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Clinical Immunology (2011) 141, 133–142

Th3 Immune responses in the progression of leprosyvia molecular cross-talks of TGF-β, CTLA-4 and Cbl-bSudhir Kumar a, Raza A Naqvi a, Neena Khanna b,Pankaj Pathak c, D.N. Rao a,⁎

a Department of Biochemistry, All India Institute of Medical Sciences (AIIMS), Ansari Nagar,New Delhi-110029, Indiab Department of Dermatovenerology, All India Institute of Medical Sciences (AIIMS), Ansari Nagar, New Delhi-110029, Indiac Department of Pathology, All India Institute of Medical Sciences (AIIMS), Ansari Nagar, New Delhi-110029, India

Received 27 May 2011; accepted with revision 22 June 2011Available online 1 July 2011

⁎ Corresponding author at: RoomBiochemistry, All India Institute of MeNagar, New Delhi-110029, India. Fax: +

E-mail addresses: rao.aiims@yahoodnrao311@rediffmail.com (D.N. Rao).

1521-6616/$ - see front matter. Crowdoi:10.1016/j.clim.2011.06.007

KEYWORDSTh3;Mycobacterium leprae;Leprosy;TGF-β;MLCwA

Abstract Leprosy is a chronic human disease; primarily affecting skin, peripheral nerves, eyes,testis etc. Comprehensive-expressional-profiling of Th1–Th2–Th3 associated markers (84 genes)using qRT-PCR array, negated the previously prevailing notion, Th2 bias towardsmultibacillary stageof leprosy. Highproduction TGF-β further supported thedearth of any immune response(s) in leprosyprogression. Over expression of Cbl-b, could emerge as plausible reason for contributing T cellhyporesponsiveness, possibly by degradation of T cells signaling molecules. Anti-TGF-β treatmentsfurther confirm the TGF-β-dependent-Cbl-b overexpression in multibacillary patients. Diminished

Cbl-b expression in CTLA-4 knockout studies using siRNA, provided other evidence towards T cellhyporesponsiveness. Further, high T cell proliferation and IL-2 production in PBMC cultures treatedwith anti-TGF-β and siRNA offers here a strategy to revert T cell hyporesponsiveness bydownregulating Cbl-b expression in leprosy. Thus, this study negates Th2 bias and substantiatesmolecular cross-talk amongst TGF-β-CTLA-4-Cbl-b eventually leads to M. leprae persistence.Crown Copyright © 2011 Published by Elsevier Inc. All rights reserved.

No. 3029, Department ofdical Sciences (AIIMS), Ansari91-11-26593545..com,

n Copyright © 2011 Published

1. Introduction

Leprosy is one of the most dreading diseases hauntingmankind since time immemorial. Myobacterium leprae,causative agent of leprosy, infects only human and nine-banded armadillos. To date it has never been successfully

by E

cultivated artificially in laboratory media [1]. Due to itsunique clinical spectrum leprosy provided a better opportu-nity for immunologists to unveil the bridging between innateand adaptive immunological-signaling-mechanism. Leprosypatients with a high cell-mediated immunity (i.e., pauciba-cilli, relatively resistance to pathogen, unique expression ofTh1 cytokines), localized infection, central hypopigmenta-tion and hypoesthesia are classified as paucibacillary i.e.,borderline tuberculoid (BT)/tuberculoid (TT). On otherhand, patients with defective CMI (no apparent resistance

lsevier Inc. All rights reserved.

134 S. Kumar et al.

to M. leprae), numerous but poorly demarcated, raised ornodular lesions on all parts of the body, foamy macrophagesin the dermis (due to very large numbers of bacilli), areknown as multibacillary i.e., borderline lepromatous (BL),and lepromatous (LL) [1,2].

The underlying mechanism of observed T cell hyporespon-siveness and clonal anergy in leprosy patients towards highbacteriological index of M. leprae is still a challenge. In thisconnection, numerous efforts from our laboratory, as well asother international groups, strived to work out this problemand expected Th2 bias towards advanced stages of leprosy [3–6] but the recent reports of our group depicting the losses tomacrophages to TCR signaling, less possibilities of formation ofimmunological synapses and over expression of variousnegative regulators of TCR signaling towards BL/LL poles ofleprosy [7] enforced us to rethink the phenomenon of Th2 biasin the disease progression. Therefore, we comparativelyanalyzed the Th1, Th2, Th3 profiling in the BT/TT and BL/LLand healthy controls (HCs) and considered a total number of 84respective genes in our study.

Molecular mechanisms mediating the stimulation of theTCR via synapse formations, between antigen presenting cellsand T cells, eventually leading to the differentiation of naive Tcell precursors either in Th1 or Th2 direction is a matter ofintensive investigations over recent years. Scientific reportsshowed that the differentiation of uncommitted CD4+ T cellseither in Th1 or Th2 lineages is regulated by a special networkof transcription factors. Thus, we next decided to analyze theexpression of Th1/Th2 specificmaster regulatory factors in thedisease progression. Since T-bet and GATA-3 have now beenestablished as master regulators for Th1 and Th2, respectively[8–10], therefore, we also evaluated the expression dynamicsof T-bet and GATA-3 in BT/TT and BL/LL forms of leprosy.

