TLR5 signalling is hyper-responsive in porcine cystic fibrosis airways epithelium

Excessive lung inﬂammation and airway epithelium damage are hallmarks of cystic ﬁbrosis (CF) disease. It is unclear whether lung inﬂammation is related to an intrinsic defect in the immune response or to chronic infection. We aimed to determine whether TLR5-mediated response is defective in the CF airway epithelium. We used a newborn CF pig model to study intrinsic alterations in CF airway epithelium innate immune response. Airway epithelial cells (AECs) were stimulated with ﬂagellin or lipopolysaccharide to determine responses speciﬁc for TLR5 and TLR4, respectively. We observed a signiﬁcant increase in cytokine secretion when CF AECs were stimulated with ﬂagellin compared to wild type (WT) AECs. These results were recapitulated when AECs were treated with an inhibitor of CFTR channel activity. We show that TLR5-signalling is altered in CF lung epithelium at birth. Modulation of TLR5 signalling could contribute to better control the excessive inﬂammatory response observed in CF lungs.


Introduction
Cystic fibrosis (CF), the most common lethal genetic disease in the Caucasian population, is a recessive genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene, rendering the protein non-functional [1] .It is characterized by chronic bacterial infection, mainly Pseudomonas aeruginosa , persistent inflammation with exacerbated recruitment of polymorphonuclear neutrophils (PMNs) into the lungs, excessive release of proteases and ultimately lung tissue destruction [1] .
There is a long-standing debate regarding the links between CFTR mutations and the development of persistent infection and inflammation [1] .While some suggest that CFTR dysfunction leads to intrinsic inflammation [2] , others hypothesize that excessive inflammation is rather the result of a lung environment that favours bacterial colonization [3] .The development of the mutated CFTR F508/ F508 pig [4] and the CFTR −/ − knockout pig [5] , which mimic the human CF symptoms show that there is no intrinsic airways inflammation in naive, non-infected lungs [4] .However, their immune response upon a bacterial challenge seems to be altered facilitating lung colonization by the pathogen and consequently a pro-inflammatory lung environment [6] .
Understanding the processes leading to this altered inflammatory response would be crucial to improve CF therapies.Toll-like receptors (TLRs) are the main family of pattern recognition receptors (PRRs) of the innate immune system.In the CF context, TLR4 and TLR5, which interact with LPS and flagellin, respectively, are known to be major factors in the immune response to P. aeruginosa [7] .In particular, TLR5 response to flagellin has been suggested to play a role in the excessive inflammation observed in CF airway epithelial cells [8] .Here, we aimed to determine whether TLR5 signalling is defective in the CF airway epithelium using primary AECs from newborn CFTR −/ − piglets.

Ethics statement
All experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee at IN-RAE.All experimental procedures were evaluated by the Ethics Committee of the Val de Loire (CEEA VdL, committee N °19) and approved by the Ministry of Higher Education and Research (APAFIS#1166-2015071615392426 Notification and APAFIS#10125-20170602162555 Notification).

Production of recombinant flagellin
The recombinant flagellin FliC 174-400 came from S. enterica serovar Typhimurium FliC and was produced with an histidine tag, as described previously [10] .

Development of a differentiated primary culture of airway epithelia (AECs) from newborn cystic fibrosis piglets
Bronchial epithelial cells from newborn CFTR + / + and CFTR −/ − piglets were differentiated under air-liquid-interface conditions as described in [11] .

cDNA pre-amplification and RT-qPCR on the BioMark HD real-time PCR platform
Extracted total RNA was converted to cDNA by reverse transcription of 20 ng of RNA using the Nucleospin® RNA XS kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's protocol.Pre-amplification was performed with cDNA diluted 1:10 in TE (10 mM Tris, 0.1 mM EDTA, pH 8.0, Fluka Bio-Fig.2. Real-time qPCR analysis of differentiated airway epithelial cells from newborn CFTR + / + and CFTR −/ − piglets stimulated with LPS.Real-time RT-qPCR analysis of airway epithelial cells from newborn CFTR + / + and CFTR −/ − piglets stimulated for 5 h with 200 μl of 1 μg/ml of apical LPS.Gene expression was normalized against the unstimulated (Mock) CFTR + / + group.Data were analysed by two-way ANOVA, using pig genotype and LPS stimulation as factors, followed by Tukey's HSD post hoc test.Data are representative of three experiments.chemika #93283) using a Preamp Master Mix (Fluidigm, # 100-5581).The pre-amplification thermal cycle conditions were: 95 °C for 2 min followed by 14 cycles of 95 °C for 15 seconds and 60 °C for 4 min.Real-time quantitative PCR was performed in a 48 × 48 Dynamic Array Integrated Fluidic Circuits (Fluidigm).The quantitative RT-PCR data were analyzed using the 2 Ct method.The results are expressed as relative fold change (Fc) in comparison with the wild type unstimulated control.

Statistical analysis
Statistical analysis were performed using the statistical package R version 4.0.1 and the packages ggplot2 and ggpubr.Intergroup differences were analysed using two-way analysis of variance (ANOVA) followed by Tukey's HSD post hoc test.A p-value < 0.05 was considered statistically significant.

