature.Dilution buffer (PBS-10% FCS) was used as a negative control.The enzyme–substrate reaction was developed after 20 minusing a O-phenylenediamine (OPD) (Sigma) substrate solution.BCG-specific IgG titers were expressed as optical density (OD)measured at 492 nm and multiplied by the dilution factor.

2.7. Protein determination with BCA

Total protein content in BAL (average 0.3 mg/ml; range0.04–2.2 mg/ml), pleura fluid (average 41.4 mg/ml; range11.6–57.9 mg/ml) and plasma (average 71.6 mg/ml; range11.9–106 mg/ml) samples obtained from the study subjectswas determined using the bicinchoninic acid (BCA) assay(Pierce, Thermo Scientific, Rockford, USA) according to themanufacturer's instructions. To adjust for dilution of the BALfluid, the total protein content in BAL samples was used tonormalize data obtained with the multiplex luminex assay[18]. Normalized data of the BAL fluid samples are expressedas pg/mg of total protein.

2.8. mRNA extraction and real-time PCR

Cell samples were stored in RNAlater until RNA wasextracted using the Ambion RiboPure extraction kit (LifeTechnologies) according to the manufacturer's instructions.RNA was reverse transcribed to cDNA using superscriptreverse transcriptase (Invitrogen). Amplification of targetgenes using primers (Sigma Genosys, St. Louis, MO) forβ-actin (Forward: 5′-TTGCCGACAGGATGCAGAA-3′, Reverse:5′-GCCGATCCACACGGAGTACT-3′), SOCS1 (Forward: 5′-TTTT

88 S. Ashenafi et al.

TCGCCCTTAGCGTGA-3′, Reverse: 5′-AGCAGCTCGAAGAGGCAGTC-3′) and SOCS3 (Forward: 5′-TGAGCGCGGCTACAGCTT-3′, Reverse: 5′-TCCTTAATGTCACGCACGATTT-3′) wasperformed using SYBR Green JumpStart (Sigma, St. Louis,MO) and the ABI PRISM 7700 Sequence Detection Systemincluding the 7500 software v2.0.6 (Applied Biosystems,Foster City, CA). The specificity of the PCR reactions wasverified by post-amplification melting-curve analysis. The Ct(cycle threshold) values for SOCS1 and SOCS3 mRNA werenormalized to β-actin to provide the delta Ct values. Therelative mRNA expression was determined using the Livakmethod (the 2−ΔΔCT method for real-time PCR). The dataare presented as fold change of mRNA in the pulmonary orpleural TB groups compared to controls.

2.9. Human primary cells

PBMCs were isolated from ten healthy blood donors(Karolinska University Hospital Blood Center, Stockholm,Sweden) using density gradient centrifugation (Lymphoprep,Axis-Shield, Norway). Monocyte Enrichment solution fromRosetteSep (Stemcell Technologies, Grenoble, France) wasused for purification of monocytes from six of the healthydonors. Isolated monocytes were differentiated to macro-phages in DMEM medium (Invitrogen) supplemented withrecombinant human M-CSF (50 ng/ml) (Stemcell Technolo-gies, Reston, VA) for 6 days.

2.10. Bacterial cultures and infection of human cells

M. tuberculosis strain H37Rv (American Type CultureCollection) was cultured in Middlebrook 7H9 broth supple-mented with 10% OADC (BD), 0.05% Tween-80 (Sigma) and0.5% glycerol (Sigma-Aldrich) for 2–3 weeks at 37 °C. Thebacterial suspension was washed twice in PBS-0.05%Tween-80 before the pellet was resuspended in plain DMEMmedium. To obtain a single cell suspension, the bacteriawere repeatedly vortexed and sonicated using an ultrasoundsonication bath. The bacterial concentration was deter-mined from the OD of the culture obtained at 600 nm, as afunction of colony forming units per milliliter. PBMCs werecultured in 24-well plates (2 × 106 cells/well) in thepresence (MOI:5 and MOI:10) or absence of bacteria whilehuman primary macrophages were cultured in 24-well plates(2 × 106 cells/well) and infected with the bacteria (MOI:5and MOI:10) for 1–2 h before the cells were washed toremove extracellular bacteria. Macrophage as well as PBMCcultures were incubated in RPMI-10% FCS (GIBCO, Invitrogen)at 37 °C for 4 h and 24 h before harvest of the cells formRNA analysis.

