Nucleoside Diphosphate Kinase and Flagellin from Pseudomonas aeruginosa Induce Interleukin 1 Expression via the Akt/NF-κB Signaling Pathways

Abstract

Inflammatory responses are a first line of host defense against a range of invading pathogens, consisting of the release of proinflammatory cytokines followed by attraction of polymorphonuclear neutrophils (PMNs) to the site of inflammation. Among the many virulence factors that contribute to the pathogenesis of infections, nucleoside diphosphate kinase (Ndk) mediates bacterially induced toxicity against eukaryotic cells. However, no study has examined how Ndk affects inflammatory responses. The present study examined the mechanisms by which Pseudomonas aeruginosa activates inflammatory responses upon infection of cells. The results showed that bacterial Ndk, with the aid of an additional bacterial factor, flagellin, induced expression of the proinflammatory cytokines interleukin-1α (IL-1α) and IL-1β. Cytokine induction appeared to be dependent on the kinase activity of Ndk and was mediated via the NF-κB signaling pathway. Notably, Ndk activated the Akt signaling pathway, which acts upstream of NF-κB, as well as caspase-1, which is a key component of inflammasome. Thus, this study demonstrated that P. aeruginosa, through the combined effects of Ndk and flagellin, upregulates the expression of proinflammatory cytokines via the Akt/NF-κB signaling pathways.

INTRODUCTION

Airway cells, including epithelial cells and macrophages, act as the primary interface between the host and invading pathogens, and the airways are a crucial site at which innate immune responses are initiated to recruit polymorphonuclear neutrophils (PMNs) to the site of infection, where they clear the microbes from the system (1). However, chronic inflammatory responses mediated by excessive production of proinflammatory cytokines such as interleukin-1β (IL-1β) and IL-6 recruit a very high number of neutrophils, which can result in damage to the host tissues (2). Therefore, tight control of inflammatory responses is important for host defense; such control is achieved by regulating the expression of proinflammatory and anti-inflammatory cytokines (3).

IL-1α and IL-1β are proinflammatory cytokines produced by diverse cell types, including epithelial cells, endothelial cells, fibroblasts, and macrophages (4). Host defense responses mediated by the release of these cytokines play a role in resolving infections by recruiting inflammatory cells and by stimulating both local and systemic immune responses via the production of various enzymes, such as phospholipase A2, cyclo-oxygenase 2, metalloproteinases, and inducible nitric oxide synthase (5, 6). A previous study showed that prolonged increases in IL-1α and IL-1β levels occurred during Pseudomonas aeruginosa infections (7).

P. aeruginosa is an opportunistic bacterial pathogen that causes morbidity and mortality in immunocompromised patients and in individuals with cystic fibrosis (8). P. aeruginosa possesses a number of pathogenic virulence factors and secretory systems, but no studies to date have examined the role played by the bacterial nucleoside diphosphate kinase (Ndk; PA3807) in inducing host inflammatory responses, although Ndk is cytotoxic when incubated with eukaryotic cells (9, 10). Here, we show that bacterial Ndk, with the aid of flagellin, a well-known pathogen-associated molecular pattern (PAMP), induced the expression of IL-1α and IL-1β. Cytokine induction appeared to be dependent on the kinase activity of Ndk and was mediated via the Akt/NF-κB signaling pathways. Thus, the present report provides new insights into the roles of Ndk and flagellin in inducing the expression of proinflammatory cytokines during pseudomonas infections.

MATERIALS AND METHODS

Reagents.

Lipopolysaccharide (LPS; L9143) from P. aeruginosa, ultrapure flagellin (FLA-ST), MG132, and LY294002 were purchased from Sigma-Aldrich (St. Louis, MO), InvivoGen (San Diego, CA), A.G. Scientific (San Diego, CA), and Cell Signaling Technology (Danvers, MA), respectively.

Bacterial strains and culture conditions.

