Signaling mediated by toll-like receptor 5 sensing of pseudomonas aeruginosa Flagellin Influences IL-1ß and IL-18 production by primary fibroblasts derived from the human cornea

ORIGINAL RESEARCH published: 19 April 2017 doi: 10.3389/fcimb.2017.00130 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 1April 2017 | Volume 7 | Article 130 Edited by: David Dockrell, University of Edinburgh, UK Reviewed by: Gill Diamond, University of Florida, USA Helen Maria Marriott, University of Sheffield, UK *Correspondence: Myron Christodoulides [email protected] †These authors have contributed equally to this work. Received: 10 November 2016 Accepted: 29 March 2017 Published: 19 April 2017 Citation: Cendra MdM, Christodoulides M and Hossain P (2017) Signaling Mediated by Toll-Like Receptor 5 Sensing of Pseudomonas aeruginosa Flagellin Influences IL-1βand IL-18 Production by Primary Fibroblasts Derived from the Human Cornea. Front. Cell. Infect. Microbiol. 7:130. doi: 10.3389/fcimb.2017.00130 Signaling Mediated by Toll-Like Receptor 5 Sensing of Pseudomonas aeruginosa Flagellin Influences IL-1β and IL-18 Production by Primary Fibroblasts Derived from the Human Cornea Maria del Mar Cendra1, Myron Christodoulides1*†and Parwez Hossain2 † 1Division of Clinical and Experimental Sciences, Department of Molecular Microbiology, Faculty of Medicine, University of Southampton, Southampton, UK, 2Eye Unit, Division of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK Pseudomonas aeruginosa is the principal cause of bacterial keratitis worldwide and overstimulation of the innate immune system by this organism is believed to contribute significantly to sight loss. In the current study, we have used primary human corneal fibroblast (hCF) cells as an ex vivo model of corneal infection to examine the role of P. aeruginosa flagellum and type three secretion system (TTSS) in inducing inflammasome-associated molecules that trigger IL-1βand IL-18 production during the early stages of the infection. Our results show that P. aeruginosa infection stimulated the non-canonical pathway for IL-1βand IL-18 expression and pathway stimulation was influenced predominantly by the flagellum. Both IL-1βand IL-18 cytokines were expressed intracellularly during bacterial infection, but only the former was released and detected in the extracellular environment. We also investigated the signaling pathways in hCFs mediated by Toll-Like Receptor (TLR)4 and TLR5 sensing of P. aeruginosa, and our data show that the signal triggered by TLR5-flagellin sensing significantly contributed to IL-1βand IL-18 cytokine production in our model. Our study suggests that IL-18 expression is wholly dependent on extracellular flagellin sensing by TLR5, whereas IL-1βexpression is also influenced by P. aeruginosa lipopolysacharide. Additionally, we demonstrate that IL-1βand IL-18 production by hCFs can be triggered by both MyD88-dependent and -independent pathways. Overall, our study provides a rationale for the development of targeted therapies, by proposing an inhibition of flagellin-PRR-signaling interactions, in order to ameliorate the inflammatory response characteristic of P. aeruginosa keratitis. Keywords: Pseudomonas aeruginosa, fibroblasts, human cornea, cytokine, NLRC4, flagellin, MyD88, TLR5 INTRODUCTION Bacterial keratitis is a global health problem in which pathogen-induced cytotoxicity and overstimulation of the host innate inflammatory response can lead to severe visual impairment and sight lost (Taube et al., 2015). In the human eye, superficial damage and surface laceration are often associated with contact lens wear. Thus, when the corneal epithelium barrier and Bowman’s Cendra et al. TLR5 Sensing of Pseudomonas Flagellin membrane located between the epithelium and the stroma in the cornea are physically breached, microbes can penetrate and establish infection in the stroma. Keratitis is characterized by the production of inflammatory cytokines, the subsequent influx of polymorphonuclear leukocytes (PMNL) and extensive tissue damage. As a consequence of microbial penetration into the stroma, resident keratocytes can respond by transforming into corneal fibroblasts that exacerbate the inflammatory response (O’brien, 2003). Pseudomonas aeruginosa is the leading cause of bacterial keratitis associated with contact lens wear (Shah et al., 2011; Stapleton and Carnt, 2012). Stimulation of the host innate immune response is initiated by pattern recognition receptors (PPR), which are immune receptors that recognize pathogen-associated molecular patterns (PAMPs) of infectious agents and trigger different types of cellular responses. Expression of cell wall lipopolysaccharide (LPS) O-antigen, the exotoxin injector type three secretion system (TTSS) and flagellum are key P. aeruginosa PAMPs (Hauser, 2011; Le Berre et al., 2011). The TTSS is a molecular machine encoded by pathogenic gram-negative bacteria, which is used to meditate interactions with eukaryotic host cells. This bacterial complex consists of a syringe-like “injectisome” that enables active translocation of bacterial proteins into the host cell cytoplasm, in order to alter eukaryotic cell biology during infection (Notti and Stebbins, 2016). Intracellular delivery of exotoxins is sensed by the host immune system: intracellular PPRs sense conserved bacterial determinants that have been internalized, e.g., components of the injectisome, thus activating a host response to infection (Puhar and