TGF-β molecules have now appeared as the sole factorexerting anti-proliferative effects on Th1 and Th2 cells andeventually leading to Th3 immune response, by perturbing thedifferentiation of either Th1 or Th2 and inhibition of pro-inflammatory cytokine production, [11–15]. Also, we recentlyreported over expression of TGF-β is associated with high M.leprae load [5]. Taking together these evidences, wehypothezised here that M. leprae progression drives Th3, afavorable immune suppressive environment for its survival andmultiplication. To prove thiswemanipulated the expression ofTGF-β in PBMCs of leprosy patient with high bacteriologicalindex by anti-TGF-β effects in vitro and tried to assess therelease of TGF-β induced cytokines in this study.

Additionally, our laboratory also reported the overexpression of CTLA-4 molecules on T cell surface in leprosypatients showing T cell hyporesponsiveness [5]. CTLA-4negatively regulates the TCR signaling processes throughSHP2 recruitment at its cytoplasmic domain [16]. CTLA-4demonstrated high affinity toward B7 molecule and bycompeting with B7-1/B7-2 engagement processes with CD28perturbs the effector functions of T cells [16,17]. In ourrecent report we have shown that dephosphorylation anddegradation of the T cell signaling components (Lck, ZAP-70etc.) via cross-talk between CTLA-4 and SHP2 and overexpression of Cbl-b, respectively, major driving forcescontributing T cell anergy/hyporesponsiveness to M. lepraetotal cell wall derived antigen (MLCwA). Cbl-b is a memberof mammalian Cbl family proteins and contains an N-terminal tyrosine kinase binding domain, a RING finger, and

C-terminal proline-rich sequences. RING finger domainrecruits ubiquitin (Ub)-loaded Ub conjugation enzymes orE3 to help transfer Ub to target proteins for theirdegradation [18,19]. Therefore, in this study, we tried todraw the parallel relationship, if existing, between TGF-β,CTLA-4 and Cbl-b.

2. Materials and methods

2.1. Patients

A total of one hundred leprosy patients, of paucibacillary(PB, TT/BT; N=50) and multibacillary (MB, BL/LL; N=50)were recruited from Department of Dermatovenerology,AIIMS, New Delhi, India. Table 3 describes the demographicprofiles of the patients recruited in this study. The patientswere categorized in the above groups based on the clinical,histopathological and bacteriological parameters of Ridley–Joplin classification [2]. This study was approved by theInstitute Ethics Committee, All India Institute of MedicalSciences (AIIMS), New Delhi. Also, equal numbers of healthyvolunteers were recruited after getting their writtenconsent.

2.2. Antigen, immunomodulators and liposomepreparation

M. leprae derived total cell wall antigen (MLCwA) was a kindgift from Dr. Patrick Brennan, Colorado State University,Colorado, USA, and NIH, NAIAID “Tuberculosis ResearchMaterials and Vaccine Testing organization”. Murabutide,an analog of muramyl dipeptide (MDP; the smallest bioactiveunit of bacterial peptidoglycan) (InvivoGen, USA, USA)and in house synthesized T cell peptide of Trat protein(NH2-GLQGKIADAVKAKG-COOH) Escherichia coli membrane,were used as immunomodulators (IMs) in this study. Theoptimal amount of MLCwA was delivered to the PBMC culturesin three formulations, and with twomodes of delivery systems:MLCwA alone, MLCwA+immunomodulators (soluble form),MLCwA+immunomodulators (liposome form). Freeze andthaw technique was used to prepare liposomes for entrapmentof antigenswith both the immunomodulators [4,5]. Percentageentrapment was assessed by BCA Assay Kit (Sigma Aldrich) andwas found to be in the range 49–56%.

2.3. Antibodies

Mouse monoclonal anti human CD3 Ab (clone UCHT1) andanti-CD28 Ab (clone CD28.2) were obtained from BDPharMingen, San Diego, CA. Rabbit polyclonal anti humanTGF-β (ab66043) was obtained from Abcam, USA. Rabbitmonoclonal anti human Cbl-b (clone E160) was obtained fromAbcam, USA. Mouse monoclonal anti human CTLA-4 Ab (Clone14D3), anti human T-bet Ab (Clone eBio4B10) and anti-GATA-3 (clone TWAJ) were obtained from eBiosciences, USA. Goatpolyclonal anti-rabbit HRP were obtained from JacksonImmunoResearch, USA. Rabbit polyclonal anti human β-actin and rabbit polyclonal anti-mouse HRP were obtainedfrom Abcam, USA.

135Th3 Immune responses in the progression of leprosy via molecular cross-talks

2.4. Isolation and culture of PBMCs

Peripheral blood mononuclear cells (PBMC) were isolatedfrom approximately, 15 ml of heparinised venous blood ofleprosy patients and the healthy controls, by centrifuga-tion over Ficoll–Hypaque (Sigma-Aldrich) [20]. Aboveseparated PBMCs (1×106 cells/well) were then culturedin 96 well tissue culture in triplicate at 37 °C (5% CO2), for72 h in complete RPMI 1640 medium (Sigma-Aldrich, USA)with 10% fetal calf serum (Sigma-Aldrich, USA), 100 U/mlpenicillin, 50 μg/ml streptomycin (Sigma-Aldrich). Duringthe last 12–16 h of the culture, 0.5 μCi of 3H-thymidine(specific activity 6.7 Ci/m mol Bhabha Atomic ResearchCentre, Trombay, Mumbai) was added to each well.Cultures were harvested onto glass fiber disk andincorporation of radioactivity was measured by β-liquidscintillation counter. Results were expressed as cell countper minute (c.p.m.). Standardized amounts of MLCwA werepresented to the respective culture, in various combina-tions and formulations were: MLCwA alone (1 μg/well),MLCwA+murabutide+Trat peptide (1:2:2 μg/well) in solu-ble and liposome form.