The bacterial strains used in this study are listed in Table 1. P. aeruginosa was grown in Luria (L) broth or on L agar plates at 37°C. To obtain supernatants and pellets, bacterial cells were harvested by centrifugation at 10,000 × g for 20 min at 4°C after overnight broth culture growth. The culture supernatant was filtered through a membrane (Sartorius, Goettingen, Germany) (0.22 μm pore size) to completely remove bacteria. The bacterial pellet was resuspended in phosphate-buffered saline to obtain live bacteria or heated to 65°C for 10 min to obtain heat-killed (Hk) bacteria.

TABLE 1.

Bacterial strains and plasmids

Strain or plasmid	Descriptiona	Reference or source
P. aeruginosa strains
PAKΔ7	PAK derivative with chromosomal deletion of exoS, exoT, exoY, popN, xcpQ, lasR, and lasI	16
PAKΔ8	PAKΔ7 with chromosomal deletion of ndk	16
PAKfliC	PAK with chromosomal disruption of the fliC locus; Gmr	11
Plasmids
pcDNA3.1(+)	Eukaryotic expression vector containing CMV promoter; Apr	Invitrogen
pDNNDK	ndk from P. aeruginosa in pcDNA3.1(+); Apr	16
pDNNDKH117Q	pDNNDK with kinase null ndk mutant; Apr	16

^aAp^r, ampicillin resistance; CMV, cytomegalovirus; Gm^r, gentamicin resistance.

Cell culture.

All of the media described below were supplemented with 10% heat-inactivated fetal bovine serum (FBS) (HyClone; Thermo Scientific), penicillin (100 units/ml), and streptomycin (0.1 mg/ml). A549 (human alveolar epithelial) and THP-1 (human macrophage) cells were cultured in RPMI 1640 (HyClone; Thermo Scientific). Wild-type (WT) mouse embryonic fibroblasts (MEFs), IκB kinase β knockout (IKKβ^−/−) MEFs (12), and BEAS-2B cells (immortalized primary human bronchial epithelial cells) were cultured in Dulbecco's modified Eagle's medium (DMEM) (HyClone; Thermo Scientific). Unless specified otherwise, cells were exposed to bacteria for 4 h at various multiplicities of infection (MOIs). Cells were maintained at 37°C in a humidified 5% CO₂ air-jacketed incubator.

Plasmids and transfections.

The expression plasmids used in this study are listed in Table 1. pDNNDK [Ndk cloned into the eukaryotic expression vector pcDNA3.1(+)], pDNNDKH117Q, and IKK (IκB kinase) β (dominant negative [DN]) (13) were prepared using an EndoFree Plasmid Maxi kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Cells were transfected with 1.5 μg of plasmid DNA by electroporation using a pipette-type microporator (Neon transfection system; Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Transfected cells were incubated for 48 h in RPMI 1640 supplemented with 10% FBS at 37°C.

Real-time qRT-PCR analysis.

Total RNA was isolated using TRIzol reagent (Invitrogen, Grand Island, NY) according to the manufacturer's instructions. Quantitative reverse transcription-PCR (qRT-PCR) was performed using SYBR green PCR master mix (Kapa Biosystems, Woburn, MA). cDNA was synthesized from total RNA using a ReverTra Ace qRT-PCR kit (Toyobo, Japan). Primer sequence information is as follows: human IL-1α, 5′-GTCTCTGAATCAGAAATCCTTCTATC-3′ and 5′-CATGTCAAATTTCACTGCTTCATCC-3′; human IL-1β, 5′-AAACAGATGAAGTGCTCCTTCCAGG-3′ and 5′-TGGAGAACACCACTTGTTGCTCCA-3′; mouse IL-1α, 5′-TCGGGAGGAGACGACTCTAA-3′ and 5′-GGCAACTCCTTCAGCAACAC-3′. Reactions were amplified and quantified using a CFX96 real-time PCR system (Bio-Rad, Hercules, CA) and the following thermal conditions: stage 1, 50°C for 2 min and 95°C for 10 min; stage 2, 95°C for 15 s and 60°C for 1 min. Stage 2 was repeated for 40 cycles. The relative quantities of mRNA were calculated using the comparative threshold cycle (C_T) method, and the amount of RNA used in each reaction was normalized according to the expression of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (primers 5′-CCCTCCAAAATCAAGTGG-3′ and 5′-CCATCCACAGTCTTCTGG-3′) or mouse GAPDH (primers 5′-TGTGTCCGTCGGGATCTGA-3′ and 5′-CCTGCTTCACCACCTTCTTGAT-3′).