Sansonetti, 2014). For P. aeruginosa, the PopB, PopD and PcrV are essential TTSS proteins for the exotoxin delivery mechanism. Possession of the TTSS has been suggested to be indispensable for PAO1 strain in promoting tissue damage and keratitis in animal models (Pier, 2007; Hauser, 2009). P. aeruginosa flagellin (FliC) is the protein product of the fliC gene, which becomes the flagellum filament when it is polymerized (Hayashi et al., 2001; Berg, 2003; Ghosh, 2004). Toll-like receptors (TLRs) are a family of PPRs and classified depending on whether they are found anchored within the cell membrane or located intracellularly (Janeway and Medzhitov, 2002; Kawasaki and Kawai, 2014). In the current study, we have focused on investigating cell membrane-associated TLR2, which senses bacterial lipoproteins; TLR4, which is activated by LPS, a classical inducer of host inflammatory processes; and TLR5, which is activated by extracellular flagellin that can subsequently be delivered within the cytosol (Kawasaki and Kawai, 2014). When stimulated, TLRs recruit TIR1domain-containing adaptors, such as MyD882, TRIF3, TIRAP4/MAL5, or TRAM6, and activate subsequent signaling pathways that lead to transcription of NF-κβ7and 1TIR: Toll/interleukin-1 receptor. 2MyD88: Myeloid differentiation primary response gene 88. 3TRIF: Toll/interleukin-1 receptor (TIR) domain containing adapter inducing interferon-β. 4TIRAP: Toll/interleukin-1 receptor (TIR) domain containing adapter protein. 5MAL: MyD88 adapter-like. 6TRAM: TRIF-related adaptor molecule. 7NF-κβ: Nuclear Factor Kappa-light-chain-enhancer of activated B cells. IRFs8to orchestrate a wide range of inflammatory responses and cytokine expression (O’neill et al., 2013; Kawasaki and Kawai, 2014). P. aeruginosa has been reported to activate TRIF and MyD88 adaptor molecules in mouse macrophages, but not TIRAP (Sun et al., 2010). Nod-like receptors (NLRs) are another family of PPRs, but they are found within the cytosol. NLRs sense intracellular PAMPS and promote the assembly of a multi-protein scaffold called the “inflammasome” (Chen et al., 2009; O’neill et al., 2013). The inflammasome recruits and activates certain caspases for maturing interleukin (IL)-1βand IL-18 cytokine pro-forms, and also induces a cell-death program called “pyroptosis” (Schroder and Tschopp, 2010; Latz et al., 2013). To date, numerous inflammasome-associated proteins have been described including NLRP91, NLRP2, NLRP3, AIM210, and NLRC114 (Schroder and Tschopp, 2010; Ozaki et al., 2015). P. aeruginosa flagellin and TTSS have been reported to be important for the intracellular assembly of the non-canonical inflammasome NLRC4 in mouse models (Sutterwala et al., 2007; Miao et al., 2010; Zhao et al., 2011). It is generally accepted that PAMP recognition by host cell PRR leads to the production of cytokines as well as other innate immune mediator signals (Mogensen, 2009). Herein, we have studied the role of the flagellum, TTSS and LPS PAMPs in the activation of inflammasome-associated signaling molecules, and consequential IL-1βand IL-18 production by human primary stromal corneal fibroblasts (hCFs), during the early stages of P. aeruginosa infection. The rationale for studying IL-1βand IL-18 expression in our in vitro model of hCFs is based on our observation that infection of these cells with wild-type P. aeruginosa is characterized predominantly by the production of these two cytokines, with the expression of other cytokines, chemokines, and immune mediator molecules not significant (manuscript submitted elsewhere). Furthermore, we have tested the hypothesis that inhibition of intracellular signaling pathways during P. aeruginosa infection can lead to reduced pro-inflammatory cytokine expression in hCFs. MATERIALS AND METHODS Human Primary Stromal Corneal Fibroblast (hCF) Corneal buttons were collected aseptically from patients at the eye unit of a large hospital in the south of England. After removing the corneal epithelium, the stromal layer was dissected and digested with collagenase Type-1 (1 mg/mL; Life Technologies, Warrington, UK) for 3 h at 37◦C. Digested stroma was cultured in Corneal Culture Medium (CCM) and hCF were characterized as described previously (Wong et al., 2011). Human monocytic cell line THP-1 was cultured in RPMI 1640 containing glutamine (Lonza, Biologics, Tewkesbury, UK), 1% (v/v) Penicillin-Streptomycin and 10% (v/v) decomplemented 8IRF: Interferon regulatory factor. 9NLRP: NLR family pyrin domain containing. 10AIM2: Absent in melanoma 2. 11NLRC: NLR family CARD domain containing. Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 2April 2017 | Volume 7 | Article 130 Cendra et al. TLR5 Sensing of Pseudomonas Flagellin Fetal Calf Serum (dFCS; Life Technologies, Warrington, UK). Cells were cultured in a humidified incubator at 37◦C and 5% (v/v) CO2. Ethics Statement Patients provided written informed consent to use surplus corneal tissue specimen for research via the NHS Blood Transplant Eye Retrieval Service based at Southampton General Hospital (Southampton, UK). Protocols were approved by the NRES Committee South Central-Berkshire 06/Q1602/56. Bacterial Strains, Plasmids, and Growth Conditions Pseudomonas aeruginosa strain PAO1 (Holloway1C Stanier131) was obtained from the National Collection of Industrial, Food and Marine Bacteria, UK. P. aeruginosa strain PA14 and PA14 1flgK and PA14 1popB mutants were provided by George O’Toole (Dartmouth Medical School, Dartmouth, USA). PA14 