https://orcid.org/0000-0003-2787-8895
Figures
Abstract
Objective
The airway epithelium provides a first line of defense against pathogens by release of antimicrobial factors and neutrophil-attracting chemokines. Pseudomonas (P.) aeruginosa, a Gram-negative bacterium that expresses flagellin as an important virulence factor, is a common cause of injurious airway inflammation. The aim of our study was to determine the contribution of flagellin to the inflammatory, antimicrobial, and metabolic responses of the airway epithelium to P. aeruginosa. Furthermore, as we previously showed that targeting mTOR limited the glycolytic and inflammatory response induced by flagellin, we assessed the effect of rapamycin on human bronchial epithelial (HBE) cells stimulated with flagellated and non-flagellated P. aeruginosa.
Methods
Primary pseudostratified HBE cells, cultured on an air-liquid-interface, were treated on the basolateral side with medium, vehicle or rapamycin, exposed on the apical side with flagellated or flagellin-deficient P. aeruginosa, and analyzed for their inflammatory, antimicrobial, and glycolytic responses.
Results
Flagellin augmented the P. aeruginosa-induced expression of antimicrobial factors and secretion of chemokines by HBE cells but did not further increase the glycolytic response. Treatment of HBE cells with rapamycin inhibited mTOR activation in general and flagellin-augmented mTOR activation in particular, but did not affect the glycolytic response. Rapamycin, however, diminished the flagellin-augmented inflammatory and antimicrobial response induced by Pseudomonas.
Conclusions
These results demonstrate that flagellin is a significant factor that augments the inflammatory and antimicrobial response of human airway epithelial cells upon exposure to P. aeruginosa and suggest that mTOR inhibition by rapamycin in the airway epithelium diminishes these exaggerated responses.
Citation: van Linge CC, Hulme KD, Peters-Sengers H, Kullberg RF, de Jong MD, Russell CA, et al. (2025) mTOR inhibition impacts the flagellin-augmented inflammatory and antimicrobial response of human airway epithelial cells to Pseudomonas aeruginosa. PLoS One 20(5): e0321462. https://doi.org/10.1371/journal.pone.0321462
Editor: Daniela Flavia Hozbor, Universidad Nacional de la Plata, ARGENTINA
Received: November 14, 2024; Accepted: March 6, 2025; Published: May 8, 2025
Copyright: © 2025 van Linge et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: European Union H2020, FAIR project, grant number 847786 - The European Research Council [NaviFlu 818353].
Competing interests: The authors have declared that no competing interests exist.
1. Introduction
Pseudomonas (P.) aeruginosa is a flagellated Gram-negative bacterium and one of the most frequent causative pathogens in pneumonia [1]. Patients at risk include those who are hospitalized and/or suffer from chronic lung disorders, such as cystic fibrosis or chronic obstructive pulmonary disease, which are associated with airway colonization by Pseudomonas [1]. Furthermore, P. aeruginosa was defined as one of the six leading pathogens causing antimicrobial resistance-associated deaths worldwide [2], emphasizing the importance of developing alternative therapies.
The respiratory epithelium in the lung is responsible for the first line of defense against inhaled pathogens and can be activated through different pattern recognition receptors, such as Toll-like receptors (TLRs) [3–5]. TLR2, TLR4, and TLR5 are involved in the recognition of P. aeruginosa in the lung, as exemplified by reduced host defense against this pathogen in mice deficient for these TLRs or the common adaptor myeloid differentiation primary response-88 [6–9]. TLRs can be activated by specific Pseudomonas-derived molecular patterns, including lipoproteins (TLR2), lipopolysaccharide (LPS; TLR4) and flagellin (a structural component of the flagellum; TLR5) [9–12]. Previously, we and others have shown that primary human bronchial epithelial (HBE) cells can be activated by flagellin and by P. aeruginosa [11–15], leading to the expression and secretion of neutrophil-attracting chemokines and antimicrobial proteins [12,14,15]. However, the contribution of flagellin to the Pseudomonas-induced epithelial response has not yet been described in HBE cells.
Glycolysis is a crucial metabolic pathway that converts glucose into pyruvate and lactate, providing cells with energy for the production and release of inflammatory cytokines. Mechanistic target of rapamycin (mTOR) plays a central role in regulating cellular metabolism and stimulates glycolysis [16,17]. Recently, we found that inhibition of mTOR activation by rapamycin, a drug prescribed for various chronic diseases and prevention of transplant rejection [18], impeded glycolysis and limited the secretion of chemokines by flagellin-stimulated human airway epithelial cells [14]. Yet, whether mTOR inhibition by rapamycin could reduce inflammation during P. aeruginosa infection is currently unknown.
