Interleukin (IL)-1 family cytokine in primary immune thrombocytopenia: a comparison with systemic lupus erythematosus-associated thrombocytopenia

Background : Primary immune thrombocytopenia (ITP) is an autoimmune-mediated disorder characterized by decreased platelet count. Systemic lupus erythematosus (SLE) is also an autoimmune disease which thrombocytopenia is a common hematologic manifestation. Interleukin proinflammatory and immunoregulatory mediators. This study aimed to investigate the role of IL-1 cytokines in patients with ITP and SLE and the potential pathophysiologic mechanism to differentiate SLE-associated thrombocytopenia (SLE-TP) from ITP. Methods : Multiplex cytokine assay and real-time polymerase chain reaction (RT-PCR) were used to measure the IL-1 cytokines in 17 newly diagnosed ITP patients, 17 SLE-TP patients, 19 SLE patients without thrombocytopenia (SLE-NTP) and 10 healthy controls. Results : The serum levels of IL- 1β , IL-18, IL-36 α , IL-36 β , IL-36 γ and IL-33 were decreased significantly in ITP patients as compared with SLE-TP, SLE-NTP patients and healthy controls ( p <0.05). There was no significantly difference in the serum level of IL-37 between ITP and SLE-TP patients, however, there is a positive correlation between platelet count with IL-37 level in ITP patients. Our data suggested that serum IL- 1β , IL-18, IL-36 α , IL-36 β , IL-36 γ , IL-33 and IL-37 were involved in the pathogenesis of ITP. Conclusions : Serum IL- 1β , IL-18, IL-36 α , IL-36 β , IL-36 γ and IL-33 could be considered biomarkers to differentiate SLE-TP from ITP patients.


Background
Primary immune thrombocytopenia (ITP) is an autoimmune-mediated acquired bleeding disorder, defined as a platelet count less than 100× 1 0 9 /L without other causes of isolated thrombocytopenia [1,2]. The etiology of ITP is complex and heterogeneous. At present, the diagnosis of ITP is still exclusive diagnosis, and there is lack of molecular markers specific to diagnosis. Multiple mechanisms have been SLE is a complex autoimmune disease and is usually associated with hematological abnormality [3], also with thrombocytopenia. Prevalence of thrombocytopenia in SLE patients has been reported to be 7%-30% [4]. Conversely, prevalence of SLE in all ITP cases in adult is approximately 5%, which is the most common cause of secondary ITP [5]. Sometimes in the early stages of SLE, when there are only symptoms of thrombocytopenia, it is difficult to differentiate between the ITP with SLE [5]. SLE-associated thrombocytopenia (SLE-TP) is defined as a platelet count less than 100×10 9 /L in the absence of any other identifiable cause [6].
The pathogenesis of thrombocytopenia in SLE is heterogeneous and multifactorial.
However, it is widely accepted that increased platelet clearance mediated by autoantibody against platelet contributes to the pathogenesis, which is analogous to the mechanism of ITP. Different from primary ITP, clinical treatment for thrombocytopenia secondary to an identifiable cause is often targeted toward the ongoing disorder. However, the biomarker to differentiate SLE-TP from I TP is still not very clear [7][8][9].
Recently, IL-1 family cytokines have gained extra attention. It has been reported that blocking IL-1, particularly IL-1β, is now the standard of therapy for autoinflammatory diseases [16,17]. Moreover, IL-1β, driving tumor-promoting inflammation in cancer, can be targeted in patients by using an IL-1 receptor antagonist which can be considered a checkpoint inhibitor [18,19]

Patients and controls
Seventeen patients with newly diagnosed primary ITP (10 females and 7 males, Committee and the American College of Rheumatology criteria [5], respectively. Also 10 age-and sex-matched healthy controls were enrolled in the study (6 females and 4 males, median age 40.5 years, range 24-68 years). The study was approved by local Medical Ethics Committees of Zhongshan Hospital of Fudan University.
Written informed consent was obtained from each patient in accordance with the declaration of Helsinki before being included in the study.