2.5. Th1/Th2/Th3 gene profiling using real-time PCR

RNA from PBMCs of leprosy patients was subjected toreverse-transcription using SuperScript reverse transcrip-tase (Invitrogen). Specific Th1/Th2/Th3 PCR array (Super-Array, SA Biosciences, USA) were used to examine themRNA levels of several genes according to the manufac-turer's protocol. Relative gene expressions amongst varioussamples were evaluated by on-line support of SABiosciences.

2.6. CTLA-4 silencing using siRNA

To knock out CTLA-4 gene expression, PBMCs from BL/LLpatients were treated with 300 nmol/L small interfering RNA(siRNA) targeting CTLA-4 or control siRNA together withLipofectamine™ (Invitrogen) for 48 h. The following siRNAsequences were used (5′→3′): CTLA-4 sense: CCCAAAUUAC-GUGUACUAC-UdTdT; CTLA-4 antisense: GUAGUA CAC-GUAAUUUGGG -CdTcT ; s c r amb l ed RNA s en s e :UUCUCCGAACGUGUCACG-UdTdT; antisense: ACGUGACAC-GUUCGGAGA-AdTdT.

2.7. Western blotting

Whole cell extracts were obtained from PBMC cultures(~1×106 cells) using their respective lysis buffers (Ther-mo Fisher Scientific Inc.). The presence of specificproteins was elucidated by western blotting with theirrespective mono or polyclonal antibodies followed by theappropriate HRP-conjugated secondary antibody. Immuno-blots were then detected by ECL detection system(Millipore, USA). For subsequent reprobing, membraneswere stripped off with stripping buffer (62.5 mM Tris–HCl, 2% SDS, and 100 mM 2-mercaptoethanol, pH 6.7) at56 °C for 20 min.

2.8. ELISA

The culture supernatants collected after 72 h were assayedby sandwich ELISA using BD OptEIA set (BD Biosciences, USA),as per manufacturer's instructions, to measure the produc-tion of IL-2.

2.9. Statistical analysis

Statistical analysis was performed by ANOVA with a post-hocanalysis using the Bonferroni test. Data represented as ±SDand differences were considered significant when the P valuewas less than 0.05.

3. Results

3.1. Profiling of Th1 responsive genes in leprosypatients

To examine the previously existing notion, leprosy pro-gresses with the inhibition of Th1 immune responses [4,5],led us to evaluate the comparative pattern of expression offollowing genes (associated with Th1 immune responses) inleprosy patients: CCR5, CD28, CSF2, CXCR3, HAVCR2, IFNγ,IGSF6, IL12B, IL12RB2, IL-18, IL18R1, IL2, IL2RA, IRF1,SOCS1, SOCS5, STAT1, STAT4, TBX21, TLR4, TLR6, TNF andGLMN. mRNA expression of these said genes was analyzedfrom BT/TT to BL/LL poles of leprosy. CCR5, IFNγ, IL12B,IL12RB2, IL18, IL18R1, IL2, STAT1 and TNF gene showedsignificant down regulation from BT/TT to BL/LL poles ofleprosy. However, other Th1 specific genes also showedremarkable down-regulation but their fold changes werenot found as significant. Amongst first category, IL12B,SOCS1, STAT4, and IFNγ were found to be maximally down-regulated towards BL/LL poles. Furthermore, analysis ofwell-known Th1 genetic switch formed by the combinato-rial actions of IL18, IL12, IL12RB1, IFNγ, STAT1/4 andTBX21 genes further validated the reduction or loss of Th1immune responses towards multibacillary leprosy (BL/LL).Moreover, IL2RA (CD25) gene showed a remarkable upre-gulation towards BL/LL, thereby suggesting the presence ofimmunosuppressive environment made by T reg cells(Table 1).

3.2. Profiling of Th2 responsive genes in leprosypatients

Our next goal was to confirm that whether in the absence ofTh1 immune responses, is there any Th2 bias as advocatedearlier in leprosy? Similar to Th1, we also analyzedunderlying genes, as their concerted actions eventuallydrive Th2 responses. In this pursuit, we evaluated CCL11,CCL5, CCL7, CCR2, CCR3, CCR4, CEBPB, NFATC2IP, GATA3,GFI1, GPR44, ICOS, IL13, IL13RA1, IL1R1, IL1R2, IL4, IL4R,IL5, IL9, IRF4, JAK1, MAF, NFATC1, NFATC2, PCGF2, STAT6,and TMED1 genes in BT/TT and BL/LL group of leprosypatients in association with healthy controls (HCs). Of theabove evaluated genes, we found significant down regula-tion of the following major Th2 regulating genes towardsBL/LL poles of leprosy: CCL11, CCL5, CCR3, CEBPB,

Table 1 Detailed analyses of Th1/Th2 signatures, T cell activation markers and T regs markers in BT/TT and BL/LL patients ofleprosy.