The current study had two main objectives: first, to determine the contribution of flagellin to the P. aeruginosa-induced inflammatory and antimicrobial response by primary HBE cells, and second, to establish whether targeting of mTOR by rapamycin can modulate the inflammatory and antimicrobial responses of the human airway epithelium induced by flagellated and non-flagellated Pseudomonas. For this purpose, we studied primary HBE cells, cultured in a pseudostratified manner on an air-liquid-interface, after treatment on the basolateral side with rapamycin and exposure at the apical side with flagellated P. aeruginosa (PAK) or flagellin-deficient (PAKΔF) bacteria. The results of our study indicate that flagellin augments the P. aeruginosa-induced expression of chemokines and antimicrobial factors, and that these responses are diminished by inhibition of mTOR activation.
2. Materials and methods
2.1. Preparation of bacterial stocks
Wild-type P. aeruginosa (PAK) and flagellin-deficient PAK, generated by deletion of the fliC gene (PAK ∆ fliC), hereafter designated PAKΔF, were cultured as described [19,20]. Briefly, bacteria were grown to mid-logarithmic phase in Luria broth at 37°C with shaking, washed with 0.9% NaCl, and resuspended in phosphate-buffered saline (PBS). Bacterial numbers were determined by plating serial dilutions on blood agar plates. Bacteria were heat-killed for 20 minutes at 70°C, stored at 4°C, and used the next day. We previously showed that heat treatment did not affect the stimulatory capacity of flagellin [21].
2.2. HBE cell culture and stimulation
Primary human bronchial epithelial (HBE) cells were cultured as previously described [12]. Briefly, primary bronchial epithelial cells were isolated from healthy lung tissue obtained anonymously from patients undergoing a lobectomy for lung cancer (Amsterdam UMC approved study protocol 2015–122#A2301550). Written informed consent was obtained before surgery and tissue was sampled between July 1st 2015 to December 30th of 2018. Primary epithelial cells were isolated and differentiated following Fulcher’s protocol [22]. Passage 2–4 primary HBE cells were viably frozen between 2015 and 2018. Cells were thawed for the current study. At this point, cells were differentiated on Transwell inserts (Corning) [22]. When the cells on the inserts reached confluency, an air-liquid interface was formed with PneumaCult-ALI medium (StemCell Technologies) on the basolateral side, and removal of culture medium from the apical side. For bacterial stimulation, HBE cells were inoculated on the apical side with 4*106 colony-forming units of PAK or PAKΔF per insert in a volume of 20 μl (or 20 μl PBS as control) and incubated at 37°C. After 24 hours, the apical surface was washed with 200 μl PBS. Basolateral medium and apical wash were stored at -20°C until further analysis. HBE cells were stored in RNA lysis/binding buffer at -80°C for RNA isolation. For inhibition of mTOR, HBE cells were treated with 10 nM rapamycin (Cayman chemical) in the basolateral culture medium 1 hour prior to bacterial stimulation, as described [14].
2.3. Cytokine and lactate measurements
Chemokine CXC motif ligand (CXCL)1, CXCL8, CC chemokine ligand (CCL)20, granulocyte-colony stimulating factor (G-CSF), and calprotectin were measured by ELISA according to the manufacturer’s instructions (R&D Systems). Lactate was measured as described [14] using an assay in which lactate was oxidized and the resulting hydrogen peroxide was coupled to the conversion of Amplex Red reagent (Thermo Fisher Scientific) to fluorescent resorufin by horseradish peroxidase.
2.4. mRNA analysis
Total mRNA was isolated from HBE cells using the High Pure RNA Isolation Kit (Roche), following the manufacturer’s instructions. cDNA synthesis and qPCR were performed as previously described [14]. Data were analyzed with LinRegPCR software based on PCR efficiency values derived from amplification curves. HPRT was used as a housekeeping gene for normalization. Primers for HPRT, CXCL1, CXCL8, CCL20, S100A8, and S100A9 were similar as described [12]. All other primers are listed in Table 1.
2.5. Immunoblot analysis
Western blot analysis was performed as previously described [14]. Briefly, HBE cells were lysed in RIPA buffer supplemented with HALT protease and phosphatase inhibitor (Thermo Fisher Scientific) and heat-denatured in reducing sample buffer (Laemmli buffer) prior to 12.5% polyacrylamide gel electrophoresis. Blots were first incubated with antibodies against β-Actin (4967L; Cell Signaling Technologies) and phosphor-S6 Ribosomal Protein (Ser235/236) (2211S; Cell Signaling Technologies) and subsequently with horse radish peroxidase-conjugated goat anti-rabbit IgG (7074S; Cell Signaling Technologies). Blots were incubated with the Lumi-Light detection kit (Roche) and pictures were taken using a ChemiDoc MP (Bio-Rad). Western blot results were quantified by densitometry analysis using Adobe Photoshop version 24.3.0 (Adobe).
