NLRC4 Deficiency Leads to Enhanced Phosphorylation of MLKL and Necroptosis

Innate immunity plays a crucial role in detecting and eliminating pathogens from an infected host. Programmed cell death (PCD) is a vital innate immune mechanism used by a host. Multiple PCD pathways, such as pyroptosis, apoptosis, and necroptosis, have been described with clear links to innate immunity. More recently, evidence of extensive cross-talk between cell death signaling molecules has been found, which has led to the conceptualization of PANoptosis. PANoptosis is an innate immune inflammatory PCD pathway dependent on PANoptosomes, which are innate immune danger-sensing complexes that activate inflammatory cell death. These complexes contain caspases with or without inflammasome components and receptor interacting protein homotypic interaction motif-containing proteins. This cell death pathway cannot be accounted for by pyroptosis, apoptosis, or necroptosis alone (1–19).

Several bacterial, viral, and fungal pathogens induce PANoptosis (7, 12, 14–18, 20). Salmonella enterica serovar Typhimurium, a Gram-negative bacterium, is one such pathogen that has been shown to trigger PANoptosis in macrophages (14, 20). Although S. Typhimurium robustly activates the NLR family apoptosis inhibitory protein (NAIP)-NLR family CARD domain-containing protein 4 (NLRC4) inflammasome to activate the inflammasome and pyroptosis (21, 22), deletion of pyroptotic molecules, as in Casp1/11^−/− or Gsdmd^−/− cells, only partially protects from death (14, 20). Cell death is only rescued when PANoptosis is inhibited via combined deletions of caspases-1, -11, and -8 with Ripk3 (14, 20).

Similar to Salmonella, Pseudomonas aeruginosa, an opportunistic Gram-negative bacteria that causes severe infections in humans (23), can be detected in the cytosol by NLRC4 and activate pyroptosis (22). Upon Gram-negative bacteria infection, host NAIP5 interacts with flagellin and triggers a NAIP5–NLRC4 interaction that activates caspase-1 (24, 25). Activated caspase-1 cleaves gasdermin D (GSDMD) and proinflammatory cytokines, IL-1β and IL-18, which can then be released via GSDMD pores (26–30). The interplay between pyroptosis and apoptosis has been studied in several NLRC4 engaging Gram-negative bacteria (20, 22, 31); however, the roles of necroptosis as well as PANoptosis have not been well studied in this infection model.

Understanding PCD pathways and cross-talk between them helps identify new drug targets and strategies to circumvent host pathogenesis upon infection. Each bacterial, viral, and fungal trigger can induce a unique immune response; therefore, a comprehensive characterization of cell death, in response to a variety of immune triggers, is warranted. In this study, we show that in primary mouse macrophages, P. aeruginosa infection can induce inflammatory cell death with key features of PANoptosis and that cell death was rescued in PANoptosis-deficient bone marrow–derived macrophages (BMDMs). In addition, we demonstrate that BMDMs deficient in inflammasome activation—NAIP5- or NLRC4-deficient cells—show enhanced activation of phosphorylated MLKL (MLKL) and phosphorylated RIPK1 (RIPK1). This, along with experimental evidence showing that caspase-1 deficiency does not fully rescue BMDMs from cell death, indicates that the P. aeruginosa–induced cell death pathway involves key components of pyroptosis, apoptosis, and necroptosis and that MLKL-mediated cell death may be acting as a compensatory mechanism in the absence of NLRC4.

C57BL/6J (wild-type [WT]), Naip5^−/− (32), Nlrc4^−/− (33), Nlrc4^−/−Nlrp3^−/− (34), and Casp1/11^−/−Casp8^−/−Ripk3^−/− (19) mice have been described previously. All mice were bred and maintained in a specific pathogen–free facility at the Animal Resources Center at St. Jude Children’s Research Hospital and were backcrossed to the C57BL/6 background (J substrain) for at least 10 generations. Age- and sex-matched male and female 6- to 12-wk-old mice were used. Animal studies were conducted under protocols approved by the St. Jude Children’s Research Hospital Committee on the Use and Care of Animals.

