Pseudomonas aeruginosa Mediates Host Necroptosis through Rhl-Pqs Quorum Sensing Interaction Open Access

Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative and ubiquitous bacterium (1). It is a leading cause of severe acute and chronic infections such as pneumonia, sepsis, urinary tract infections, and respiratory tract infections in immunocompromised patients and the major cause of morbidity and mortality in patients with cystic fibrosis (2–5). As one of the most versatile and opportunistic bacteria, P. aeruginosa possesses a broad range of virulence factors that facilitate its infections and adaptations to host immune defenses (6). Owing to its intrinsic resistance to a wide spectrum of antibiotics, as well as acquisition of adaptive mutations during chronic infections, P. aeruginosa has become one of the most difficult bacteria to be eradicated in patients (3–5). Thus, novel treatments and antibiotic development are urgently needed to treat infections induced by P. aeruginosa (4).

P. aeruginosa has multiple mechanisms to facilitate its survival and rapid adaptation to various conditions. Quorum sensing (QS) is one such mechanism that allows bacteria to dynamically coordinate their behavior in response to changes in cell density and surrounding environments (1, 7, 8). P. aeruginosa uses multiple types of autoinducers and related receptors in the QS system to regulate this communication system (9, 10). QS is involved in various P. aeruginosa activities, including biofilm formation, virulence factor production, and subsequent release (2). Recently, the relationship between QS and host immune response has become increasingly critical in antimicrobial research. Three hierarchical P. aeruginosa QS systems are extensively investigated, including las, rhl, and pqs (9, 10). A recent study revealed that the mammalian aryl hydrocarbon receptor can recognize the autoinducers released by the las and pqs system to adjust host immune response upon P. aeruginosa infection (1). Another study reported that las-regulated protease, LasB, impairs host inflammatory responses by targeting lung epithelial cystic fibrosis transmembrane regulator signaling (11). Therefore, accumulative evidence indicates a critical role of QS in regulating the host immune response.

Programmed cell death (PCD) is a pivotal innate immune mechanism in protecting against acute bacterial infection, thus limiting their spread by removing infected cells (12, 13). Apoptosis, necroptosis, and pyroptosis are the three most common types of PCD, known as PANoptosis, which plays a critical role in host defense during bacterial infection (13). Through recognizing pathogen-associated molecular patterns released by bacteria with multiple sensors, a protein complex formation, the inflammasome, will be initiated and functions as a hub to interact with and activate PCD machinery (13, 14). It has been reported that the NLR family apoptosis inhibitory protein 5 is activated by flagellin and induces NLR family CARD domain-containing 4 (NLRC4) to initiate inflammasome activation, leading to pyroptosis (15–17). Additional studies reported that QS molecule N-(3-oxododecanoyl) homoserine lactone (C₁₂-HSL) released by P. aeruginosa triggers apoptosis in human macrophages (18, 19). Despite the well-known function of PCD in anti–P. aeruginosa response, whether QS affects host PCD during P. aeruginosa infection remains unclear.

In this study, we discovered that rhl-deficient P. aeruginosa caused an enhanced PCD in mouse macrophages during infection. Both pyroptosis and necroptosis were robustly elevated, as evidenced by the cleavage of gasdermin D (GSDMD) and phosphorylation of receptor-interacting protein kinase-3 (RIPK3) and mixed-lineage kinase-like protein (MLKL), respectively. We identified that necroptosis increased by rhl-deficient P. aeruginosa was caused by the upregulation of the downstream P. aeruginosa pqs system. Rhl-deficient P. aeruginosa–induced necroptosis was abolished by depleting the pqsA, suggesting, to our knowledge, a novel, rhl–pqs axis controlling macrophage necroptosis, which may represent a therapeutic target for anti–P. aeruginosa treatment strategies.