Cell ELISA.

The production of IL-1 was measured by using a human IL-1α or IL-1β enzyme-linked immunosorbent assay (ELISA) kit (Pierce, Rockford, IL) according to the manufacturer's instructions.

Immunoblot analysis.

Antibodies specific for IKKβ, IκBα, p-Akt, Akt, IL-1β (D3U3E), and caspase-1 (D7F10) (Cell Signaling Technology, Danvers, MA) were used to probe proteins present in total cell lysates according to the manufacturers' instructions. A polyclonal anti-β-actin antibody was purchased from Santa Cruz Biotech (Dallas, TX).

Statistical analysis.

All experiments were performed in triplicate, and the results were expressed as means ± standard deviations (SD). Statistical analysis was performed using Student's t test, and P < 0.05 was considered significant.

RESULTS

Ndk induces the expression of IL-1α and IL-1β.

Ndk has been reported to cause cytotoxicity in macrophages by disrupting ATP concentrations (14); however, it is not clear whether Ndk is capable of inducing inflammatory responses. To examine this further, we quantified the expression of IL-1α and IL-1β in cells incubated with P. aeruginosa strains PAKΔ7 and PAKΔ8. The PAKΔ8 strain is an ndk deletion derivative of the PAKΔ7 strain, which is PAK deleted of type III secreted effectors (exoS, exoT, exoY, and popN), quorum sensing regulators (lasI and lasR), and type II secretion gene xcpQ (Table 1). As shown in Fig. 1A, PAKΔ7 (but not PAKΔ8) induced the expression of the IL-1α and IL-1β genes in A549 cells at 4 h posttreatment. The induction was further examined and confirmed in BEAS-2B and THP-1 cells as shown in Fig. 1B to E. Taken together, these data suggest that Ndk is involved in the induction of IL-1 expression.

FIG 1.

Ndk induces the expression of IL-1α and IL-1β. (A to C) A549 (A), BEAS-2B (B), or THP-1 (C) cells were treated with either the PAKΔ7 or PAKΔ8 strain at an MOI of 5 for 4 h (A) or at the indicated MOIs for 4 h (B) or for 24 h (C), and mRNA levels were measured by qRT-PCR. (D and E) BEAS-2B (D) or THP-1 (E) cells were treated with either the PAKΔ7 or PAKΔ8 strain at an MOI of 20 for 24 h, and protein levels were measured by either immunoblot assay (D) or ELISA analysis (E). Data are expressed as the means ± SD (n = 3) and are representative of the results of three separate experiments. *, P < 0.05 versus PAKΔ8. CON, control; MOI, multiplicity of infection.

Ndk-induced expression of the IL-1α and IL-1β genes requires other P. aeruginosa-derived molecules.

P. aeruginosa secretes Ndk via a type I secretion system (T1SS) (15); therefore, we next incubated A549 cells with culture supernatants from Pseudomonas strains PAKΔ7 and PAKΔ8. However, the supernatants did not show any clear difference in their abilities to induce cytokine expression, except that the supernatant from PAKΔ8 induced even more (Fig. 2A). This implies that there might be released factors other than Ndk involved in the expression and that Ndk released into the culture supernatant via T1SS does not play a role in the expression. It is thought that Ndk is translocated into cells via T3SS (16), a protein delivery mechanism that translocates effector molecules directly from the bacterial cytoplasm into the host cell. On the basis of this finding, we then investigated the effects of transfecting A549 cells with pDNNDK (Table 1), a plasmid carrying ndk cloned into an eukaryotic expression vector, pcDNA3.1(+). However, we did not observe the induction of cytokine expression in transfected cells, suggesting that Ndk might require additional factors for induction. We next treated pDNNDK-transfected cells with a heat-killed PAKΔ8 (Hk Δ8) strain. Indeed, the expression of the IL-1α and IL-1β genes increased in an MOI-dependent manner (Fig. 2B), implying that Ndk-mediated induction requires additional bacterial factors which may be present on the bacterial membrane. Taken together, these results suggest that Ndk requires additional bacterial factors to induce the expression of IL-1α and IL-1β.

