1241 - Differences in Gene Expression Profiles in Rheumatoid Arthritis Peripheral Blood Mononuclear Cells Suggest Apoptosis As Relevant Mechanism for Response to Methotrexate Monotherapy

Monday, November 7, 2011: 9:00 AM-6:00 PM
Hall F2 - Poster Hall (McCormick Place West)
RD Oliveira1, V. Fontana, C. Macedo, CM Junta, Eduardo A. Donadi3, GA Passos and Paulo Louzada-Junior1, 1School of Medicine of Ribeirao Preto - University of Sao Paulo, Ribeirao Preto, Brazil, 2Faculty Med of Ribeirao Preto, Ribeirao Preto, Brazil
Presentation Number: 1241

Background/Purpose: Methotrexate (MTX) remains the standard therapy for rheumatoid arthritis (RA), although about 30% of RA patients do not respond to MTX monotherapy. A better understanding of the mechanisms of MTX action may be useful in identifying those RA patients who are most likely to have benefit from MTX treatment. The aim of the study was to evaluate the gene expression profile in peripheral blood mononuclear cells (PBMC) in RA patients who did not respond to MTX monotherapy.

Method: We evaluated 17 MTX-non-responders (MTX-NR) RA patients using MTX monotherapy (15-25 mg/week). As a control group, we also evaluated eight MTX-responders (MTX-R) RA patients, also under MTX monotherapy (15-25 mg/week). All patients were using stable dose of prednisone (5 mg/day). The classification in responders and non-responders to MTX fulfilled the EULAR response criteria for RA. Total blood was collected and RNA was extracted from peripheral blood mononuclear cells (PBMC) and the complementary DNA microarray was performed. We selected the genes presented at least in 80% of the microarray hybridizations.

Result: Clinical and laboratorial characteristics of the RA patients are shown in Table 1. Microarray analysis showed 535 differentially expressed genes when we compared MTX-NR with MTX-R and the hierarchical clustering analysis showed an unambiguous distinction. To identify general mechanisms of action, we selected genes whose magnitude of difference (fold change) in the gene expression between MTX-NR and MTX-R were higher than 1.3 or lower than 0.7. For the MTX-NR group, we observed four down-regulated genes involved in apoptosis induction, four up-regulated genes involved in apoptosis inhibition, and one up-regulated gene involved with immune response and inflammation (Table 2).

Conclusion: Using analysis of the gene expression profile from PBMC, we observed a unique distinction between MTX-NR and MTX-R patients. These results suggest apoptosis as relevant mechanism involved in the non-response to MTX, providing new gene insights in the pharmacological actions of MTX in the treatment of RA.

Table 1. Clinical and laboratorial characteristics of the RA patients.

Features

MTX-R  (n=8)

MTX-NR (n=17)

p

 Age, mean years (range)

54.7 (37-71)

51 (29-67)

Nsa

Female/Male

4/1

3/1

Nsb

Time of disease (years)

6.6

5.4

Nsa

Smoking (%)

38

53

Nsb

+ RF (%)

88

65

Nsb

 + ACPA (%)

75

82

Nsb

+ HLA-SE(%)

75.0

70

Nsb

DAS28      (mean ± SD)

1.87+0.76

6.47+1.1

<0.0001a

MTX-R: responders to methotrexate; MTX-NR: non-responders to methotrexate; +RF: positive rheumatoid Factor; +ACPA: positive antibodies to citrullinated protein antigen; + HLA-SE: presence of shared epitope of Human Leukocyte Antigen; DAS28: disease activity index including a 28-joint count; Ns= not significant.

Statistical tests: atwo-sample t test,bexact Fisher test

Table 2. Differentially regulated genes in MTX non-responder group when compared with MTX responder group

Gene Symbol

Gene Name

Process involved

Fold change       (MTX-NR vs MTX-R)

Apoptosis

 

 

 

 

HTRA2

HtrA serine peptidase 2

Induction of apoptosis

0.69

CAV1

Caveolin 1

Induction of apoptosis

0.65

CASP8AP2

Caspase 8 associated protein 2

Induction of apoptosis

0.54

PRKDC

Protein kinase, DNA-activated, catalytic polypeptide

Induction of apoptosis

0.49

BCL2A1

BCL2-related protein A1

Inhibition of apoptosis

1.62

MXD1

MAX dimerization protein 1

Inhibition of apoptosis

1.43

TNIP1

TNFAIP3 interacting protein 1

Inhibition of apoptosis

1.35

BTG2

 

BTG family, member 2

Inhibition of apoptosis

1.32

Immune response

 

 

 

 

 

 

 

CCL4

 

Chemokine (C-C motif) ligand 4

Inflammatory response

1.89


Keywords: apoptosis, methotrexate (MTX) and rheumatoid arthritis, treatment

Disclosure: R. Oliveira, None; V. Fontana, None; C. Macedo, None; C. Junta, None; E. A. Donadi, None; G. Passos, None; P. Louzada-Junior, None.