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Multiparameter phospho-flow analysis of lymphocytes in early rheumatoid arthritis: implications for diagnosis and monitoring drug therapy.

Galligan CL, Siebert JC, Siminovitch KA, Keystone EC, Bykerk V, Perez OD, Fish EN - PLoS ONE (2009)

Bottom Line: Stratification by medications revealed that patients receiving leflunomide, systemic steroids or anti-TNF therapy had significant reductions in phospho-specific activation compared with patients not receiving these therapies.Correlative trends between medication-associated reductions in the levels of phosphorylation of specific signaling effectors and lower disease activity were observed.Notably, phosphorylation of these signaling effectors did not distinguish ERA from late RA, suggesting that the activation status of discrete cell populations is already established early in disease.

View Article: PubMed Central - PubMed

Affiliation: Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada.

ABSTRACT

Background: The precise mechanisms involved in the initiation and progression of rheumatoid arthritis (RA) are not known. Early stages of RA often have non-specific symptoms, delaying diagnosis and therapy. Additionally, there are currently no established means to predict clinical responsiveness to therapy. Immune cell activation is a critical component therefore we examined the cellular activation of peripheral blood mononuclear cells (PBMCs) in the early stages of RA, in order to develop a novel diagnostic modality.

Methods and findings: PBMCs were isolated from individuals diagnosed with early RA (ERA) (n = 38), longstanding RA (n = 10), osteoarthritis (OA) (n = 19) and from healthy individuals (n = 10). PBMCs were examined for activation of 15 signaling effectors, using phosphorylation status as a measure of activation in immunophenotyped cells, by flow cytometry (phospho-flow). CD3+CD4+, CD3+CD8+ and CD20+ cells isolated from patients with ERA, RA and OA exhibited activation of multiple phospho-epitopes. ERA patient PBMCs showed a bias towards phosphorylation-activation in the CD4+ and CD20+ compartments compared to OA PBMCs, where phospho-activation was primarily observed in CD8+ cells. The ratio of phospho (p)-AKT/p-p38 was significantly elevated in patients with ERA and may have diagnostic potential. The mean fluorescent intensity (MFI) levels for p-AKT and p-H3 in CD4+, CD8+ and CD20+ T cells correlated directly with physician global assessment scores (MDGA) and DAS (disease activity score). Stratification by medications revealed that patients receiving leflunomide, systemic steroids or anti-TNF therapy had significant reductions in phospho-specific activation compared with patients not receiving these therapies. Correlative trends between medication-associated reductions in the levels of phosphorylation of specific signaling effectors and lower disease activity were observed.

Conclusions: Phospho-flow analysis identified phosphorylation-activation of specific signaling effectors in the PB from patients with ERA. Notably, phosphorylation of these signaling effectors did not distinguish ERA from late RA, suggesting that the activation status of discrete cell populations is already established early in disease. However, when the ratio of MFI values for p-AKT and p-p38 is >1.5, there is a high likelihood of having a diagnosis of RA. Our results suggest that longitudinal sampling of patients undergoing therapy may result in phospho-signatures that are predictive of drug responsiveness.

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Drug therapy affects the phosphorylation levels of signaling effectors in ERA PBMC.Hierarchical clustering heatmap showing the level of statistical significance, as a p value, for the difference in MFI values for each indicated phospho-signaling effector comparing patients on therapy versus patients not on therapy. Statistical differences were calculated by Student's t test.
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pone-0006703-g006: Drug therapy affects the phosphorylation levels of signaling effectors in ERA PBMC.Hierarchical clustering heatmap showing the level of statistical significance, as a p value, for the difference in MFI values for each indicated phospho-signaling effector comparing patients on therapy versus patients not on therapy. Statistical differences were calculated by Student's t test.