1flgK mutant is unable to synthesize a functional flagellum and the PA14 1popB mutant lacks the PopB protein, an essential protein without which the TTSS cannot deliver exotoxins within eukaryotic cells (Berg, 2003; Hauser, 2009). Green Fluorescence Protein (GFP) was inserted into PAO1, PA14 and mutant strains by transformation of the PAO1/GFP plasmid provided by Alice Prince (Columbia University, New York, USA) (Parker and Prince, 2013). To complement PA14 1flgK and PA14 1popB mutation, the flgK and popB coding region sequences from the PA14 genome were amplified using the primers listed in Supplementary Table 1 and cloned into the HindIII and BamHI sites of the shuttle cloning vector pUCP19 (Schweizer, 1991). pUCP19-flgK and pUCP19-popB were transformed into Escherichia coli DH5αand into PA14 1flgK and PA14 1popB strains (Irani and Rowe, 1997). Carbenicillin (Sigma-Aldrich, Poole, UK) at 100 and 300 µg/mL was added to the medium for plasmid maintenance for E. coli and P. aeruginosa, respectively. Bacterial strains were grown in Luria-Bertani broth and agar (Oxoid, Basingstoke, UK) at 37◦C. Transfection of hCFs with TLR5-siRNA (siTLR5-hCFs) hCFs were grown to 30–50% of confluence in a 24-well plate and transfected with four TLR5 siRNA (FlexiTube GeneSolution, Qiagen, Manchester, UK) using INTERFERin R  (Polyplus transfection, Illkirch-Graffenstaden, France). Mock RNA was used as a transfection control. A total of 1 nM of TLR5-siRNA per well was mixed with 100 µl of dFCSfree CCM and 2 µl of INTERFERin R . After 10 min at room temperature (RT), the transfection mix was added to the CCM (500 µl) already present in the wells and incubated for 4 h. Transfection solution was then removed and 1 mL of fresh CCM was added per monolayer and cells were incubated for 48 h. Mock transfection with an irrelevant siRNA was done to ensure that TLR5 expression remained unaltered. TLR5 silencing was checked by measuring TLR5 expression in PA14-stimulated hCFs by Real Time quantitative Polymerase Chain Reaction (RT-qPCR) and western-blot. Expression and Purification of Recombinant (r) FliC Protein of P. aeruginosa The fliC (flagellin) coding region of PAO1 is 100% identical to that in PA14, and it was amplified using the primers listed in Supplementary Table 1 and cloned into the NdeI and XhoI restriction sites of the T7 promoter vector pET22b(+) (Merck Millipore, Hertfordshire, UK). After checking pET22b-fliC by PCR with T7 universal oligonucleotides (Supplementary Table 1) and sequencing, the plasmid was transformed into E. coli BL21 using ampicillin (Sigma-Aldrich, Poole, UK) at 50 µg/mL to maintain the plasmid. BL21 pET22b-fliC culture was grown to an OD λ600 nm =0.5 and induced with isopropyl-β-D-1thiogalactopyranoside (IPTG; 1 mM) for 4 h, harvested and lysed. Cell supernatant and pellet from the lysed culture were analyzed by SDS-PAGE, which demonstrated presence of the recombinant (r)FliC in the supernatant. rFliC was purified from the soluble fraction of harvested cultures using nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography (Qiagen, Manchester, UK) under native conditions, as described previously (Hung et al., 2015). Bound rFliC was eluted with 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole buffer (pH 8.0) and elution fractions analyzed by SDS-PAGE. Pooled fractions were dialyzed against PBS, pH7.4 for 2 days. Lipopolysaccharide (LPS) content was quantified with the Pierce LAL Chromogenic Endotoxin Quantitation Kit (Thermo Fisher Scientific, Basingstoke, UK) following the manufacturer’s instructions. SDS-PAGE of purified rFliC confirmed the purity and molecular mass (∼49 KDa) of the protein (Supplementary Figure 1). Infection of hCFs with P. aeruginosa Strains and Different Stimulatory Conditions hCFs were seeded into 24-well culture plates (Greiner bio-one, Stonehouse, UK, ∼105cells/well) and infected over time with P. aeruginosa at a Multiplicity Of Infection (MOI) of 10 in dFCSfree DMEM medium (Sigma-Aldrich, Poole, UK). Stimulatory conditions included 100 ng/mL of P. aeruginosa LPS (SigmaAldrich, Poole, UK) and 5 µg/mL of purified P. aeruginosa rFliC protein for 6 h and when required, streptolysin-O (SLO), was used for the intracellular delivery of the molecule as described previously (Walev et al., 2001). Bacterial association to hCFs was quantified over time as described previously (Hardy et al., 2000). Briefly, monolayers were washed gently 4 times with phosphate buffered saline (PBS), pH7.4 and lysed with 250 µl of PBS containing saponin [1% (w/v); Sigma-Aldrich, Poole, UK] and dFCS (1% w/v) for 15 min. Determination of colony forming units was done by serial dilution and viable counting on agar plates. Signaling Pathway Inhibition Inhibition experiments were done using 50 µM of Myd88 peptide inhibitor (MyD88i, Bio-techne, Abington, UK) and 1 µM of TAK-242 (Merck Millipore), respectively (Loiarro et al., 2005; Takashima et al., 2009; Matsunaga et al., 2011). hCFs were treated with MyD88i and TAK-242 for 24 and 12 h, respectively, prior Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 3April 2017 | Volume 7 | Article 130 Cendra et al. TLR5 Sensing of Pseudomonas Flagellin to hCFs stimulation with P. aeruginosa strains, rFliC or LPS for 6 h. Uninfected hCFs monolayers in inhibited and non-inhibited conditions were used as controls. Total RNA Extraction and Real Time Quantitative Polymerase Chain Reaction (RT-qPCR) In this study, total RNA was extracted from