FIG 2.

Ndk-induced expression of the IL-1α and IL-1β genes requires other P. aeruginosa-derived molecules. (A) A549 cells were treated with supernatants (Sup) from cultures of PAKΔ7 or PAKΔ8 Pseudomonas strains. (B) A549 cells were transfected with pDNNDK [Ndk cloned into the eukaryotic expression vector pcDNA3.1(+)]. After transfection, cells were exposed to Hk Δ8 at an MOI of 20 or 50 and mRNA levels were measured by qRT-PCR. Data are expressed as means ± SD (n = 3) and are representative of the results of three separate experiments. *, P < 0.05 versus the control group (A) or the vector-transfected group (B).

Flagellin is also required for the induction of the IL-1α and IL-1β genes.

P. aeruginosa is a Gram-negative bacterium that expresses a number of PAMPs on the cell membrane, including LPS. To determine whether LPS plays a role in bacterially induced cytokine expression, we transfected A549 cells with pDNNDK and then exposed them to LPS. As shown in Fig. 3A, exposing pDNNDK-transfected cells to LPS did not increase IL-1 gene expression, so we next tested the effect of flagellin, another important PAMP for the pathogenesis of P. aeruginosa, by exposing cells to a heat-killed PAK isogenic fliC-deficient mutant (Hk fliC mt). As shown in Fig. 3B, exposing cells to the Hk fliC mt led to a reduction in the expression of both the IL-1α and IL-1β genes, indicating that flagellin is required for the induction of cytokine expression. When Ndk-transfected cells were treated with purified flagellin, expression of both the IL-1α and IL-1β genes increased (Fig. 3C), thereby confirming that flagellin is required for the bacterially induced cytokine expression. Taken together, these results clearly show that both flagellin and Ndk are required for the induction of IL-1α and IL-1β expression.

FIG 3.

Flagellin is also required for the induction of expression of the IL-1α and IL-1β genes. Cells were transfected with pDNNDK. (A and C) Transfected cells were treated with either LPS (A) or flagellin (C) at the indicated concentrations. (B) Transfected cells were treated with either Hk Δ8 or Hk fliC mt, each at an MOI of 20 or 50, and mRNA levels were measured by qRT-PCR. Data are expressed as means ± SD (n = 3) and are representative of the results of three separate experiments. *, P < 0.05 versus the Hk Δ8-treated group (B) or cells transfected with empty vector (C).

Expression of the IL-1α and IL-1β genes requires kinase activity of Ndk.

Ndk catalyzes the exchange of phosphate groups between different nucleoside diphosphates (17), and the kinase activity of Ndk is the cause of cytotoxicity in macrophages mediated by disrupting ATP concentrations (14). Thus, we examined whether the kinase activity of Ndk plays a role in inducing cytokine expression by comparing the effects of Ndk (pDNNDK) with those of a kinase-deficient Ndk (pDNNDKH117Q) generated by the site-directed mutagenesis of a conserved histidine residue (H117) within P. aeruginosa Ndk. H117, which is conserved in all known Ndks, is phosphorylated during the generation of nucleoside triphosphates (NTPs) (18). Compared to A549 cells transfected with pDNNDK, cells transfected with pDNNDKH117Q showed a clear reduction in the expression of the IL-1α and IL-1β genes (Fig. 4), indicating that cytokine induction is dependent on the kinase activity of Ndk.

FIG 4.

The expression of the IL-1α and IL-1β genes requires kinase activity of Ndk. A549 cells were transfected with either pDNNDK or pDNNDKH117Q and were then treated with the Hk Δ8 strain at an MOI of 20 or 50. mRNA levels were then measured by qRT-PCR. Data are expressed as means ± SD (n = 3) and are representative of the results of three separate experiments. *, P < 0.05 versus cell transfected with pDNNDK.

Ndk induces the expression of the IL-1α and IL-1β genes via the NF-κB signaling pathway.