Mentions: We next examined the effects of specific disease modifying anti-rheumatic drugs (DMARDs) on the phosphorylation status of 12 phospho-epitopes in ERA patients. As a first screen, patients were grouped according to whether they were receiving a particular DMARD or not, regardless of whether a patient was on monotherapy or not. All patients in the drug study cohort were on some form of drug therapy (refer to Table S1). The effects of the non-steroidal anti-inflammatory drugs (NSAID), methotrexate (MTX), sulphasalazine (SSZ), plaquenil (HCQ) and leflunomide (LEF), systemic steroids (predominantly prednisone), TNF inhibitors (Enbrel or Humira), and intra-articular (IA) steroids, were assessed. Notably, TNF inhibitors, LEF, systemic steroids and MTX therapy resulted in decreased activation of multiple phospho-eptiopes (Figure 6). Significant differences in MFI values were observed in the PBMC of patients on LEF (Figure 6, Figure S2) or systemic corticosteroids (Figure 6, Figure S3), compared to those patients not taking these specific DMARDs. Notably, 3 of the 5 patients on LEF were also receiving Enbrel. Interestingly, the phospho-activation signatures for each drug were not identical and patients on LEF exhibited reductions in p-STAT6 in their CD20+ B cells and reduced p-PLC-γ in their CD4+ and CD8+ T cells, while patients on systemic steroids showed decreases in p-BTK and p-JNK in their CD4+ cells, p-ERK, p-p38 and p-STAT3 in their CD20+ cells, and p-STAT4 in their CD4+ and CD8+ cells. Patients on TNF inhibitors (Enbrel or Humira) exhibited significant reductions in levels of p-AKT, p-BTK, p-ERK, p-JNK, p-p38, p-PLCγ, pSTAT1, p-STAT3, p-STAT5 and p-STAT6 in their CD4+ cells (Figure 6, Figure S4). Reductions in MFI values for p-AKT, p-p38, p-JNK, p-PLCγ, and p-STAT5 were also observed in their CD8+ cells and reductions in p-p38 were observed in their CD20+ cells.


Multiparameter phospho-flow analysis of lymphocytes in early rheumatoid arthritis: implications for diagnosis and monitoring drug therapy.

Galligan CL, Siebert JC, Siminovitch KA, Keystone EC, Bykerk V, Perez OD, Fish EN - PLoS ONE (2009)

Drug therapy affects the phosphorylation levels of signaling effectors in ERA PBMC.Hierarchical clustering heatmap showing the level of statistical significance, as a p value, for the difference in MFI values for each indicated phospho-signaling effector comparing patients on therapy versus patients not on therapy. Statistical differences were calculated by Student's t test.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2724743&req=5

pone-0006703-g006: Drug therapy affects the phosphorylation levels of signaling effectors in ERA PBMC.Hierarchical clustering heatmap showing the level of statistical significance, as a p value, for the difference in MFI values for each indicated phospho-signaling effector comparing patients on therapy versus patients not on therapy. Statistical differences were calculated by Student's t test.
Mentions: We next examined the effects of specific disease modifying anti-rheumatic drugs (DMARDs) on the phosphorylation status of 12 phospho-epitopes in ERA patients. As a first screen, patients were grouped according to whether they were receiving a particular DMARD or not, regardless of whether a patient was on monotherapy or not. All patients in the drug study cohort were on some form of drug therapy (refer to Table S1). The effects of the non-steroidal anti-inflammatory drugs (NSAID), methotrexate (MTX), sulphasalazine (SSZ), plaquenil (HCQ) and leflunomide (LEF), systemic steroids (predominantly prednisone), TNF inhibitors (Enbrel or Humira), and intra-articular (IA) steroids, were assessed. Notably, TNF inhibitors, LEF, systemic steroids and MTX therapy resulted in decreased activation of multiple phospho-eptiopes (Figure 6). Significant differences in MFI values were observed in the PBMC of patients on LEF (Figure 6, Figure S2) or systemic corticosteroids (Figure 6, Figure S3), compared to those patients not taking these specific DMARDs. Notably, 3 of the 5 patients on LEF were also receiving Enbrel. Interestingly, the phospho-activation signatures for each drug were not identical and patients on LEF exhibited reductions in p-STAT6 in their CD20+ B cells and reduced p-PLC-γ in their CD4+ and CD8+ T cells, while patients on systemic steroids showed decreases in p-BTK and p-JNK in their CD4+ cells, p-ERK, p-p38 and p-STAT3 in their CD20+ cells, and p-STAT4 in their CD4+ and CD8+ cells. Patients on TNF inhibitors (Enbrel or Humira) exhibited significant reductions in levels of p-AKT, p-BTK, p-ERK, p-JNK, p-p38, p-PLCγ, pSTAT1, p-STAT3, p-STAT5 and p-STAT6 in their CD4+ cells (Figure 6, Figure S4). Reductions in MFI values for p-AKT, p-p38, p-JNK, p-PLCγ, and p-STAT5 were also observed in their CD8+ cells and reductions in p-p38 were observed in their CD20+ cells.