2.6. Statistical analysis
Statistical analysis was performed using a mixed model with a random intercept on the level of experiment and donor, with 3–4 technical replicates from 2 donors. Mixed models were adjusted for donor by including it as a covariate. Outcomes were log-transformed for analyses. All (co)variances were assumed equal and equivalent to a compound symmetry covariance structure. If the homogeneity of variances across groups was violated, as determined by Levene’s test (p < 0.05), we used non-parametric Mann-Whitney U tests to compare groups that did not meet this assumption. In case the overall group difference was significant, determined by type-III Wald test in the mixed model analyses (p < 0.05), then each stimulation was compared with the reference category to identify differences between groups. We used package “nlme” (version 3.1–164) for mixed models in the R statistical framework. Single-donor data from the rapamycin experiment were analyzed using GraphPad Prism version 9 (GraphPad Software). To this end, parametric variables were analyzed using a 2-way analysis of variance (comparison between 3 or more groups) or a Student’s t-test (2-group comparison). Non-parametric variables were analyzed using Kruskall-Wallis and Mann-Whitney U tests. In graphpad, multiple comparisons were Benjamini-Hochberg-corrected for multiple testing. Statistical significance is shown as *p < 0.05, **p < 0.01, ***p < 0.001.
3. Results
3.1. Flagellin augments P. aeruginosa-induced chemokine release by HBE cells
We showed earlier that both P. aeruginosa and flagellin induced an inflammatory response in pseudostratified HBE cells characterized by the secretion of CXCL1, CXCL8, CCL20 [12], and G-CSF [14]. To determine the contribution of flagellin to the Pseudomonas-induced epithelial response, we treated HBE cells on the apical side with either PAK, PAKΔF, or medium alone and measured the expression and secretion of inflammatory mediators 24 hours later. Both PAK and PAKΔF induced the expression of CXCL1, CXCL8, CCL20, and GCSF compared to medium control, yet PAK induced significantly higher mRNA levels compared to PAKΔF (Fig 1A). Similarly, both PAK and PAKΔF induced the secretion of CXCL1, CXCL8, and CCL20 in the basolateral medium, with higher chemokine concentrations after exposure to PAK compared to PAKΔF (Fig 1B). G-CSF levels in the apical wash were increased after stimulation with PAK and PAKΔF but reached significance only for PAK (Fig 1B). These findings indicate that the expression and secretion of chemokines by HBE cells in response to Pseudomonas is augmented by flagellin.
HBE cells were exposed at the apical side to vehicle (CTR), 4*106 CFUs of flagellated P. aeruginosa (PAK) or flagellin-deficient PAK (ΔF) for 24 hours, at which point samples were collected. mRNA levels of CXCL1, CXCL8, CCL20, and GCSF, normalized to HPRT (A), CXCL1, CXCL8, CCL20 levels in the basolateral medium and G-CSF in the apical wash (B). Data are presented as box and whiskers from 2 donors with 4 technical replicates per group. *Indicates significance between the indicated groups. *p < 0.05; **p < 0.01; ***p < 0.001.
3.2. Flagellin increases the P. aeruginosa-induced expression of antibacterial defense genes by HBE cells
Upon encountering bacteria, the bronchial epithelium secretes antimicrobial peptides that aid in host defense by exerting direct antimicrobial effects and recruiting other immune cells to the site of infection [5,23]. We determined expression levels of genes encoding β-defensin 4a (DEFB4A) and calprotectin (S100A8 and S100A9), factors involved in antibacterial defense, after 24 hours of exposure to PAK or PAKΔF. Both PAK and PAKΔF induced the expression of DEFB4A and S100A8 compared to control, with significantly higher levels after PAK stimulation compared to the flagellin-deficient strain (Fig 2A). In contrast to increased expression of S100A8, no differences were found in calprotectin protein levels in apical wash upon exposure to PAK or PAKΔF (Fig 2B). These data suggest that flagellin augments the PAK-induced expression of antibacterial defense genes.
HBE cells were exposed to vehicle (CTR), flagellated (PAK) or flagellin-deficient (ΔF) P. aeruginosa as described in Fig 1. Relative mRNA levels of DEFB4A, S100A8, and S100A9 after 24 hours (A), and protein levels of calprotectin in apical wash (B). Data are presented as box and whiskers from 2 donors with 4 technical replicates per group. *Indicates significance between the indicated groups. For DEFB4A, the homogeneity of variance could not be assumed, because of the minimal variance in the control group. Therefore, we compared the PAK and PAKΔF group separately. *p < 0.05; **p < 0.01; ***p < 0.001.
3.3. Exposure to P. aeruginosa induces a glycolytic response in HBE cells
Previously, we reported that purified flagellin triggered glycolysis in HBE cells after 24-hour stimulation [14]. To determine whether flagellin impacted the glycolytic response of HBE cells evoked by P. aeruginosa, we measured the mRNA expression of hexokinase-2 (HK2), the rate-limiting enzyme required for the first step in glycolysis [14], in HBE cells and the release of lactate in the basolateral medium after exposure to PAK or PAKΔF. Both PAK and PAKΔF increased mRNA levels of HK2 (Fig 3A) and the release of lactate compared to unstimulated cells (Fig 3B). Flagellin, however, did not affect the glycolytic response of HBE cells as HK2 mRNA levels and lactate concentrations were not different after exposure to PAK or PAKΔF. These results suggest that P. aeruginosa induces an increase in glycolytic activity in the human airway epithelium independent of flagellin.
