Atypical Activation of the Unfolded Protein Response in Cystic Fibrosis Airway Cells Contributes to p38 MAPK-Mediated Innate Immune Responses

Abstract

Inflammatory lung disease is the major cause of morbidity and mortality in cystic fibrosis (CF); understanding what produces dysregulated innate immune responses in CF cells will be pivotal in guiding the development of novel anti-inflammatory therapies. To elucidate the molecular mechanisms that mediate exaggerated inflammation in CF following TLR signaling, we profiled global gene expression in immortalized human CF and non-CF airway cells at baseline and after microbial stimulation. Using complementary analysis methods, we observed a signature of increased stress levels in CF cells, specifically characterized by endoplasmic reticulum (ER) stress, the unfolded protein response (UPR), and MAPK signaling. Analysis of ER stress responses revealed an atypical induction of the UPR, characterized by the lack of induction of the PERK–eIF2α pathway in three complementary model systems: immortalized CF airway cells, fresh CF blood cells, and CF lung tissue. This atypical pattern of UPR activation was associated with the hyperinflammatory phenotype in CF cells, as deliberate induction of the PERK–eIF2α pathway with salubrinal attenuated the inflammatory response to both flagellin and Pseudomonas aeruginosa. IL-6 production triggered by ER stress and microbial stimulation were both dependent on p38 MAPK activity, suggesting a molecular link between both signaling events. These data indicate that atypical UPR activation fails to resolve the ER stress in CF and sensitizes the innate immune system to respond more vigorously to microbial challenge. Strategies to restore ER homeostasis and normalize the UPR activation profile may represent a novel therapeutic approach to minimize lung-damaging inflammation in CF.

Rescue of Dysfunctional Autophagy Attenuates Hyperinflammatory Responses from Cystic Fibrosis Cells

“…Furthermore, blockade of autophagy initiation with 3-MA, which would lead to further CFTR accumulation in the ER, induced proinflammatory responses to FliC (Fig. 3A), consistent with our recent observation that elevated ER stress is present in CF cells and tissues, and predisposed CF airway cells to exhibit hyperinflammatory responses to FliC (19). On this basis, we therefore propose that the specific disruption of autophagosome clearance by FliC in CF cells decreases their ability to deal with their ER stress burden by causing the system to back up, leading to activation of proinflammatory ER stress pathways that amplify subsequent TLRelicited responses.…”

“…5). We recently demonstrated that ER stress is elevated at baseline in a variety of CF tissues and cell types, causing activation of downstream pathways that synergize with canonical TLR5 signaling to induce exaggerated inflammatory responses to FliC (19). Given that ER stress promotes autophagy as a negative-feedback system to limit misfolded CFTR accumulation in the ER through autophagosome-lysosome-mediated degradation (13,66), we propose that therapeutic interventions that promote normal autophagy in CF cells could potentially limit inflammation by reducing the burden of ER stress in CF cells.…”

“…Activation of ER stress pathways by oxidative stress and misfolded CFTR accumulation correlates with inflammation in CF (14,15). Autophagy has recently been iden-tified as dysfunctional in CF (16)(17)(18), which might lead to heightened activation of ER stress pathways with inflammatory consequences (19); however, a direct link between autophagy and inflammation in CF has not yet been established.…”

The Role of Short-Chain Fatty Acids, Produced by Anaerobic Bacteria, in the Cystic Fibrosis Airway

“…However, overexpression of these miRNAs failed to reduce GPR41 mRNA and protein expression in CFBE41o 2 cells (data not shown), excluding the possibility of regulation of GPR41 expression by these miRNAs. Accumulation of misfolded F508del-CFTR in the ER causes ER stress that activates UPR, a signal transduction pathway to alleviate this stress and restore ER homeostasis (32,45,46). Exposure of 16HBE14o 2 cells to tunicamycin or thapsigargin, well-known inducers of ER stress and UPR, increased protein expression of GPR41 ( Figure 6A), implying that the ER stress and UPR may also, independent of CFTR activity, contribute to GPR41 up-regulation.…”

Chronic Inflammation in CF Airways - A Persistent Issue for A20

“…Global gene expression in immortalized human CF and non-CF airway cells showed an inflammatory gene signature involving MAPK signalling and the hyper-inflammatory phenotype in response to microbial stimulation was found to be dependent on p38 activity [46]. CF lung biopsies showed increased immunoreactivity for p38 MAPK activity markers and CFTR Phe508del/Phe508del airway epithelial cells lines (NuLi) confirmed a further increase in p38 activity when stimulated with P. aeruginosa [47].…”