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ORIGINAL ARTICLE
Year : 2016  |  Volume : 41  |  Issue : 3  |  Page : 116-120

Role of interleukin-27 in immune thrombocytopenic purpura and its impact on disease response


Clinical Hematology, Oncology and Bone Marrow Transplantation Unit, Internal Medicine Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt

Date of Submission15-May-2016
Date of Acceptance09-Jun-2016
Date of Web Publication27-Dec-2016

Correspondence Address:
Amro M.S. El-Ghammaz
37 Mohamed Korayem Street, Nasr City, Cairo
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-1067.196177

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  Abstract 

Background Aberrant cytokine profiles play important roles in immune thrombocytopenia (ITP) pathogenesis. Interleukin-27 (IL-27) has pleiotropic immunomodulatory effects. However, the role of IL-27 in ITP and its impact on disease response are still controversial.
Patients and methods This study included 60 adult ITP patients [20 de-novo patients (group 1), 20 with complete response (CR) to corticosteroids (group 2), and 20 with refractory ITP (group 3)]. Serum IL-27 level was assessed in all patients using enzyme-linked immunosorbent assay.
Results The mean IL-27 for all patients was significantly higher than that for controls (P<0.001). There were significant differences in mean IL-27 levels between group 1 and group 2 (P=0.002) and between group 2 and group 3 (P=0.030). There was a significant negative correlation between IL-27 level in all studied patients and platelet count (P=0.003). A serum level of IL-27 of 32 pg/ml had a sensitivity of 100% and specificity of 90% in differentiating de-novo ITP patients from healthy controls as detected by means of receiver operating characteristic curve. Moreover, a serum level of IL-27 of 72.5 pg/ml had a sensitivity of 65% and specificity of 75% in differentiating refractory patients from those with CR.
Conclusion Serum IL-27 is significantly elevated in ITP patients (de novo, in CR, and refractory) and it can be used as a predictor for disease occurrence and to a lower extent for its responsiveness to corticosteroid therapy.

Keywords: immune thrombocytopenic purpura, interleukin-27, prediction, response state


How to cite this article:
Gad Allah HA, Moussa MM, El-Ghammaz AM, Ali BS. Role of interleukin-27 in immune thrombocytopenic purpura and its impact on disease response. Egypt J Haematol 2016;41:116-20

How to cite this URL:
Gad Allah HA, Moussa MM, El-Ghammaz AM, Ali BS. Role of interleukin-27 in immune thrombocytopenic purpura and its impact on disease response. Egypt J Haematol [serial online] 2016 [cited 2020 Aug 5];41:116-20. Available from: http://www.ehj.eg.net/text.asp?2016/41/3/116/196177


  Introduction Top


Primary immune thrombocytopenia (ITP) is a common autoimmune disorder characterized by immune-mediated accelerated platelet destruction and suppressed platelet production and resulting in isolated thrombocytopenia [1]. It has a significant incidence of about 3.3 per 100 000 adults/year [2]. ITP is rarely fatal. Adult ITP typically has an insidious onset and rarely resolves spontaneously [3]. Concepts surrounding the mechanisms of thrombocytopenia in ITP have shifted from the traditional view of increased platelet destruction mediated by autoantibodies to more complex mechanisms in which impaired platelet production, T-cell-mediated effects, and disturbed cytokine profiles play a role [4],[5].

Interleukin-27 (IL-27), a heterodimeric cytokine composed of Epstein–Barr virus-induced gene 3 (Ebi3) and IL-27p28, belongs to the IL-6/IL-12 family cytokines. Its receptor is composed of gp130 and IL-27 receptor α chain (IL-27Rα, also known as WSX1 or TCCR) that activates the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway and the mitogen-activated protein kinase (MAPK) pathway [6]. IL-27 is a cytokine with both proinflammatory and anti-inflammatory effects. There is accumulating evidence suggesting various roles of IL-27 in autoimmune diseases in humans (e.g. rheumatoid arthritis, Crohn’s disease, psoriasis, and multiple sclerosis) [7]. Many studies focused on the role of IL-27 in ITP [8],[9],[10]. However, the role of IL-27 in ITP and its impact on disease response have not been fully elucidated and are still controversial. In this study, we assessed the serum level of IL-27 in adult ITP patients at diagnosis and in different states of response in comparison with healthy controls to evaluate its impact on treatment response.


  Patients and methods Top


Patients

This study comprised 60 adult ITP [20 de-novo patients (group 1), 20 patients with complete response (CR) to corticosteroids (group 2), and 20 patients with refractory ITP (group 3)]. Twenty age-matched and sex-matched controls were also included in the study. All patients were diagnosed according to the diagnostic criteria of ITP described in the international consensus report published in 2010 [5]. The patients were enrolled in this study between March 2015 and January 2016. The study excluded patients with acute or chronic inflammation, associated autoimmune disease and/or malignancy, patients with Evans syndrome, and patients with secondary ITP.

Definitions

CR was defined as a platelet count of at least 100×109/l measured on two occasions more 7 days apart and the absence of bleeding [11]. Refractory ITP was defined as failure of achievement of response to initial treatment with glucocorticoids and also to splenectomy together with the presence of a risk for bleeding that in the opinion of the attending physician requires therapy [12].