Bottom Line: Stratification by medications revealed that patients receiving leflunomide, systemic steroids or anti-TNF therapy had significant reductions in phospho-specific activation compared with patients not receiving these therapies.Correlative trends between medication-associated reductions in the levels of phosphorylation of specific signaling effectors and lower disease activity were observed.Notably, phosphorylation of these signaling effectors did not distinguish ERA from late RA, suggesting that the activation status of discrete cell populations is already established early in disease.

View Article: PubMed Central - PubMed

Affiliation: Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada.

ABSTRACT

Background: The precise mechanisms involved in the initiation and progression of rheumatoid arthritis (RA) are not known. Early stages of RA often have non-specific symptoms, delaying diagnosis and therapy. Additionally, there are currently no established means to predict clinical responsiveness to therapy. Immune cell activation is a critical component therefore we examined the cellular activation of peripheral blood mononuclear cells (PBMCs) in the early stages of RA, in order to develop a novel diagnostic modality.

Methods and findings: PBMCs were isolated from individuals diagnosed with early RA (ERA) (n = 38), longstanding RA (n = 10), osteoarthritis (OA) (n = 19) and from healthy individuals (n = 10). PBMCs were examined for activation of 15 signaling effectors, using phosphorylation status as a measure of activation in immunophenotyped cells, by flow cytometry (phospho-flow). CD3+CD4+, CD3+CD8+ and CD20+ cells isolated from patients with ERA, RA and OA exhibited activation of multiple phospho-epitopes. ERA patient PBMCs showed a bias towards phosphorylation-activation in the CD4+ and CD20+ compartments compared to OA PBMCs, where phospho-activation was primarily observed in CD8+ cells. The ratio of phospho (p)-AKT/p-p38 was significantly elevated in patients with ERA and may have diagnostic potential. The mean fluorescent intensity (MFI) levels for p-AKT and p-H3 in CD4+, CD8+ and CD20+ T cells correlated directly with physician global assessment scores (MDGA) and DAS (disease activity score). Stratification by medications revealed that patients receiving leflunomide, systemic steroids or anti-TNF therapy had significant reductions in phospho-specific activation compared with patients not receiving these therapies. Correlative trends between medication-associated reductions in the levels of phosphorylation of specific signaling effectors and lower disease activity were observed.

Conclusions: Phospho-flow analysis identified phosphorylation-activation of specific signaling effectors in the PB from patients with ERA. Notably, phosphorylation of these signaling effectors did not distinguish ERA from late RA, suggesting that the activation status of discrete cell populations is already established early in disease. However, when the ratio of MFI values for p-AKT and p-p38 is >1.5, there is a high likelihood of having a diagnosis of RA. Our results suggest that longitudinal sampling of patients undergoing therapy may result in phospho-signatures that are predictive of drug responsiveness.

Show MeSH
Related in: MedlinePlus