Genename

Description Refseq BL/LL P-value BT/TT P-valueaFoldregulation

aFoldregulation

Th1 signaturesCCR5 Chemokine (C-C motif) receptor 5 NM_000579 −3.1023 0.05 −3.6427 0.05CD28 CD28 molecule NM_006139 −73.4099 NS −32.4318 NSCSF2 Colony stimulating factor 2 (granulocyte-macrophage) NM_000758 −21.1121 NS −15.2951 NSCXCR3 Chemokine (C-X-C motif) receptor 3 NM_001504 −3.1095 NS −18.5713 0.005HAVCR2 Hepatitis A virus cellular receptor 2 NM_032782 −1.4044 NS −1.0317 NSIFNg Interferon, gamma NM_000619 −83.0937 0.0004 −5.3943 0.0004IGSF6 Immunoglobulin superfamily, member 6 NM_005849 −1.4241 NS −2.0069 0.05IL12B Interleukin 12B (natural killer cell stimulatory factor 2,

cytotoxic lymphocyte maturation factor 2, p40)NM_002187 −982.2865 0.00441 −192.449 0.004476

IL12RB2 Interleukin 12 receptor, beta 2 NM_001559 −1.4743 0.000104 −1.5105 NSIL-18 Interleukin 18 (interferon-gamma-inducing factor) NM_001562 −8.2059 0.003301 −2.488 0.016113IL18R1 Interleukin 18 receptor 1 NM_003855 −73.6868 0.000002 −1.6189 0IL2 Interleukin 2 NM_000586 −548.748 0.005 −12.7728 NSIL2RA Interleukin 2 receptor, alpha NM_000417 8.2641 0.001 2.5099 NSIRF1 Interferon regulatory factor 1 NM_002198 −18.0426 NS −15.015 NSSOCS1 Suppressor of cytokine signaling 1 NM_003745 −538.6982 NS −58.2829 NSSOCS5 Suppressor of cytokine signaling 5 NM_144949 −162.3916 NS −3.5186 NSSTAT1 Signal transducer and activator of transcription 1, 91 kDa NM_007315 −62.3939 0.000965 −3.4462 NSSTAT4 Signal transducer and activator of transcription 4 NM_003151 −392.5321 NS −77.0826 NSTBX21 T-box 21 NM_013351 −43.5118 NS −39.5334 NSTLR4 Toll-like receptor 4 NM_138554 −4.084 0.00016 −2.9794 NSTLR6 Toll-like receptor 6 NM_006068 −10.7779 NS −8.2535 NSTNF Tumor necrosis factor (TNF superfamily, member 2) NM_000594 −2062.245 0.0001 −145.513 0.003GLMN Glomulin, FKBP associated protein NM_053274 −1506.1677 NS −5.5597 NS

Th2 signaturesCCL11 Chemokine (C-C motif) ligand 11 NM_002986 −1.6625 0.000569 −1.521 NSCCL5 Chemokine (C-C motif) ligand 5 NM_002985 1.4306 0.000018 1.1607 NSCCL7 Chemokine (C-C motif) ligand 7 NM_006273 −1.0546 NS −1.3613 NSCCR2 Chemokine (C-C motif) receptor 2 NM_000648 −1.8575 0.000005 −1.6994 NSCCR3 Chemokine (C-C motif) receptor 3 NM_001837 −2.5257 NS −2.2894 NSCCR4 Chemokine (C-C motif) receptor 4 NM_005508 −2.362 NS −2.2115 NSCEBPB CCAAT/enhancer binding protein (C/EBP), beta NM_005194 −3.5064 0.000287 −3.1932 NSNFATC2IP Nuclear factor of activated T-cells, cytoplasmic,

calcineurin-dependent 2 interacting proteinNM_032815 −4.8121 0.000014 −4.8736 NS

GATA3 GATA binding protein 3 NM_002051 1.6805 0.005 1.1711 NSGFI1 Growth factor independent 1 transcription repressor NM_005263 −3.0314 0.000618 −2.558 NSGPR44 G protein-coupled receptor 44 NM_004778 −3.6893 0.000065 −3.8238 NSICOS Inducible T-cell co-stimulator NM_012092 −1.651 NS −1.3613 NSIL10 Interleukin 10 NM_000572 2.555 0.018027 −3.2982 0.009961IL13 Interleukin 13 NM_002188 −1.2454 0.675872 −1.6377 0.000736IL13RA1 Interleukin 13 receptor, alpha 1 NM_001560 −1.1865 NS 1.2614 NSIL1R1 Interleukin 1 receptor, type I NM_000877 −1.2805 NS −1.0905 NSIL1R2 Interleukin 1 receptor, type II NM_004633 −1.3755 NS 1.0105 NSIL4 Interleukin 4 NM_000589 −1.9908 0.001043 −2.3161 0.000349IL4R Interleukin 4 receptor NM_000418 −1.021 NS 1.1527 NSIL5 Interleukin 5 (colony-stimulating factor, eosinophil) NM_000879 −2.4794 0.009146 −3.0916 0.00164IL9 Interleukin 9 NM_000590 −2.0326 NS −1.4158 NSIRF4 Interferon regulatory factor 4 NM_002460 −41.6429 NS −36.1269 NSJAK1 Janus kinase 1 NM_002227 −2.4453 0.002793 −2.0209 NSMAF V-maf musculoaponeurotic fibrosarcoma oncogene

homolog (avian)NM_005360 −1.7411 0.03325 −1.5529 NS

NFATC1 Nuclear factor of activated T-cells, cytoplasmic,calcineurin-dependent 1

NM_172390 −4.005 0.000019 −2.558 NS

136 S. Kumar et al.

Table 1 (continued)