Serum interleukin-27 level assessment

Serum level of IL-27 was assessed using IL-27 subunit α ELISA kit (E0385h, EIAab, Wuhan, China). Briefly, the microtiter plate provided had been precoated with an antibody specific to IL-27 subunit α. The samples were then added to the microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for IL-27 subunit α, and avidin conjugated to horseradish peroxidase was added to each microplate well and incubated. Thereafter, a tetramethyl benzidine substrate solution was added to each well. Only those wells containing IL-27 subunit α, biotin-conjugated antibody, and enzyme-conjugated avidin exhibited a change in color. The enzyme–substrate reaction was terminated by the addition of a sulfuric acid solution and the color change was measured spectrophotometrically at a wavelength of 450±2 nm. The concentration of IL-27 subunit α in the samples was then determined by comparing the optical density of the samples with the standard curve. The detection range was 15.6–1000 pg/ml.

Statistical methods

Descriptive statistical analysis of variables was carried out (mean, SD, median, range, number and percentage). Comparison of different variables was made using the analysis of variance test (for continuous variables) and the χ2-test (for categorical variables). Spearman correlation coefficients were used to assess the relation between two quantitative parameters in the same group. Receiver operating characteristic (ROC) curve was used to assess best cutoff point for serum level of IL-27 to discriminate between de-novo ITP patients and controls and between refractory patients and those in CR together with calculation of area under the curve (AUC), sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of serum IL-27. Statistical significance was determined at the 0.05 level. All P values were two-sided. Standard computer program SPSS for Windows, release 17.0 (SPSS Inc., Chicago, IL, USA) was used for data entry and analysis.

Ethical issues

All procedures followed were in accordance with the ethical standards of the ethical committee of Faculty of Medicine of Ain Shams University on human experimentation and with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was obtained from all participants included in the study.


  Results Top


Baseline patient characteristics

The mean age of patients was 31.37±5.99 years and that of controls was 30.95±7.19 years (range=20–44 years). There were 53 female patients (88.3%) and 16 female controls (80%). The patients and controls were matched as regards age and sex (P=0.798 and 0.349, respectively). The baseline patient characteristics are summarized in [Table 1].
Table 1 Baseline patient characteristics

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Serum interleukon-27 in patients and controls

The mean IL-27 for all patients was 113.4 pg/ml (range=22.5–270 pg/ml) and that for controls was 13.9 pg/ml (range=2–69 pg/ml). There was a significant difference between the two levels (P<0.001) ([Figure 1]). The mean IL-27 level was 145.25 pg/ml (range=37.5–270 pg/ml) for group 1, 70.15 pg/ml (range=22.5–270 pg/ml) for group 2, and 124.8 pg/ml (range=22.5–270 pg/ml) for group 3. There were significant differences in mean IL-27 levels between group 1 and group 2 (P=0.002). Moreover, there was a significant difference in mean IL-27 between group 2 and group 3 (P=0.030). However, there was a nonsignificant difference in mean IL-27 between group 1 and group 3 (P=0.452). However, there were significant differences in mean IL-27 levels between groups 1, 2 and 3 and controls (P<0.001, P<0.001, and P<0.001, respectively).
Figure 1 Comparison of serum interleukin-27 (IL-27) in patients and controls [mean IL-27=113.4 vs. 13.9 pg/ml (P<0.001)].

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Correlation of serum interleukin-27 with platelet count and other variables

There was a significant negative correlation between IL-27 level and platelet count (r=−0.375; P=0.003) ([Figure 2]). However, there was no significant correlation between IL-27 and platelet count in variable groups (for group 1, r=−0.082 and P=0.732; for group 2, r=−0.357 and P=0.122; and for group 3, r=−0.081 and P=0.733). As regards other variables, there was a nonsignificant correlation between IL-27 level and age, total leukocytic count, and hemoglobin (r=−0.037 and P=0.781; r=−0.102 and P=0.437; and r=−0.044 and P=0.741, respectively).
Figure 2 Correlation of serum interleukin-27 (IL-27) with platelet count in immune thrombocytopenia (ITP) patients. There was a significant negative correlation (r=−0.375; P=0.003).

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Sensitivity, specificity, positive predictive value, and negative predictive value of interleukin-27 as a screening test for immune thrombocytopenia diagnosis and response to first-line treatment

A serum level of IL-27 of 32 pg/ml had a sensitivity of 100% and specificity of 90% in differentiating de-novo ITP patients from healthy controls as detected by means of ROC curve, with a PPV of 90.9% and NPV of 100% (AUC=0.985; 95% confidence interval=0.000–1.000; P<0.001) ([Figure 3]). Moreover, a serum level of IL-27 of 72.5 pg/ml had a sensitivity of 65% and specificity of 75% in differentiating patients in CR from refractory patients as detected by means of ROC curve, with a PPV of 72.2% and NPV of 68.2% (AUC=0.683; 95% confidence interval=0.514–0.851; P=0.048) ([Figure 4]).
Figure 3 Receiver operating characteristic (ROC) curve for serum level of interleukin-27 (IL-27) in de-novo immune thrombocytopenia (ITP) patients and healthy controls. The optimum cutoff point of IL-27 for maximum sensitivity (100%) and specificity (90%) is 32 pg/ml.

