free paper presentation - exposure to mycobacterial cord factor enhances in vitro il6 production in...
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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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