NF-κB is a key transcriptional factor that initiates host inflammatory responses upon infection by microbes (19). We previously reported that P. aeruginosa activates the NF-κB signaling pathway in A549 cells (13). To determine whether cytokine induction by P. aeruginosa strain PAKΔ7 is mediated via the NF-κB signaling pathway, we pretreated cells with MG132 (a chemical inhibitor of NF-κB) and then exposed them to P. aeruginosa. We found that the PAKΔ7-mediated expression of the IL-1α and IL-1β genes was significantly reduced by pretreatment with MG132 in a dose-dependent manner (Fig. 5A). To confirm this result, we further used a more specific approach to inhibit NF-κB signaling. Figure 5B shows that overexpressing IKKβ DN abrogated cytokine expression in A549 cells. Further confirmation was obtained in an experiment using WT and IKKβ^−/− MEFs (Fig. 5C, upper panel) in which the absence of IKKβ protein was confirmed by immunoblotting (Fig. 5C, lower panel). The absence of cytokine expression in IKKβ^−/− cells suggests that P. aeruginosa-induced expression is mediated by the NF-κB pathway. In addition, immunoblot analysis showed that PAKΔ7, but not PAKΔ8, potently increased the degradation of IκBα (Fig. 5D). Taken together, these results suggest that P. aeruginosa PAKΔ7 mediates the expression of IL-1α and IL-1β via the NF-κB pathway.

FIG 5.

Ndk induces the expression of the IL-1α and IL-1β genes via the NF-κB signaling pathway. (A) A549 cells were pretreated with the indicated concentrations of MG132 for 1 h followed by treatment with either PAKΔ7 or PAKΔ8 at an MOI of 5. mRNA levels were measured by qRT-PCR. (B) Overexpression of IKKβ DN repressed the PAKΔ7-mediated induction of the IL-1α and IL-1β genes expression. (C) PAKΔ7 induces the transcription of the mouse IL-1α (mIL-1α) gene in WT MEFs but not in IKKβ^−/− MEFs (upper panel). The absence of IKKβ was confirmed by immunoblot analysis (lower panel). (D) PAKΔ7 induced IκBα degradation in a time-dependent manner as assessed by immunoblot analysis. Data in panels A to C are expressed as means ± SD (n = 3), and data are representative of the results of three separate experiments. *, P < 0.05 versus mock-treated (A and B) or WT (C) cells.

Ndk activates the Akt signaling pathway and caspase-1.

Since IL-1 expression appeared to be mediated via the NF-κB signaling pathway, we were interested in identifying upstream signaling molecules activated by Ndk. To determine this, we inhibited the activity of several signaling mediators using specific chemical inhibitors. PAKΔ7-mediated IL-1 gene expression was significantly reduced in a dose-dependent manner when cells were pretreated with LY294002, a chemical inhibitor for PI3 kinase-dependent Akt (protein kinase B) phosphorylation (Fig. 6A). This suggests that the Akt signaling pathway is also involved in the induction of IL-1 expression in response to P. aeruginosa PAKΔ7. To examine the effect of Ndk on Akt, we examined Akt phosphorylation by immunoblotting analysis (Fig. 6B). The results show that Ndk potently increased the phosphorylation of Akt. Next, we examined the effect of Ndk on IκBα degradation. As shown in Fig. 6C, transfection of Ndk alone did not increase IκBα degradation; however, a clear increase in degradation was observed when cells were exposed to PAKΔ8 after transfection. Moreover, mature IL-1β is produced by a caspase-1-mediated process (20), and, notably, Ndk was capable of activating caspase-1 in THP-1 cells (Fig. 7A) but was barely capable of doing so in BEAS-2B cells (Fig. 7B). Taken together, these data suggest that Ndk-mediated Akt phosphorylation is not sufficient for NF-κB activation but is capable of amplifying the activity of NF-κB, which may be weakly stimulated by PAKΔ8.

FIG 6.