Flagellin induces an inflammatory response in airway epithelial cells that is exacerbated in cystic fibrosis
Airway epithelial cells from WT piglets (cultured under ALI conditions) were treated for 24 h with GlyH-101 (a CFTR inhibitor) and then stimulated apically with the recombinant flagellin FliC 174-400 .We observed that inhibition of CFTR activity with GlyH-101 upregulated the expression of genes coding for inflammatory cytokines ( CCL2, CXCL2 ) and regulation of the immune response ( NFKBIA, NFKBIE ) after flagellin administration ( Fig. 1 A).We also observed that a 24h supplementation of the AECs with GlyH-101 was enough to increase the gene expression of the evaluated genes in the absence of an inflammatory stimulus ( Fig. 1 A).
Our data suggest that TLR5 signalling could be altered after pharmacological inhibition of the CFTR channel.However, GlyH-101 could also affect other types of Cl -conductance [12] .To confirm the data, we used AECs from newborn CFTR + / + or CFTR −/ − pigs.Stimulation of CFTR −/ − AECs with FliC 174-400 resulted in a significantly increased expression of CXCL2 CXCL8 , which are known to play an important role in the recruitment of neutrophils into the lungs, NFKBIA and NFKBIZ genes ( Fig. 1 B).This excessive response to an inflammatory stimulus seemed to be specific for TLR5 since LPS stimulation did not induce a significant overexpression of inflammatory genes in CFTR −/ − cells ( Fig. 2 ).In addition, no differences were found in either TLR4 or TLR5 gene expression between CFTR + / + or CFTR −/ − AECs ( Fig. 3 ).

Discussion
Regulation of innate immunity in CF has been a topic of controversy throughout the years.We have investigated this question in a relevant translational model of cystic fibrosis, the CF pig model [5] .Porcine lungs share many physiological features with those of humans.Moreover, the pig model also displays a similar lung inflammation upon infection by P. aeruginosa, the main CF pathogen, as observed in humans [13] .Also, recent findings show that newborn CF piglets do not present intrinsic inflammation in the lungs, which spontaneously develops later in life [4] .This feature makes the newborn CF pig model especially well-suited to evaluate early defects in innate immune signalling, avoiding confounding effects that could modify the airway epithelium response to an inflammatory stimulus.Our data, however, points to an exacerbated inflammatory response in the airway epithelium driven by TLR5 signalling dysregulation that does not seem to be related with a receptor overexpression.This is in line with earlier studies that describe an overproduction of pro-inflammatory cytokines airway epithelia from CF patients [14] .This fits also with the known major role played by TLR5 signalling in the exaggerated cytokine production [8] .In addition, a recent study showed a decreased secretion of airway surface liquid in response to flagellin-expressing P .aeruginosa in the CF pig airways in vivo, suggesting a possible defect in TLR5 signaling [15] .No differences were observed when CFTR −/ − AECs were stimulated with LPS, which could be related to a decreased sensitivity of AECs to LPS [16] .A possible mechanism explaining the CFTR −/ − AECs increased response to flagellin could be the existence of endoplasmic reticulum (ER) stress in the CF airways.ER-stress have been linked to chronic inflammatory diseases, such as CF [17] .In this regard, in vitro induction of ER-stress in the AECs increases their inflammatory response to flagellin [18] .Moreover, we observed that thapsigargin-induced ER-stress in the AECs leads to increased response to flagellin but not LPS (unpublished results).
Our results differ somehow from a study by Bartlett et al [6] who analysed the impact on airway epithelium of heat-killed Staphylococcus aureus [6] , which does not express flagellin [15] , and which is therefore unable to activate TLR5 signalling.Very consistently, a recent study showed opposite effect in the induction of IL-8 by S. aureus and P. aeruginosa in BEAS-2B airway epithelial cells, with P. aeruginosa strongly inducing IL-8 secretion, and S. aureus having a minimal effect [19] .
In conclusion, our results suggest that TLR5 response is dysregulated in the CF airway epithelium.We point to TLR5 signalling as a key player in this exacerbated inflammatory response.Our data open new leads toward the design of novel immune-modulatory therapies targeting TLR5 that could improve the management of lung inflammation in CF.

Declaration of Competing Interest
JC Sirard is inventor or co-inventor of Patent Applications WO2009156405, WO2011161491, WO2015011254 and

Fig. 1 .
Fig. 1.Newborn CFTR −/ − piglets present an exacerbated TLR5 response.(A) Gene transcription analysis in airway epithelial cells from wild-type pigs treated with 50 μM of apical GlyH-101 or the vehicle for 24 h and stimulated for 5 h with 200 μl of either 100 ng/ml of apical FliC 174-400 or the vehicle.Gene expression levels are shown relative to the mock group (treated with the vehicle).(B) Real-time RT-qPCR analysis of airway epithelial cells from newborn CFTR + / + and CFTR −/ − piglets stimulated for 5 h with either 200 μl of 100 ng/ml of apical FliC 174-400 or the vehicle.Data were analysed by two-way ANOVA, using pig genotype and flagellin stimulation as factors, followed by Tukey's HSD post hoc test.Data are representative of four experiments.

Fig. 3 .
Fig. 3. Real-time qPCR analysis of TLR4 and TLR5 expression in differentiated airway epithelial cells from newborn CFTR + / + and CFTR −/ − piglets.Gene expression was normalized against the CFTR + / + group.Data were using the Mann-Whitney-Wilcoxon test.Data are representative of four independent experiments.