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Differential expression of pro-inflammatory cytokines IL-15Ralpha, IL-15, IL-6 and TNFalpha in synovial fluid from rheumatoid arthritis patients.

Santos Savio A, Machado Diaz AC, Chico Capote A, Miranda Navarro J, Rodríguez Alvarez Y, Bringas Pérez R, Estévez del Toro M, Guillen Nieto GE - BMC Musculoskelet Disord (2015)

Bottom Line: We found higher and significant levels of TNFalpha, IL-6 and IL-15Ralpha but not of IL-15 in RA compared with the OA group.In addition we found correlation between the value of IL-15Ralpha in SF and disease activity score, DAS28.Finally, we found a correlation between IL-15Ralpha-IL-6, IL-15Ralpha-IL-15, but we did not find any correlation between other pairs of studied cytokines in SF.

View Article: PubMed Central - PubMed

Affiliation: Pharmaceutical Division, Center for Genetic Engineering and Biotechnology, Havana, CP 10600, Cuba. alicia.santos@cigb.edu.cu.

ABSTRACT

Background: Pro-inflammatory cytokines are directly implicated in the pathogenesis of Rheumatoid arthritis (RA). Variable clinical response to cytokine targeted therapies as TNFalpha and IL-6, strongly highlights the heterogeneity of inflammatory process in RA. Another cytokine, IL-15 has also been related to the inflammatory process in RA. Recently we described for the first time, the presence of its specific receptor, IL-15Ralpha, in synovial fluid (SF). The aim of this work was to compare the expression profile of IL-15Ralpha, its ligand IL-15, TNFalpha and IL-6 and how these cytokines are correlated in SF from RA patients taking as a reference Osteoarthritis (OA), an articular but not autoimmune disease.

Methods: Synovial fluids were obtained from the knee joints of 60 patients, 30 with confirmed diagnosis of RA and 30 with OA diagnosis. The levels of TNFalpha, IL-6, IL-15 and IL-15Ralpha were measured by ELISA. A statistical analysis was performed with GraphPad Prism v5.0 using the Mann-Whitney U test and Spearman's rank correlation. A cluster analysis was run in MeV software v4.9.0 and differences across clusters were evaluated by an ANOVA including post-test analysis.

Results: We found higher and significant levels of TNFalpha, IL-6 and IL-15Ralpha but not of IL-15 in RA compared with the OA group. Additionally, a high inter-individual variability in the levels of these 4 cytokines was observed in RA, although we identified 4 patients' subgroups by cluster analysis of cytokines concentration in SF. We also found a positive correlation between IL-15Ralpha-IL-6 and IL-15Ralpha-IL-15, but not for other pairs of cytokines in RA. In addition we found correlation between the value of IL-15Ralpha in SF and disease activity score, DAS28.

Conclusions: In our current work we found a high inter-individual variability in the levels of TNFalpha, IL-6, IL-15 and IL-15Ralpha in SF of RA patients and were identified four principal clusters of cytokines concentration in SF, suggesting the importance of identifying disease subset of patients for personalized treatment. Finally, we found a correlation between IL-15Ralpha-IL-6, IL-15Ralpha-IL-15, but we did not find any correlation between other pairs of studied cytokines in SF.

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Cluster analysis of cytokines concentration in SF from RA patients. Hierarchical Cluster analysis of cytokines expression profiles obtained for 30 RA patients (Spearman rank correlation and single linkage algorithm). Dendrogram resulting from clustering the RA SFs (rows) identified four principal groups of patients (C1-C4). The most intense red blocks represent highly over-expressed cytokines and most intense green blocks represent the cytokines with greatest degree of under-expression. Black indicates cytokines whose expression is similar in the AR and OA reference samples. At the right side C3A and C3B denote two subclusters of cluster 3 showing different levels of IL-15Ralpha expression. RF+ indicates patients which are positive to Rheumatoid factor.
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Fig2: Cluster analysis of cytokines concentration in SF from RA patients. Hierarchical Cluster analysis of cytokines expression profiles obtained for 30 RA patients (Spearman rank correlation and single linkage algorithm). Dendrogram resulting from clustering the RA SFs (rows) identified four principal groups of patients (C1-C4). The most intense red blocks represent highly over-expressed cytokines and most intense green blocks represent the cytokines with greatest degree of under-expression. Black indicates cytokines whose expression is similar in the AR and OA reference samples. At the right side C3A and C3B denote two subclusters of cluster 3 showing different levels of IL-15Ralpha expression. RF+ indicates patients which are positive to Rheumatoid factor.