infectedand stimulatedhCF cells during different experimental conditions including hCF infection with the PAO1, PA14 wild-type and 1flgK,1popB mutant strains for 1, 3 or 6 h, and after stimulation with rFliC (5 µg/mL) and P. aeruginosa LPS (100 ng/mL) for 6 h. Total RNA was extracted from siTLR5-hCFs monolayers after 6 h of similar stimulatory and infection conditions. RNA extraction was done using the RNeasy Mini Kit (Qiagen, Manchester, UK). Control conditions were uninfected and un-stimulated hCFs or siTLR5-hCFs. RNA samples were treated with DNAse-50 (PrimerDesign, Chandler’s Ford, UK) and total concentration was quantified using a NanoDrop spectrophotometer (ND-1000, NanoDrop, Thermo Fisher Scientific, Basingstoke, UK). cDNA was synthetized from 1 µg of RNA using the RT-nanoscript system (PrimerDesign, Chandler’s Ford, UK) and the reverse oligonucleotides listed in Supplementary Table 1. RT-qPCRs were done with 1 µl of each cDNA in a Rotorgene-Q 5plex HRM (Qiagen, Manchester, UK) using the Power SYBR green PCR Master Mix (Life Technologies, Warrington, UK) and oligonucleotides listed in Supplementary Table 1. β-actin was used as an endogenous control and melting curves were done to ensure specific gene amplification. The 2−11CT method was used for data analyses (Livak and Schmittgen, 2001). Protein Extraction and Western Blot Analysis Intracellular protein analysis of TLR2, TLR4, TLR5, NLRC4, caspase-1, caspase-4, and the cytokines IL-18 and IL-1βwas done after 6 h in similar infection and stimulatory conditions, including the experiments with siTLR5-hCFs cells, as for RTqPCR experiments. hCFs infected with complemented PA14 1flgK pUCP19-flgK and PA14 1popB pUCP19-popB strains were included for protein analyses. Intracellular mature IL-18 and IL-1βwere also measured in hCFs treated with TAK-242, MyD88i and TAK-242/MyD88i and infected with PA14 and mutant strains. Cells were washed in PBS and total protein extract was obtained by using CytobusterTM protean extraction reagent (Merck Millipore, Nottingham, UK). Total protein (5 µg) was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred onto polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Amersham, UK). Membranes were blocked for 1 h in PBS containing 0.05% (v/v) Tween20 (PBST) and 5% (v/v) dry milk powder. Antibodies used were rabbit monoclonal antiTLR2, -TLR4, -TLR5; rabbit polyclonal anti-NLRP3, -Caspase4, -IL-18, and -IL-1β(Abcam, Bristol, UK); rabbit polyclonal anti-NLRC4 (Bio-techne Ltd, Abingdon, UK). All antibodies were used at the manufacturers’ recommended dilutions and left overnight at 4◦C. Membranes were washed three times in PBST and incubated for 1 h with goat anti-Rabbit IgG (H&L)- HRP pre-adsorbed secondary antibody (Abcam, Bristol, UK) according to the manufacturer’s recommendations. Rabbit antiβ-actin polyclonal antibody (Abcam, Bristol, UK) was used as an endogenous and loading control. Immuno-detection was carried out in a Versadoc 4000 and data analyzed with Quantity One 4.6.9 software (BioRad, Langford, UK). Cytokine Release Analysis Extracellular IL-18 and IL-1βcytokines were quantified with the Meso Scale Discovery (MSD) electro-chemiluminescence assays (Meso Scale Diagnostics, Gaithersburg, USA.) and by western blot after protein precipitation with trichloroacetic acid (TCA). Supernatants from triplicate hCF monolayers infected with wildtype PAO1, PA14, mutant PA14 1flgK, and PA14 1popB strains (MOI =10) were collected after 6 h of infection. Confocal Microscopy For visual comparison of bacterial association with hCFs, hCF monolayers, grown on borosilicate glass cover slips, were infected for 9 h with GFP-expressing wild-type PAO1, PA14 and mutant PA14 1flgK and PA14 1popB strains (MOI =10). After infection, cells were washed three times with warmed DMEM medium and live-stained with Wheat Germ Agglutinin (1/1,000) for 10 min at RT. Monolayers were washed with PBS and fixed with paraformaldehyde (4% v/v) for 15 min at RT. After three more PBS washes, cells were permeabilized with PBS containing saponin 0.1% (v/v) and 10% (v/v) dFCS for 2 h at 4◦C and counterstained with PBS containing DAPI (1/1,000) for 15 min at RT. hCFs monolayers were washed three times in PBS and examined with a Leica SP5 LSCM confocal microscope (Leitz). Statistics Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison test. P<0.05 denoted significance. RESULTS Pseudomonas aeruginosa Flagellum Influences Bacterial Attachment to hCFs In preliminary experiments, hCFs were infected over time with different MOI (1-100) of P. aeruginosa. These experiments demonstrated that a MOI of 10 was optimal for infecting hCFs in order to study both logarithmic association of P. aeruginosa to hCFs and maximal gene and protein expression in intact cell monolayers (data not shown). Adhesion patterns of P. aeruginosa wild-type bacteria and mutant strains for flagellum (1flgK) and TTSS (1popB) to hCFs were first examined as an initial step required for cellular PRR stimulation. No significant differences (P>0.05) in the association dynamics of wild-type PAO1 and PA14 strains and the PA14 1popB mutant with hCF monolayers were observed (Figure 1A). By contrast, the PA14 1flgK mutant showed a significant ∼1 log reduction in bacterial association with hCFs compared to the wild-type and 1popB strains at every time point examined (P<0.05). The complemented PA14 1flgK pUCP19flgK strain showed a similar association pattern (P>0.05) as the Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 4April 2017 | Volume 7 | Article 130 Cendra et al. TLR5 Sensing of Pseudomonas Flagellin FIGURE 1 | Expression of flagellum contributes to P. aeruginosa adhesion to hCFs. (A) Cells were infected with MOI =10 of PAO1, PA14, 1flgK,1popB and complemented PA14 1flgK p-flgK, PA14 1popB p-popB strains and association to hCFs monolayers was quantified by viable counting after 1, 3, 6 and 9 h. The symbols represent the means and bars the standard error of the mean (SEM) of n=3 independent experiments. PA14 1flgK and PA14 1popB strains carrying the cloning vector pUCP19 without insert were tested in order to exclude possible vector interferences: no differences were observed when compared with their respective mutant strains in their association capabilities (data not shown). Statistical significance was calculated with a one-way ANOVA with Dunnett’s multiple comparison test and it is represented by asterisks as follows: *P<0.05 and **P<0.01. (B) Confocal microscopy images of GFP-expressing PAO1, PA14 and mutant strains after 9 h of infection. Arrows denote the positions of bacteria and images are representative of n=3 independent experiments. wild-type PA14 strain by up to 6 h, but by 9 h there was an ∼1 log divergence in adherence of the complemented strain compared with the parent strain (Figure 1A). There was no significant difference in adherence between the PA14 wild-type strain, the 1popB mutant and the complemented PA14 1popB pUCP19popB strain over the 9 h time period (P>0.05; Figure 1A). Representative confocal microscopy images confirmed visually that the numbers of PA14 1flgK bacteria associated to hCFs monolayers were reduced compared to PA14 wild-type strain and 1popB mutant (Figure 1B). Bacterial growth rates were similar for all the strains (Supplementary Figure 2), except for the PA14 1flgK mutant, which was marginally slower between 0 and 3 h compared with the wild-type PA14 strain, although the growth rates of both were similar thereafter (P>0.05). In addition, cytotoxicity induced by P. aeruginosa infection (MOI =10) as determined by release of lactate dehydrogenase enzyme was not significant by 9 h (<5% observed enzyme release, data not shown). Death of the cell monolayers occurred between 9 and 24 h of infection, regardless of phenotype (data not shown). Pseudomonas aeruginosa Flagellum and TTSS Enhance Non-canonical Inflammasome Signaling Molecule Expression Induction of inflammasome-associated signaling molecules in hCFs during the early stages of infection by P. aeruginosa and the effects caused by the 1flgK and 1popB mutations were analyzed. Expression of the tlr2, tlr4, tlr5, nlrp3 and nlrc4 receptor genes, il-18 and il-1βcytokine genes and caspase-1 and caspase-4 genes was quantified by RT-qPCR after 1, 3 and 6 h of bacterial infection (Figure 2A). Each point in the graph represents the fold-change in expression caused by the wild-type and mutant bacteria compared against uninfected cells, using the 2−11CT method. No significant changes in expression were observed for tlr2, nlrp3 or caspase-1 (data not shown). The highest levels of gene induction were observed 6 h after infection with wild-type PAO1 and PA14 strains (Figure 2A), with significant increases at 3 h observed for il-18 and il-1βonly with PA14 (P<0.05). A oneway ANOVA with Dunnett’s test was used to compare the foldchanges for these genes induced by the PA14 wild-type compared with the PAO1 wild-type, and the fold-changes induced by PA14 1flgK and 1popB mutants compared with their parental PA14 strain. At 3 h, the levels of il-18 and il-1βcytokine genes induced by PA14 wild-type infection of hCF were ∼3-fold higher than the levels induced by PAO1 wild-type (P<0.05; Figure 2A). At 6 h, PA14 induced statistically significant ∼4-fold higher levels of tlr4 (P<0.05) compared with PAO1, whereas for the other genes, the observed fold-increases induced by both strains were similar (P>0.05). hCF infection with the PA14 1flgK and 1popB mutants, showed that at 3 h, there were significantly lower (∼13-fold) levels of il-18 (P<0.001) and il-1β(P< 0.01) cytokine gene induction, compared with the PA14 wild-type strain (Figure 2A). By 6 h, infection with the PA14 1flgK mutant showed significantly decreased levels (∼5–10-fold) of tlr4, tlr5, nlrc4,casp-4, il-18, and il-1βcytokine gene expression compared with PA14 wild-type (P<0.05; Figure 2A). By contrast, infection Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 5April 2017 | Volume 7 | Article 130 Cendra et al. TLR5 Sensing of Pseudomonas Flagellin FIGURE 2 | Inflammatory responses of human corneal fibroblast cells (hCFs) toward P. aeruginosa infection. (A) RT-qPCR of immune signaling molecules after 1, 3 and 6 h hCFs infection with PAO1 and PA14 wild-type and PA14 1flgK and PA14 1popB mutant strains at a MOI =10. Symbols represent the mean and bars the SEM of the log of gene expression from infected hCFs compared with uninfected controls from n=10 independent hCF samples. Validation of the oligonucleotides used for RT-qPCR examination of gene expression in infected hCFs was done by demonstrating the same signals in THP-1 cells stimulated with 100 ng/mL of P. aeruginosa LPS (data not shown). Statistical significance was calculated with a one-way ANOVA with Dunnett’s multiple comparison test and it is represented by asterisks as follows: *P<0.05, **P<0.01, ***P<0.001. (B) Cells infected with MOI =10 of PAO1 and PA14 wild-type, PA14 1flgK and