Protease-activated receptor 1 in the pathogenesis of cystic fibrosis

Abstract

Background

The most common cause of death in those with cystic fibrosis (CF) is respiratory failure due to bronchiectasis resulting from repeated cycles of respiratory infection and inflammation. Protease-activated receptor 1 (PAR1) is a cell surface receptor activated by serine proteases including neutrophil elastase, which is recognised as a potent modulator of inflammation. While PAR1 is known to play an important role in regulating inflammation, nothing is known about any potential role of this receptor in CF pathogenesis.

Methods

PAR1 (PAR1^-/-) and intestinal-corrected CFTR (Cftr^-/-) deficient mice were crossed to generate double knock-out (DKO) mutants lacking both PAR1 and CFTR, as well as matching sibling single mutant and wildtype (WT) littermate controls. Mice were weighed weekly to 15 weeks of age; then, the lungs and intestines were examined.

Results

Cftr-deficient mice gained body weight at a significantly slower rate than WT controls and presented with no lung inflammation, but had increased weights of their ilea and proximal colons. DKO mice (lacking both CFTR and PAR1) gained body weight at a similar rate to Cftr^-/- mice but only gained weight in their proximal colons. Weight gain in the ilea of Cftr^-/- but not DKO mice was associated with increased ileal levels in the pro-inflammatory cytokine interleukin (IL)-6.

Conclusions

This study provides the first evidence of PAR1 contributing to the pathological effects of Cftr deficiency in the intestine and suggests a possible effect of PAR1 on the regulation of IL-6 in CF pathogenesis.

WHAT IS ALREADY KNOWN ON THIS TOPIC

• Cystic fibrosis (CF) is a multi-organ disease where multiple cycles of infection-related inflammation are responsible for significant morbidity. Protease-activated receptor 1 (PAR1) is a cell surface receptor and an important modulator of inflammation.

WHAT THIS STUDY ADDS

• This study shows for the first time that PAR1 contributes to the pathological effects of the chloride channel defect inherent in CF and suggests a possible effect of PAR1 on the regulation of the pro-inflammatory cytokine, interleukin-6, in CF pathogenesis.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

• This study highlights the role of PAR1 in mediating inflammation in CF and should be further studied to examine its potential as a therapeutic target for attenuating infection-related inflammation in CF.

Introduction

The most common cause of death in those with cystic fibrosis (CF) is respiratory failure as a consequence of bronchiectasis. The damage to the lungs results from a repeated and sometimes chronic cycle of lower respiratory infection and pulmonary inflammation. The presence of pulmonary inflammation during infancy and preschool years is associated with a worse nutritional status,¹ the presence of micro-organisms in the lower respiratory airways,^{2 3} bronchiectasis that is detectable on chest CT4,6 and lung function abnormalities.^{7 8} Early pulmonary inflammation is, therefore, a suitable and desirable target for the treatment even in the era of highly effective modulator treatment, meaning factors that regulate inflammation in the lungs are of potential therapeutic interest.^{9 10}

Protease-activated receptors (PAR) are a family of G-protein-coupled cell surface receptors, expressed by endothelial, epithelial and immune cells that are well recognised as modulators of inflammation.^{11 12} These receptors have a unique mechanism of activation: specific serine protease(s) released during inflammation, tissue damage or infection cleaves an extracellular domain on the PAR molecule unmasking a specific peptide sequence on a tethered ligand that autologously binds to the body of the PAR molecule. This results in receptor activation and a range of downstream signalling effects depending on the PAR molecule, cell type and activating protease.¹³ PAR activation can trigger a variety of cell responses, including proliferation, apoptosis or modified cytokine production,¹¹ and has been shown to play important roles in the inflammatory responses in many infections and diseases.¹²

PAR-activating proteases come from many sources, including epithelial and inflammatory cells (ie, neutrophil elastase), coagulation factors (ie, thrombin) and bacteria (including Pseudomonas aeruginosa).14,16 A role for neutrophil-derived serine proteases in the development of early lung disease in CF is well established.¹⁷ Free neutrophil elastase activity is detectable in bronchoalveolar lavage in up to 30% of infants and predicts CT-scan-diagnosed bronchiectasis by the age of 3 years.^{5 18 19} These studies suggest that the serine anti-protease host defence mechanisms are overwhelmed in CF, creating the potential for enzymatic degradation of the lung structural integrity.²⁰

