Chi Wong

Previously, we showed that serum and monocytes from patients with CF exhibit an enhanced NLRP3-inflammasome signature with increased IL-18, IL-1β, caspase-1 activity and ASC speck release (Scambler et al. eLife 2019). Here we show that CFTR modulators down regulate this exaggerated proinflammatory response following LPS/ATP stimulation. In vitro application of ivacaftor/lumacaftor or ivacaftor/tezacaftor to CF monocytes showed a significant reduction in IL-18, whereas IL-1β was only reduced with ivacaftor/tezacaftor. Thirteen adults starting ivacaftor/lumacaftor and eight starting ivacaftor/tezacaftor were assessed over three months. Serum IL-18 and TNF decreased significantly with treatments, but IL-1β only declined following ivacaftor/tezacaftor. In (LPS/ATP-stimulated) PBMCs, IL-18/TNF/caspase-1 were all significantly decreased and IL-10 was increased with both combinations. Ivacaftor/tezacaftor alone showed a significant reduction in IL-1β and pro-IL-1β mRNA. This study demonstrates that these CFTR modulator combinations have potent anti-inflammatory properties, in addition to their ability to stimulate CFTR function, which could contribute to improved clinical outcomes.

Cystic Fibrosis (CF) is a monogenic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, resulting in defective CFTR-mediated chloride and bicarbonate transport, with dysregulation of epithelial sodium channels (ENaC). These changes alter fluid and electrolyte homeostasis and result in an exaggerated proinflammatory response driven, in part, by infection. We tested the hypothesis that NLRP3 inflammasome activation and ENaC upregulation drives exaggerated innate-immune responses in this multisystem disease. We identify an enhanced proinflammatory signature, as evidenced by increased levels of IL-18, IL-1β, caspase-1 activity and ASC-speck release in monocytes, epithelia and serum with CF-associated mutations; these differences were reversed by pretreatment with NLRP3 inflammasome inhibitors and notably, inhibition of amiloride-sensitive sodium (Na⁺) channels. Overexpression of β-ENaC, in the absence of CFTR dysfunction, increased NLRP3-mediated inflammation, indicating that dysregulated, ENaC-dependent signalling may drive exaggerated inflammatory responses in CF. These data support a role for sodium in modulating NLRP3 inflammasome activation.

Cystic Fibrosis (CF) is a recessive genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR mutations cause dysregulation of channel function with intracellular accumulation of misfolded proteins and endoplasmic reticulum (ER) stress, with activation of the IRE1α-XBP1 pathway that regulates a subset of unfolded protein response (UPR) genes. This pathway regulates a group of genes that control proinflammatory and metabolic responses in different immune cells; however, the metabolic state of immune cells and the role of this pathway in CF remain elusive. Our results indicate that only innate immune cells from CF patients present increased levels of ER stress, mainly affecting neutrophils, monocytes, and macrophages. An overactive IRE1α-XBP1 pathway reprograms CF M1 macrophages toward an increased metabolic state, with increased glycolytic rates and mitochondrial function, associated with exaggerated production of TNF and IL-6. This hyper-metabolic state, seen in CF macrophages, is reversed by inhibiting the RNase domain of IRE1α, thereby decreasing the increased glycolic rates, mitochondrial function and inflammation. Altogether, our results indicate that innate immune cells from CF patients are primarily affected by ER stress. Moreover, the IRE1α-XBP1 pathway of the UPR is responsible for the hyper-metabolic state seen in CF macrophages, which is associated with the exaggerated inflammatory response. Modulating ER stress, metabolism and inflammation, by targeting IRE1α, may improve the metabolic fitness of macrophages, and other immune cells in CF and other immune-related disorders.

Introduction In Cystic Fibrosis (CF), infection and local hypoxia causes increased cell death, cytokine release and damage-associated molecular patterns (DAMPS) which results in hyper-inflammation. The accumulation of misfolded CFTR protein within the cell, along with loss of function leads to excessive cellular stress, defective autophagy and a disruption of ionic balance, all of which contribute to this hyper-inflammatory state. The accumulation of the faulty CFTR, along with the lack of expression of a functional CFTR channel on the cell membrane, leads to perturbation of autophagy in epithelial cells. This, in turn, leads to increased proteasomal degradation of the peroxisome proliferator activated receptor (PPAR)-γ, an anti-inflammatory protein that downregulates expression of genes involved in inflammation. Method Serum cytokines were measured in patients with CF and controls. In vitro assays were used to characterise effects of small molecules on NLRP3 inflammasome activation in airway epithelial cells and immune cells. A p valueResults Levels of IL-18 were significantly increase in patients with CF vs controls. Immune and epithelial cells harbouring CF mutations had increased IL-1Β and IL-18 cytokine production, which was blocked by the addition of inhibitors targeting the NLRP3. The activation of NLRP3 appeared to be mediated by increased K⁺ efflux following dysregulation of ENaC and high sodium flux. Lumacaftor/ivacaftor significantly reduced the levels of inflammatory cytokines in patients within three months of treatment (n=8) as well as in immune and epithelial cells in vitro. The addition of Na⁺ channel inhibitor, Amiloride, and S18 (peptide based on endogenous ENaC inhibitor SPLUNK1) in vitro inhibited secretion of cytokines processed by the NLRP3 inflammasome. This potential mechanism was further corroborated by the failure of EIPA which targets all Na⁺ channels except ENaC to replicate the anti-inflammatory effects. The addition of the small molecule, cysteamine, recovered autophagy and resulted in down-regulation of inflammasome components and decrease in cytokine secretion. Conclusion Collectively, we have shown that auto-inflammation in CF can be restored with the use of small molecule therapies which target CFTR and ENaC. Data also suggests that correction and potentiators of CFTR will help reverse auto-inflammation in this complex disease