High expression of S100 calgranulins

genes in peripheral blood mononuclear

cells of patients with Takayasu arteritis

Jayakanthan Kabeerdoss, Ruchika Goel, Hindumathi M,

Debashish Danda

Department of Rheumatology

Christian Medical College

Vellore – 632 004

Introduction

• Takayasu arteritis (TA) is inflammatory large-vessel vasculitis

• Immunopathogenesis of TA is not clear – T cell mediated (Th1 & Th17)

TLR4 and TLR5 induce distinct types of vasculitis Circ Res. 2009 Feb 27; 104(4): 488–495.

Cellular and humoral immune responses to mycobacterial heat shock protein-65 and its human homologue in Takayasu's arteritis Clin Exp Immunol. 2004 Dec; 138(3): 547–553.

Hypothesis

Lumen

Intima

Media

Adventitia

?

VEGF,IL-6,MCP-1

inflam

matio

n

TLR4

hsp65HMGB-1

VEGF,IL-6,MCP-1, ???

AimTo examine expression of endogenous TLR4 ligands

MethodologyPatients and Controls recruitment

PBMC separation

RNA isolation & cDNA conversion

Quantification of mRNA expression by sequence specific primers & SYBR green in real time PCR

Endogenous TLR4 ligands: S100 Calcium Binding Protein A8  (S100A8), S100A9, S100A12, high-mobility group box-1 (HMGB-1), fibronectin-1 (FN-1), heat shock protein -70 & tenascin-C

diagnosed according to ACR 1990 criteria

Clinical Details of TA Patients (n=24) ParametersAge in years 25.5 (19-58)Gender (Male: Female) 6:18Duration of symptoms in months 6 (0.25-120)Age of onset in years 25 (13-57)Angiotype

Type I

Type IIb

Type III

Type IV

Type V

 

4 (16.67%)

1 (4.17%)

1 (4.17%)

1 (4.17%)

17 (70.84%)ESR at 90 38 (6-71)CRP mg/dl 13 (1-91)ITAS 2 (0-16)Dei.Tak 11 (4-16)Treatment

Treatment naïve

Glucocorticoids

DMARDS

Biological DMARDs

 

12 (50%)

12 (50%)

11(45.84%)

1 (4.17%)

Results

mRNA expression of TLR4 & RAGE in PBMCs

Healthy Controls (HC) (n=19); TA (n=24) ; Median age of controls is 30 (22-48) years and male: female ratio is 1:6.34.

Expressions of mRNA values are shown in the box plot are median (central line), interquartile range (limit of the box) and range (whiskers)

Number of denotes strength of significance; =p<0.05,=p<0.01

0.0001

0.001

0.01

0.1 HCTA

TLR4 RAGE

Rel

ativ

e ex

pres

sion

Endogenous TLR4 ligands

Expressions of mRNA values are shown in the box plot are median (central line), interquartile range (limit of the box) and range (whiskers)

Both left and right y-axes are used, expression levels of S100A9 & S100A12 are depicted using the right y-axis

Number of denotes strength of significance; =p<0.05,=p<0.01

0.1

1

10

100

1000

0.01

0.1

1

10

100

HC

TA

S100A8 S100A9 S100A12

Rel

ativ

e ex

pres

sion

Relative expression

Endogenous TLR4 ligands & Inflammatory mediators

No difference in expression levels of high-mobility group box-1 (HMGB-1) & fibronectin-1 (FN-1)

0.0001

0.001

0.01

0.1

1

10

HMBG1 FN1 HSP70

Rel

ativ

e ex

pres

sion

0.0001

0.001

0.01

0.1

1

0.1

1

10

100

1000

10000

100000

HC

TA

Interleukin-6 Interleukin-8

Rel

ativ

e ex

pres

sion

Relative expression

S100A8/A9 (MRP8/14)(Calprotectin) levels in serum

Serum S100A8/A9 levels in Healthy controls (HC) (n=19) and patients with Takayasu arteritis (TA) (n=24) were measured by commercial ELISA kit from Biolegend

Values are shown in the box plot are median (central line), interquartile range (limit of the box) and range (whiskers)

Serum MRP8/14

HC TA0

2000

4000

6000

8000

10000HCTA

MR

P8/1

4 (n

g/m

l)

S100A12 levels in serum

Serum S100A12 levels in Healthy controls (HC) (n=19) and patients with Takayasu arteritis (TA) (n=24) were measured by commercial ELISA kit CirculexTM

Values are shown in the box plot are median (central line), interquartile range (limit of the box) and range (whiskers)

Serum S100A12

HC TA0

500

1000

1500HCTA

S100

A12

(ng/

ml)

Correlation between S100A8/A9 (MRP8/14)(Calprotectin) and S100A12

r- 0.738p<0.0002

Correlation of Serum S100A8/A9 & S100A12

0 500 1000 15000

5000

10000

15000

S100A12 (ng/ml)

S100

A8/9

(ng/

ml)

• Is S100A12 involved in intracellular function or defect in secretion in TA?

• LPS induced secretion of S100A12 in cell culture supernatant by PBMCs

Am J Respir Crit Care Med (2013) 187(12),1324–1334

LPS induced S100A12 secretion by PBMCs

Delta S100A12 values were calculated by LPS/Control of each subjects

Each circle represents each subjects and connecting line represents their corresponding S10012 secretion levels in Control (without LPS) and LPS treatment on PBMCs

Number of denotes strength of significance; =p<0.05

Relative induction of S100A12

HC TA0

5

10

15

Del

ta S

100A

12

Healthy Controls

Umstimulated LPS0

2000

4000

6000

8000

10000

S100

A12

(pg/

ml)

TA

Umstimulated LPS0

5000

10000

15000

20000

25000

S100

A12

(pg/

ml)

ConclusionIncreased expression of TLR4 and its endogenous TLR4 ligands – S100A8, S100A9, S100A12 in PBMCs of TA

acknowledgement

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