Mentions: Two-dimensional Hierarchical cluster analysis of expression patterns in SF of the four studied cytokines was performed (Figure 2). Four principal clusters of patients were identified and also a high correlation between IL-15Ralpha and IL-6 cytokines. Comparison of the SF cytokine concentrations in each cluster was carried out using the Kruskal-Wallis test with Dunn’s post-test analysis. Cluster 1 includes 7 patients which showed low levels of all cytokines (more significantly IL-6 and IL-15Ralpha) or only high level in one of them; Cluster 2 includes 8 patients expressing significantly high concentration of TNFalpha (C2vs.C3:P < 0.001; C2 vs.C4:P < 0.05) and IL-6 (C2vs.C1:P < 0.001; C2vs.C4:P < 0.01) and most of them express high or mid concentration of IL-15Ralpha and/or IL-15. Cluster 3 includes 12 patients expressing significantly low levels of TNFalpha (C3vs.C2:P < 0.001), significantly high levels of IL-6 (C3vs.C1:P < 0.05) and IL-15Ralpha (C3vs.C1:P < 0.05) and high levels of IL-15. This cluster includes 8 out of 15 patients RF+. Cluster 4 includes 3 patients expressing significantly low levels of TNFalpha (C4vs.C2:P < 0.05) and IL-6 (C4vs.C2:P < 0.01), and positive to IL-15, IL-15Ralpha and RF. Analysis of variance showed significant differences across clusters, which may be indicative of heterogeneity of the inflammation process.Figure 2


Differential expression of pro-inflammatory cytokines IL-15Ralpha, IL-15, IL-6 and TNFalpha in synovial fluid from rheumatoid arthritis patients.

Santos Savio A, Machado Diaz AC, Chico Capote A, Miranda Navarro J, Rodríguez Alvarez Y, Bringas Pérez R, Estévez del Toro M, Guillen Nieto GE - BMC Musculoskelet Disord (2015)

Cluster analysis of cytokines concentration in SF from RA patients. Hierarchical Cluster analysis of cytokines expression profiles obtained for 30 RA patients (Spearman rank correlation and single linkage algorithm). Dendrogram resulting from clustering the RA SFs (rows) identified four principal groups of patients (C1-C4). The most intense red blocks represent highly over-expressed cytokines and most intense green blocks represent the cytokines with greatest degree of under-expression. Black indicates cytokines whose expression is similar in the AR and OA reference samples. At the right side C3A and C3B denote two subclusters of cluster 3 showing different levels of IL-15Ralpha expression. RF+ indicates patients which are positive to Rheumatoid factor.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4359511&req=5