An important member of the PAR family is PAR1, which is activated by several proteases produced in the CF lung, including neutrophil elastase.¹⁵ While we and others have shown PAR1 to be a critical regulator of infection-driven inflammation, the role of PAR1 can vary depending on the infection or site of infection. For example, PAR1 is potently anti-inflammatory in chronic Helicobacter pylori infection of the stomach^{21 22} but pro-inflammatory during influenza infection in the lungs²³ or in colitis.²⁴ PAR1 has been shown to affect the functionality of important immune cells in the lung, including alveolar macrophages and neutrophils,^{25 26} and to play a regulatory role in lung infections.^{23 27} Therefore, PAR1 could be crucial in modifying the inflammatory responses in CF lung. However, despite the demonstration of an important role for PAR1 in the lung and other organs, as well as the known ability of neutrophil elastase to activate this receptor,¹⁵ virtually nothing is known about the role of PAR1 in CF.

We hypothesised that PAR1 might play an important role in CF disease progression. Here, a combined Cftr crossed Par1 knock-out mouse model was used to examine whether PAR1 activation could play an important role in modifying the inflammatory response in CF. In this model,²⁸ transgenically modified Cftr null mice differentially express the human CFTR protein in the gut to prevent the fatal intestinal obstruction associated with a complete lack of CFTR, thus allowing us to study the contribution of PAR1 to the pathological effects of Cftr deficiency in the intestine.

Materials and methods

Mice

Animal experimentation was performed under institutional guidelines and with the approval from the Murdoch Children’s Research Institute Animal Ethics Committee (Application ID A797). Experimental analysis was performed according to ARRIVE guidelines 2.0.²⁹ The mice were housed under specific pathogen-free conditions with food and water ad libitum. Post weaning, all mice were maintained on a high-fat diet and given ColonLYTELY (Dendy Pharmaceuticals, Australia) in drinking water to mitigate the effects of intestinal blockages that are characteristic of the Cftr null mice.²⁸

Cftr null mice, transgenically modified to express the human CFTR gene in the gut (Cftr^tm1Unc Tg(FABPCFTR),³⁰ were kindly provided by Professor D. Parsons, The University of Adelaide. PAR1^-/- mice³¹ were kindly provided by Professor S. R. Coughlin, The University of California San Francisco. To generate animals for this study (n=35), Cftr^tm1Unc and PAR1^-/- mice were crossed with each other and wildtype (WT) C57BL/6 mice through several generations, then genotyped, to produce matched sibling littermate mice that were either Cftr^+/+ PAR1^+/+ (WT controls, n=8), Cftr^tm1Unc PAR1^+/+ (Cftr knock-outs, n=9), Cftr^+/+ PAR1^-/- (PAR1 knock-outs, n=10) or Cftr^tm1Unc PAR1^-/- (double knock-outs; DKO, n=8). Based on prior experience with the Cftr null mice, the sample size (8–10 animals per group) was calculated with the power of the experiment set to 80%. All healthy offspring resulting from the above breeding scheme were included in the study, and the entire cohort was confirmed by genotyping to carry the Tg(FABPCFTR) transgene. On the generation of sufficient animals of appropriate genotypes, both male and female mice were left until 15 weeks of age before analysis. The mice were weighed using a Scout Portable Balance (Ohaus, NJ, USA). Randomisation was carried out as follows: males and females were housed in separate cages, and the mice were identified by an ear clip identification system. Cages and mice were randomly selected for routine weighing. Each animal (or tissue from each) was considered an experimental unit for further analysis. The primary outcome of this study will be mouse body weights.