2.11. Statistical analysis

The multiplex luminex protein data are presented as log10values in scatter dot plot graphs showing the median foreach individual while the mRNA data are presented as log10values in box and whisker plots showing the median andrange. Non-parametric analyses were used to calculatethe indicated p-values for non-normally distributed data(D'Agostino & Pearson omnibus normality test) and includedthe Kruskal–Wallis test and Dunn's post-test or the Mann

Whitney test. Spearman's correlation test was used for thecorrelation analysis. A value of r = 1, indicates a perfectpositive correlation whereas r = −1 indicates a perfectnegative correlation. Statistical analyses were performedin GraphPad Prism-5.

3. Results

3.1. Elevated pro-inflammatory and Th2 cytokineresponses in the absence of a Th1 response locallyin the lung of patients with active pulmonary TB

The clinical demographics of pulmonary and extrapulmonaryTB patients as well as controls are summarized in Table 1. TBwas confirmed using clinical and pathologic–anatomic diag-noses and/orM. tuberculosis culture. Approximately one thirdof the patients were TB/HIV co-infected and showed a lowperipheral CD4+ T cell count (Table 1). Multiplex proteinanalysis revealed that the pro-inflammatory cytokines IL-1β,IL-6 and TNF-α were significantly higher (p b 0.05–0.001) inBAL fluid samples from patients with active pulmonary TBcompared to the controls (Fig. 2A), while only IL-6 wassignificantly elevated (p b 0.01–0.001) in plasma samplesfrom the TB patients (Fig. 2A). Despite the elevation of thesepro-inflammatory markers in the lung of patients with activeTB, we could not detect elevated levels of IL-12 or IL-2 (datanot shown). Similarly, no increase in IFN-γ or IL-17 wasobserved in the lungs of patients with active TB (Fig. 2B).However, the multiplex analysis revealed higher plasma levelsof IFN-γ in TB/HIV co-infected patients compared to controlsbut lower plasma levels of IL-17 in TB/HIV co-infected patientsas compared to TB infected patients (Fig. 2B). In contrast, IL-4was significantly elevated in both BAL (p b 0.01) and plasma(p b 0.05) samples from TB patients as compared to thecontrols (Fig. 2B). Altogether, we found an 8–10 fold increasein IL-1β and IL-6 and a 2–3 fold increase in TNF-α and IL-4 inBAL fluid from patients with pulmonary TB, which denotepotent pro-inflammation and a predominant Th2 or a mixedTh1/Th2 response in active TB.

3.2. Local chemokine responses in the lung ofpatients with active pulmonary TB demonstrateselevated levels of CXCL9, CXCL10 and CCL4 but lackof CCL5

Consistent with the observed pro-inflammatory signature(Fig. 2A), we also detected increased levels of the CXCinflammatory chemokine CXCL8 (IL-8) in all TB patients (datanot shown). Despite the lack of IFN-γ up-regulation in the lungof patients with active TB, the IFNγ-inducible inflammatorychemokines, CXCL9 (MIG) and CXCL10 (IP-10), were increased(p b 0.05–0.001) in both BAL (15–20 fold increase) and plasma(6–8 fold increase) from pulmonary TB patients (Fig. 3).However, the amounts of both CXCL9 and CXCL10 weresignificantly higher (p b 0.05–0.01) in plasma samples fromTB/HIV co-infected patients (Fig. 3), which is in line with theincreased plasma levels of IFN-γ (3-fold increase) observed inthese patients (Fig. 2B). Among the CC chemokines, only CCL4(MIP-1β) was significantly higher (p b 0.01) in BAL samplesfrom patients with active TB compared to controls (Fig. 3).

Table 1 Clinical demographics of included study subjects.

Clinical features Pulmonary TB patients and controls Pleural TB patients and controls

HIV-neg HIV-pos Controls HIV-neg HIV-pos Controls

Numbers 22 13 10 16 7 5Age in years(median with range)

24 (20–51) 38 (18–54) 26 (18–46) 25 (18–72) 35 (28–56) 40 (25–60)

Male/female gender 12/10 7/6 5/5 9/7 2/5 2/3Clinical TB diagnosis(no. (%)) a

22 (100%) 13 (100%) 0 (0%) 16 (100%) 7 (100%) 0 (0%)

Abnormal chest X-ray(no. (%)) b

22 (100%) 13 (100%) 0 (0%) 16 (100%) 7 (100%) 5 (100%)

Positive Mtb culture ofBAL (no. (%)) c

16 (73%) 8 (62%) 0 (0%) nd nd nd

Positive Mtb cyto-histopathology(no. (%)) d

nd nd nd 16 (100%) 7 (100%) 0 (0%)

CD4 T cell counts e

(mean with range)685(183–1333)

130(10–279)

693(593–806)

418(166–906)

196(49–457)

