Cytokine Fluctuations in Immune Thrombocytopenia (ITP) over Time; Insights into the Pathogenesis and Evolution of the Disease
Authors: Adams George , Graebner Lennart , Sayed Anwar , Vandrovcova Jana , Glaser Alice , Paul Deena , Bussel James B. , Cooper Nichola 
Published: October 2019

Abstract
Background: Immune thrombocytopenia (ITP) is an autoimmune condition characterised by an isolated thrombocytopenia at the exclusion of any other causes. The prevailing paradigm for the pathogenesis of ITP is that it is caused by IgG auto-antibodies against platelet receptors. There is however good evidence that dysregulation of the cellular immune system is a major contributing factor. In particular it has been hypothesised that the cellular immune system has a role in perpetuating the disease although how this occurs is unclear. For this reason by understanding the cellular immune system in ITP we may be able to develop more targeted treatments for patients with refractory or resistant disease. Our aim was to investigate the alterations in the cellular immune system by measuring a selection of cytokines known to be associated with different T-cell immune responses (Th1, Th2, Th17 helper cell responses) in patients with ITP and look at how these changed over the course of the disease. Methods: Using a chromogenic assay we measured serum cytokine concentrations from 51 samples from 37 different patients with ITP and 15 healthy control subjects. Each sample was processed in duplicate. We selected to measure cytokines associated with key T-helper cell responses. These included IFNγ, IL-12, IL-2 (Th1 helper cell responses), IL-4, IL-5, IL-10, IL-13 (Th2 helper responses), IL-17 (Th17 helper responses) as well as TNFα, IL6, IL1b, EGF, VEGF, GCSF and GM-CSF. The platelet counts in our patient cohort ranged from 1 to 345x109/L. Samples were taken at different time points, ranging from 5 days to 12,753 days from diagnosis. Primary component analysis (PCA) was used to explore cytokine patterns within the ITP population and directed analysis using partial least square-discriminate analysis (PLS-DA) was used to identify which cytokines altered with the platelet count. Factor Analysis (FA) was used to distinguish which cytokines showed redundant variance. Finally Least-square non-parametric regression was used to plot cytokines levels (pg/ml) versus time from diagnosis (days) in a model curve.
Results: Multivariate data analyses (PCA, PLS-DA) showed that patient samples had particularly marked differences in IL-12, IL-17, VEGF and IFNα when compared to controls. IL-17 and VEGF were much higher compared to controls (IL17 vs. controls; 17.2±5.0 vs. 4.75±0.0 pg/ml, VEGF vs. controls; 11.3 ±4.3 vs. 4.3 ±3.0 pg/ml) whereas IL-12 and IFNα where suppressed (IL-12 vs. controls; 119±100 vs. 554± 190pg/ml, IFNα vs. controls; 50±38 vs. 176±74pg/ml). Using Factor Analysis (FA) we also identified that IL-12, IFNα, IFNγ and TNFα had significant co-variance suggesting a common link whereas IL-17 was independent of other cytokines. From our PLS-DA model IL-1b and RANTES had the strongest correlation with platelet count, with both cytokines falling with the platelet count. The remaining cytokines did not show a strong correlation with platelet count. When plotted against time from diagnosis (in days) a subset of cytokines (IFNγ, IL-7, TNFα, GCSF, VEGF and IL-6) showed a marked rise from diagnosis with a continued increase only to peak after 2-3 years from diagnosis. They then declined slowly, reaching a baseline 10 years after diagnosis (Figure 1). In contrast, the remaining cytokines within the panel remained predominately flat (although still higher than controls) and unvaried over the time-course.
Conclusion; Our findings suggests that the cell-mediated immune system is significantly altered in ITP with a particularly polarised cytokine pattern in keeping with a suppressed Th1 profile (including IL-12 and IFNγ level) and a heightened Th17 response (IL-17). This is different from published results of a skew towards a Th1 profile and may reflect the high proportion of patients with chronic disease in this cohort. We also demonstrate for the first time that there is a fluctuating pattern in the cytokine profile of patients with ITP over time with a sharp onset at the point of diagnosis presumably in response to some unknown stimulus and this subsequent rise continues (despite treatments) and evolves over years as opposed to weeks or months. These findings cast a new light onto a disease which until recently has been thought to be largely antibody mediated. Further evaluation is underway to establish whether a specific cell subset is responsible for the cytokine release and how this contributes to disease.

Disclosures
Bussel: Physicians Education Resource: Speakers Bureau; Boehringer Ingelheim: Research Funding; Prophylix Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Immunomedics: Research Funding; Symphogen: Membership on an entity's Board of Directors or advisory committees; BiologicTx: Research Funding; Momenta Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Protalex: Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Research Funding; Sysmex: Research Funding; Cangene: Research Funding; UpToDate: Patents & Royalties; Rigel Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ligand: Membership on an entity's Board of Directors or advisory committees, Research Funding; Shionogi: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Cooper:Amgen Inc.: Consultancy, Honoraria, Other: Received honoraria for speaking at educational meetings ; GlaxoSmithKline: Consultancy, Honoraria, Other: Received honoraria for speaking at educational meetings ; Eisai: Consultancy, Honoraria, Other: Received honoraria for speaking at educational meetings; Imperial College BRC: Research Funding; National Organization for Rare Disorders: Research Funding; UK ITP support association: Research Funding.