Mouse lung pathology

For the assessment of lung inflammation, the mice were euthanised under deep sedation with inhaled isoflurane (oxygen flow metre set at 0.6 L/min and isoflurane vaporiser set to 5% gas delivery) followed by cardiac bleeds and cervical dislocation. The lungs were perfused via the trachea with 50% OCT compound (Scigen, Gardenia, California, USA) to preserve the lung architecture. Perfused lungs were embedded in OCT and frozen at −80°C. 10 µm sections were cut, mounted on Superfrost slides, fixed in 100% ice-cold ethanol and stained with H&E. Blinded lung sections were graded for cellular infiltration as follows: Grade 0 (none); Grade 1 (mild)—focal aggregates of immune cells, usually around bronchioles and blood vessels; Grade 2 (moderate)—dense cuffs of immune cells surrounding the blood vessels and airways; Grade 3 (marked)—moderate infiltrate of immune cells, extending into surrounding tissue; and Grade 4 (severe)—severe infiltration with extensive areas of lung tissue affected.

Analyses of murine intestines

The entire intestines were dissected out, and intestinal contents were gently removed with a curved dissecting probe. Intestines were then weighed and laid flat on the dissection surface alongside a ruler. Tissue sections corresponding to the ileum, proximal colon and distal colon were removed (respective length measurements were kept standard between mice) and weighed separately. Finally, all masses were calculated relative to individual mouse body weight. For the analysis of cytokines, longitudinally halved intestinal sections were homogenised in phosphate-buffered saline (T10 homogeniser, IKA-Werke).

Quantification of cytokines by ELISA

Tissue homogenates were centrifuged to remove cells/debris prior to the determination of cytokines by ELISA as previously described,³² using primary antibodies: anti-mouse interferon gamma (IFNγ; 0.1 µg/well; BD Biosciences, USA), interleukin 17A (IL-17A; 0.5 µg/well; eBioscience, USA), interleukin 6 (IL-6; 0.05 µg/well; eBioscience, USA) and macrophage inflammatory protein) (MIP-2; 0.05 µg/well; R&D Systems, USA); secondary antibodies: anti-mouse IFNγ (0.05 µg/well), IL-17A (0.025 µg/well), IL-6 (0.05 µg/well) and MIP-2 (0.003 µg/well) (same manufacturers as respective capture antibody). Cytokine concentrations in samples were measured against a standard curve with known concentrations of recombinant cytokines (same manufacturers as antibodies).

Statistical analyses

Statistical analyses were performed using Graphpad Prism version 10.1.2. All data were compared using one-way analysis of variance with Tukey’s post hoc analysis, except for changes in body weight, which were compared by area under the curve (AUC) analysis. P values of less than 0.05 were considered statistically significant.

Patient and public involvement

Patients and members of the public were not involved in the design, conduct and analyses of the study.

Results

PAR1 contributes to body weight loss in Cftr-deficient mice

To examine a potential role for PAR1 in CF, mutant mice were bred that were deficient in both Cftr and Par1 genes (Cftr^tm1Unc Par1^-/-; DKO); these were compared with matching littermates deficient in either Par1 alone (Par1^-/-) or Cftr alone (Cftr^tm1Unc). Littermate mice homozygous positive for both Par1 and Cftr were used as WT controls. All mice were positive for the transgene expressing human CFTR in the gut (FABP-hCFTR). Mice were monitored until 15 weeks of age; after which. they were euthanised and tissues were removed for analysis.

Cftr^-/- mice gained body weight at a slower rate than their WT and Par1^-/- littermates (figure 1a). DKO mice also gained weight slowly, at a rate similar to Cftr-deficient mice (figure 1). While weight gain in the DKO mice (lacking both Cftr and Par1) appeared slightly higher than in Cftr mice at 15 weeks (figure 1b), this was not significantly different by the AUC analysis for the entire observation period (data not shown).

Figure 1. Protease-activated receptor 1 (PAR1) contributes to body weight loss in Cftr-deficient mice. (a) Littermate wildtype (WT; four males, four females), Cftr-deficient Cftr^tm1Unc (Cftr^-/-; four males, five females), Par1 deficient (Par1^-/-; four males, six females) and double knock-out mice lacking both Par1 and Cftr (DKO; two males, six females) were weighed weekly from 7 to 15 weeks of age. The percentage of weight gain (compared with starting weights at week 7) was plotted over time. Cftr^-/- and DKO mice gained significantly less weight compared with WT and Par1^-/- mice (area under the curve analysis). Markers show group averages with SEM at each timepoint. (b) Gains in body weights relative to WT controls (expressed as %) at 15 weeks. Cftr^-/- and DKO mice had significantly lower body weight gain compared with WT (*p<0.0001 and p<0.05, respectively; t-test). Par1^-/- mice had significantly higher body weight gain compared with Cftr^-/-. Box and whisker plots show the minimum and maximum values within each group.