659(298–1123)

CD8 T cell counts e

(mean with range)576(102–1902)

669(57–1773)

599(234–1288)

302(84–845)

642(124–2000)

940(433–1712)

HIV viral load f

(median with range)nd 40,000

(2.6 × 103–1.2 × 106)

nd nd 18,000(0.3 × 103–0.4 × 106)

nd

Mtb, M. tuberculosis; nd, not determined.a Diagnosis of clinical TB was based on clinical symptoms of more than 4 weeks duration (persistent cough, general illness, fever, sweating,

and weight loss) and response to standard anti-TB treatment (clinical recovery and resolution of lesions evident on the chest X-ray).b Abnormal radiologic chest X-ray findings included pulmonary infiltrates/dense lesions or pleural effusions.c Bronchoscopy was performed not only on pulmonary TB patients with an abnormal chest X-ray, but also on individuals with a normal

chest X-ray who had a minor hemoptysis caused by a minor bronchial wall irritation.d Pleural controls with pleural diseases other than TB all had abnormal chest X-ray finings and included malignancies (n = 3), congestive

heart failure (n = 1) and systemic lupus erythematosus (n = 1).e T cell counts in peripheral blood were determined as cells/mm3.f HIV viral load in plasma from TB/HIV co-infected patients was determined as number of viral copies/ml.

89A Th2 immune signature and elevated SOCS3 in active TB

Although the T cell chemoattractant CCL5 (RANTES) could bedetected in all patients with active TB, the levels were notsignificantly different compared to the control group (Fig. 3).Hence, CCL4 was found to be the only chemokine selectivelyup-regulated (5-fold increase) in the local environment of theM. tuberculosis-infected lung.

3.3. Elevated levels of IL-4 and CCL4 are associatedwith more severe forms of clinical TB disease

In order to analyze whether specific cytokine/chemokineresponses could be linked to severity of TB, pulmonary TBpatients were grouped into mild or moderate-to-severedisease based on their chest X-ray results. A greater proportionof patients with moderate-to-severe TB disease were culturepositive for M. tuberculosis (81%) compared to patients withmild TB disease (58%). Interestingly, we observed that IL-1β(data not shown), IL-4 and CCL4 were significantly increased(p b 0.05–0.01) at the site of infection in HIV-negativepatients with moderate-to-severe compared to mild TBdisease (Fig. 4A). Although we detected significantly elevatedlevels of both IL-4 and CCL4 in TB/HIV co-infected patients(Fig. 2B, 3), there was no difference in IL-4 or CCL4 levels inmoderate-to-severe compared to mild TB disease in thesepatients (Fig. 4A). This may be due to limitations in the

radiological scoring system in TB/HIV co-infected patients.Next, we assessed clinical samples from patients with extra-pulmonary pleural TB (Table 1), which is generally consideredto be a less severe disease compared to pulmonary TB [17].Interestingly, multiplex analysis of pleural fluid samplesrevealed that both IL-4 and CCL4 remained unaltered inpatients with extrapulmonary pleural TB as compared to thecontrols, while IFN-γ and CCL5 were significantly increased(p b 0.05–0.001) in both HIV-negative (15-fold and 3-foldincrease, respectively) and HIV-positive (10-fold and 2-foldincrease, respectively) patients with a local TB pleuritis(Fig. 4B). These findings suggest that the immune response isshifted towards a Th2 profile in TB, particularly in patientswith more advanced clinical TB disease.

3.4. A predominant Th2 response in patients withactive pulmonary TB correlates to aM. tuberculosis-specific antibody response

To study whether a shift towards a Th2 profile in patientswith active pulmonary TB concurred with a humoral immuneresponse, we assessed the presence of mycobacteria-specific IgG antibodies in TB patients and controls. Inter-estingly, significantly elevated levels of BCG-specific IgGantibodies were detected in BAL (p b 0.05–0.01) and

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Figure 2 Elevated pro-inflammatory and Th2 cytokine responses in patients with active pulmonary TB (PTB). Assessment of (A)pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) and (B) effector cytokines (IFN-γ, IL-17 and IL-4) in BAL fluid and plasma samplesfrom HIV-negative and HIV-positive patients with active PTB (circles) and controls (triangles). Graphs are presented as scatterdot-plots and the solid bars indicate the median values for each group. The statistical significance of differences in cytokineexpression in vivo between the different groups was determined using the Kruskal–Wallis test, *p b 0.05, **p b 0.01 and ***p b 0.001.One out of three similar experiments is shown.