HBE cells were exposed to vehicle (CTR), flagellated (PAK) or flagellin-deficient (ΔF) P. aeruginosa as described in Fig 1. Relative mRNA levels of HK2 (A) and lactate levels in basolateral medium (B) after 24 hours stimulation. Data are presented as box and whiskers from two donors with 4 technical replicates per group. *Indicates significance between the indicated groups. **p < 0.01.
3.4. Rapamycin inhibits P. aeruginosa-induced mTOR activation in HBE cells but has little impact on lactate release
Since mTOR is a central regulator of cellular metabolism in flagellin-treated HBE cells and targeting of mTOR by rapamycin inhibited the flagellin-induced glycolytic and inflammatory response after 24-hour stimulation [14], we next assessed the effect of mTOR inhibition on the response of HBE cells to P. aeruginosa. To this end, we treated HBE cells with rapamycin before exposure to PAK or PAKΔF and first analyzed the phosphorylation of ribosomal protein S6, a direct downstream target of mTOR [18], by Western blot analysis (Fig 4A). PAK, in contrast to PAKΔF, tended to increase the expression of phospho-S6 in HBE cells (Fig 4B). Treatment with rapamycin significantly inhibited the activation of the mTOR pathway, as revealed by lower levels of phospho-S6 in HBE cells exposed to medium, PAK, or PAKΔF. Next, we determined whether rapamycin also decreased the glycolytic response upon stimulation with P. aeruginosa by analysis of HK2 expression and lactate release. Rapamycin did not impact the expression of HK2 or the release of lactate, compared to vehicle, without bacterial stimulation. Upon exposure to PAK or PAKΔF, rapamycin impeded the increased expression of HK2 (Fig 4C). Strikingly, however, increased lactate levels in the supernatant of HBE cells after stimulation with PAK and PAKΔF were not affected by rapamycin treatment (Fig 4D). These data indicate that rapamycin inhibits the augmented mTOR activation in HBE cells exposed to flagellated P. aeruginosa, but does not affect their glycolytic flux as revealed by lactate release.
HBE cells were treated with vehicle (Veh) or rapamycin (Rapa) and, after 1 hour, exposed to vehicle (CTR), flagellated (PAK), or flagellin-deficient (ΔF) P. aeruginosa. Western blot of phosphorylated ribosomal protein S6, a downstream target of mTOR, and β-actin as a control after 2 hours of stimulation (A). Ratio of mean intensity of pS6 and β-actin bands determined by densitometry (B). Relative mRNA levels of HK2 (C), and lactate levels in basolateral medium (D) 24 hours after exposure. Data are presented as box and whiskers from one donor with 8 technical replicates per group. *Indicates significance between the indicated groups. *p < 0.05; **p < 0.01; ***p < 0.001.
3.5. Inhibition of mTOR activation diminishes the inflammatory and antimicrobial response induced by flagellated P. aeruginosa
After having established that rapamycin inhibits mTOR activation in Pseudomonas-exposed HBE cells, we determined the effect of rapamycin on the PAK- and PAKΔF-induced inflammatory response of HBE cells. Rapamycin inhibited the flagellin-augmented expression of CXCL8, CCL20, and GCSF, but not of CXCL1 (Fig 5A). After rapamycin treatment, PAK and PAKΔF induced similar mRNA levels of these genes in HBE cells. In addition, rapamycin reduced the secretion of CXCL1, CCL20, and G-CSF upon exposure to PAK (not CXCL8 secretion), but did not impact PAKΔF-induced secretion of these mediators, except for CCL20 (Fig 5B). Next, we determined the effect of rapamycin on the PAK- and PAKΔF-induced antimicrobial response of HBE cells. Rapamycin decreased the flagellin-augmented mRNA levels of DEFB4A and S100A8 upon exposure to Pseudomonas, as well as S100A8 mRNA levels induced by PAKΔF exposure (Fig 5C). The flagellin-augmented secretion of calprotectin was diminished in rapamycin-treated HBE cells (Fig 5D). Taken together, these data indicate that mTOR inhibition by rapamycin diminishes both the flagellin-augmented inflammatory and antimicrobial response induced by P. aeruginosa.
HBE cells were treated with vehicle (Veh) or rapamycin (Rapa) and, after 1 hour, exposed to vehicle (CTR), flagellated (PAK), or flagellin-deficient (ΔF) P. aeruginosa for 24 hours, at which point samples were collected. Relative mRNA levels of CXCL1, CXCL8, CCL20, and GCSF (A), chemokine levels in the basolateral medium and G-CSF in the apical wash (B), relative mRNA levels of DEFB4A, S100A8 and S100A9 (C), and calprotectin levels in apical wash (D). Data are presented as box and whiskers from one donor with 8 technical replicates per group. *Indicates significance between the indicated groups. *p < 0.05; **p < 0.01; ***p < 0.001.