PAR1 contributes to increased ileal weights in Cftr-deficient mice

Mouse models involving mutations in Cftr are known to poorly reflect the lung pathology that occurs in CF, but they do typically present with intestinal symptoms. Hence, as expected, while histological analysis revealed no overt pathology in the lungs of any of the mouse strains including no significant differences in immune cell infiltrate (figure 2a), Cftr^-/- mice did present with a significant increase in their intestinal weights (figure 2b), despite the expression of the hCFTR transgene in the gut. DKO mice lacking Par1 in addition to a deficiency in Cftr had an intermediate increase in intestinal weights; greater than WTs but significantly less than Cftr^-/- mice, which possessed functional PAR1. The examination of individual intestinal regions revealed that while the weights of ilea (figure 2c) and proximal colons (figure 2d) were significantly greater in Cftr^-/- mice as compared with WT littermate controls, only the weights of proximal colons were elevated in DKO mice. Distal colon weights were similar between all groups (figure 2e). Hence, these data indicated that PAR1 expression contributes to the increased ileal weights observed in Cftr mice, supporting a role for PAR1 in the pathogenic effects of Cftr deficiency in the ileum, but not the colon.

Figure 2. Protease-activated receptor 1 (PAR1) contributes to increased ileal weight in Cftr-deficient mice. Wildtype, Cftr-deficient Cftr^tm1Unc (Cftr^-/-), Par1-deficient (Par1^-/-) and double knock-out littermates lacking both Par1 and Cftr were housed for 15 weeks before analysis. (a) H&E-stained lung sections were analysed in a blinded manner for histopathology grading of inflammation by reviewing immune cell infiltration into the lung tissue. (b) The entire intestines, (c) ilea, (d) proximal colons and (e) distal colons were removed and weighed, and their masses were calculated relative to individual mouse body weight. P values as shown on graphs (analysis of variance with Tukey’s post-hoc analysis). The loss of PAR1 in the double mutant mice reduced the effect of CFTR deficiency on increased intestinal weight, particularly in the ileum. Box and whisker plots show the minimum and maximum values within each group.

Association of PAR1 regulation of ileal inflammation in the Cftr-deficient mouse with IL-6

We profiled the levels of key proinflammatory cytokines in the ileum and proximal colon of these mice (cytokines in the distal colon were not analysed given the lack of increased organ weight). Neither IL-17A (Th17) or MIP-2 were increased in the ilea or proximal colons of Cftr mice (figure 3a,b). There were no changes in the levels of IFN-γ as well. IL-17A was significantly elevated in the ilea of DKO mice (figure 3a), but this did not correlate with any observed increase in organ weight. However, an increase in IL-6 levels was observed in the ilea of Cftr but not DKO mice (figure 3b), which correlated with the increase in organ weight seen in figure 1.

Figure 3. Effects of protease-activated receptor 1 (PAR1) on intestinal cytokines. Cytokine levels in homogenised ilea and proximal colons from 15-week-old wildtype, Cftr-deficient Cftr^tm1Unc (Cftr^-/-), Par1 deficient (Par1^-/-) and double knock-out (Par1^-/- Cftr^-/-) littermates were quantified by ELISA. *Significantly greater than other groups (p<0.05; analysis of variance with post hoc analysis). Box and whisker plots show the minimum and maximum values within each group.

Discussion

This study provides the first evidence that PAR1 plays a role in regulating inflammatory processes that characterise CF. Intestinal pathology is one of the serious consequences of CF and the main effect of Cftr mutations in mice. Here, by the generation of compound mice deficient in both CFTR and PAR1, we show that PAR1 was required for the significant increase in weight in the ilea of Cftr^-/- mice. The analysis of tissue cytokine levels identified an association between this effect of PAR1 and the potential regulation of ileal IL-6.