90 S. Ashenafi et al.

plasma (p b 0.01–0.001) samples from both HIV-negativeand HIV-positive TB patients (Fig. 5A). Accordingly, therewas a significant correlation between BCG-specific IgGlevels and IL-4 in BAL fluid (r = 0.55; p = 0.008) and plasma(r = 0.42; p = 0.05) samples from HIV-negative patientswith active pulmonary TB (Fig. 5A). There was also asignificant correlation between BCG-specific IgG levels andCCL4 in BAL fluid (r = 0.47; p = 0.03), but not in plasmafrom HIV-negative TB patients (Fig. 5B), which mayillustrate preferential recruitment of IgG secreting cells tothe site of M. tuberculosis infection in the lung. We alsofound that BCG-specific IgG levels determined in plasmawere significantly higher (p = 0.0003) in patients withpulmonary TB compared to patients with extrapulmonarypleural TB (Fig. 5C), which is consistent with higher levels ofIL-4 and CCL4 in pulmonary TB. Pleural TB patients did notpresent elevated BCG-specific IgG titres compared to thecontrols (data not shown). These data support a bias

towards a Th2-driven antibody response, particularly inHIV-negative patients with pulmonary TB.

3.5. A Th2 response in patientswith active pulmonaryTB is associated with an elevated expression of SOCS3

To explore whether expression of SOCS was associated withimmune polarization towards an IL-4/CCL4 response signaturein patients with active TB, we analyzed the mRNA accumula-tion of SOCS1 and SOCS3 in BAL and pleural cells as well asPBMCs samples from patients with active TB and controlindividuals. Strikingly, both SOCS1 and SOCS3 mRNA levelswere significantly up-regulated (p b 0.05–0.01) in BAL cellsfrom pulmonary TB patients (Fig. 6A); while no increase inSOCS was evident in the PBMCs (data not shown). However,SOCS1 expression was not associated with severity ofpulmonary TB disease, whereas SOCS3 expression was

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Figure 3 Induced chemokine expression of CXCL9, CXCL10 and CCL4 but not CCL5 in patients with active pulmonary TB (PTB).Assessment of chemokine (CXCL9, CXCL10, CCL4 and CCL5) responses in BAL fluid and plasma samples from HIV-negative andHIV-positive patients with active PTB (circles) and controls (triangles). Graphs are presented as scatter dot-plots and the solid barsindicate the median values for each group. The statistical significance of differences in chemokine expression in vivo between thedifferent groups was determined using the Kruskal–Wallis test, *p b 0.05, **p b 0.01 and ***p b 0.001. One out of three similarexperiments is shown.

91A Th2 immune signature and elevated SOCS3 in active TB

significantly enhanced (p b 0.05–0.01) in HIV-negative pa-tients with moderate-to-severe compared to mild TB disease(Fig. 6B). Importantly, there was a significant correlationbetween SOCS3 mRNA expression in BAL cells and IL-4 as wellas CCL4 in BAL fluid samples (r = 0.37; p = 0.03 and r = 0.48;p = 0.004, respectively) (Fig. 6), but not between SOCS1 andIL-4 or CCL4 (data not shown). In line with these findings,pleural fluid cells from patients with extrapulmonary pleuralTB had increased SOCS1 but not SOCS3 mRNA levels (Fig. 6D),which also supports the conclusion that SOCS3 is a marker ofmore severe or advanced forms of clinical TB disease. Insummary, SOCS1 was up-regulated in both pulmonary TB(Fig. 6A) and pleural TB (Fig. 6D), while SOCS3 was onlyup-regulated in pulmonary TB (Fig. 6A and D) and also inmoderate-to-severe forms of pulmonary TB disease (Fig. 6B).These results confirm an association between IL-4, CCL4 andSOCS3 in patients with active pulmonary TB.

3.6. M. tuberculosis induces both SOCS1 and SOCS3expression in human PBMCs and macrophages

Since increased levels of SOCS mRNA were only evident incells from the site of M. tuberculosis infection, we isolatedPBMCs or monocytes from healthy individuals to investigateSOCS mRNA expression in response to mycobacteria in vitro.Exposure of PBMCs or infection of blood-derived macro-phages with virulent H37Rv revealed that SOCS1 and SOCS3were significantly induced (p b 0.01–0.001) in both PBMC(Fig. 7A) and macrophage (Fig. 7B) cultures. The induction ofboth SOCS molecules were 100–1000 fold higher in macro-phages compared to PBMCs (Fig. 7), which imply that SOCSare rapidly and strongly up-regulated primarily in macro-phages productively infected with M. tuberculosis.