C57BL/6J (000664) mice were obtained from The Jackson Laboratory. Gsdmd^−/− (20), Ripk3^−/− (21, 22), Nlrp3^−/− (23), Nlrc4^−/− (17), Casp1/11^−/− (24), and Casp1/11^−/−Ripk3^−/−Casp8^−/− (25) mice have been described previously. All mice were housed in specific pathogen-free facilities at The Ohio State University. Eight- to 12-wk-old, sex-matched mice were used in animal experiments. All in vivo experiments were performed according to the guidelines established by The Ohio State University and the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the institutional animal care and use committee (protocol 2018A00000022-R2).

Bone marrow–derived macrophages (BMMs) were generated from the indicated genotypes of mice in the presence of L-929 conditioned medium, as described in our previous studies (22, 26). After 6-d culture, BMMs were harvested and seeded at a concentration of 0.2 × 10⁶ cells into 96-well plates or at a concentration of 1 × 10⁶ cells into 12-well plates and incubated overnight for subsequent assays.

All bacterial strains, plasmids, and primers are listed in Table I. Gene deletion constructs were incorporated into the P. aeruginosa genome using homologous recombination as previously described (27). P. aeruginosa was grown in 5 ml Luria broth (LB) overnight at 37°C with 200-rpm shaking, diluted 1:5 (v/v), and grown for 2 h to reach the exponential phase. Bacteria were washed and resuspended with Dulbecco’s PBS (DPBS) for subsequent experiments.

Resuspended P. aeruginosa was diluted and applied to BMMs at a multiplicity of infection (MOI) of 10 for 2 h. BMMs were then washed with sterile DPBS twice to remove excess P. aeruginosa. The medium was replaced with 200 µg/ml gentamicin-containing medium. After additional indicated periods, cells and supernatants were collected for further experiments. For autoinducer costimulation assays, BMMs were pretreated with N-butyryl-L-homoserine lactone (C₄-HSL) (Sigma-Aldrich, SML3427, 100 µM), 2-heptyl-3-hydroxy-4(1H)-quinolinone (PQS) (Cayman, 29186, 100 µM), and 2-heptyl-4-quinolone (HHQ) (Sigma-Aldrich, SML0747, 100 µM) 2 h before P. aeruginosa stimulation.

BMMs seeded into 96-well plates were infected with different strains of P. aeruginosa at an MOI of 10 for 2 h. The cells were then washed twice with DPBS and lysed in 200 µl 0.1% Triton X-100 in DPBS. Tenfold serial dilutions of the lysates were mixed with DPBS, and 10 µl was applied on an antibiotic-free LB agar plate. Colonies were counted after 8-h incubation at 37°C. Each experiment was conducted in triplicate wells and repeated three times.

Supernatants were collected from 96-well plates of bacterial stimulation experiments, and lactate dehydrogenase (LDH) activity was determined with the Cytotoxicity Detection Kit (LDH) (11644793001, Roche). Cells left untreated or treated with 1% Triton X-100 were used as negative and positive controls, respectively.

For immunoblotting, cells were collected and lysed with radioimmunoprecipitation assay buffer containing a protease inhibitor mixture. Electrophoresis of proteins was performed by using the NuPAGE system (Invitrogen) according to the manufacturer’s protocol. Proteins were transferred to nitrocellulose membranes (Bio-Rad Laboratories). Primary Abs were applied to membranes after 1 h of blocking with 5% skim milk for overnight incubation at 4°C. Appropriate HRP-conjugated secondary Abs were used, and proteins were detected using ECL reagent (Thermo Scientific). The images were acquired with the ChemiDoc MP System (Bio-Rad Laboratories). Primary Abs for immunoblotting included GSDMD (10026, 1:1000) from Genentech; caspase-1 (AG-20B-0042, 1:1000) from AdipoGen, caspase-8 (8592, 1:1000), caspase-7 (9492, 1:1000), p-RIPK3 (57220, 1:1000), and p-MLKL (62233, 1:1000) from Cell Signaling Technology; total MLKL (AP14272b, 1:1000) from Abgent; total RIPK3 (NBP1-77299, 1:1000) from Novus; and HRP-conjugated anti–β-actin (sc-47778, 1:3000) from Santa Cruz Biotechnology. Secondary Abs for immunoblotting included anti-rabbit HRP-linked IgG (7074, 1:2000) and anti-mouse HRP-linked IgG (7076, 1:2000) from Cell Signaling Technology and anti-rat HRP-linked IgG (sc-2006, 1:3000) from Santa Cruz Biotechnology.

