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Exposure to mycobacterial cord factor enhances in vitro IL6 production by peripheral blood mononuclear cells

from patients with Takayasu arteritis

Nikhil Gupta, Jayakanthan Kabeerdoss, Hindumathi M, Ruchika Goel, Debashish Danda

Department of Clinical Immunology & Rheumatology, Christian Medical College, Vellore, Tamil Nadu

Disclosures

• None

Evidence of link between TA & TB

Retrospective analysis- Active pulmonary tuberculosis in 20% patients of TA

Case control study TA, TST positivity was higher in TA group vs. control (P = 0.008)

Mwipatayi BP et al. Takayasu's arteritis: clinical features and management: report of 272 cases. ANZ J Surg.

2005;75:110e117. Karadag O et al. Assessment of latent tuberculosis infection in Takayasu's arteritis with tuberculin skin test and Quantiferon-

TB Gold test. Rheumatol Int. 2010;30:1483e1487.

Evidence of link between TA & TB (cont….)

Increased humoral and cellular immune response to mTB antigens in TA

Chauhan SK, Tripathy NK, Singh M, Nityanand S. Cellular and humoral immune responses to mycobacterium heat shock protein-64 and its human homologue in Takayasu's arteritis. Clin Exp Immunol. 2004;138:547e553.

Aggarwal A, Chang M, Sinha N, Naik S. Takayasu's arteritis : role of Mycobacterium tuberculosis and its 65 kda heat shock protein. Int J Cardiol. 1996;55:49e55.

MYCOBACTERIAL CELL WALL GLYCOLIPID

• Trehalose-6,6- dimycolate (TDM) – cord factor

• Trehalose-6,6-dibehenate (TDB) – synthetic analogue

• We hypothesized that TA patients exposed previously to TB antigens may show high cellular immune responses on re-exposure of same antigens

Tera V. Guidry, Robert L. Hunter, Jeffrey K. Actor. Mycobacterial glycolipid trehalose 6,6 -dimycolate-induced hypersensitive ′granulomas: contribution of CD4+ lymphocytes. Microbiology. 2007 Oct; 153(Pt 10): 3360–3369.

Aim

To study and compare the cellular immune responses against

Mycobacterial cord factor synthetic analog trehalose-6,6-dibehenate (TDB) in patients with type 5 TA and healthy controls

MATERIALS AND METHODS

Patients

INCLUSION CRITERIA • > 18 yrs of age should

• ACR (1990) for TA

• Angiography – type 5

EXCLUSION CRITERIA

• Co morbidities other than hypertension and dyslipidemia

• Active infection

• Past history of tuberculosis

Controls

• Age and sex matched healthy controls were recruited Exclusion Criteria

• Individual previously diagnosed as TB and other granulomatous diseases

• Family history of autoimmune diseases

• Individuals who were on immunosuppression or taking steroids

Blood collection from subjects

PBMCs separated (Ficoll Hypaque

gradient and centrifugation)

PBMCs cultured for 48 hrs and supernatant separated

Cultured PBMCs stimulated with

TDB

IL-6 , TNF- , IL17 concentration measured in supernatant

METHODOLOGY

Fold change post stimulation/ basal

concentration calculated

STATISTICAL ANALYSIS

• Cytokine levels between TDB stimulated and unstimulated cells were compared by wilcoxon signed rank test

• TNF-, IL-6 and IL-17 Cytokine levels between groups was compared using Unparied t-test

• P value <0.05 was considered statistically significant

RESULTS

Baseline characteristics, laboratory parameters and disease activity of study cohort

Cases (n=22) Controls (n=21)

Median duration of disease (months)

8 (1-60) ---

CRP median (range) mg/l 6.81 (0.9-81.1) ---

ESR median (range) mm 32 (6-75) ---

Median ITAS 2010 4.5 (0-20) ---

Duration of immunosuppression among cases

Treatment Number of patients(%)

Treatment naive 5 (22.7)

Treatment duration less than 1 month 8 (36.4)

Treatment duration for 1month-2 years 5 (22.7)

Treatment duration 2-4 years 4 (18.2)

Inflammatory cytokines following stimulation with TDB (5µg/ml)

ELISA Unstimulated TDB (5µg/ml) p Value

IL-6 HC (pg/ml) (n=21) 789(77.73-1834) 2052(452.2-4067) < 0.0001

IL-6 TA (pg/ml) (n=22) 158(41-1948) 2313(554.5-7368) 0.0007

TNF-α HC (pg/ml) (n=21) 20(7.5-33.5) 67(25-103) < 0.0001

TNF-α TA (pg/ml) (n=22) 15(7.25-36.5) 52.5(31.25-102) 0.0037

IL-17 was undetectable

Inflammatory cytokines following stimulation with TDB (50µg/ml)

ELISA Unstimulated TDB (50µg/ml) p ValueIL-6 HC (pg/ml) (n=10) 1236(246-1836) 2192(613.2-3492) 0.0057

IL-6 TA (pg/ml) (n=11) 129.4(50.51-1100) 5096(996.8-15061) 0.0012

TNF-α HC (pg/ml) (n=10) 23.5(10.25-42.25) 268(35-563) 0.0010

TNF-α TA (pg/ml) (n=11) 12.5(7.25-22.75) 205(33.25-463.3) 0.0017

IL-17 was undetectable

Relative fold changes of IL-6 & TNF-α after stimulation with TDB (50 µg/ml) of PBMCs of healthy controls and TA patients

MINCLE (CLEC4E)

SIGNALLING

TDB

MINCLE (CLEC4E)

FcR-Ȣ

Syk

BCL 10 Cytokines & chemokines

Blood collection from subjects

PBMCs separated (Ficoll Hypaque

gradient and centrifugation)

PBMCs cultured for 48 hrs

Cultured PBMCs stimulated with

50 µg/ml of TDB

Fold change post stimulation/ basal

concentration calculated

mrna expression of CLECE4,

BCL10 in PBMC

Relative fold changes of CLEC4E & BCL10 after stimulation with TDB (50 µg/ml) of PBMCs of healthy controls and TA patients

Gene ExpressionTDB50/

unstimulatedHC (n=8) TA (n=9) p Value

CLEC4E 0.43(0.36-0.57) 1.03(0.623-1.346) 0.0111

BCL10 0.33(0.28-1.62) 0.97(0.77-1.243) 0.2991

Discussion & Conclusion

• On exposure to mycobacterial cord factor antigen TA patients showed

Increased IL-6 responses

Increased expression of MINCLE ( CLEC4E)

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