PA14 1popB mutant strains, and the complemented PA14 1flgK p-flgK, PA14 1popB p-popB and control PA14 1flgK pUCP19 and PA14 1popB pUCP19 strains were lysed and intracellular protein levels of TLR4, TLR5, NLRC4, active caspase-4 and matured forms of IL-18 and IL-1βwere detected by western blot. Western blot image is representative of three independent experiments. (C) Western blot images show protein levels of proand matured forms of caspase-4, IL-18 and IL-1βin hCFs infected with PAO1 and PA14 wild-type and PA14 1flgK and PA14 1popB mutant strains. Protein expression for uninfected hCFs is also shown. The rabbit polyclonal anticaspase-4, -IL-18, and -IL-1βantibodies are specific for binding to the mature form of each respective protein and with less affinity for the pro-forms. Respective molecular weights of pro and mature forms are denoted with arrows. A representative image from n=5 independent experiments is shown. with the PA14 1popB mutant showed significant reductions (∼ 4-fold) in expression of tlr4,tlr5,nlrc4, and il-18 genes (P<0.05) compared to PA14 wild-type, but not significantly different for casp-4 and il-1βgenes (P>0.05). Protein levels detected by western blot were consistent with the levels of gene expression quantified by RT-qPCR. After 6 h of hCFs infection with wild-type PAO1 and PA14, significant levels of TLR4, TLR5, NLRC4, native form of caspase4 and IL-1βand IL-18 mature-formed proteins were detected by western blot with quantification of protein densitometry ratios relative to endogenous β-actin (Figure 2B, Supplementary Table 2). Significantly lower levels of production of these molecules were induced by the PA14 1flgK and 1popB mutant strains (P<0.05) and the percentage reductions in inflammatory molecule protein densitometry values are shown in Supplementary Table 2. In comparison with wild-type bacteria, infection with the complemented PA14 1flgK pUCP19-flgK and PA14 1popB pUCP19-popB strains restored to varying degrees the protein levels of TLR4, TLR5, NLRC4, caspase-4 and mature IL-1βand IL-18 (Figure 2B, Supplementary Table 2). Thus, complementation of 1flgK restored ∼50, 85, 60, 40, 32, and 61% of TLR4, TLR5, NLRC4, Caspase-4, IL-18, and IL-1βprotein levels, respectively (Supplementary Table 2). Complementation of 1popB restored ∼65, 60, 62, 57 37, and 73% of TLR4, TLR5, NLRC4, Caspase-4, IL-18, and IL-1βprotein levels, respectively (Supplementary Table 2). Protein levels of the caspase-4 pro-enzyme, and IL-18 and IL-1βpro-forms were also analyzed by western blot. Caspase4 pro-enzyme and pro-IL-18 protein expression was found to be constitutive, whereas pro-IL-1βwas induced by P. aeruginosa Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 6April 2017 | Volume 7 | Article 130 Cendra et al. TLR5 Sensing of Pseudomonas Flagellin wild-type infection, reduced by infection with the 1popB mutant and very weakly induced by infection with the PA14 1flgK mutant (Figure 2C). hCFs Infected with P. aeruginosa Release Extracellular IL-1βbut Not IL-18 The inflammatory cytokine profile of hCFs infected with wildtype P. aeruginosa is characterized predominantly by the production of IL-1βand IL-18 with the expression of other cytokines and chemokines not significant (manuscript submitted elsewhere). The early release of mature IL-1βand IL-18 was quantified by MSD-ELISA from the supernatants of hCFs infected with wild-type PAO1 and PA14 strains and isogenic PA14 1flgK and 1popB mutants after 6 h. No significant differences in IL-1βwere found between PAO1 and PA14 (P> 0.05). Both P. aeruginosa wild-type strains induced ∼30 pg/mL of IL-1βsecretion in hCFs after 6 h of infection (Figure 3A). By contrast, a significant reduction in IL-1βsecretion to levels of ∼5 pg/mL was observed from hCFs infected with the PA14 1flgK mutant strain. These lower levels of extracellular IL-1βdetected in the supernatants of hCFs infected with the PA14 1flgK are in agreement with the low intracellular IL-1βlevels shown in Figure 2B under the same infection conditions. There were no significant differences (P>0.05) in the levels of IL-1βsecretion induced by the PA14 1popB mutant (∼20 pg/mL) compared to wild-type PA14 after 6 h (Figure 3A). Complementation of each mutation restored the levels of IL-1βrelease similar to those induced by the PA14 wild-type strain. Measurement of IL-18 in infected hCFs supernatants revealed levels of extracellular IL-18 of ≤0.25 pg/mL for all infection conditions, which was similar to uninfected controls (P>0.05; Figure 3B). Western blotting of IL-18 protein precipitated from infected hCF-supernatants collected after 6 h infection did not show the presence of mature IL-18 (Figure 3C), which was in agreement with the MSD-ELISA data. Signal Pathway Inhibition in hCFs Appears to Influence IL-1βand IL-18 Expression and Release Pathway inhibition was used to target key intracellular signaling molecules stimulated in hCFs by P. aeruginosa flagellum and TTSS that potentially could contribute to IL-18 and IL-1β production. Since our results showed similar cytokine production in hCFs caused by PAO1 and PA14 infection, only infection with PA14 was used for the subsequent inhibition experiments. Confluent monolayers were treated with MyD88i, TAK-242, and MyD88i/TAK-242 inhibitors and then infected with PA14 wild-type and PA14 1flgK and 1popB mutant strains for 6 h. MyD88i blocks the signal through the MyD88 molecule and TAK-242 interferes with the association between the TIRAP and