Differential expression of coagulation and inflammation genes in ITP patients before and after treatment with thrombopoietin receptor agonists Free

Introduction Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by low platelet count resulting from immune-mediated increased platelet destruction and decreased production. Despite increased bleeding risk, ITP patients also appear to have increased thrombotic risk, and treatment with thrombopoietin receptor agonists (TPO-RA) can further increase that risk. Different immune and hemostatic dysregulations have been reported in ITP on cellular and protein levels; however, few studies have investigated differential expression of coagulation and inflammation genes in ITP patients.

Aim To explore the differential expression of coagulation and inflammation genes in ITP and to study changes in their expression with TPO-RA treatment.

Method: Global gene expression profiling was performed in 16 adult patients with chronic ITP scheduled for TPO-RA and 10 healthy age- and sex-matched controls using Clariom™ D microarrays. Whole blood samples were collected in PAXgene RNA tubes prior to TPO-RA initiation and 2, 6 and 12 weeks after treatment. In total 54 blood samples were collected (baseline n=16, 2 weeks n=14, 6 weeks n=13, 12 weeks n=10). We used limma un-paired t-test to conduct a differential gene expression analysis of 60 coagulation and inflammation genes, comparing patients before and after treatment with controls. Temporal gene expression patterns were visualized using self-organizing maps.

Results Median age for patients was 56 years and for controls 55 years; 56% of patients and 60% of controls were males. Nine patients started romiplostim and 7 eltrombopag; 10 patients were on steroids at TPO-RA initiation. Of the16 patients, 12 (75%) responded (platelet count > 50·10⁹/L).

Before initiating TPO-RA treatment, 10 genes were significantly (p < 0.05) upregulated in ITP patients compared with controls:RNASE2, RNASE3, F11, IL10, IL1R2, BCL2L1, BCL2A1, SERPINF1, SERPINB2 and GP9, while 15 genes were significantly downregulated: ST6GAL1 (encodes beta-galactoside alpha-2,6-sialyltransferase 1), IL2RB, TGFB1, BCL2, PDGFD, CX3CR1, CREB1, CXCR6, NFKB1, STAT5A, HIF1A, TBX21, PROCR, F7 and MTHFR.

Two weeks after TPO-RA treatment, several upregulated genes became downregulated: RNASE2, RNASE3, BCL2L1 and IL10, while several downregulated genes became upregulated: CXCR6, ST6GAL1, TGFB1, SELP (encodes P-selectin), PROS1, STAT5A, BCL2 and CX3CR1. After 6 weeks of treatment TGFB1, SELP, CXCR6, PDGFD, PROS1, F8 and MTHFR were upregulated while IL10 and F11 remained downregulated. After 12 weeks of treatment TGFB1, SERPINE1 (encodes PAI-1), HIF1A, CXCR6, PROS1 and F8 were upregulated, and RNASE2, RNASE3 remained downregulated.

Discussion and conclusions Before initiating TPO-RA treatment, upregulation of BCL2L1, BCL2A1 and RNASE2/3 could collectively be associated with suppression of T-regulatory cells, increased survival of the autoreactive T/B-lymphocytes, and autoantibody production promoting immune activation and inflammation, while upregulation of IL10 and IL1R2 may be a compensatory mechanism to limit inflammation. Downregulation of IL2RB, TGFB1, STAT5A, NFKB1, and BCL2 could contribute to impaired T-regulatory cells and immune intolerance promoting autoantibody production. Downregulation of ST6GAL1 could contribute to increased platelet desialylation thus increased platelet clearance. These were all in line with changes in cytokines and immune phenotypes seen in ITP. Upregulation of F11, SERPINs and GP9 could be a compensatory mechanism to prevent bleeding.

After TPO-RA treatment, upregulation of TGFB1, STAT5A and ST6GAL1 could collectively contribute to less suppression of T-regulatory cells and enhanced survival of T-cells, enhanced platelet production and decreased platelet clearance. The upregulation of F13A1, F8, SELP, and SEPINE1 reflect coagulation and platelet activation and impaired fibrinolysis, in line with multiple reports of increased coagulation activation, soluble P-selectin and PAI-1 in ITP leading to a procoagulant environment.

Our findings support immune and hemostatic dysregulation in ITP on the gene expression level leading to an unbalanced immune response promoting inflammation, autoantibody production and increased platelet clearance and compensatory upregulated hemostasis. Changes in gene expression after TPO-RA treatment reflected a shift towards greater immune tolerance leading to increased platelet counts, but also a more procoagulant state which could contribute to increased thrombotic risk.