Genename

Description Refseq BL/LL P-value BT/TT P-valueaFoldregulation

aFoldregulation

NFATC2 Nuclear factor of activated T-cells, cytoplasmic,calcineurin-dependent 2

NM_012340 −5.7149 0.000017 −4.4847 NS

PCGF2 Polycomb group ring finger 2 NM_007144 −65.7993 NS −43.2611 NSSTAT6 Signal transducer and activator of transcription 6,

interleukin-4 inducedNM_003153 1.4373 NS −1.3482 NS

TMED1 Transmembrane emp24 protein transport domaincontaining 1

NM_006858 −12.4955 0.000001 −10.9663 NS

CD4+ T-cell markersCD4 CD4 molecule NM_000616 −22.5231 NS −22.8639 NSCD40LG CD40 ligand NM_000074 −2.1535 NS −3.3909 NSCD80 CD80 molecule NM_005191 −116.1625 NS −2.7798 NSCD86 CD86 molecule NM_006889 −159.4176 NS −1.9031 NSCREBBP CREB binding protein NM_004380 −1.3134 NS −1.3996 NSCTLA4 Cytotoxic T-lymphocyte-associated protein 4 NM_005214 3.2283 NS 1.0069 NSFASLG Fas ligand (TNF superfamily, member 6) NM_000639 −12.2666 NS −10.0213 NSIL15 Interleukin 15 NM_000585 −197.6312 NS −21.0391 NSIL6 Interleukin 6 (interferon, beta 2) NM_000600 −34.3761 NS −15.2951 NSIL6R Interleukin 6 receptor NM_000565 −10.8278 NS −11.5115 NSIL7 Interleukin 7 NM_000880 1.2198 NS 1.5422 NSJAK2 Janus kinase 2 NM_004972 −107.1385 NS −1.6189 NSLAG3 Lymphocyte-activation gene 3 NM_002286 −46.7426 NS −48.0012 NSLAT Linker for activation of T cells NM_014387 −53.6932 NS −48.3351 NSMAP2K7 Mitogen-activated protein kinase kinase 7 NM_145185 −33.2819 NS −39.3511 NSMAPK8 Mitogen-activated protein kinase 8 NM_002750 −84.0592 NS −4.5473 NSPTPRC Protein tyrosine phosphatase, receptor type, C NM_002838 −33.3589 NS −30.5903 NSTFCP2 Transcription factor CP2 NM_005653 −28.7735 NS −27.5695 NSTGFB3 Transforming growth factor, beta 3 NM_003239 12.5432 0.000136 2.5758 NSCD27 CD27 molecule NM_001242 8.5479 0.021129 1.5422 NSTNFRSF8 Tumor necrosis factor receptor superfamily, member 8 NM_001243 13.8144 NS 11.2122 NSTNFRSF9 Tumor necrosis factor receptor superfamily, member 9 NM_001561 6.6735 NS 1.8637 NSTNFSF4 Tumor necrosis factor (ligand) superfamily, member 4 NM_003326 2.3457 NS 1.7471 NSTYK2 Tyrosine kinase 2 NM_003331 −5.8835 0.000016 −2.8626 NSYY1 YY1 transcription factor NM_003403 −2.4909 0.002152 −2.1214 NS

Other Treg networkIL17A Interleukin 17A NM_002190 1.0718 NS −1.0461 NSIL23A Interleukin 23, alpha subunit p19 NM_016584 −14.3535 0.000022 −7.8083 NS

NS indicating non significantP values.N=50 subjects fromeach groupswere taken in to account for this analysis. All P-valueswere calculated on95% CI.a Fold regulation and P value were calculated by online data analysis tool of Super array biosciences.

Th2 signatures

137Th3 Immune responses in the progression of leprosy via molecular cross-talks

NFATC2IP, GFI1, GPR44, IL10, IL13, IL4, IL5, JAK1, MAF,NFATC1, NFATC2 and TMED1. Out of these, PCGF2 (Poly-comb group ring finger 2) and TMED1 (Transmembraneemp24 protein transport domain containing 1) showedmaximum down regulation, both in BL/LL and BT/TTgroups. Additionally, the rest of the genes were alsodown-regulated but their levels were found to be insignif-icant. However, feeble upregulation of GATA-3 and overexpression of IL10 towards BL/LL pole furthermorehighlighted the suppression of Th2 immune responses.Furthermore mixed type of results of other imperative Th2inducing genes viz IL13, IL4, IL5, STAT6 and GATA-3,

strongly advocated Th2, which further substantiated ourresults (Table 1).

