E-Poster with pre-recorded video presentation Lorne Infection and Immunity 2021

Therapeutic targeting of the myofibroblast NLRP3 inflammasome as a novel approach to treating fibrosis (#258)

Anita A Pinar 1 , Felipe Tapia-Caceres 1 , Alexander Yuferov 1 , Tracey A Gaspari 1 , Chrishan S Samuel 1
  1. Pharmacology, Monash Biomedicine Discovery Institute, Clayton, VIC, Australia

Background: The NLRP3 inflammasome is a key immunomodulatory platform induced by the immune system to produce the pro-inflammatory cytokines, interleukin(IL)-1β and IL-18 following tissue injury. However, the chronic activation of IL-1β and IL-18, by pro-fibrotic stimuli such as transforming growth factor (TGF)-β1 and toll-like receptor (TLR)-4 can drive fibrosis (tissue scarring) progression1. The anti-fibrotic hormone, relaxin, has been found to limit the pro-fibrotic effects of TGF-β1, TLR-4 and/or IL-1β on myofibroblast differentiation and excessive collagen deposition.     

Aim: Hence, the extent to which the anti-fibrotic effects of recombinant human relaxin (RLX) targeted the myofibroblast NLRP3 inflammasome was determined.

Methods: Primary human cardiac (HCFs)2 or dermal (HDFs)3 fibroblasts (1.5x105 cells/well in 12 well-plates) were left untreated or stimulated with TGF-β1 (T; 2 ng/mL; to promote myofibroblast differentiation) + LPS (L; 100 ng/mL; to prime the NLRP3 inflammasome) + ATP (A; 5mM; to activate the NLRP3 inflammasome) for 8h or 72h; in the absence/presence of RLX (100ng/mL)2,3. HDFs were also subjected to lipofectamine-siRNA-induced knockdown of NLRP3 inflammasome priming components (NLRP3, ASC and caspase-1)3, and treated with/without RLX (100ng/mL) for 72h (n=6-8) in vitro. 8-10 week-old-male 129/sv mice were subjected to isoprenaline (ISO, 25 mg/kg, s.c)-administration over 5 consecutive days, then left for 9 days for fibrotic healing to occur. Subgroups of ISO-injured mice were either left untreated or treated with RLX (0.5 mg/kg/day) or the NLRP3 inflammasome inhibitor, MCC950 (10 mg/kg/day) from days 7-14 post-injury (n=4-6/group) in vivo.

Results: T+L+A-stimulation of HCFs2 or HDFs3in vitro significantly increased TLR-4, measures of NLRP3 inflammasome priming (NLRP3, ASC, caspase-1) and activity (IL-1β, IL-18), α-SMA (myofibroblast differentiation) and collagen-I (interstitial fibrosis) after 8h or 72h. Likewise, repeated ISO-administration promoted all these measures in the left ventricle of mice after 14-days of injury in vivo2. These T+L+A- or ISO-stimulated effects were significantly inhibited in vitro or ameliorated after 7 days in vivo by RLX. However, these NLRP3 inflammasome-inhibitory and anti-fibrotic effects of RLX were abrogated when caspase-1 was knocked-down.

Conclusion and Significance: Targeting inducers (TGF-β1, TLR-4) and/or components (caspase-1) of the myofibroblast NLRP3 inflammasome may represent a previously unrecognised approach for treating IL-1β- and IL-18-mediated fibrosis progression.

  1. 1 Pinar AA*, et al., Pharmacol Ther. 209:107511 (2020).
  2. 2 Cáceres FT, et al., FASEB J. 33(12):14717-14733 (2019).
  3. 3 Pinar AA*, et al., Front Pharmacol 11:1201 (2020).