All experiments were performed with at least three independent replications. GraphPad Prism version 9.0 software was used for data analysis. Data were analyzed as mean ± SD. The means of two groups were compared with a Student unpaired t test. Comparisons between multiple groups were analyzed by repeated-measures ANOVA with Bonferroni posttests. Survival analyses were performed by the Kaplan–Meier method and log-rank (Mantel–Cox) test for significance. A p value less than 0.05 was considered statistically significant.

To better define the role of P. aeruginosa rhl in macrophage cell death, we employed two P. aeruginosa rhl transposon mutation strains (rhl mutant), rhlI::Tn and rhlR::Tn, derived from the comprehensive mutant library (28). These strains, along with the isogenic wild-type (WT) strain PAO1, were used to infect mouse BMMs. We observed a significant increase of cell death induced by rhl mutants compared with PAO1, whereas no difference existed between the two transposon mutants (Fig. 1A). To further investigate if cell death promoted by rhl mutants was specific to pyroptosis, we conducted stimulation experiments with BMMs generated from Gsdmd^−/− mice, which are deficient in the execution of pyroptosis (20, 29, 30). Deletion of GSDMD in macrophages failed to rescue the increased cell death induced by rhl mutants (Fig. 1B). Additional gene deletion cells including Nlrp3^−/−, Nlrc4^−/−, and Casp1/11^−/− BMMs, could not reverse the enhanced cell death induced by rhl mutants (Supplemental Fig. 1). These results suggest that increased cell death stimulated by rhl mutants was not specific to pyroptosis. We next examined Ripk3^−/− BMMs and obtained similar results, indicating that this cell death promoted by rhl mutants was not specific to necroptosis (Fig. 1C). Furthermore, we pretreated BMMs with zVAD-fmk, a pan-caspase inhibitor, before P. aeruginosa infection, and cell death still existed (Fig. 1D). To test that the cell death stimulated by rhl mutants was PANoptosis (13), we repeated those experiments with Casp1/11^−/−Casp8^−/−Ripk3^−/− BMMs and observed a completely suppressed cell death induced by both WT and rhl mutant P. aeruginosa, indicating that cell death induced by rhl-deficient P. aeruginosa was PANoptosis (Fig. 1E) (13). We also examined macrophage phagocytosis of bacteria and found no difference between PAO1 and rhl mutants engulfed by macrophages after 2 h of incubation (Fig. 1F). These results suggest that increased PANoptosis induced by rhl-deficient P. aeruginosa was not caused by altered bacterial phagocytosis. In sum, we found that rhl deficiency in P. aeruginosa induces an elevated PANoptosis, which could not be blocked in macrophages with the deficiency in any single PCD pathway. Gsdmd^−/− macrophages blocked the pyroptosis pathway elicited by P. aeruginosa, whereas necroptosis and apoptosis remain activated during infection. As a pan-caspase inhibitor, zVAD-fmk greatly impeded caspase protein cleavage, which in turn suppressed both the apoptosis and pyroptosis pathways during infection, resulting in decreased cell death.

We next sought to examine individual cell death pathways upon P. aeruginosa infection with immunoblotting. Increased cleavage of caspase-1 (P20) and cleavage of GSDMD (P30) were detected in BMMs with rhl mutants compared with those with PAO1 bacteria (Fig. 2A). We also detected cleaved caspase-8 (P18) and cleaved caspase-7 (P20) in both PAO1 and rhl mutant groups. Stimulation with Rhl mutants elevated the cleavage of these apoptosis-related caspases, showing that apoptosis was also increased to a low extent (Fig. 2A). In addition, we found markedly enhanced phosphorylation of RIPK3 and MLKL under the stimulation of rhl-deficient P. aeruginosa compared with PAO1 (Fig. 2B). Together, these results demonstrate that rhl-deficient P. aeruginosa promotes host PCD, especially pyroptosis and necroptosis.