4. Discussion
Flagellin has been implicated as a potent activator of the pulmonary epithelium by inducing the secretion of immune cell-attracting chemokines and antimicrobial proteins [11]. In the present study, we investigated the contribution of flagellin to the inflammatory and antimicrobial response of pseudostratified HBE cells in the context of Pseudomonas infection. We demonstrate that flagellin potentiated chemokine secretion and to some extent also the expression of antimicrobial proteins and metabolic response evoked by P. aeruginosa in the human airway epithelium. Moreover, our study reveals that mTOR inhibition by rapamycin diminished this flagellin-augmented inflammation to the level evoked by non-flagellated P. aeruginosa. These findings indicate that the flagellin-induced airway epithelial response to P. aeruginosa is regulated through mTOR.
Our findings that flagellated P. aeruginosa induced higher expression and secretion of chemokines by HBE cells compared to flagellin-deficient P. aeruginosa are in line with earlier studies by our group using HBE cells and purified flagellin [12,14,15], and findings of others showing that soluble bacterial ligands derived from flagellated P. aeruginosa, but not of flagellin-deficient P. aeruginosa, increased the release of CXCL8 by HBE cells after 24-hour stimulation [24]. Furthermore, a study with primary pseudostratified tracheal epithelial cells on an air-liquid interface also showed that flagellated P. aeruginosa increased the release of CXCL8 to the basolateral medium compared to a flagellin-deficient strain [25]. These findings support the notion that flagellin is a potent activator of inflammatory responses by lung epithelial cells during Pseudomonas infection in the lung.
The expression of TLR5 by airway epithelial cells is regarded as crucial for host defense against P. aeruginosa [26–28], both for induction of antimicrobial factors and the release of cytokines and chemokines triggering the attraction and activation of immune cells including neutrophils. Analysis of TLR5 mRNA levels in HBE cells revealed that neither PAK, PAK ∆ F or rapamycin impacted on its expression (data not shown). Our finding that PAK lacking the TLR5 ligand flagellin triggered a substantial inflammatory response in HBE cells, however, may be explained by activation of pattern recognition receptors via other ligands of Pseudomonas. Previously, we demonstrated that HBE cells, besides TLR5, express other TLRs including TLR1, TLR2, and TLR3 [12]. However, HBE cells do not express TLR4 and are unresponsive to lipopolysaccharide (LPS) [14]. Both PAK and PAKΔF have been found to activate TLR2 [13], and several components of P. aeruginosa have been identified as TLR2 ligands [9,29,30]. In agreement, lung epithelial cells derived from mice deficient in both TLR2 and TLR4 displayed severely hampered chemokine release upon PAKΔF exposure[6,31]. Thus, although the inflammatory response of HBE cells upon exposure to P. aeruginosa is vastly augmented by flagellin-mediated triggering of TLR5, other receptors also contribute to the P. aeruginosa-induced activation.
In line with our earlier study with flagellin-stimulated HBE cells [14], we found that P. aeruginosa induces a glycolytic response in the airway epithelium, as revealed by increased levels of hexokinase 2 mRNA and secreted lactate. Flagellin expression by Pseudomonas, however, did not impact this response, indicating that ligands other than flagellin induce the glycolytic response of HBE cells in the context of P. aeruginosa exposure. Also consistent with our former study investigating the effect of purified flagellin [14], was the finding that PAK, but not PAKΔF, triggered the activation of the mTOR pathway in HBE cells, as indicated by increased phospho-S6 protein levels. These findings corroborate a recent study showing that P. aeruginosa upregulated the expression of genes involved in mTOR signaling in human airway organoids [32]. Strikingly, we found that rapamycin, although reducing HK2 mRNA levels, did not diminish lactate secretion by HBE cells upon exposure to P. aeruginosa, opposite from our previous result that mTOR inhibition impeded glycolysis after 24-hour exposure to flagellin [14]. Nevertheless, consistent with the effect of rapamycin on flagellin-stimulated HBE cells [14], rapamycin diminished the expression of inflammatory and antimicrobial factors upon exposure to PAK. One study investigated the effect of mTOR-inhibition on the inflammatory response of a mouse macrophage cell line and found that rapamycin decreased flagellin-induced cytokine production by preventing the activation of p65 and STAT3, downstream of the TLR5-mTOR signaling pathway [33]. These latter findings suggest that mTOR inhibition can directly prevent the inflammatory response induced by flagellin, regardless of glycolytic activation.
The effect of rapamycin on lung inflammation and bacterial infection in experimental models in vivo has been variable. We previously showed that rapamycin treatment reduced pulmonary chemokine production during flagellin-induced lung inflammation in mice [14]. Others showed that rapamycin treatment in mice inhibited LPS-induced lung inflammation, but augmented lung injury [34,35] Rapamycin treatment ameliorated lung inflammation and host defense against pulmonary Burkholderia cenocepacia infection in a mouse model of cystic fibrosis [36]. Rapamycin, however, did not impact host defense against pulmonary Streptococcus pneumoniae infection and exacerbated Staphylococcus aureus pneumonia [37,38]. These studies highlight the varying effects of rapamycin on the host response in the lung evoked by different bacteria and bacterial ligands and warrant further studies on rapamycin in P. aeruginosa pneumonia.