3.3. Evaluation of CD4+ T-cell signatures in leprosypatients

Cell mediated immunity (CMI) in leprosy patients was alsoexamined in terms of expression of CD4 T cell markers. Herein,we thoroughly examined the expression of following markers:CD4, CD40LG, CD80, CD86, CREBBP, CTLA4, FASLG, IL15, IL6,IL6R, IL7, JAK2, LAG3, LAT, MAP2K7, MAPK8, PTPRC, TFCP2,

138 S. Kumar et al.

TGFB3, CD27, TNFRSF8, TNFRSF9, TNFSF4, TYK2 and YY1. Ofthese genes CD27, TYK2 and YY1, showed significant downregulation. Therefore, this study strongly pinpoints towardsthe presence of immunosuppressive environment during thedisease progression. Remarkable up-regulation of TGF β,CTLA4 and IL7 further substantiated our hypothesis that highM. leprae load leads to the development of Th3 immuneresponses (Table 1).

Figure 1 Immunoblot analysis of TGF-β and CTLA-4, and Th1 (T-bLane 1–4: number of subjects; HC: healthy individuals; BT/TT: borderesults are expressed as mean±S.D. All semiquantitative analyses wN=50 from each group were taken for this analysis. *Pb0.05; **Pb0

3.4. Effect of anti TGF-β and siRNA CTLA-4 on Cbl-bexpression and T cell hyporesponsiveness in leprosypatients

Transcriptional down-regulation of Th1, Th2 and CD4+activation markers and concomitant over expression TGF-βsuggested the possibilities of Th3 immune responses. In thiscontinuation we performed immunoblot analysis of Th1, Th2

et)/Th2 (GATA-3) specific master regulators in leprosy patients.rline tuberculoid and BL/LL: borderline lepromatous leprosy. Allere performed after normalizing the expressions with β-actin..005; ***Pb0.0005.

Figure 2 TGF-β and CTLA-4 dependent expressional dynamicsof Cbl-b in multibacillary leprosy patients. Lane No treatment:expression of Cbl-b from PBMC cultures without treatment(either anti-TGF-β or siRNA CTLA-4); Lane anti-TGF-β: Cbl-bexpression PBMC cultures treated with anti-TGF-β; Lane scrRNA: Cbl-b expression after treatment with scrambled RNA;Lane si-CTLA-4: Cbl-b expression after siRNA treatment (48 h).For this experiment N=30 BL/LL patients were taken intoaccount. All results are expressed as mean±S.D. *Pb0.05;**Pb0.005; ***Pb0.0005.

Figure 3 Effects of anti-TGF-β and siCTLA-4 on T cellproliferation and subsequent IL-2 production in multibacillaryleprosy patients. (A) Assessment of IL-2 levels upon varioustreatments of PBMC cultures. (B) Schematic of T cell prolifer-ation (3H incorporation; expressed as count per minute) underthe effect of anti-TGF-β and siRNA treatment, with and withoutanti-CD3/28. PHA is used as a positive control to examine the Tcell proliferation. For this experiment N=50 BL/LL patients weretaken into account. All results are expressed as mean±S.D.*Pb0.05; **Pb0.005; ***Pb0.0005.

139Th3 Immune responses in the progression of leprosy via molecular cross-talks

and markers of immune suppressive environment i.e. T-bet,GATA-3, TGF-β, and CTLA-4 genes, respectively (Fig. 1).Immunoblotting results further authenticated the possibili-ties of Th3 environment. Previously, we have demonstratedthat the over expressions of TGF-β and Cbl-b were plausiblereasons contributing to T cell hyporesponsiveness in leprosyprogression [7]. Therefore, we treated PBMC culturesobtained from BT/TT and BL/LL patients by anti-TGF-βantibody. To our surprise, we found significant reduction ofCbl-b in treated cells. Next, we evaluated: does overexpression of CTLA-4, as reported by our laboratorypreviously [5], contribute to the Cbl-b over expression inmultibacillary patients showing T cell hyporesponsivenesstowards MLCwA. With this intent, we knocked out CTLA-4expression in PBMC cultures of BL/LL patients using siRNAtechnology. BL/LL patient cultures without transfection andcells transfected with scrambled RNA (scr RNA) were used asthe negative control. The efficiency of siRNA transfectionwas checked using immunoblot of CTLA-4. Our resultsdemonstrated that siRNA delivery after 48 h led to significantreduction (Pb0.05) in CTLA-4 (Fig. 2). The efficiency ofCTLA-4 silencing was observed as 43.3%±6.36 after 48 h inimmunoblot analysis. Reprobing of this blot with anti-Cbl-b,in turn showed a remarkable reduction in Cbl-b expression(Fig. 2). Therefore, over expression of TGF-β and CTLA-4 isassociated with the over expression of Cbl-b, in turncontributes to T cell hyporesponsiveness. To ascertain theeffect of Cbl-b on T cell hyporesponsiveness, we performed Tcell proliferation studies in PBMCs cultures treated with anti-TGF-β and siRNA CTLA-4. In this study, the PBMC cultures of

BL/LL patients were stimulated with anti-CD3/CD28. PHAtreatment was used as proliferation control. In both cases(anti-TGF-β and siCTLA-4) a remarkable increase of T cellproliferation was observed (Fig. 3A). Interestingly, thisincrease was found to be more prominent in anti-TGF-βtreatment group. Furthermore, when we treated the CTLA-4silenced PBMC cultures with anti-CD3/CD28, a significantincrease in 3H incorporation was observed (Fig. 3A). Theabruptly high presence of IL-2 in anti-TGF-β treated culturesfurther substantiated the reversal T cell anergy. Further wemeasured IL-2 production by ELISA in PBMC culture superna-tant of anti TGF-β and siRNA CTLA-4 treated cultures ofBL/LL patients. A marginal increment of IL-2 production wasobserved in both treated PBMC groups (Fig. 3B).