4. Discussion

To get a proper reflection of M. tuberculosis-specific immuneresponses induced at local sites, human TB disease needs to be

explored using relevant clinical materials obtained from thesite of infection or from areas close to the infected sites[3,8,17,19,20]. In this study, we show for the first time thatthe local immune response in the lung of patients withpulmonary TB is associated with impaired Th1/Th17 cytokineresponses but elevated levels of IL-4, CCL4 and the regulatoryproteins SOCS1 and SOCS3. This Th2-like immune responsesignature correlates to enhanced mycobacteria-specific IgGresponses. Furthermore, IL-4, CCL4 and SOCS3 were elevatedin HIV-negative patients with moderate-to-severe pulmonaryTB as compared tomild disease, which support the notion of anaberrant immune response in the progression of clinical TB.Importantly, we observed an induction of a Th1 response in theabsence of Th2 response in less severe forms of extra-pulmonary pleural TB disease. Accordingly, pleural TB patientsalso lacked up-regulation of SOCS3 and mycobacteria-specificIgG. In summary, our results suggest that the local immuneresponse in theM. tuberculosis infected lung is dominated by aTh2 type inflammation in combination with SOCS3 that maycontribute to the failure of immune control in patients withprogressive clinical TB.

Despite strong pro-inflammation including high locallevels of IL-1β, IL-6 and TNF-α, there was a decreasedTh1/Th2 cytokine ratio in patients with active TB. Wedemonstrate that IL-6 and IL-4 were up-regulated both inperipheral blood and locally in the M. tuberculosis-infectedlung, where we also found a significant correlation (r = 0.40;p b 0.01) between these cytokines. Similar to IL-12, IL-6 isabundantly produced by APCs and may therefore participatein the early events of Th1/Th2 development. However, incontrast to IL-12, IL-6 has been described to polarize naiveCD4+ T cells into IL-4 producing Th2 effector cells [21,22]and IL-6 could also inhibit Th1 differentiation and interferewith IFN-γ signaling by specific induction of SOCS1 inactivated CD4+ T cells [22]. Similarly, IL-1β can also actdirectly on CD4+ T cells to promote the differentiation ofIL-4-producing Th2 cells upon infection with virulentM. tuberculosis [23]. Interestingly, while TNF-α clearly hasa protective role in TB, the presence of IL-4 may enhance

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Figure 4 Increased expression of IL-4 and CCL4 in patients with moderate-to-severe pulmonary TB (PTB) disease but not in patientswith extrapulmonary TB (EPTB). (A) Comparison of IL-4 and CCL4 expression in BAL fluid samples from HIV-negative and HIV-positivepatients with mild PTB (open circles) to patients with moderate-to-severe PTB (gray circles). Disease severity in HIV-negative PTBwith mild (n = 12) or mod–sev (n = 10) disease and in HIV-positive PTB patients with mild (n = 7) or mod–sev (n = 6) disease wasdetermined using chest X-ray. (B) Assessment of cytokine (IL-4 and IFN-γ) and chemokine (CCL4 and CCL5) responses in pleural fluidsamples from HIV-negative and HIV-positive EPTB patients with a local TB pleuritis (circles) and controls (triangles). Graphs arepresented as scatter dot-plots and the solid bars indicate the median values for each group. The statistical significance of differencesin cytokine/chemokine expression in vivo between the different groups was determined using the Kruskal–Wallis test. *p b 0.05,**p b 0.01 and ***p b 0.001. One out of three similar experiments is shown.

92 S. Ashenafi et al.

TNF-α mediated immunopathology in progressive disease[24,25].

Previous studies that have investigated the Th1/Th2balance in patients with active TB do not provide a consistentview on cytokine polarization in human TB [26–32]. Theobserved discrepancies may be due to differences in patients'ethnicity and clinical form of TB disease, analysis of cells orserum samples, ex vivo analysis or recall T cell responses invitro, the method of analysis used etc. Consistent with ourresults, increased levels of IL-4 have previously been found inthe blood [30,33,34] and BAL [31,35] of TB patients with

advanced compared tomild-to-moderate lung disease and IL-4levels were also associated with elevated antibody responsesin the serum [34]. Progression of clinical TB disease could alsoinvolve a mixed Th1/Th2 response that may impair bacteri-cidal function and promote pulmonary fibrosis [7,8,17,25,36].It was recently described that patients with multi-drugresistant TB (MDR-TB) had down-regulated expression ofIFN-γ and IL-2 in the lung, but instead up-regulated IL-4,IL-6, and TNF-α expression during progressive disease [37].Accordingly, certain MDR outbreak strains induce a weak IFN-γbut strong IL-4 response, which correlates to reduced CTL