Proteases, including serine proteases, are commonly released at sites of infection and inflammation where they can potentially either contribute to, or protect against, the pathological processes. The inflammation occurring in the CF lung is no different, with key serine proteases typically released into the milieu including the host enzymes neutrophil elastase, proteinase 3 and cathepsin G³³. Anti-protease inhibitors including secretory leucocyte protease inhibitor, elafin and alpha-1 antitrypsin are also present, but they appear unable to control the large amounts of proteases released into the CF lung both by the inflammatory response and infecting pathogens.^{14 33 34}

Due to the release of these enzymes, the mammalian host has developed cell surface sensors called PARs, which detect extracellular serine proteases and signal cells as to their presence. PARs are well-known regulators of the host inflammatory response, with PAR2 being the most extensively studied among them. A link between neutrophil elastase and PAR2 is well established, with elastase increasing PAR2 expression by the airway epithelial cell line Calu-3, an effect associated with increased secretion of the mucin Muc5AC.³⁵ Neutrophil elastase can also act as a bias agonist for PAR2, modulating cell signalling by deactivating conventional G-protein-coupled signalling, while triggering activation of a MAP kinase pathway.³⁶ Interestingly, some strains of P. aeruginosa produce an elastase that can cleave and inactivate PAR2, and it has been suggested that this potentially confers an advantage to the pathogen by modulating the host immune response.³⁴ It has been proposed that the interaction of neutrophil elastase and PAR2 can influence receptor expression on neutrophils and that this might have implications in CF.³⁷

In contrast to PAR2, very little is known about the potential contribution to CF pathogenesis of its close family member PAR1. The effect of PAR1 activation appears more complex than PAR2 and can either increase or decrease the pathological response to infection in vivo, depending on the infection model. For example, in mouse models, PAR1 expression reduces gastritis caused by infection with H. pylori²¹ but increases the severity of pathology caused by influenza infection.²³ This indicates a system that might act as an inflammatory rheostat and could therefore be potentially targeted for therapeutic modification of inflammation.

Evidence suggests that neither neutrophil elastase nor proteinase 3 activate PAR1 via its conventional cleavage site,³⁸ but rather induce a biased agonistic activation of PAR1 that prevents activation by thrombin (generally considered the main activator of PAR1) while triggering an alternative non-canonical activation of MAP kinase signalling pathways.^{15 39} Moreover, neutrophil elastase has been shown to increase the response of peripheral blood γδ T cells to anti-CD3 stimulation via the activation of PAR1,⁴⁰ suggesting a role for this enzyme on this receptor on immune cells. Hence, available data indicate serine proteases present in the CF lung, such as neutrophil elastase, could activate PAR1, at least in cells in the culture.

To examine the in vivo importance of PAR1 in CF, we employed a well-characterised mouse model of the disease.³⁰ Although different CF mouse models exhibit varying degrees of abnormal electrophysiology in the upper respiratory tract, these do not spontaneously develop the exaggerated lung pathology typical of the human disease.⁴¹ This may be in part due to the expression of non-CFTR, calcium-activated anion channels and non-gastric H⁺/K⁺ ATPase that maintain the pH of the air-surface liquid in murine lungs.⁴² However, akin to extrapulmonary manifestations of CF in humans, CF mice can develop moderate to severe intestinal complications, although postnatally.

