Longitudinal Patient Analysis Suggests a Positive Correlation of IL-10/IL-6 and FVIII Inhibitor Amount

Abstract

Hemophilia A (HA) is the most common X-linked bleeding disorder caused by a deficiency in coagulation factor VIII (FVIII). Inhibitor development in up to 30% of HA patients is the most challenging complication in HA treatment and has been associated with several patient-related risk factors such as gene mutation and residual plasma factor VIII concentrations. Treatment related risk factors are under debate. However, HCV infections were recently shown to correlate with low level inhibitor abundance, potentially as a result of chemokine and cytokine regulation. Therefore, the aim of this study was to evaluate a potential connection between proinflammatory cytokine levels and FVIII-specific B cell responses in HA patients without infections, but with other comorbidities that might modulate cytokine levels. Thus, several clinical parameters and concentrations of plasmatic proinflammatory cytokines (IL-1ß, IL-6, IL-8, IL-10, IL-12p70, TNF, IFNa, IFNg, TNFa, MCP-,1 IL-17A, IL-18, IL-23 and IL-33) were quantified from different patient samples. At first, the influence of proinflammatory cytokines on inhibitor development was assessed in a HA patient, who had an intron 22 inversion and was also diagnosed with type 1 diabetes. Prophylactic treatment began at the age of 9 months with 30IU/kg Octanate, and after four days of exposure, the formation of inhibitors was initially detected. Longitudinal cytokine analysis in plasma samples of that patient revealed differential regulation patterns of cytokines. IL-1b, IFNg, TNFa, IL-6, IL-10 and IL-12p70 tended to increase upon inhibitor occurrence, whereas other cytokines remained unchanged (MCP-1, IL-8, IL-17A, IL-18 and IL-23) or decreased (IFNa2 and IL-33). Next, a correlation analysis was performed, assuming that proinflammatory cytokines might stimulate B cells to produce antibodies. This analysis involved all cytokines that were found to be elevated upon inhibitor formation. The comparison of clinical and laboratory data from that patient revealed a strong association of inhibitor levels and IL-10 expression (Pearson r = 0,9391; R squared = 0,8818) as well as with IL-6 (Pearson r = 0,9290; R squared = 0,8630). Furthermore, a modest correlation was observed for IL-1b (Pearson r = 0,5277; R squared = 0,2785), IFNg (Pearson r = 0,3413; R squared = 0,1165), IL-12p70 (Pearson r = 0,3547; R squared = 0,1258), and TNFa (Pearson r = 0,3070; R squared = 0,09422). Other proinflammatory cytokines with a rather stable profile upon inhibitor initiation like MCP-1 (Pearson r = -0,06810; R squared = 0,004638) and IL-18 (Pearson r = 0,2245; R squared = 0,05042) seemed not to be directly correlated with inhibitor occurrence. Finally, we aim to investigate the potential impact of IL-10 and IL-6 on inhibitor production and/or FVIII immune tolerization across various patient groups and conditions. Therefore, we will compare patients with detectable inhibitor titers, those who have successfully eradicated inhibitors, and individuals without inhibitor titers (with normal recovery). Preliminary data suggest a positive correlation between both cytokines and active inhibitor presence. However, to reach a definitive conclusion, the sample size will be/should be increased.