Contribution of Autophagy-Notch1-Mediated NLRP3 Inflammasome Activation to Chronic Inflammation and Fibrosis in Keloid Fibroblasts

Abstract

Keloid is a representative chronic fibroproliferative condition that occurs after tissue injury. Emerging evidence showed that activation of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome is involved in the pro-inflammatory response in injured tissues. However, the role of NLRP3 inflammasome in keloid progression remains unclear. Notch signaling, which activates NLRP3 inflammasome, is known to contribute to scar formation in keloid, but the cause of enhanced Notch signaling in keloid is not clear. We sought to investigate whether autophagy regulates Notch1 signaling in keloid fibroblasts and determine whether Notch1 signaling might regulate NLRP3 inflammasomes and myofibroblast differentiation. An in vitro model of keloid was established by culturing primary keloid fibroblasts from patients. Expression levels of Notch1, NLRP3 inflammasome proteins, pro-inflammatory cytokines, and myofibroblast markers in keloid fibroblasts were examined and compared with those in normal fibroblasts. Autophagy known to mediate Notch1 degradation was also monitored in fibroblasts. Small interfering RNA (siRNA) targeting Notch1 was used to transfect keloid fibroblasts to further examine the role of Notch signaling in NLRP3 inflammasome activation. Expression levels of Notch1 and NLRP3 inflammasome in keloid fibroblasts increased compared to those in normal fibroblasts. Such increases were accompanied by increased LC3 levels and reduced autophagic flux. Notch1 silencing in keloid fibroblasts by siRNA transfection significantly suppressed increased levels of overall NLRP3 inflammasome complex proteins, NF-kB, and α-smooth muscle actin. Autophagy induction by rapamycin treatment in keloid fibroblasts effectively suppressed expression levels of Notch1 and NLRP3 inflammasome proteins. Decreased autophagy activity in keloid can result in Notch1-mediated myofibroblast activation and NLRP3 inflammasome signaling activation which is critical for chronic inflammation. Collectively, these results identify Notch1 as a novel activator of NLRP3 inflammasome signaling leading to chronic tissue damage and myofibroblast differentiation in keloid progression.

Downregulation of hsa_circ_0002198 inhibits keloid fibroblast activities in vitro by reducing NLRP3 inflammasome activity

“…Moreover, in vitro experiments demonstrated that overexpression of NLRP3 could partially reverse the effects of hsa_circ_0002198 on keloid fibroblast cell proliferation, migration, and invasion, as well as the levels of TGF-β, Col I, α-SMA, and caspase 3 proteins in human keloid fibroblasts. A recent study by Lee et al (2020) not only showed that NLRP3 inflammasomes are activated in keloid fibroblasts and involved in chronic inflammatory processes, but also that NLRP3-dependent IL-1β release may aggravate tissue damage, prolong inflammatory responses, and adversely affect remodeling by inducing continuous myofibroblast differentiation in keloids (Lee et al, 2020). Accumulated evidence has elucidated how circRNAs help to regulate NLRP3 inflammasomes in various diseases, such as depression (Zhang et al, 2019), preeclampsia (Li et al, 2020b), diabetes mellitus (Cheng et al, 2019), and traumatic brain injury (Li et al, 2020a).…”

Role of oleanolic acid in relieving psoriasis and its underlying mechanism of action

“…The highly conserved Notch gene family was first discovered in Drosophila melanogaster by Morgan et al in 1917, and comprises four transmembrane receptors, Notch1, Notch2, Notch3 and Notch4, which are involved in regulating the viability, proliferation and differentiation of cells and the development of organs (Nantie et al 2014;Ebens & Maillard, 2013). Previous studies have reported that the upregulated expression of Notch1 promoted the autophagy of cells (Xu et al 2019, Lee et al 2020. Similarly, the results of the present study also showed that the protein expression levels of Notch1 were significantly increased Ole treated groups.…”

Notch-ing up knowledge on molecular mechanisms of skin fibrosis: focus on the multifaceted Notch signalling pathway

“…The dermis of KD is characterized by abundant thick hyalinized collagen bundles, also known as “keloidal collagen”, and by the persistence of myofibroblasts, and is surmounted by a thickened epidermis which undergoes accelerated differentiation [ 166 ]. Though Notch signalling has been less investigated in KD than in other fibrotic skin diseases, available experimental evidences support its involvement in KD pathomechanisms [ 174 , 175 ]. A study by Syed and Bayat revealed that (i) NOTCH1, NOTCH2 and JAG1 mRNA and protein levels are significantly up-regulated in KD skin biopsies and primary FBs from KD patients (KD FBs) with respect to healthy tissues and FBs, respectively; (ii) Notch pathway stimulates cell proliferation, migration, invasion and angiogenetic properties of cultured KD FBs; and (iii) Notch activation positively correlates with inflammatory degree in KD tissues, suggesting that immune cells might turn on Notch cascade in vivo [ 174 ].…”

“…Furthermore, a recent study reported that KD FBs from subjects affected with active KD ( i.e. patients with a recent keloid and complaining keloid pruritus and pain) exhibit a more prominent NOTCH1 activation as compared to KD patients with stable lesions [ 175 ]. In addition, KD FBs were hallmarked by: (i) a reduced autophagic flux, which has been associated with a reduced autophagy-mediated degradation of Notch, and (ii) a NOTCH1-mediated induction of α-SMA, TGF-β3 and NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome, which primes the inflammatory cascade in KD.…”

“…In addition, KD FBs were hallmarked by: (i) a reduced autophagic flux, which has been associated with a reduced autophagy-mediated degradation of Notch, and (ii) a NOTCH1-mediated induction of α-SMA, TGF-β3 and NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome, which primes the inflammatory cascade in KD. Finally, treatment of KD FBs with rapamycin—an inducer of autophagy—determined NOTCH1, and in turn NLRP3, down-regulation in KD FBs [ 175 ]. Deregulation of the Notch pathway in KD has been recently described by an integrative analysis of mRNA and miRNA expression levels at the wound site of KD-prone individuals [ 176 ].…”