Therefore, to dissect the role of PAR1 in CF, we bred Par1^-/- x Cftr^-/- DKO mice in an experiment tightly controlled by the use of sibling littermates that were genotyped to be Par1 or Cftr single knock-outs and WTs. The analysis of the lungs revealed no evidence of pulmonary inflammation in the mice, which was not unexpected as Cftr-modified mice, including this strain, do not typically develop significant lung pathology. Mice completely devoid of Cftr exhibit extremely severe intestinal effects resulting in early death, which is why Cftr^tm1Unc mice, which have had the human CFTR (hCFTR) gene re-expressed in the intestine to minimise this impact,³⁰ were used for this study. This ‘gut-corrected’ bitransgenic CF mouse model has readily detectable hCFTR expression in the intestines, but not in the lungs or nasal epithelium.³⁰ However, there are significant differences in hCFTR distribution between the small and large intestines that is distinct from the expression of endogenous murine CFTR.³⁰ Functionally, this results in the correction of goblet cell hyperplasia in some parts of the small intestine but not in the colon. Furthermore, it has been demonstrated that the small amount of hCFTR expression in the colon is not sufficient to completely rectify the transport and histologic abnormalities associated with the loss of endogenous CFTR. Thus, even with this gut correction, intestinal effects are still present, as shown by the increased weights of ilea and proximal colons in the Cftr^-/- mice. Importantly for the aims of this study, no significant increase in ilea weight occurred in the DKO mice, although it was still observed in their proximal colons. This indicates PAR1 contributes to the pathological effects of Cftr deficiency in the ileum but not colon. We also observed impaired weight gain in Cftr^-/- mice similar to the CF ‘failure to thrive’ phenotype in humans. Interestingly, the DKO mice had significantly higher body weights at the end of the study period, thus showing that the presence of PAR1 can contribute to CF-associated weight loss. Whether this has relevance to ileal abnormalities commonly present in humans with CF remains to be elucidated. Certainly, the presence of intestinal inflammation in patients with CF has received little recognition until recently, although endoscopic studies of the CF small intestine have revealed increased levels of inflammatory markers in the lumen and in biopsied tissues.⁴³ More recently, video-equipped capsule endoscopy showed that morphological abnormalities including oedema, erythema, mucosal breaks and ulcerations occur in the jejunum and ileum in >60% of CF patients and that significant elevations of faecal calprotectin (a neutrophil secretory product) occur in many CF patients, consistent with intestinal inflammation.⁴⁴ How these contribute to the clinical presentations of meconium ileus, distal intestinal obstruction syndrome, constipation and non-specific abdominal pain in CF is poorly understood. Additional investigation into the potential role of PAR1 in small bowel inflammation in CF is warranted.

We quantified the levels of proinflammatory cytokines in the ilea and proximal colons of these mice, including IL-17A, which we have previously shown, is upregulated by PAR1 in the gastrointestinal tract during colitis.²⁴ IFNγ was also analysed as a classic Th1-type cytokine and IL-6 as a typical inflammatory marker most commonly derived from macrophages. In this intestinal CF model, no association was made with the effect of PAR1 on intestinal weights and either IL-17A or IFNγ. However, the increase in ileal weights in Cftr deficient, but not DKO mice did correspond with elevated levels of the pro-inflammatory cytokine IL-6 that was only present in the Cftr^-/- mice.

CFTR defects are known to result in the upregulation of pro-inflammatory pathways in the intestine, especially nuclear factor kappa B (NF-κB)-mediated cascades, which potentiates the secretion of inflammatory cytokines such as IL-6.^{44 45} Many innate and stromal cells produce and respond to IL-6, resulting in an autocrine feedback loop that amplifies inflammation⁴⁶ and can often accelerate the transition from acute to chronic inflammation. CFTR null mice have been shown to have reduced microbial diversity and marked dysbiosis that are more pronounced in the ileum than other regions of the small intestine.⁴⁷ This microbial dysbiosis (characterised by an overabundance of more pro-inflammatory bacterial taxa) can also contribute to increased IL-6,⁴⁸ as observed in our study. However, to our knowledge, this is the first evidence of PAR1-modulating ileal IL-6 in CF mice. PAR1 is expressed throughout the gastrointestinal tract⁴⁹ and plays a critical role in regulating gut permeability in inflammatory bowel diseases.⁵⁰ The exact mechanism via which PAR1 might regulate the CF host response and IL-6 secretion warrants further investigation.

In summary, the absence of functional PAR1 significantly attenuates aspects of CF-related gut disease in a mouse model of CF, with its main focus of action appearing to be within the ileum. In particular, a possible link was indicated between PAR1 and the regulation of the pro-inflammatory cytokine IL-6. However, the expression of human CFTR in this model and its differential distribution throughout the intestine, although necessary, does limit some of the direct applicability of these findings to human CF intestinal disease. Having implicated a role for PAR1 in CF, further studies are, therefore, required to explore its role in regulating intestinal inflammation and downstream effects of PAR1 signalling, with the possibility of targeting this molecule as an approach to attenuate neutrophilic inflammation in CF.

Data availability statement

All data relevant to the study are included in the article.