Our study has several limitations. First, we used heat-killed instead of live bacteria since our primary focus was on the effect of flagellin. Since motility of PAK may contribute to virulence by enhancing invasion of epithelial cells and by translocation across epithelial barriers, further studies with live PAK and PAK ∆ F, as well as other motility mutants [39] and complementation of PAK ∆ F with purified flagellin may clarify whether detection of flagellin or motility influences the response of HBE cells. Second, our study did not control for cell death. While differences in cytokine levels between PAK- and PAK ∆ F-stimulated cells may be explained by differing rates of cell death, we regard this an unlikely explanation since bacteria were heat-killed, PAK ∆ F lack an important virulence factor and the absence of flagellin did not impact on the expression and secretion levels of several other inflammation markers. Third, since we used primary bronchial epithelial cells from two different donors, inter-donor differences could partly explain why similar experiments did not yield identical results. Moreover, slight differences in the preparation of heat-killed bacterial stocks and the pseudostratified HBE cell layer, consisting of multiple layers of different cells [4], could to some extent also impact the results. Although we consider our model with pseudostratified bronchial epithelial cells cultured in an air-liquid interface as highly relevant for the human lung, further studies are required to determine whether our findings with rapamycin also apply to other cell types in the lung [3,40]. Alveolar epithelial type II (AECII) cells are capable of secreting a variety of inflammatory mediators and antimicrobial factors [41,42], and primary mouse AECII are responsive to Pseudomonas [43] and susceptible to rapamycin [44]. Human AECII, however, are hard to culture [4]. We did not consider the alveolar epithelial cell line A549 for our experiments, despite the expression of TLR5 and other pattern recognition receptors [45], since these cells do not develop apical-basal polarity and given their aberrant cellular metabolism [40] resulting from their malignant origin. Even more interesting would be to add alveolar macrophages and neutrophils to these models, as these immune cells are known to contribute to host defense against Pseudomonas [28,46] and are known to be responsive to rapamycin [3,47,48].
In summary, we demonstrate here that flagellin augments the inflammatory and antimicrobial responses of HBE cells upon exposure to P. aeruginosa and that mTOR inhibition diminishes these exaggerated responses. These results imply that targeting mTOR in airway epithelial cells may be beneficial for the treatment of superfluous lung inflammation during Pseudomonas infection.
Acknowledgments
The authors thank Karen de Haan and Sarah van Leeuwen (Department of Medical Microbiology & Infection Prevention, Amsterdam UMC) for their technical support in the culturing of the HBE cells, and Regina de Beer (Center for Experimental and Molecular Medicine, Amsterdam UMC) for her technical support measuring mRNA expression levels.
References
- 1. Torres A, Cilloniz C, Niederman MS, Menéndez R, Chalmers JD, Wunderink RG, et al. Pneumonia. Nat Rev Dis Primers. 2021;7:25. pmid:33833230
- 2. Murray CJ, Ikuta KS, Sharara F, Swetschinski L, Robles Aguilar G, Gray A, et al. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet. 2022;399: 629–55.
- 3. Hewitt RJ, Lloyd CM. Regulation of immune responses by the airway epithelial cell landscape. Nat Rev Immunol. 2021;21:347–62. pmid:33442032
- 4. Hiemstra PS, Tetley TD, Janes SM. Airway and alveolar epithelial cells in culture. Eur Respir J. 2019;54:1–5. pmid:31515398
- 5. Leiva-Juárez MM, Kolls JK, Evans SE. Lung epithelial cells: therapeutically inducible effectors of antimicrobial defense. Mucosal Immunol. 2018;11:21–34.
- 6. Morris AE, Liggitt HD, Hawn TR, Skerrett SJ. Role of Toll-like receptor 5 in the innate immune response to acute P. aeruginosa pneumonia. Am J Physiol Lung Cell Mol Physiol. 2009;297:L1112–9. pmid:19801452
- 7. Skerrett SJ, Wilson CB, Liggitt HD, Hajjar AM. Redundant toll-like receptor signaling in the pulmonary host response to Pseudomonas aeruginosa. Am J Physiol Lung Cell Mol Physiol. 2007;292(1):L312–22. pmid:16936244
- 8. Skerrett SJ, Liggitt HD, Hajjar AM, Wilson CB. Myeloid differentiation factor 88 is essential for pulmonary host defense against Pseudomonas aeruginosa but not Staphylococcus aureus. J Immunol. 2004. Available from: http://journals.aai.org/jimmunol/article-pdf/172/6/3377/1179422/3377.pdf
- 9. McIsaac SM, Stadnyk AW, Lin T-J. Toll-like receptors in the host defense against Pseudomonas aeruginosa respiratory infection and cystic fibrosis. J Leukoc Biol. 2012;92:977–85. pmid:22892106
- 10. Jurado-Martín I, Sainz-Mejías M, McClean S. Pseudomonas aeruginosa: an audacious pathogen with an adaptable arsenal of virulence factors. Int J Mol Sci. 2021;1–37. pmid:33803907
- 11. Vijayan A, Rumbo M, Carnoy C, Sirard JC. Compartmentalized antimicrobial defenses in response to Flagellin. Trends Microbiol. 2018;26:423–35.