Fig2: Cluster analysis of cytokines concentration in SF from RA patients. Hierarchical Cluster analysis of cytokines expression profiles obtained for 30 RA patients (Spearman rank correlation and single linkage algorithm). Dendrogram resulting from clustering the RA SFs (rows) identified four principal groups of patients (C1-C4). The most intense red blocks represent highly over-expressed cytokines and most intense green blocks represent the cytokines with greatest degree of under-expression. Black indicates cytokines whose expression is similar in the AR and OA reference samples. At the right side C3A and C3B denote two subclusters of cluster 3 showing different levels of IL-15Ralpha expression. RF+ indicates patients which are positive to Rheumatoid factor.
Mentions: Two-dimensional Hierarchical cluster analysis of expression patterns in SF of the four studied cytokines was performed (Figure 2). Four principal clusters of patients were identified and also a high correlation between IL-15Ralpha and IL-6 cytokines. Comparison of the SF cytokine concentrations in each cluster was carried out using the Kruskal-Wallis test with Dunn’s post-test analysis. Cluster 1 includes 7 patients which showed low levels of all cytokines (more significantly IL-6 and IL-15Ralpha) or only high level in one of them; Cluster 2 includes 8 patients expressing significantly high concentration of TNFalpha (C2vs.C3:P < 0.001; C2 vs.C4:P < 0.05) and IL-6 (C2vs.C1:P < 0.001; C2vs.C4:P < 0.01) and most of them express high or mid concentration of IL-15Ralpha and/or IL-15. Cluster 3 includes 12 patients expressing significantly low levels of TNFalpha (C3vs.C2:P < 0.001), significantly high levels of IL-6 (C3vs.C1:P < 0.05) and IL-15Ralpha (C3vs.C1:P < 0.05) and high levels of IL-15. This cluster includes 8 out of 15 patients RF+. Cluster 4 includes 3 patients expressing significantly low levels of TNFalpha (C4vs.C2:P < 0.05) and IL-6 (C4vs.C2:P < 0.01), and positive to IL-15, IL-15Ralpha and RF. Analysis of variance showed significant differences across clusters, which may be indicative of heterogeneity of the inflammation process.Figure 2

Bottom Line: We found higher and significant levels of TNFalpha, IL-6 and IL-15Ralpha but not of IL-15 in RA compared with the OA group.In addition we found correlation between the value of IL-15Ralpha in SF and disease activity score, DAS28.Finally, we found a correlation between IL-15Ralpha-IL-6, IL-15Ralpha-IL-15, but we did not find any correlation between other pairs of studied cytokines in SF.

View Article: PubMed Central - PubMed

Affiliation: Pharmaceutical Division, Center for Genetic Engineering and Biotechnology, Havana, CP 10600, Cuba. alicia.santos@cigb.edu.cu.

ABSTRACT

Background: Pro-inflammatory cytokines are directly implicated in the pathogenesis of Rheumatoid arthritis (RA). Variable clinical response to cytokine targeted therapies as TNFalpha and IL-6, strongly highlights the heterogeneity of inflammatory process in RA. Another cytokine, IL-15 has also been related to the inflammatory process in RA. Recently we described for the first time, the presence of its specific receptor, IL-15Ralpha, in synovial fluid (SF). The aim of this work was to compare the expression profile of IL-15Ralpha, its ligand IL-15, TNFalpha and IL-6 and how these cytokines are correlated in SF from RA patients taking as a reference Osteoarthritis (OA), an articular but not autoimmune disease.

Methods: Synovial fluids were obtained from the knee joints of 60 patients, 30 with confirmed diagnosis of RA and 30 with OA diagnosis. The levels of TNFalpha, IL-6, IL-15 and IL-15Ralpha were measured by ELISA. A statistical analysis was performed with GraphPad Prism v5.0 using the Mann-Whitney U test and Spearman's rank correlation. A cluster analysis was run in MeV software v4.9.0 and differences across clusters were evaluated by an ANOVA including post-test analysis.

Results: We found higher and significant levels of TNFalpha, IL-6 and IL-15Ralpha but not of IL-15 in RA compared with the OA group. Additionally, a high inter-individual variability in the levels of these 4 cytokines was observed in RA, although we identified 4 patients' subgroups by cluster analysis of cytokines concentration in SF. We also found a positive correlation between IL-15Ralpha-IL-6 and IL-15Ralpha-IL-15, but not for other pairs of cytokines in RA. In addition we found correlation between the value of IL-15Ralpha in SF and disease activity score, DAS28.

Conclusions: In our current work we found a high inter-individual variability in the levels of TNFalpha, IL-6, IL-15 and IL-15Ralpha in SF of RA patients and were identified four principal clusters of cytokines concentration in SF, suggesting the importance of identifying disease subset of patients for personalized treatment. Finally, we found a correlation between IL-15Ralpha-IL-6, IL-15Ralpha-IL-15, but we did not find any correlation between other pairs of studied cytokines in SF.

Show MeSH
Related in: MedlinePlus