TRAM molecules to TLRs and inhibits the consequent signaling triggered by the receptor. Although TAK-242 has FIGURE 3 | Extracellular levels of IL-18 and IL-1βcytokines during hCFs infection with P. aeruginosa.IL-1βand IL-18 cytokine release was measured after 6 h hCFs infection with PAO1 and PA14 wild-type and PA14 1flgK and PA14 1popB mutant and complemented strains (MOI =10). The columns represent the mean and error bars the SEM from n=3 independent experiments. MSD-ELISA was used to quantify the levels of extracellular mature IL-1β(A) and IL-18 (B). Statistical significance was calculated with a one-way ANOVA with Dunnett’s multiple comparison test and it is represented by asterisks as follows: ***P<0.001. (C) Extracellular IL-18 detected by western blot in TCA-precipitated supernatants after 6 h infection of hCFs with P. aeruginosa wild-type and mutant strains. A total protein extract from hCFs infected with PA14 was used as a positive control for IL-18 detection (Control). Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 7April 2017 | Volume 7 | Article 130 Cendra et al. TLR5 Sensing of Pseudomonas Flagellin been reported to have selectivity for the LPS receptor TLR4, inhibitory effect has also been seen with other TLRs including TLR5 (Loiarro et al., 2005; Takashima et al., 2009; Matsunaga et al., 2011). Intracellular IL-18 and IL-1βprotein expression was examined by western blot (Figure 4A) and extracellular release of these cytokines was quantified by MSD-ELISA (Figure 4B). Treatment of hCF monolayers with MyD88i, TAK-242 alone and MyD88i/TAK-242 together, did not induce IL-18 or IL-1β production (Supplementary Figure 3). Infection with PA14 wild-type induced intracellular IL-18 protein production and protein densitometry values (from n= 3 independent western blots) revealed no significant differences in intracellular IL-18 protein expression induced by addition of any of the inhibitors (Figure 4A and Supplementary Table 3). Infection with the PA14 1popB mutant also induced mature IL-18 intracellular expression, but addition of TAK-242 (alone or in combination with MyD88i) appeared to significantly decrease its intracellular production (P<0.01; Figure 4A and Supplementary Table 3), whereas MyD88i alone had no FIGURE 4 | Effects of pathway inhibition on IL-18 and IL-1βproduction. Cells were treated with MyD88i, TAK-242, MyD88i/TAK-242 inhibitors and then infected with PAO1, PA14, 1flgK and 1popB strains (MOI =10). (A) Western blot was used to detect intracellular mature IL-18 and IL-1βcytokines after 6 h of bacterial infection. Detection of intracellular levels of β-actin was used as endogenous and loading control. (B) Quantification of extracellular IL-1βcytokine after 6 h of infection. The columns represent the means and the error bars the SEM of n=3 independent experiments. Statistical significance was calculated with a one-way ANOVA with Dunnett’s multiple comparison test and it is represented by asterisks as follows: *P<0.05, ***P<0.001. significant effect. PA14 1flgK did not induce intracellular IL-18 production, and secretory IL-18 was absent in all treated hCFs, as previously observed (Figures 3B,C). Infection with wild-type PA14 induced intracellular IL-1β protein production, which was not inhibited by MyD88i or TAK242 (Figure 4A and Supplementary Table 3). In cells infected with PA14 wild-type, levels of extracellular IL-1βwere apparently stimulated by the addition of MyD88i or the MyD88i/TAK242 combination, but no statistically significant differences were found compared to untreated cells (P>0.05, Figure 4B). PA14 1flgK mutant induced low levels of intracellular and extracellular IL-1βin agreement with the results in Figures 2B,3B, and were null with the addition of TAK-242, alone or in combination with MyD88i (Figures 4A,B and Supplementary Table 3B). Addition of TAK-242 also appeared to diminish intracellular and extracellular IL-1βproduction in hCFs infected with the PA14 1popB mutant (Figures 4A,B and Supplementary Table 3). TLR5 Silencing in hCFs Decreases IL-18 and IL-1βExpression and Release Our results showed that infection with the PA14 1flgK mutant led to reduced IL-18 and IL-1βexpression by hCFs. In order to demonstrate that the flagellum may be involved in inducing cytokine expression by these cells, we next tested the hypothesis that silencing the flagellum receptor TLR5 in hCFs would lead to a reduction of cytokine production regardless of bacterial adhesion. For these experiments, the flagellum filament protein FliC of P. aeruginosa was used as a control to demonstrate TLR5 stimulation. Recombinant (r) FliC was cloned and over-expressed in E. coli using the pET expression system (Supplementary Figure 1). Wild-type hCFs and hCFs that were TLR5-silenced using siRNA-TLR5 were stimulated with the PA14 wild-type, 1flgK mutant strain and with 5 µg/mL of rFliC, and IL-18 and IL-1βgene expression was measured by RT-qPCR. Non-stimulated cells were used as a control. Control experiments demonstrated that hCFs transfected with siRNA-TLR5 and infected with PA14 for 6 h significantly diminished TLR5 gene (P<0.0001; Supplementary Figure 4A) and protein (Supplementary Figure 4B) expression compared to control Mock transfection. Expression of il-18 gene was reduced ∼2 fold in hCF-siTLR5 compared to hCFs when stimulated with PA14 and rFliC for 6 h (Figure 5A). Infection with PA14 1flgK mutant did not induce IL-18 RNA expression and TLR5silencing did not affect IL-18 