- 12. van Linge CCA, Hulme KD, Peters-Sengers H, Sirard J-C, Goessens WHF, de Jong MD, et al. Immunostimulatory effect of flagellin on MDR-Klebsiella-infected human airway epithelial cells. Int J Mol Sci. 2023;25(1):309. pmid:38203480
- 13. Zhang Z, Louboutin J-P, Weiner DJ, Goldberg JB, Wilson JM. Human airway epithelial cells sense Pseudomonas aeruginosa infection via recognition of flagellin by toll-like receptor 5. Infect Immun. 2005;73:7151–60.
- 14. Ramirez-Moral I, Yu X, Butler JM, van Weeghel M, Otto NA, Ferreira BL, et al. mTOR-driven glycolysis governs induction of innate immune responses by bronchial epithelial cells exposed to the bacterial component flagellin. Mucosal Immunol. 2021;14:594–604. pmid:33542495
- 15. Ramirez-Moral I, Ferreira BL, Butler JM, van Weeghel M, Otto NA, de Vos AF, et al. HIF-1α stabilization in flagellin-stimulated human bronchial cells impairs barrier function. Cells. 2022;11(3):391. pmid:35159204
- 16. Weichhart T, Hengstschläger M, Linke M. Regulation of innate immune cell function by mTOR. Nat Rev Immunol. 2018;15:599–614.
- 17. Saxton RA, Sabatini DM. mTOR signaling in growth, metabolism, and disease. Cell. 2017;168:960–76. pmid:28283069
- 18.
Wang D, Eisen HJ. Mechanistic Target of Rapamycin (mTOR) Inhibitors. In: Eisen HJ, editor. Pharmacology of Immunosuppression. Cham: Springer International Publishing; 2022. pp. 53–72. https://doi.org/10.1007/164_2021_553
- 19. Qin W, Brands X, van’t Veer C, de Vos AF, Sirard JC, Roelofs JJTH, et al. Bronchial epithelial DNA methyltransferase 3b dampens pulmonary immune responses during Pseudomonas aeruginosa infection. PLoS Pathog. 2021;17:1–21. pmid:33793661
- 20. Dasgupta N, Wolfgang MC, Goodman AL, Arora SK, Jyot J, Lory S, et al. A four-tiered transcriptional regulatory circuit controls flagellar biogenesis in Pseudomonas aeruginosa. Mol Microbiol. 2003;50:809–24. pmid:14617143
- 21. Qin W, Brands X, van’t Veer C, de Vos AF, Scicluna BP, van der Poll T. Bronchial epithelial tet2 maintains epithelial integrity during acute Pseudomonas aeruginosa pneumonia. Infect Immun. 2021;89(1):1–10. pmid:33046509
- 22. Randell SH, Fulcher ML. Human nasal and tracheo-bronchial respiratory epithelial cell culture. Epithelial Cell Culture Protocols. 2012;109–21.
- 23. Invernizzi R, Lloyd CM, Molyneaux PL. Respiratory microbiome and epithelial interactions shape immunity in the lungs. Immunology. 2020;160:171–82. pmid:32196653
- 24. Ben Mohamed F, Garcia-Verdugo I, Medina M, Balloy V, Chignard M, Ramphal R, et al. A crucial role of flagellin in the induction of airway mucus production by Pseudomonas aeruginosa. PLoS One. 2012;7:e39888. pmid:22768318
- 25. Tseng J, Do J, Widdicombe JH, Machen TE. Innate immune responses of human tracheal epithelium to Pseudomonas aeruginosa flagellin, TNF-α, and IL-1β. Am J Physiol Cell Physiol. 2006;290:678–90.
- 26. Anas AA, van Lieshout MHP, Claushuis TAM, de Vos AF, Florquin S, de Boer OJ, et al. Lung epithelial MyD88 drives early pulmonary clearance of Pseudomonas aeruginosa by a flagellin dependent mechanism. Am J Physiol Lung Cell Mol Physiol. 2016;311:219–28.
- 27. Morris AE, Liggitt HD, Hawn TR, Skerrett SJ. Role of Toll-like receptor 5 in the innate immune response to acute P. aeruginosa pneumonia. Am J Physiol Lung Cell Mol Physiol. 2009;297(6):1112–9. pmid:19801452
- 28. Feuillet V, Medjane S, Mondor I, Demaria O, Pagni PP, Galá JE, et al. Involvement of Toll-like receptor 5 in the recognition of flagellated bacteria. PNAS. 2006. Available from: www.pnas.org/cgi/doi/10.1073/pnas.0605200103
- 29. Revets H, Pynaert G, Grooten J, De Baetselier P. Lipoprotein I, a TLR2/4 ligand modulates Th2-driven allergic immune responses. J Immunol. 2005;174(2):1097–103.