Multiplex cytokine assay
Blood samples were obtained from ITP patients, SLE patients and healthy controls.
Washed and resuspended in fetal bovine serum containing 10% dimethyl sulfoxide (DMSO) and stored in -80°C until RNA extraction.

Real-Time Polymerase Chain Reaction (RT-PCR)
Total RNA was isolated from PBMCs of each sample by using Trizol reagent (Invitrogen, USA) and converted into cDNA using a PrimeScript RT Reagent Kit

Statistical analysis
Analysis was performed with the SPSS 19.0 and GraphPad Prism 5.0 for Windows.
For statistical analysis, the levels of cytokine below the detection limits were arbitrarily assigned the values corresponding to the minimum limits. Data were expressed as median (interquartile range). Differences in percentages were evaluated using chi-square analysis or the Fisher's exact tests. Differences among the groups in measured values were analyzed using the one-way ANOVA with the least significant difference test for post-hoc multiple comparisons, Kruskal-Wallis test and Mann-Whitney U test as appropriate. P-values less than 0.05 were considered statistically significant.

Patient characteristics
Seventeen newly diagnosed primary ITP patients with a median platelet count 9×10 9 /L, range 1-22×10 9 /L. Seventeen SLE-TP patients with a median platelet count 66×10 9 /L, range 29-95×10 9 /L. Nineteen SLE-NTP patients with a median platelet count 183×10 9 /L, range 104-327×10 9 /L. Ten age-and sex-matched healthy controls with a median platelet count 247×10 9 /L, range 185-309×10 9 /L. Characteristics of the cases enrolled in the study are shown in Table 1. There were no significant differences in terms of distributions on age and sex among the ITP patients, SLE-TP patients, SLE-NTP patients and the controls (p>0.05).

SLE-NTP patients correlate with disease activity.
In order to understand the serum IL-1 cytokines profile in ITP patients, SLE-TP patients and SLE-NTP patients, eleven cytokines including IL-1α, IL-1β, IL-1Ra, IL-18, IL-36Ra, IL-36α, IL-37, IL-36β, IL-36γ, IL-38 and IL-33 were analyzed ( Table 2). The majority of IL-1α levels were below the detection limits of the assay (data not shown). Six pro-inflammatory cytokines were shown in Figure 1. As shown in Fig.1  To better assess the expression of IL-1 cytokines, we also analyzed the mRNA expression of IL-1 cytokines among patients. What is interesting, there were no significantly differences between ITP and SLE-TP patients, which were not similar to those that observed at the protein levels, leading us to conclude that post-transcriptional regulation such as microRNAs may take part in the pathogenesis of ITP and SLE-TP patients.
In conclusion, the present study indicates that parts of the IL-1 cytokines, such as serum IL-1β, IL-18, IL-36α, IL-36β, IL-36γ, IL-33 and IL-37 were involved in the pathogenesis of ITP. The protein levels of IL-1β, IL-18, IL-36α, IL-36β, IL-36γ and IL-33 rather than mRNA levels could be considered the biomarkers to differentiate SLE-TP from ITP patients. Considering the role of IL-1 cytokines in autoimmune and autoinflammation function, further investigations of IL-1 cytokines are warranted.

Competing interests
The authors declare that there is no conflict of interests regarding the publication of this paper.

Consent for publication
Not applicable.

Availability of data and materials
Not applicable.

Ethics approval and consent to participate
The study was in accordance with the ethical standards formulated in the Helsinki            The   The mRNA expression of IL 1 cytokines in ITP patients SLE TP patients, SLE NTP patients and healthy controls. There were no signi cant ly diffe rences between ITP patients and SLE TP patients. The expression of IL 1β mRNA was decreased signi cantly in SLE TP patients as compared with SLE NTP and healthy controls. The expression of IL 36 β and IL 38 mRNA were increased signi cantly in SLE TP patients as compared with SLE NTP and he althy controls. The expression o f IL 36 γ mRNA was increased signi cantly in ITP patients than healthy controls. The expression of IL Ra mRNA was increased signi cantly in SLE NTP patients than ITP patients, SLE TP patients and healthy controls. p 0.05 were labeled in the gure.