expression, which is in agreement with the results shown in Figure 4A. Similarly, decreased levels of IL-1βRNA expression in hCFs-siTLR5 were found following stimulation with PA14 and rFliC for 6 h (Figure 5B). Otherwise, IL-1βRNA expression was slightly increased in hCFs-siTLR5 infected with the PA14 1flgK mutant compared to no infection (Figure 5B). Secreted cytokines (IL-18 and IL-1β) were quantified from each supernatant after 6 h of hCF and hCF-siTLR5 incubation with PA14, 1flgK mutant and rFliC (Figure 5C). SLO-treatment was used to deliver rFliC intracellularly. As expected, no extracellular IL-18 was detected. Levels of secreted IL-1βprotein were significantly reduced in supernatants of hCFs-siTLR5 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 8April 2017 | Volume 7 | Article 130 Cendra et al. TLR5 Sensing of Pseudomonas Flagellin FIGURE 5 | Effect of TLR5 silencing in hCFs on IL-18 and IL-1βexpression and release. Wild-type hCFs and siTLR5-hCFs were infected with PA14 wild-type and 1flgK mutant strain at MOI =10, and stimulated with 5 µg/mL of rFliC for 6 h. LAL assay quantified that rFliC contained ∼150 pg of LPS/mg of protein (<0.00002% w/w LPS) and RT-qPCR showed that these levels of E. coli LPS did not stimulate any gene transcription in hCFs (data not shown). Non-infected monolayers were used as a control. (A,B) Gene expression of IL-18 (A) and IL-1β(B) was measured by RT-qPCR. The bars show the fold changes in gene expression in siTLR5-hCFs compared to wild-type hCFs, which were determined using the 2−11CT method (Livak and Schmittgen, 2001). (C) Extracellular IL-1βlevels measured by MSD-ELISA. rFliC was delivered intracellularly using SLO treatment. SLO treatment alone did not stimulate significant IL-1βrelease by hCFs (data not shown). The graph shows the levels of extracellular IL-1βquantified in the supernatants of hCFs and siTLR5-hCFs after 6 h following the different infection and stimulatory conditions. Statistical significance was calculated with a one-way ANOVA with Dunnett’s multiple comparison test and it is represented by asterisks as follows: *P<0.05, ****P<0.0001. compared to those detected in hCFs, both after stimulation with PA14 (P<0.05 and ∼30% of reduction). Most significantly, hCFs-siTLR5 treated with rFliC showed a reduction of IL-1β secretion of >95% (P<0.0001) compared to wild-type cells (Figure 5C), which was similar to the data obtained by RTqPCR (Figure 5B). No significant extracellular IL-1βsecretion was induced by SLO treatment alone of hCFs (∼0.7 pg/mL, data not shown). To examine if LPS was responsible, possibly, for the IL1βinduction observed in hCFs-siTLR5 infected with the PA14 1flgK mutant strain (Figure 5B), IL-1βgene expression was measured in hCFs-siTLR5 treated with the TAK-242 inhibitor. Under these inhibitory conditions, signaling through TLR5 (flagellin receptor) and TLR4 (LPS receptor) should be blocked. Repression of IL-1βexpression was detected in both hCFs and hCFs-siTLR5 infected with PA14 1flgK mutant (P< 0.05, Figure 6A) treated with TAK-242. These transcriptional results were confirmed by detecting IL-1βprotein expression under the same inhibitory conditions: addition of TAK-242 blocked the little IL-1βproduction induced in hCFs infected with the PA14 1flgK mutant (Figure 6B). In order to examine inhibitory effects on TLR4 sensing of LPS, IL-1βgene expression was also measured by RT-qPCR in hCFs that were stimulated with 100 ng/mL of pure Pseudomonas LPS, with and without TAK-242, Myd88i and TAK-242/Myd88i treatments. As shown in Figure 6C, addition of either inhibitor decreased IL-1β expression in hCFs treated with pure Pseudomonas LPS, compared to untreated cells. Similar experiments were done measuring IL-18 expression, but no increases in IL-18 expression were detected in LPS-stimulated hCFs (data not shown). No extracellular IL-1βprotein was detected after hCF stimulation with P. aeruginosa LPS alone or in the presence of any inhibitor (data not shown). Treatment of hCF Stimulated with P. aeruginosa rFliC with MyD88i and TAK-242 Inhibits IL-18 and IL-1β Expression Thus far, our data suggest that the P. aeruginosa flagellin (FliC) appears to plays an important role in inducing IL-18 and IL-1βexpression through TLR5 stimulation. We tested the hypothesis that chemical inhibition of TLR5-mediated signaling pathways reduces cytokine gene expression induced by FliC. hCF monolayers were pre-treated with MyD88i, TAK-242 and both inhibitors combined, then stimulated with rFliC for 6 h and cytokine gene expression quantified by RT-qPCR (Figure 7). Controls were hCF monolayers without chemical pre-treatment and no stimulation with rFliC. rFliC stimulation of hCF increased IL-18 gene expression ∼10 fold compared to unstimulated cells and addition of MyD88i, TAK-242 and MyD88i/TAK-242 significantly reduced gene expression (P<0.0001, Figure 7A). Similarly, rFliC stimulation increased IL-1βexpression by ∼20 fold, compared to unstimulated monolayers (Figure 7B). Inhibition with MyD88i alone reduced IL-1βgene expression by ∼40% (P< 0.05), whereas treatment with TAK-242 and MyD88i/TAK242 combined decreased IL-1βgene expression by >75% (P<0.0001) compared to untreated cells (Figure 7B). Secreted IL-1βwas quantified from supernatants of hCF stimulated with rFliC alone or in presence of the inhibitors. SLO-treatment was used to deliver the purified protein Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 9April 2017 | Volume 7 | Article 130