We next sought to determine the mechanism by which rhl mutants promoted macrophage PCD. Previous studies showed that rhl deficiency caused an increased production and secretion of flagellin in P. aeruginosa, which triggered NLRC4 inflammasome activation and pyroptosis in macrophages (31, 32). However, the mechanism by which rhl mutants promote necroptosis is still unclear. We next asked whether enhanced PCD caused by rhl mutants was due to the lack of C₄-HSL, an autoinducer produced by the rhl system (10). Pretreatment with C₄-HSL caused no significant reduction of cell death induced by WT or rhl-deficient P. aeruginosa, suggesting no involvement of C₄-HSL in PCD regulation (Fig. 3A). Previous studies have documented that rhl negatively regulates the downstream pqs system, thus repressing the production of the autoinducer PQS and HHQ (31). Pretreatment with PQS or HHQ resulted in significantly increased cell death under stimulation with PAO1 and abolished the cell death difference induced by WT and rhl mutants (Fig. 3B, 3C). These results suggest that increased PCD by rhl mutants is associated with increased production of PQS and HHQ.

To further examine the causal relationship between increased production of PQS and HHQ and elevated PCD induced by rhl-deficient P. aeruginosa, we deleted the pqsA gene in rhl-deficient background through homologous recombination (Supplemental Fig. 2). Because PqsA regulates the transcription of downstream pqsB/C/D/E genes in the pqs system, deletion of pqsA leads to defective PQS and HHQ production (8, 33). We next examined whether double-knockout (DKO) mutants, rhlI::TnΔpqsA and rhlR::TnΔpqsA, could rescue macrophage cell death. Upon P. aeruginosa challenge in BMMs, we observed that DKO mutants only partially reduced cell death increased by rhl deficiency, but they still exhibited a higher level of cell death than that of PAO1, indicating that pqs depletion indeed suppressed cell death promoted by rhl deficiency. No difference in cell death existed between PAO1 and ΔpqsA (Fig. 4A). To further identify the type of cell death reduced by DKO mutants, we tested P. aeruginosa challenge in Gsdmd^−/− BMMs. DKO mutants failed to trigger cell death when pyroptosis was blocked, suggesting that pqs depletion rescued the necroptosis induced by rhl deficiency. We also observed that ΔpqsA diminished cell death mounted by PAO1 (Fig. 4B). To determine whether pqs depletion could inhibit pyroptosis, we reconducted the challenge with Ripk3^−/− BMMs. Cell death induced by DKO mutants was slightly lower than that by rhl mutants, and no difference existed between the two PAO1 and ΔpqsA groups (Fig. 4C). Additionally, depletion of pqs in rhl mutants had no effect on phagocytosis (Fig. 4D).

To further examine whether DKO mutants suppressed necroptosis, we sought to investigate signaling pathways through immunoblotting. We observed that deletion of pqs did have some influence on pyroptosis, because the cleavage of Casp-1 (P20) and GSDMD (P30) was decreased in DKO groups compared with rhl mutant groups, although depletion of pqs seemed to have no effect on cleavage of Casp-8 (P18) (Fig. 5A). We next detected that the phosphorylation of RIPK3 and MLKL was completely suppressed by DKO mutants, which was consist with our previous cytotoxicity data showing that depletion of pqs led to the inhibition of necroptosis in macrophages (Fig. 5B). Taken together, we concluded that rhl-deficient P. aeruginosa enhanced host necroptosis through upregulation of the pqs system.

To examine the susceptibility of mice to P. aeruginosa with rhl deficiency or rhl&pqs deficiency, we employed an acute infection model with PAO1, rhlR::Tn, and rhlR::TnΔpqsA to challenge C57BL/6 mice. All bacteria were resuspended from overnight culture to achieve an exponential phase and then were centrifuged and washed with DPBS. Each mouse was administered 3 × 10⁶ CFUs of P. aeruginosa intranasally. After 9 d of observation, all mice from the rhlR::Tn group failed to survive, whereas all mice challenged with PAO1 successfully survived over the same period (Fig. 6A). Besides, mice infected with the rhl mutant exhibited fast loss of body weight and failure in recovery (Fig. 6B). However, administration of the DKO mutant significantly improved mouse survival even with rhl deficiency because 80% of mice survived after 9 d of observation. Additionally, the body weight loss of DKO had no significant difference from the PAO1 group and recovered 2 d after administration (Fig. 6A, 6B). In sum, these findings collectively demonstrated that repression of pqs could reduce the susceptibility of mice during P. aeruginosa challenge.