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Figure 4 Receiver operating characteristic (ROC) curve for serum level of interleukin-27 (IL-27) in patients in complete response (CR) and refractory patients. The optimum cutoff point of IL-27 for maximum sensitivity (65%) and specificity (75%) is 72.5 pg/ml.

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  Discussion Top


The pathophysiological significance of IL-27 has been investigated in Th1/Th17-mediated inflammatory diseases, such as rheumatoid arthritis [13], inflammatory bowel disease [14], and systemic lupus erythematosus [15]. Primary ITP is an autoimmune disease with many immune dysfunctions [16]. ITP has shown a Th1 dominant profile [17] and upregulated Th17 expression [18]. IL-27 transgenic mice exhibited an increased number of megakaryocytes [19]. However, until now the role of IL-27 in ITP has not been fully elucidated and is still controversial. A study analyzed the plasma level of IL-27 in ITP patients and demonstrated that plasma and mRNA expression levels of IL-27 in ITP patients with active disease was significantly lower than that in healthy controls or patients in remission, which was not in agreement with our results [8]. This difference can be attributed to the inclusion of patients who have received other modalities of treatment in the other study, including vincristine, danazol, rituximab, and thrombopoietin receptor agonists, raising the suspicion of the effect of these therapies on cytokine levels.

In contrast, another study indicated that the levels of IL-27 in the plasma of untreated active ITP patients were higher than that in normal controls, which is in agreement with our results [20]. Moreover, authors in the latter study evaluated the contribution of IL-27 to T-cell differentiation and found that IL-27 induced the differentiation of T helper-1 and T cytotoxic-1 cells [20]. They concluded that IL-27 might play an important role in the pathogenesis of ITP by inducing the production of proinflammatory cytokines [20]. This study points that IL-27 might be involved in the pathophysiological process of ITP and that modulation of IL-27 might provide therapeutic benefits for ITP.

Taking into consideration the heterodimeric nature of IL-27 [21] and the association of p28 promoter polymorphisms with certain diseases such as asthma [22], inflammatory bowel diseases [23], chronic obstructive pulmonary disease [24], and breast cancer [25], Zhao et al. [26] investigated the association of single nucleotide polymorphisms of the IL-27 genes with the risk for ITP. Unexpectedly, they found that there was no significant difference in genotype and allele distribution between ITP patient sand the healthy controls [26]. This indicates that the IL-27 polymorphism may not be involved in susceptibility to ITP.

We observed a significantly higher mean IL-27 level in de-novo patients than in responding patients to corticosteroids and in healthy controls. Moreover, there was a significantly higher mean IL-27 level in refractory patients than in responding patients and in healthy controls. Our results are in agreement with those of Li et al [10]. We differed from that study in that we still found a significant difference between responding patients and healthy controls. This difference can be attributed to the use of a different analysis tool in the other study (flow cytometer using a BD FACS-Aria instrument) and to the nonunified timing of sample collection for assessment of response in our study in comparison with the other study in which all blood samples were collected at 2 weeks following dexamethasone treatment [10]. This finding raises the suspicion about the possibility of relapse of those remitted patients with persistently elevated IL-27 and warrants longer follow-up. Moreover, it is in accordance with the chronic pathology of ITP.In the present study, although an inverse correlation was detected between IL-27 level and platelet count in all patients, there was no significant correlation between IL-27 and platelet count in variable groups. This finding can be attributed to the small number of patients in each group. Our finding is in agreement with that reported by Li et al. [10], who stated that there was no correlation between IL-27 level and platelet count before and after treatment in patients with de-novo active ITP. However, other studied cytokines correlated significantly with platelet count, suggesting that an interaction of many cytokines play a role in the pathogenesis of ITP [10].

Using the ROC curve, serum IL-27 is helpful as a screening test for predicting the risk for ITP occurrence. However, serum IL-27 has been found to be less useful for the prediction of response to first-line corticosteroids. To our knowledge, no other study analyzed the sensitivity and specificity of IL-27 for prediction of ITP risk or response rate. However, studies on larger number of patients are needed to confirm these results.


  Conclusion Top


IL-27 is significantly elevated in patients with ITP and this potentiates the previous suggestions about the role of IL-27 in the pathogenesis of ITP, but its exact role still requires to be elucidated. Moreover, we suggest using IL-27 as a predictor for disease occurrence and to lower extent for responsiveness to corticosteroid therapy but this needs to be confirmed in larger studies. We encourage performing studies for longer follow-up periods including patients with relapsed ITP to detect the impact of IL-27 on relapse risk. Finally, we recommend studying of IL-27 as a potential target of therapy in ITP patients especially in resistance state.

Acknowledgements

The manuscript has been read and approved by all authors. The requirements for authorship have been met. Each author believes that the manuscript represents honest work.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

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