title: Investigating the role of neutrophil extracellular traps in the pathogenesis of juvenile-onset systemic Lupus Erythematosus (JSLE)
Creators: name: Makuloluwa, Kanchani
Publisher: name: University of Liverpool Repository
Year: 2015

description: Background: JSLE is a multisystem autoimmune disorder characterised by the production of autoantibodies against nuclear self-antigens. Activated neutrophils may undergo cell death by NETosis, forming NETs comprising of DNA. Dysregulated NETosis is increasingly being investigated for their role in Lupus pathogenesis through auto-antigen exposure. Endosomally localized Toll-like receptors (TLRs) 3, 7 and 9, expressed highly in JSLE, detect nuclear antigens to induce immune responses. Our hypothesis is that NETs offer a source of nuclear autoantigens that are being detected via TLR3, TLR7 and/or TLR9, resulting in the activation of the immune system. Aim: To assess whether NETs originating from JSLE neutrophils are providing a source of nuclear autoantigens that are being detected through endosomal TLRs resulting in the activation of the immune system. Methods: Purified JSLE and paediatric control neutrophils were incubated with 10ng/ml Interferon alpha (IFNα) or 10% JSLE sera to induce NETs. NETs were dismantled using micrococcal nuclease and quantified. Quantified NETs were incubated with healthy adult peripheral blood mononuclear cells (PBMCs) pre-treated +/- 100μM Hydroxychloroquine (HCQ) to block TLR activation. Protein levels of phosphorylated Interleukin-1 Receptor-associated Kinase 1 (pIRAK1) and Interferon Regulatory Factor 3 (pIRF3) (TLR7/9 and TLR3 signalling proteins) was determined by Western blotting. The supernatant from NET-stimulated PBMCs was incubated with either control PBMCs or JSLE neutrophils pre-treated +/- 10μM Janus Kinase (JAK) inhibitor to block IFNα signalling. Protein levels of phosphorylated IFNα-signalling protein, STAT1, was measured in the control PBMCs using Western Blotting; JSLE neutrophils were visualised using confocal microscopy for NETs. Results: PBMCs incubated with IFNα or JSLE sera-induced NETs showed increased pIRAK1 and pIRF3 protein levels in both JSLE and paediatric control neutrophils-derived NETs, as compared to unstimulated PBMCs. The increase in pIRAK1 protein levels was not influenced by the source of the NETs. Incubation of NETs with PBMCs pre-treated with HCQ significantly reduced pIRAK1 protein levels (p=0.028; n=6). In control PBMCs incubated with supernatant from NET stimulated PBMCs there was increased pSTAT1 protein levels (p=0.068; n=4) compared to the control condition. pSTAT1 protein levels were reduced (p=0.068; n=4) in the control PBMCs that were pretreated with a JAK inhibitor. JSLE neutrophils released NETs following incubation with supernatant from NET stimulated PBMCs. NETs were absent in JSLE neutrophils pre-treated with a JAK inhibitor. Conclusion: We have demonstrated that IFNα and JSLE sera-induced NETs may be an important source of autoantigens in JSLE that are being detected through TLR3, TLR7 and/or TLR9, leading to activation of these receptors. HCQ was effective in inhibiting the NET-induced activation of these receptors. Supernatant of NET-stimulated PBMCs induced the activation of JAK-STAT signalling pathways of control PBMCs and stimulated JSLE neutrophils to release NETs. A JAK inhibitor was successful blocking the induction of both these responses.