140 S. Kumar et al.

3.5. Effect of MLCwA delivery in PBMC cultures ofleprosy patients

With the intent of ex-vivo immuno-therapeutics we deliv-ered multiple formulations of M. leprae derived total cellwall antigen (MLCwA) with distinct modes of antigen delivery

Table 2 Detailed analyses of Th1/Th2 signatures, T cell activation

Genes MLCwA P-value MLCwA+IMaFold regulation aFold regu

Down regulationCTLA-4 −2.37 NS −2.76CXCR3 −4.51 NS −4.54IL-10 −1.27 NS −1.34IL-13 −1.11 NS −1.78IL-2RA −3.06 NS −2.85CD40LG NC NS −1.59CCL7 NC – NCGATA-3 NC – NCIGSF6 NC – NCIL-4 NC – NCIL-5 NC – NCIL-9 NC – NCTLR6 NC – NC

Up regulationCD80 15.47 NS 20.11CD86 29.07 NS 78.43GLMN 267.18 NS 295.43IFNγ 2.89 NS 4.6IL-12B 12.07 0.01098 14.34IL-15 7.32 0.059564 10.17IL-18 2.06 0.039494 2.89IL-18R1 34.26 0.000021 42.91IL-2 30.38 NS 42.52JAK2 60.9 NS 72.17MAPK8 16.78 NS 20.87SOCS1 12.45 0.000054 14.16SOCS5 39.49 0.000001 49.45STAT-1 17.98 NS 19.07STAT-4 6.25 0.001606 4.97TNF 10.48 0.000008 19.84CD27 12.04 0.035001 12.01CD28 NC – NCCCR4 NC – NCCEBPB NC – NCCREBPB NC – NCCSF2 NC – NCHAVCR2 NC – NCIL-12RB NC – NCIL-6 NC – NCIRF-1 NC – NCT-bet NC – NCTMED1 NC – NC

NC indicating no relative change in gene expression level. NS indicating noto account for this analysis. All P-values were calculated on 95% CI.a Fold regulation and P value were calculated by online data analysi

systems. Post MLCwA delivery events facilitated the up anddown regulation of Th1 and Th3 immune responses. Ofvarious modes and formulations of antigen delivery, L-MLCwA+IMs resulted in the most prominent bias towardsTh1. Up regulation of Th1 and concomitant down regulationof Th2 and Th3 signatures are considered here as a read outof Th1 bias. Briefly, on L-MLCwA+IMs delivery we found the

markers and T regsmarkers in BL/LL patients onMLCwA delivery.

s P-value L-MLCwA+IMs P-value

lation aFold regulation

NS −2.95 NSNS −5.85 NSNS −3.64 0.019336NS −8.45 0.044426NS −3.94 NSNS −1.55 NS– −1.39 NS– −1.94 NS– −1.35 NS– −3.34 0.000822– −4.03 0.022775– −2.98 0.023361– −12.3 NS

NS 43.11 NSNS 79.52 NSNS 314.45 NS0.001775 7.27 0.0036030.000791 16.74 0.02073NS 10.75 NS0.059032 2.98 NS0.00002 43.52 0.0000530.000001 49.18 0.000002NS 75.24 NSNS 21.61 NS0.000082 19.85 0.000095NS 41.59 0.000001NS 23.48 NS0.004953 18.36 0.0000250.000001 18.32 0.0000010.057109 12.87 0.026438– 1.74 0.000063– 1.23 NS– 1.78 0.023656– 1.45 NS– 1.69 0.031394– 1.68 0.034648– 1.19 0.003318– 2.33 NS– 1.57 0.057409– 1.32 NS– 2 0.011849

n significant P values.N=30 subjects from each groups were taken in

s tool of Super array biosciences.

Table 3 Demographic details of cases and controls.

Characteristic Leprosy patients Healthy controls

BT/TT BL/LL(BI*=2–6)

(N=50) (N=50) (N=100)

Gender N (%) N (%) N (%)Male 32 (64%) 40 (80%) 25 (50%)Female 18 (36%) 10 (20%) 25 (50%)

Age group (years)b30 16 (32%) 11 (22%) 28 (56%)30–40 19 (38%) 25 (50%) 22 (44%)40–50 8 (16%) 9 (18%)>50 7(14%) 5 (10%)

BI*=Bacteriological Index, BT/TT=borderlines tuberculoid, BL/LL=borderline lepromatous.

141Th3 Immune responses in the progression of leprosy via molecular cross-talks

remarkable up regulation of CD80, CD86, GLMN, IFNγ, IL12B,IL15, IL18, IL18R1, IL2, JAK2, MAPK8, SOCS1, SOCS5, STAT1,STAT4, TNF, CD27, CD28, CCR4, CEBPB, CREBPB, CSF2,HAVCR2, IL12RB, IL6, IRF1, T-bet and TMED1 genes cumula-tively reported to induce Th1. Additionally, down regulationof Th2 and Th3 markers e.g., TGF-β, CTLA-4. CXCR3, IL10,IL13, IL2RA, CD40LG, CCL7, GATA-3, IGCF6, IL4, IL5, IL9 andTLR6 further supported Th1 bias (Table 2). MLCwA deliveryalso led to remarkably high secretions of IL2 as well as 3Hincorporation, an indicative of high T cell proliferation.However, these increases were found to be more significantin L-MLCwA treated PBMC cultures (Figs. 3A and B).