The cross-talk between P. aeruginosa and PCD in host cells has become increasingly critical in both laboratory and clinical research. Because of characteristics such as fast adaptation to the environment and a wide range of antibiotic resistance, seeking the “arsenals” from host innate immunity becomes a new strategy in battling against P. aeruginosa infection diseases. QS functions as a pivotal mechanism that facilitates P. aeruginosa adaptation and has been reported to entangle with host PCD in previous studies (18, 19, 31, 32, 34). Nevertheless, most of these studies demonstrate the interaction of QS with apoptosis and pyroptosis in innate immune cells. As for another type of PCD, necroptosis, the connection between QS and necroptosis is somewhat sparse. In this study, we identified P. aeruginosa rhl-mediated macrophage necroptosis through regulation of downstream pqs. Rhl-deficient P. aeruginosa significantly enhanced macrophage PCD during in vitro challenge, which was identified as pyroptosis and necroptosis via immunoblotting. It has been known that rhl deficiency causes increased production and secretion of flagellin in P. aeruginosa, which triggers NLRC4 inflammasome activation and pyroptosis (31, 32). Indeed, we found that in addition to Ripk3-mediated necroptosis, rhl deficiency also caused a small increase in cell death that was independent of pqsA or Ripk3, which is consistent with previous findings on increased pyroptosis due to excessive flagellin production in rhl-deficient P. aeruginosa (31, 32). RhlR had been demonstrated to suppress downstream pqs genes, and our results showed that pqs autoinducers, PQS and HHQ, robustly facilitated cell death induced by P. aeruginosa, even PAO1 (35, 36). After generating rhl/pqs DKO mutants, we observed that the overall cell death was reduced by DKO mutants compared with rhl mutants. Moreover, necroptosis was inhibited under the challenge of DKO mutants. We therefore demonstrate that rhl-deficient P. aeruginosa upregulating downstream pqs is an important mechanism underlying the activation of necroptosis.

Necroptosis is considered the best-characterized form of regulated necrosis mediated by RIPK3 and its phosphorylation substrate MLKL, which is also a major player in PCD (37, 38). Upon activation of TNF receptor 1, the downstream effector RIPK1 will be deubiquitinated and activated under the inhibition of Casp-8, which then leads to the activation and phosphorylation of RIPK3 and MLKL to finally execute necroptosis (39). Clinical studies reveal that necroptosis plays a double-edged role in diseases, both to eliminate infected cells and to contribute to pathogenesis (39, 40). In this study, mice infected with a DKO mutant had a significantly improved survival outcome when compared with mice challenged with the rhl mutant. The rhl mutant induced high intensity of cell death in macrophages as well as other immune cells, resulting in tissue damage–associated hyperinflammation and a low survival rate in mice. Through depletion of pqs, the DKO mutant reverted the outcome caused by rhl deficiency. However, further studies that identify the host receptors to monitor the elevation of pqs and pqs-related virulence factors will be important to better understand how exactly pqs triggers host necroptosis. To our knowledge, our results provide a novel aspect of a link between P. aeruginosa QS and host necroptosis. Targeting the rhl–pqs–necroptosis axis represents a potential therapeutic strategy for combating multiple P. aeruginosa involved diseases, especially cystic fibrosis.

The authors have no financial conflicts of interest.

We thank all the members of the Wen laboratory for discussion, Dr. Rebecca Tweedell from Dr. Thirumala Kanneganti’s laboratory for sharing with us Casp1/11^−/−Casp8^−/−Ripk3^−/− (QKO) BMMs, and Dr. Y. Liu from Dr. Daniel Wozniak’s laboratory for sharing pqsA primers and helping us with pqsA deletion.