Ndk activates the Akt signaling pathway. (A) A549 cells were pretreated with LY294002 at the indicated concentrations for 1 h followed by treatment with PAKΔ7 at an MOI of 5. mRNA levels were measured by qRT-PCR. (B) A549 cells transfected with pDNNDK were treated with Hk Δ8 at an MOI of 100, and Akt phosphorylation was assessed by immunoblot analysis. (C) A549 cells were transfected with pDNNDK and then treated with Hk Δ8 at an MOI of 100. The degradation of IκBα was assessed by immunoblot analysis. Data in panel A are expressed as means ± SD (n = 3), and data are representative of the results of three separate experiments. *, P < 0.05 versus mock treatment (A).

FIG 7.

Ndk activates caspase-1. THP-1 (A) and BEAS-2B (B) cells were treated with either PAKΔ7 or PAKΔ8 at an MOI of 20 for the indicated times. The activation of caspase-1 was assessed by immunoblot analysis. Data are representative of the results of three separate experiments.

DISCUSSION

Innate immune responses play a critical role in eradicating invading pathogens by the release of proinflammatory cytokines such as IL-1α and IL-1β and then the attraction of PMNs to the site of inflammation (21). Here, we showed that Pseudomonas Ndk induced the expression of IL-1α and IL-1β and that efficient expression requires additional signals initiated by bacterial flagellin. A previous study showed that infection by P. aeruginosa leads to prolonged increases in IL-1α and IL-1β expression (7), and the current study suggests that both Ndk and flagellin may be responsible for the expression.

The infection of wild-type PAK causes toxicity to cells due to the toxic effect of T3SS effectors, and the T3SS-mediated toxicity makes the effects of other virulence factors difficult to examine. The PAKΔ7 strain, a mutant with deletions of three known T3SS effectors, was purposely constructed to look for and examine other virulence factors that P. aeruginosa might have. By using this strain, we were able to identify Ndk as a novel factor involved in inducing inflammatory responses. Ndk is present in virtually all organisms and has very similar structures in prokaryotes and eukaryotes. Ndk catalyzes the exchange of phosphate groups between different nucleoside diphosphates (17). Reports from previous studies indicate that the kinase activity of Ndk is responsible for causing toxicity to eukaryotic cells (9, 10, 14). Notably, the kinase activity of Ndk was also required for the induction of IL-1 gene expression (Fig. 4). In agreement with the cytotoxicity results, we found that PAKΔ7, but not PAKΔ8, was cytotoxic to A549 cells at an MOI > 20 (data not shown). In order to allow appropriate conclusions regarding the effect of Ndk on the induction of IL-1 expression, we applied PAKΔ7 at an MOI of 5 and carefully monitored the morphology of cells after treatment with PAKΔ7 or transfection with Ndk plasmid. Under this experimental condition, we did not observe any major cytotoxicity in cells.

The Ndk-mediated expression of IL-1 required additional factors that could be present on the membrane of P. aeruginosa. One of major PAMPs is LPS, known to be a potent ligand recognized by Toll-like receptor 4 (TLR4) and to induce the expression of proinflammatory cytokines, including IL-1 (22). However, as shown in Fig. 3A, the treatment of LPS did not induce the expression in A549 cells. This could be explained by the previous reports demonstrating that A549 cells express a low level of TLR4 such that the A549 cells showed hyporesponsiveness to LPS (23, 24). Another important bacterial factor is flagellin, which is the main component of bacterial flagella and is a potent ligand recognized by TLR5 (25, 26). TLR5 is expressed by airway epithelial cells, and HEK293 cells transfected with TLR5 activate the NF-κB signaling pathway in response to P. aeruginosa flagellin (27). In addition, TLR5 engagement in macrophages plays a major role in triggering IL-1β production in response to P. aeruginosa (28). Surprisingly, a number of cell lines are only weakly responsive, or are unresponsive, to flagellin even though they express TLR5; this suggests that additional factors are required for signal transduction in the response to flagellin (29). In line with this, we found that exposing A549 cells to flagellin alone led to weak increases in IL-1 gene expression; however, expression clearly increased in the presence of both flagellin and Ndk (Fig. 3C). Also, Akt phosphorylation in Ndk-transfected cells was not sufficient to activate the NF-κB pathway; this required the additional presence of Hk Δ8 (Fig. 6C). These results suggest that Ndk and flagellin cooperate to activate NF-κB, resulting in increased cytokine expression.