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Figure 5 Enhanced BCG-specific IgG antibody response in patients with active pulmonary TB (PTB). (A) Assessment of BCG-specificIgG titres in BAL fluid and plasma samples from HIV-negative and HIV-positive patients with active PTB (circles) and controls(triangles). The statistical significance of differences in IgG titres in vivo between the different groups was determined using theKruskal–Wallis test, *p b 0.05, **p b 0.01 and ***p b 0.001. (B) Correlation analysis between BCG-specific IgG titres and IL-4 or CCL4protein levels in BAL fluid and plasma samples from HIV-negative PTB patients. Spearman's correlation test was used to determine thecorrelation coefficient rs and the indicated p-values (ns p N 0.05). (C) Comparison of BCG-specific IgG titres in plasma samples frompatients with PTB to EPTB patients with a local TB pleuritis (circles). The statistical significance of differences in IgG titres betweenPTB and EPTB patients was determined using the Mann–Whitney test, ***p b 0.0003. Graphs are presented as scatter dot-plots andthe solid bars indicate the median values for each group. One out of two similar experiments is shown.

93A Th2 immune signature and elevated SOCS3 in active TB

activity of these MDR-TB patients [38]. Consequently, thevirulence of different M. tuberculosis strains may affectcytokine polarization and disease outcome [39]. A patholog-ical role of IL-4 is also evident in TB-susceptible contacts,where a rise in serum IL-4 concurrent with low IFN-γ isassociated with development of active TB disease [40].Likewise, pleural TB is characterized by local production ofIFN-γ and a predominant Th1 response, while miliary orsystemic TB is characterized by local IL-4 production in thelung and a shift towards a Th2 response [8]. WhetherTh2-driven inflammation is a cause or effect of TB disease isnot known. Individuals with a higher basal production of Th2cytokines may be predisposed to develop active TB disease,which has been observed in patients with helminth infections[41]. Accordingly, Th2 responses that are experimentallyintroduced in mice before infection with M. tuberculosis willresult in enhanced disease severity and death, which supportsthe notion that the Th2 response is a cause rather than aneffect of TB [42,43]. However, enhanced Th2 polarizationmayalso be the consequence of progressive TB disease, whenimpairment of Th1 responses results in a failure to antagonizeTh2-induced immunity.

The increased antibody response we observed in TB/HIVco-infected patients did not seem to correlate with IL-4production, which may suggest that humoral immunity in

this group of patients, is regulated by factors other thanIL-4. High serum levels of total [44] and M. tuberculosis-specific [45] IgG antibodies have previously been shown inpatients with TB/HIV co-infection. Interestingly, a splicevariant and competitive antagonist of IL-4, IL-4delta2, hasbeen described to be expressed at elevated levels in BALfluid but not in peripheral blood from TB/HIV co-infectedpatients [46]. These results imply that there may be aselective increase of IL-4 in the periphery of TB/HIV co-infected patients [46]. Although our multiplex analysis couldnot separate IL-4 from IL-4delta2 expression, it is temptingto speculate that a higher IL-4/IL-4delta2 ratio in the lung ofTB single-infected compared to TB/HIV co-infected patientscould undermine the efficacy of a local Th1 response andpromote antibody responses, particularly in the lung of HIV-negative patients with pulmonary TB. The IL-4/IL-4delta2ratio has been shown to be decreased in individuals withlatent TB [47], thus this cytokine ratio may be useful as amarker for disease activity.

Analysis of the chemokine response in our study cohortrevealed that the inflammatory chemokines CXCL8, CXCL9and CXCL10 (IP-10) were significantly higher in patients withactive pulmonary TB. Importantly, even very low levels ofIFN-γ can promote high levels of CXCL10 [48], which couldexplain high levels of CXCL9 and CXCL10 in the absence of

94 S. Ashenafi et al.

IFN-γ up-regulation. Alternatively, an early transient rise ofIFN-γ in the lung of M. tuberculosis-infected patients may besufficient to induce CXCR3 ligands, but insufficient to establishprotective Th1 immunity. Furthermore, it has been shown thatCXCR3 ligands can be induced by other cytokines than IFN-γ,including IL-27, TNF-α [49], IL-12 [48] and IFNα/β [50]. Here,increased levels of IL-27 correlated with enhanced CXCL10

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Figure 7 Induction of SOCS1 and SOCS3 mRNA expression in PBMCs exposed to M. tuberculosis as well as in M. tuberculosis-infectedhuman primary macrophages. (A) PBMCs were isolated from the blood of healthy donors (n = 10) and exposed to M. tuberculosisH37Rv for 4 h or 24 h before assessment of SOCS mRNA. (B) In parallel, primary macrophages were differentiated from the samedonors (n = 6) and infected with H37Rv for 1–2 h before extracellular bacteria were washed away and SOCS mRNA was assessed after4 h and 24 h. mRNA data are presented in box and whisker plots showing the median and range from n = 6–10 donors in each group.The statistical significance of differences in SOCS mRNA expression in vitro between the different groups was determined using theKruskal–Wallis test, ns p N 0.05, **p b 0.01 and ***p b 0.001.

