Melissa Kleinau1, Mohammed Massumi2, Lysa Langevin2, Paul Tsoukas2, Harper Cheng2, Suzanne Tam2, Trang Duong2 and Rae Yeung3, 1University of Toronto, Toronto, ON, Canada, 2Peter Gilgan Centre for Research and Learning, Toronto, ON, Canada, 3The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada
Background/Purpose: Kawasaki Disease (KD) is the leading cause of acquired heart disease in children in developed countries characterized by inflammation of the coronary arteries leading to aneurysms. Multiple genome wide association studies (GWAS) have implicated single nucleotide polymorphisms (SNPs) in B-lymphoid tyrosine kinase (BLK) to increased susceptibility in KD. Common disease-associated SNPs are known to cause a 20-70% decrease in the expression of BLK, however, the impact that this loss has in the development of inflammatory diseases is unknown. BLK is a non-receptor tyrosine kinase, part of the Src family kinases (SFKs), most studied in the context of B-cells. It is well-established that the innate immune system – particularly monocytes – play a critical role in the pathogenesis of KD. Here, we examine BLK -/- mice in a model of KD to gain insight into its undetermined role in disease pathogenesis.
Methods: Bone marrow derived macrophages (BMDMs) from BLK+/+, BLK +/- and BLK -/- mice were stimulated with various TLR agonists. Production of IL6 and TNF were measured via ELISA and RNA was prepared for mRNA sequencing by Novogene Inc. In vivo, KD was induced by intraperitoneal Lactobacillus casei cell wall extract (LCWE) injection. Coronary artery inflammation was scored by a pathologist blinded to experimental conditions. To examine intrinsic versus extrinsic effect of the loss of BLK, bone marrow chimeras (BMCs) were used. Briefly, BLK +/+ hematopoietic stem cells were reconstituted via intravenous injection into irradiated BLK -/- recipient mice and vice versa followed by monitoring of engraftment via flow cytometry.
Results: At steady state, BLK -/- mice have significantly lower leukocyte counts - specifically monocytes - in peripheral circulation compared to BLK +/+ littermates. Immunophenotyping via flow cytometry elucidated a trend in decreased percentage of pro-inflammatory monocytes (CD11b+Ly6C+CCR2+) that egress from the bone marrow in the absence of Blk. In response to stimulation with all TLR agonists used, BLK -/- BMDMs produce increased IL6 and TNFα. In vivo, post-LCWE injection, BLK -/- mice develop larger sized coronary artery lesions due to significantly increased inflammation. IHC staining of serial sectioned hearts demonstrate lesions consist mainly of neutrophils and macrophages. BMC experiments demonstrate that the absence of Blk in stromal cells is necessary in developing LCWE induced carditis as increased lesion size and inflammation is seen in these mice.
Conclusion: Our data supports the importance of BLK in the pathogenesis of KD through the increased production of proinflammatory cytokines despite lower percentages of monocytes in the periphery. In animals with decreased expression or absence of Blk, there is increased severity of LCWE induced carditis. Our data also highlights an unknown, important contribution of Blk expression in stromal cells in macrophage development and function. These observations will provide important insight into the future for treatment of patients with KD. Understanding the role of Blk will not only elucidate its role in the pathogenesis of KD but will also provide insight into its role in other autoimmune diseases.
Disclosures: M. Kleinau, None; M. Massumi, None; L. Langevin, None; P. Tsoukas, None; H. Cheng, None; S. Tam, None; T. Duong, None; R. Yeung, None.
