Differential Expression of IL-17 Correlates with Clinical and Pathological Features of ITP

Introduction:

Immune thrombocytopenic purpura (ITP) is an acquired autoimmune disorder, which is characterized by megakaryocyte (MgK) inhibition/destruction in the bone marrow (BM) and/or platelet in the spleen and liver. Abnormal MgKs, which is recognized by autoantibodies, are phagocytosed by macrophages. Recently, it has been demonstrated that interleukin-(IL-)17-mediated cells contribute to the imbalance of cellular immunity in the pathogenesis of ITP. Studies of pathological findings in BM of patients with ITP using immunohistochemistry (IHC) are limited. Therefore, we examined samples of BM clots to test the hypothesis that IL-17-mediated immunological changes are involved in the BM of patients with ITP.

Methods:

We enrolled 35 patients with a median age of 61 years (range, 19-91 years), who were retrospectively selected at random and were referred to our hospital between 2005 and 2016. All patients were diagnosed with chronic ITP. Twenty-five patients underwent first- or second-line therapies such as glucocorticosteroids, cyclosporine A, azathioprine, danazol and intravenous immunoglobulin and/or splenectomy as conventional therapies. Twenty-three patients who could not undergo those therapies were administered with eltrombopag. BM clots were obtained before treatment and stained with hematoxylin and eosin for morphological examination. Formalin-fixed and paraffin-embedded specimens were used for IHC using antibodies for the following markers: CD3, CD4, CD8, CD20 (L26), CD25, CD68, CD163, and IL-17. Pathological findings and clinical information including laboratory data were compared between the patients with ITP and control subjects, which were obtained from 11 untreated malignant lymphoma patients without the infiltration of lymphoma cells in BM. We also compared those between 2 groups: an IL-17-high-expression group and IL-17-low-expression group.

Results:

A univariate analysis revealed increased cells expressing the following markers in patients with ITP than in the control subjects: CD20 (5.80 ± 3.46% vs. 3.06 ± 1.90%; p= 0.02), CD68 (11.3 ± 11.1% vs. 3.14 ± 3.23%; p= 0.02), CD163 (5.10 ± 2.74% vs. 2.13 ± 1.10%; p= 0.001), and IL-17 (3.13 ± 1.48% vs. 1.49 ± 1.00%; p= 0.002) and decreased cells expressing following markers: CD4 (0.33 ± 0.71% vs. 1.96 ± 1.13%; p < 0.0001) and CD25 (0.09 ± 0.18% vs. 0.23 ± 0.10%; p= 0.01). The difference of cells expressing CD3 (p= 0.96) and CD8 (p= 0.41) were not statistically significant. There were significant correlations between CD68 and CD163 expressions (r= 0.61). The expressions of both CD68 and CD163 showed correlations with IL-17 expressions (r= 0.61, 0.51). We found significant increased expressions of CD3 (3.98 ± 2.96% vs. 6.37 ± 3.33%; p= 0.03), CD25 (0.05 ± 0.08% vs. 0.17 ± 0.22%; p= 0.03), CD68 (7.84 ± 5.09% vs. 17.3 ± 15.6%; p= 0.01), and CD163 (4.23 ± 2.37% vs. 6.57 ± 2.77%; p= 0.01) in the IL-17-high-expression group (N = 13) compared with IL-17-low-expression group (N = 22). The expressions of CD4 (p= 0.98), CD8 (p= 0.54), and CD20 (p= 0.85) were not statistically significant. Responses to eltrombopag were significantly better in the IL-17-low-expression group than in the IL-17-high-expression group (p= 0.03).

Conclusions:

By IHC analysis, we report for the first time increased CD68- and/or CD163-expressing cells in the BM of patients with ITP. Those are possibly associated with the pathogenesis of ITP. Our results indicate that CD68- and/or CD163-expressing macrophages and monocytes, but not lymphocytes are involved in deregulated expression of IL-17 in ITP. We demonstrate that the response of eltrombopag is significantly better in the IL-17-low-expression group. These results indicate that patients with ITP, achieving remission early, might belong to the category of the low-IL-17, CD68, and CD163 expressions. We suggest that IHC staining in patients with ITP may be a potential indicator to understand the mechanism of ITP and to predict the prognosis for treatment.