95A Th2 immune signature and elevated SOCS3 in active TB

and migration of effector CD8+ T cells [53] as well asM. tuberculosis-infected macrophages [54] to the lung. Thus,the lack of CCL5 as well as CCL2 and CCL3, which are keychemoattractants of monocytes, neutrophils and T cells, maypartly explain the impaired Th1 immune response observed inpatients with active pulmonary TB [55]. Recent data alsosuggest that IL-17-dependent induction of CXCL13 in theM. tuberculosis-infected lung may enhance Th1 effectorfunctions andmacrophage activation in TB inflammatory lesions[56]. CXCL13 promotes local recruitment of CXCR5+ T cells that

Figure 6 Elevated mRNA expression of SOCS1 and SOCS3 in patientlevels in BAL cell samples from HIV-negative and HIV-positive patieSOCS3 mRNA expression in BAL cell samples from HIV-negative and Hmoderate-to-severe PTB (gray bars). Disease severity in HIV-negatHIV-positive PTB patients with mild (n = 7) or mod–sev (n = 6) disebetween SOCS3 mRNA and IL-4 or CCL4 protein levels in BAL cell andSpearman's correlation test was used to determine the correlation coand SOCS3 mRNA levels in cell samples from pleural fluid obtained fpleuritis and controls. mRNA data are presented in box and whisker psignificance of differences in SOCS mRNA expression in vivo betweetest, ns p N 0.05, *p b 0.05, **p b 0.01 and ***p b 0.001. One out of t

express high levels of Th1 cytokines including IFN-γ, TNF-α andIL-2 in the M. tuberculosis-infected lung [57], which underlinethe importance of continued studies of both chemokines andtheir receptor expression in the local tissue environment.

Although we did not find a major impact of HIV infection onthe local immune response in the lung of patients with activeTB, plasma levels of CXCL9 and CXCL10 were particularly highin TB/HIV co-infected patients. Accordingly, we found anegative correlation between CXCL9 and CXCL10 levelsand peripheral CD4 T cell counts (r = −0.69; p b 0.0001 and

s with active pulmonary TB (PTB). (A) Assessment of SOCS mRNAnts with active PTB and controls. (B) Comparison of SOCS1 andIV-positive patients with mild PTB (open bars) to patients withive PTB with mild (n = 12) or mod–sev (n = 10) disease and inase was determined using chest X-ray. (C) Correlation analysisfluid samples from HIV-negative and HIV-positive PTB patients.efficient rs and the indicated p-values. (D) Assessment of SOCS1rom HIV-negative and HIV-positive EPTB patients with a local TBlots showing the median and range for each group. The statisticaln the different groups was determined using the Kruskal–Walliswo similar experiments is shown.

96 S. Ashenafi et al.

r = −0.75; p b 0.0001). Previously it has been shown that thenumber of CD4+ T cells is also significantly reduced in BALfluid from HIV-positive compared to HIV-negative patientswith pulmonary TB [58], which may result in reduced locallevels of IFN-γ and prevent the induction of an effectiveimmune response. However, high CXCL10 levels in TB/HIVco-infected subjects are less dependent on CD4 T cell countsthan IFN-γ levels [59,60], perhaps due to CXCL10 productionby monocytes and macrophages rather than T cells.

In the panel of chemokines tested, we found a predominantlocal expression of CCL4 in the BAL fluids obtained from M.tuberculosis-infected lungs. Interestingly, the CCL4 genedemonstrates significant association with increased suscepti-bility to TB [61] and accumulating data provide evidence of animmunoregulatory role of CCL4 [14,62]. IL-4 specificallyelevates CCL4 expression and supports polarization of regula-tory Th2 cells in vivo, which protects diabetic mice fromdeveloping inflammation and destructive disease [63–65].Accordingly, diabetic TB patients who experienced a fastdeterioration in their clinical TB disease showed a bias towardsa Th2 response [66]. Remarkably, islet-specific Th1 cells wereable to produce CCL2, CCL3, CCL4 and CCL5, while Th2 cellsonly produced CCL4 [67]. IL-4-induced CCL4 suppressesTh1-mediated pathogenesis by decreasing the recruitment ofactivated CD8+ T cells into pancreatic islets lesions, whileenhancing the recruitment of antigen-specific CD4+ Treg cells[14,68]. Similarly, in vivo expression of CCL4 induced T cells toproduce less IFN-γ and more IL-4 [14], which resulted indecreased local CTL responses but increased peripheral IgGand IgA antibody responses after mucosal immunization[69,70]. Other studies have shown that CCL4 is the mostpotent chemoattractant of CD4+CD25+ Treg cells [71], whilehuman Treg cells secreting CCL4 can inhibit microbe-specific Tcell activation [62]. Interestingly, such CD8+LAG3+CCL4+ Tregcells were shown to be enriched 20-fold in granulomas of M.tuberculosis-infected lymph nodes [62].

