Association Of Cardiac Iron By T2* With Innate Immune Markers In Transfusion-Dependent Thalassemia Patients Undergoing Combined Chelation Therapy
Authors: Walter Patrick B , Higa Annie , Ng Vivian , Weyhmiller Marcela G , Slater Nick R , Evans Patricia , Porter John B , Sweeters Nancy , Price Jackson , Manji Alisha , Killilea David , Lal Ashutosh , Vichinsky Elliott 
Published: October 2019

Abstract

Introduction
The thalassemias are inherited anemias sometimes characterized by severe transfusion dependence that can lead to extra-hepatic cardiac iron overload, causing cardiomyopathy. Despite improved chelation therapies, patients with transfusion-dependent thalassemia still endure cardiomyopathy and chronic inflammation. The innate immune system provides the first line of defense against infection and specificity depends on pattern recognition receptors (PRRs) specific to microbial pathogens. One class of PRR called the toll-like receptors (TLRs) interacts with CD14 on innate immune cells transducing the signal for bacterial lipopolysaccharide. Another cell surface protein that is not a PRR, but aids phagocytosis and is important in granulocytes function and chemotaxis is the adhesive polysaccharide antigen, CD15. The role that excess iron plays in determining expression level of these innate immune proteins is unknown. Thus, the goal in these studies is to investigate the relationship of cardiac iron overload and its chelation to innate immune cell expression of TLR4 and CD15 in patients with transfusion-dependent thalassemia.

Patients and Methods
Eighteen patients with transfusion dependent thalassemia (11 – 29 years old) (participating in the Novartis sponsored CICL670AUS24T) were enrolled in a substudy investigating innate immunology (Novartis sponsored CICL670AUS42T). Patients were investigated at baseline, then after 6 months and one year of combined chelation therapy with deferasirox and deferoxamine. Fasting blood samples were obtained after a 72 hr washout with no chelators. Fourteen healthy controls (10 - 35 yrs old) were also enrolled. Changes in LIC (ferritometer), cardiac function (MRI) and myocardial iron (MRI T2*) were monitored. Peripheral blood mononuclear cells (PBMCs) and granulocytes were isolated from blood samples using density gradients. Monocytes and granulocytes were further purified using antibody-linked magnetic microbeads. Highly enriched populations of CD14+ monocytes and CD15+ granulocytes were verified by flow cytometry. The expression level of CD15 and TLR4 was determined.

Results
Previously we found that transfusion-dependent thalassemia patients had 37% higher TLR4+ neutrophils than control patients and a smaller percentage of CD15+ neutrophils. We have also observed a decrease in TLR4 expression during the course of combined chelation therapy on neutrophils but not monocytes, indicating that TLR4 is differentially modulated on neutrophils compared to monocytes. Now we find that these flow cytometry parameters show significant relationships to markers of iron burden. The percentage of TLR4+ monocytes was related to liver iron concentration (r=- 0.49, p = 0.039), ferritin concentration (r=-0.47, p = 0.049), serum iron level (r=0.61, p = 0.008), and total iron binding capacity (TIBC; r=0.51, p = 0.021), while the percentage of CD15 positive neutrophils predicted myocardial iron, as measured by MRI T2* (r= 0.69, p<0.001), and left ventricular ejection fraction (LVEF; r=0.50, p = 0.022). Lastly, analysis of covariance, controlling for age and gender, revealed that the number of CD15+ neutrophils increased significantly from baseline (90.84%) to 52 weeks (95.09%) of combined chelation therapy (p = 0.007).

Conclusions
This study found evidence that the innate immune system may be modulating iron trafficking not only to the liver but to the heart as well. The negative correlation between LIC and TLR4 expression suggests that severe iron overload may lead to heptocellular damage causing monocyte dysfunction, the pathology of which could be due to altered TLR4 expression. This relationship may also be driving the positive correlation observed between TLR4 expression and TIBC. CD15 seems to play an important role in cardiac health as it is positively correlated to LVEF and MRI T2*. This relationship is further validated by our previous finding that transfusion-dependent thalassemia patients had a smaller percentage of CD15+ neutrophils, which we now show improved during one year of combination chelation therapy. Taken together, this suggests that chelation therapy may enhance cardiac health by increasing the percentage of CD15+ neutrophils.

Disclosures:
Walter: Novartis: Research Funding. Porter:Novartis: Consultancy, Honoraria, Research Funding; Shire: Consultancy, Honoraria; Celgene: Consultancy. Vichinsky:Novartis: Honoraria, Research Funding.