*Purpose: Immunological mechanisms leading to chronic rejection, bronchiolitis obliterans syndrome (BOS), following human lung transplantation remains unclear though a strong correlation have been identified between development of Abs to mismatched donor HLA and development of BOS. Recent studies have identified receptor interacting protein kinase (RIPK) 3 as cardinal regulator of necroptosis. However, the precise roles of RIPK3 in anti-human leukocyte antigen (HLA) mediated lung injury leading to BOS remain poorly understood. The goal of this study is to investigate the role of RIPK3 in anti-HLA mediated lung injury in human bronchial epithelial cells.

*Methods: Towards this we employed human bronchial epithelial cells (Beas2b), ligated antigens by Abs specific to HLA (HiPRA) (1:50 dilution) and murine monoclonal antibody (mAb) to HLA framework determinants (20µg/ml). Beas2b cells supplemented with RPMI media were incubated with HiPRA or mAb to HLA for 3 and 6 hrs in normoxic conditions. Normal sera (1:50 dilution) and isotype murine Ab C1.18.4 (20µg/ml) served as controls. Protein supernatants were extracted from cell homogenates after 3 and 6 hr incubation using RIPA buffer and proteins collected were analyzed for necroptosis markers and lung associated self-antigens, Collagen V (Col-V) and Kα1Tubulin (Kα1T), using western blotting with specific Abs.

*Results: We demonstrated increased protein expression of RIPK3 in Beas2b cells challenged with anti-HLA treatment at both 3 hrs (0.35 vs 0.19 1.8 fold change) and 6 hrs (0.44 vs 0.32 1.4 fold change). We also demonstrated increased expression of Col-V and Kα1T in response to anti-HLA treatment in Beas2b cells at both 3 hrs (Col-V 0.44 vs 0.14 3.0 fold change; Kα1T 1.49 vs 0.15 9.61 fold change) and 6 hrs (Col-V 0.7 vs 0.13 5.22 fold change; Kα1T 0.51 vs 2.81 fold change). Further, the basal expression and phosphorylation of SMAD3 were increased in response to anti-HLA treatment in Beas2b cells at both 3 hrs (SMAD3 0.48 vs 0.36 1.3 fold change; p-SMAD3 0.51 vs 0.26 1.9 fold change) and 6 hrs (SMAD3 0.55 vs 0.38 1.4 fold change; p-SMAD3 0.51 vs 0.3 1.7 fold change).

*Conclusions: Together our data suggests that there is an association between necroptotic protein RIPK3 and SMAD3 signaling pathway resulting in the induction and/or expression of lung self-antigens, Col-V and Kα1T, ultimately leading to immune responses to self-antigens in the immunopathogenesis of BOS following human lung transplantation.

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