Our findings show that both SOCS1 and SOCS3 inhibitoryproteins were up-regulated in pulmonary TB, while SOCS3was more associated with severe forms of TB. Both SOCS1and SOCS3 can be rapidly induced by i.e. TLR4-ligands andmycobacteria can also trigger rapid production of pro-inflammatory cytokines i.e. IL-1β and IL-6 that up-regulateSOCS-expression [72]. It has previously been determinedthat SOCS1 expression is 5-fold higher in Th1 than in Th2cells, whereas Th2 cells contain 23-fold higher levels ofSOCS3 [73]. SOCS1 and SOCS3 can be induced in murinemacrophages upon infection with avirulent mycobacteria[74,75], and our data suggest that SOCS1 and SOCS3 wereup-regulated in PBMCs as well as in macrophages uponin vitro exposure or infection with virulent M. tuberculosis.mRNA expression profiles in sputum samples from TBpatients demonstrated significantly higher levels of anti-inflammatory mediators including both SOCS1 and SOCS3[76]. In addition, gene expression profiles of human bloodsamples revealed an accumulation of SOCS3 in activeTB compared to latent TB [77,78]. SOCS3 was reducedsimultaneously as IFN-γ was increased upon successfulchemotherapy in patients with active TB [76,77]. Interest-ingly, previous research in both mice and humans has found amechanistic link between increased levels of SOCS3 and thedevelopment and maintenance of Th2 cells. T cells frompatients with Th2-mediated allergy have a high SOCS3

expression that correlates with disease pathology, includingthe induction of humoral immunity [79]. Accordingly, Th2responses were strongly elevated in SOCS3 transgenic mice[79], while mice with SOCS3-deficient T cells produced lessIL-4 as well as antigen-specific IgG1 and IgE responses [80].Deficient SOCS3-binding also results in an increased CCL5expression and T cell recruitment to the site of inflammation[81]. Consistently, SOCS3-deficient macrophages have astronger capacity to induce Th1 and Th17 cell differentiation[82], while induction of SOCS3 in CD4+ T cells force a Th2profile by preventing IL-12-induced Th1 differentiation [83].SOCS3 inhibits IL-12-mediated STAT4 activation by binding tothe IL-12Rβ2 subunit and thus promotes Th2 polarization [84].Contrary to these reports, recently published data suggestthat SOCS3-deficient mouse macrophages are converted toalternatively activated M2-type [85], and that SOCS3 is criticalfor resistance to M. tuberculosis infection in mice [86].Apparently, further consensus regarding the role of SOCSproteins in the establishment of TB as well as progression ofactive disease is required, particularly in humans.

5. Conclusions

Altogether, the findings from this study demonstrate that alow Th1-mediated immune response may be the result of aninappropriate Th2-like immune response signature involvingenhanced expression of IL-4 and CCL4 in combination withSOCS3. Excess expression of regulatory SOCS proteins inhuman TB may contribute to Th2 immune polarization andloss of important Th1 effector functions that could promoteintracellular persistence of M. tuberculosis. Th2 and SOCS3-regulated immune response pathwaysmay be further exploredas potential targets for future clinical interventions.

Financial support

This work was supported by funding from the SwedishResearch Council (VR), the Heart- and Lung Foundation(HLF), the Swedish Society for Medical Research (SSMF), theSwedish International Development Cooperation Agency(Sida), the Swedish Civil Contingencies Agency (MSB) and theSwedish Foundation for Strategic Research (SSF).

Conflict of interest statement

The authors do not have a commercial or other associationthat might pose a conflict of interest.

Acknowledgments

We thank the research nurses, laboratory staff, and techni-cians at the Black Lion University Hospital and ArmauerHansen Research Institute, Addis Ababa, Ethiopia, for excel-lent collaboration and support during this study.

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