JSLE and the NLRP3 inflammasome – a novel therapeutic target


Gamble, J ORCID: 0000-0001-7302-3412 (2017) JSLE and the NLRP3 inflammasome – a novel therapeutic target. In: 2017 Annual Congress of PReS, 2017-9-14 - 2017-9-17, Athens.

[img] Text
s12969-017-0185-x.pdf - Published version
Download (1MB)
[img] Text
Jo updated 1.1 - JSLE and the NLRP3 Inflammasome.docx - Submitted version
Download (15kB)

Abstract

Introduction: Juvenile-onset Systemic Lupus Erythematosus (JSLE) is a severe autoimmune disease causing organ damage and long-term morbidity. Impaired clearance of nuclear debris from cells and upregulated circulating damage-associated molecular patterns (DAMPs) and cytokines are thought to trigger cell death and thus, further increase release of pro-inflammatory molecules following the cytosolic assembly of inflammasomes. Objectives: To investigate the role of pyroptotic cell death in JSLE, which occurs following the assembly and activation of the NLRP3 inflammasome, which may be a promising target in the treatment of JSLE and other inflammatory diseases. Methods: THP-1 cell line-derived macrophages (Mφs) were primed using lipopolysaccharide (LPS; 10 ng/ml) and interferon (IFN) γ (20 ng/ml) in complete media at 37 °C for 24 hours, and subsequently treated with 10 mM Adenosine triphosphate (ATP) for 30 min. Primed Mφs not treated with ATP were used as a negative control. Some Mφs were incubated with 10% JSLE patient sera or NETosis-derived material (10 ng/ml) from PMA-treated neutrophils. Primed Mφs were tested for cell surface markers, HLA-DR, CD282 (TLR2), and CD68 using flow cytometry. ATP-treated Mφs were collected and either assayed for pyroptosis marker, lactate dehydrogenase (LDH) activity, cleaved caspase-1 with immunofluorescence (IF), or lysed for cleaved caspase-1 using western blotting. Results: Primed Mφs showed an M1 phenotype with geometric means ± SEM of HLA-DR, TLR2 and CD68 expression respectively of: 1177 ± 1.15, 613 ± 0.9, and 1549 ± 0.9 gMFI, respectively compared with un-primed Mφs of 597 ± 1.0, 122 ± 0.88, and 1225 ± 0.9 gMFI, respectively (n = 3; p < 0.05). ATP-treated Mφs showed increased LDH activity compared to controls (3.4x10-3 ± 0.12x10-3) compared to 0.00 milliunits/ mL, respectively; n = 3; p < 0.05). Furthermore, a greater increase in LDH activity was observed in Mφs that were incubated with JSLE serum and NET material (3.7x10-3 ± 0.5x10-3 and 7.6x10-3 ± 0.5x10-3, respectively, compared to 0.00 milliunits/mL; n = 3-6; p < 0.05). IF was positive for cleaved caspase-1 in ATP treated Mφs; and this was confirmed in lysed cells, using western blotting.

Item Type: Conference or Workshop Item (Unspecified)
Depositing User: Symplectic Admin
Date Deposited: 24 Nov 2017 15:58
Last Modified: 19 Jan 2023 06:51
URI: https://livrepository.liverpool.ac.uk/id/eprint/3011285
CORE (COnnecting REpositories)