We were also interested to find out whether Ndk induces the expression of TLR5, because if this were the case, then TLR5 would bind flagellin and activate the NF-κB pathway. However, there was no increase in TLR5 expression in A549 cells transfected with Ndk (data not shown). Instead, Ndk activated the Akt signaling cascade, which subsequently linked into the NF-κB pathway (Fig. 6B and C). This is consistent with a previous report demonstrating that Akt acts upstream of the NF-κB signaling (30). In addition to the effects of Ndk and flagellin, P. aeruginosa seems to release other factors involved in the induction of IL-1 gene expression, as shown in Fig. 2A. We looked for major proteins secreted by P. aeruginosa in supernatants and identified multiple proteins by analysis using mass spectrometry. We expect that some of these proteins might play a role in inducing the IL-1 expression. We are currently conducting investigations to find the responsible proteins required for the induction.

We have examined the protein level of IL-1 induced by PAKΔ7 by applying ELISA and immunoblot assays. We could clearly measure the secretion of IL-1 by ELISA in THP-1 macrophage cells (Fig. 1E) but not in A549 epithelial cells (data not shown). With the BEAS-2B cells, we still could not detect the production of mature IL-1β by immunoblot assays but we were able to detect the production of pro-IL-1β (Fig. 1D). The production of mature IL-1β requires the proteolytic maturation catalyzed by caspase-1, a key component of the inflammasome (20, 31). In a line with this, Ndk-mediated activation of caspase-1 was clearly observed in THP-1 cells (Fig. 7A) but was barely observed in BEAS-2B cells (Fig. 7B). Human cancer cells are known to generally not produce caspase-1 (32), and bronchial epithelial cells do not significantly activate caspase-1 (33). A549 cells are type II human alveolar epithelial cancer cells and are known to not produce caspase-1 (32). In addition, BEAS-2B cells are immortalized primary human bronchial epithelial cells and are known to weakly activate caspase-1 (34). These could be among the reasons explaining why we failed to detect the production and secretion of mature IL-1β in epithelial cells.

The innate immune system is equipped with dual-signaling pathways operating via TLR5 for extracellular flagellin and Nod-like receptor Ipaf for cytosolic flagellin (35, 36). P. aeruginosa activates caspase-1 via Ipaf to induce IL-1β secretion in infected macrophages (37, 38). The activation requires a functional T3SS but does not require all of the known T3SS effectors, ExoS, ExoT, and ExoY, implying the presence of additional effector molecules responsible for the activation (37–39). Although PAKΔ7 is a mutant with deletions of all three of the known T3SS effectors (ExoSTY), PAKΔ7, but not PAKΔ8, still induced IL-1 expression (Fig. 1) and activated caspase-1 (Fig. 7A). This suggests that Ndk could be the T3SS effector responsible for the activation, since Ndk has been identified as an additional effector translocated into cells via T3SS (16). PAKΔ8 did not activate caspase-1 (Fig. 7A), but the treatment induced a certain level of IL-1 expression in THP-1 cells as shown in Fig. 1C and E. It has been reported that IL-1β usually requires caspase-1-mediated cleavage for secretion, but additional proteases such as caspase-8, myeloblastin, and granzyme A are also involved in the maturation of pro-IL-1β (40–42), indicating the possible effects of other proteases during PAKΔ8 infection. We are currently conducting investigations to understand the mechanism of Ndk-mediated caspase-1 activation.

Hosts have developed a variety of strategies to facilitate the efficient clearance of invading pathogens, including the induction of inflammatory responses that form the first line of defense during the early stages of infection. TLR5- or Ipaf-mediated recognition of flagellin is one mechanism that activates pulmonary defenses against P. aeruginosa (36, 43); however, the contribution of TLR5 or Ipaf to the innate immune response to P. aeruginosa is still unclear. Here, we showed that Ndk and flagellin cooperate to induce IL-1 expression by activating the Akt/NF-κB signaling pathway, suggesting cross talk between Ndk- and flagellin-mediated signaling pathways during host-Pseudomonas interactions. To better understand this cross talk and the pathogenesis of this important human pathogen, we need to further examine the molecular mechanisms underlying bacterially induced cytokine expression.