4. Discussion

The present study was performed to ascertain thedetailed view of immunological responses in the progres-sion of leprosy. Detailed analysis of Th1, Th2, T cellactivating genes and CD4+ cell markers, enforced here tostate that leprosy progresses with depressed mode ofimmunological responses. Unchanged expressional patternsof IL-4 and IL-13, major players of Th2 immune responsestowards BL/LL, pinpointed the absence of Th2 bias withhigh bacteriological index. Thus, this study negates thepreviously prevalent notion that leprosy progresses withTh2 bias [3,6]. Also, relatively steady expression of T-betand GATA-3; master players of Th1 and Th2 immuneresponses throughout this study further validated thesame notion.

Next we looked for the possibilities of Th3 environmenti.e. presence of immune suppressive environment in thepresence of high TGF-β secreting T regs [13]. Nextquestion is, from where is this high amount of TGF-βreleased during the infection process? We recentlyreported that M. leprae infection to the macrophageschanges their surface phenomenon by utilization ofcholesterol from the membrane lipid raft. These immatureare the rich sources of TGF-β, thereby releasing highamount of TGF-β during the M. leprae infection [7].Significant high expression of TGF-β towards BL/LL pole, inthis study, further validated it.

Our previous laboratory reports suggested the overexpression of Cbl-b and CTLA-4 in the progression of leprosy[5,7]. Does TGF-β enriched environment in multibacillaryleprosy lead to increased expression of Cbl-b? Significantlyhigh and low expression of Cbl-b in TGF-β treated PBMCcultures of healthy controls and anti-TGF-β treated PBMCcultures from BL/LL patients, respectively, strongly give aclue of TGF-β mediated expression of Cbl-b in advancedstages of leprosy. We recently reported that Cbl-b, beingnegative regulator of T cell signaling, increased the proteinturnover of TCR signaling components viz. Lck, ZAP-70,ITAMs etc. [7]. Taking together the results of both studies,we can say that TGF-β enriched environment via Cbl-b overexpression leads to T cell hyporesponsiveness, by degrada-tion of imperative players of T cell signaling, in advancedstages of leprosy. The over representation of pSmad3 inhealthy controls and BT/TT on TGF-β treatment further triedto substantiate the presence of TGF-β mediated signaling(unpublished data).

Also, our earlier laboratory report has demonstrated theover expression of CTLA-4 on T cells leading to T cellhyporesponsiveness against MLCwA antigen [5]. We nextaddressed, is Cbl-b over expression associated with CTLA-4levels in leprosy progression? Diminished expression of Cbl-bin CTLA-4 knockout PBMC cultures after 48 h supported ourhypothesis affirmatively. As for proper immunologicalsynapsing and activation of T cell signaling cascade, cross-talk between B7 molecules (on APC) and CD28 on T cells areindispensable [16]. Therefore, being a competitor of CD28,CTLA-4 overexpression at T cell surface is expected toscavenge most of the B7 molecules of APCs, leading toblockade of CD28 mediated signaling transduction andeventually T cell hyporesponsiveness/anergy. Recently thedetailed mechanism was shown by Qureshi et al., whereinthey proposed that CTLA-4 expressing cells, by a process oftrans-endocytosis, removes CD80/86 mediated signaling bycapturing its ligands from opposing cells which eventuallyresults in impaired costimulation (via CD28) and TCRsignaling [21].

High production of IL-2 and T cell proliferation in PBMCcultures treated siRNA CTLA-4 and anti-CD3/28, in this studyfurther supported this argument. That CTLA4-Ig inhibited theresponse of T cells and prolonged the allograft survival inseveral rodent and organ transplantation models furthersupported our results [22]. Also, its SHP2 mediated dephos-phorylation of Lck and ZAP-70 further poses the one morejustification for T cell hyporesponsiveness in leprosy pro-gression. Our recent reports showing the over expression ofSHP2 and concomitant down regulation of miR-181a furthersupported this notion [7].

5. Conclusion

Thus, we may conclude that M. leprae progresses with Th3immune responses (absence of Th1 and Th2). Secondly, wemay infer here that the over expression of TGF-β and CTLA-4leads to T cell hyporesponsiveness, a major hallmark ofleprosy, by increasing the expression of Cbl-b. Reversal of Tcell hyporesponsiveness by treating PBMC cultures with anti-TGF-β and CTLA-4 gives us a clue for the development ofappropriate therapeutic regimes. Whether it could lead a

142 S. Kumar et al.

novel strategy for M. leprae progression, needs furtherinvestigation.

Conflict of interest

The authors have no conflict of interest.

Acknowledgments

The authors are thankful to ICMR and CSIR for providingfinancial support to carry out this work. We are also thankfulto Mr. Pankaj Pathak, Department of Pathology, AIIMS, forreal time PCR analysis in this study.

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