- 30. Epelman S, Stack D, Bell C, Wong E, Neely GG, Krutzik S, et al. Different domains of Pseudomonas aeruginosa exoenzyme S activate distinct TLRs. J Immunol. 2004;173:2031–40. pmid:15265938
- 31. Raoust E, Balloy V, Garcia-Verdugo I, Touqui L, Ramphal R, Chignard M. Pseudomonas aeruginosa LPS or flagellin are sufficient to activate TLR-dependent signaling in murine alveolar macrophages and airway epithelial cells. PLoS One. 2009;4(10):e7259. pmid:19806220
- 32. Tang M, Liao S, Qu J, Liu Y, Han S, Cai Z, et al. Evaluating bacterial pathogenesis using a model of human airway organoids infected with Pseudomonas aeruginosa biofilms. Microbiol Spectr. 2022;10(6):e0240822. pmid:36301094
- 33. Bao W, Wang Y, Fu Y, Jia X, Li J, Vangan N, et al. mTORC1 regulates flagellin-induced inflammatory response in macrophages. PLoS One. 2015;10:1–14. pmid:25942007
- 34. Hu Y, Lou J, Mao Y-Y, Lai T-W, Liu L-Y, Zhu C, et al. Activation of MTOR in pulmonary epithelium promotes LPS-induced acute lung injury. Autophagy. 2016;12:2286–99.
- 35. Fielhaber JA, Carroll SF, Dydensborg AB, Shourian M, Triantafillopoulos A, Harel S, et al. Inhibition of mammalian target of rapamycin augments lipopolysaccharide-induced lung injury and apoptosis. J Immunol. 2012;188:4535–42.
- 36. Abdulrahman BA, Khweek AA, Akhter A, Caution K, Kotrange S, Abdelaziz DHA, et al. Autophagy stimulation by rapamycin suppresses lung inflammation and infection by Burkholderia cenocepacia in a model of cystic fibrosis. Autophagy. 2011;7:1359–70.
- 37. Yu F-Y, Zheng K, Wu Y-F, Gao S-W, Weng Q-Y, Zhu C, et al. Rapamycin exacerbates Staphylococcus aureus pneumonia by inhibiting mTOR-RPS6 in macrophages. J Inflamm Res. 2023;16:5715–28. pmid:38053607
- 38. Hinojosa CA, Mgbemena V, Van Roekel S, Austad SN, Miller RA, Bose S, et al. Enteric-delivered rapamycin enhances resistance of aged mice to pneumococcal pneumonia through reduced cellular senescence. Exp Gerontol. 2012;47: 958–965.
- 39. Toutain CM, Zegans ME, O’Toole GA. Evidence for two flagellar stators and their role in the motility of Pseudomonas aeruginosa. J Bacteriol. 2005;187:771–7.
- 40. Mahieu L, Van Moll L, De Vooght L, Delputte P, Cos P. In vitro modelling of bacterial pneumonia: a comparative analysis of widely applied complex cell culture models. FEMS Microbiol Rev. 2024;48(2):fuae007. pmid:38409952
- 41. Davis JD, Wypych TP. Cellular and functional heterogeneity of the airway epithelium. Mucosal Immunol. 2021;14:978–990. pmid:33608655
- 42. Whitsett JA, Alenghat T. Respiratory epithelial cells orchestrate pulmonary innate immunity. Nat Immunol. 2015;16:27–35. pmid:25521682
- 43. Kannan S, Huang H, Seeger D, Audet A, Chen Y, Huang C, et al. Alveolar epithelial type II cells activate alveolar macrophages and mitigate P. aeruginosa infection. PLoS One. 2009;4:e4891.
- 44. Chung EJ, Sowers A, Thetford A, McKay-Corkum G, Chung SI, Mitchell JB, et al. Mammalian target of rapamycin inhibition with rapamycin mitigates radiation-induced pulmonary fibrosis in a murine model. Int J Radiat Oncol Biol Phys. 2016;96: 857–66.
- 45. Hou YF, Zhou YC, Zheng XX, Wang HY, Fu YL, Fang ZM, et al. Modulation of expression and function of Toll-like receptor 3 in A549 and H292 cells by histamine. Mol Immunol. 2006;43:1982–92.
- 46. Muggeo A, Coraux C, Guillard T. Current concepts on Pseudomonas aeruginosa interaction with human airway epithelium. PLoS Pathog. 2023;19(3):e1011221. pmid:36996043
- 47. Hussell T, Bell TJ. Alveolar macrophages: plasticity in a tissue-specific context. Nat Rev Immunol. 2014;14:81–93. pmid:24445666
- 48. Li X, Han X, Llano J, Bole M, Zhou X, Swan K, et al. Mammalian target of rapamycin inhibition in macrophages of asymptomatic HIV+ persons reverses the decrease in TLR-4-mediated TNF-α release through prolongation of MAPK pathway activation. J Immunol. 2011;187:6052–8. pmid:22025552