Primary mouse BMDMs were generated from the bone marrow of WT and indicated mutant mice. Cells were grown for 5–6 d in IMDM (Thermo Fisher Scientific, 12440053) supplemented with 1% nonessential amino acids (Thermo Fisher Scientific, 11140-050), 10% FBS (Biowest, S1620), 30% L929 conditioned media, and 1% penicillin and streptomycin (Thermo Fisher Scientific, 15070-063). BMDMs were then seeded into antibiotic-free media at a concentration of 1 × 10⁶ cells into 12-well plates and incubated overnight.

BMDMs were infected with the PAO1 strain of P. aeruginosa at a multiplicity of infection (MOI) of 1. After 2 h of infection, 50 µg/ml gentamicin (Thermo Fisher Scientific, 15750-060) was added to kill extracellular bacteria, and BMDMs were then incubated for 12 h.

The kinetics of cell death were determined using the Incucyte SX5 (Sartorius) live-cell automated system. BMDMs (5 × 10⁵ cells/well) were seeded in 24-well tissue culture plates. Cells were infected with P. aeruginosa at an MOI of 1 and stained with propidium iodide (Life Technologies, P3566) following the manufacturer’s protocol. The plate was scanned, and fluorescent and phase-contrast images (four image fields/well) were acquired in real time every 1 h from 0 to 12 h postinfection. Propidium iodide–positive dead cells were marked with a red mask for visualization. The image analysis, masking, and quantification of dead cells were performed using the software package supplied with the Incucyte imager.

Cell lysates and culture supernatants were combined in caspase lysis buffer (containing 1× protease inhibitors, 1× phosphatase inhibitors, 10% Nonidet P-40, and 25 mM DTT) and 4× sample loading buffer (containing SDS and 2-ME) for immunoblot analysis of caspases. For immunoblot analysis of signaling components, supernatants were removed, and cells were washed once with Dulbecco’s PBS, followed by lysis in RIPA buffer and sample loading buffer. Proteins were separated by electrophoresis through 8–12% polyacrylamide gels. Following electrophoretic transfer of proteins onto polyvinylidene difluoride membranes (Millipore, IPVH00010), nonspecific binding was blocked by incubation with 5% skim milk, and then membranes were incubated with primary Abs against the following: caspase-8 (AdipoGen Life Sciences, AG-20T-0138-C100, 1:1000), cleaved caspase-8 (Cell Signaling Technology [CST], 8592, 1:1000), caspase-7 (CST, 9492, 1:1000), cleaved caspase-7 (CST, 9491, 1:1000), caspase-3 (CST, 9662, 1:1000), cleaved caspase-3 (CST, 9661, 1:1000), caspase-1 (AdipoGen, AG-20B-0042, 1:1000), caspase-11 (Novus Biologicals, NB120-10454, 1:1000), GAPDH (CST, 5174, 1:1000), pMLKL (CST, 37333, 1:1000), total MLKL (Abgent, AP14272b, 1:1000), pRIPK1 (CST, 65746, 1:1000), total RIPK1 (CST, 3493, 1:1000), GSDMD (Abcam, ab209845, 1:1000), and β-actin HRP (Santa Cruz Biotechnology, sc-47778, 1:5000). Membranes were then washed and incubated with the appropriate HRP-conjugated secondary Abs (Jackson ImmunoResearch Laboratories, anti-rabbit [111-035-047; 1:5000], anti-rat [112-035-143; 1:5000], and anti-mouse [315-035-047; 1:5000]). Proteins were visualized using Immobilon Forte Western HRP substrate (Millipore, WBLUF0500).

TNF cytokine was measured from BMDM culture supernatant according to the manufacturer’s instructions (Invitrogen, BMS607-3).

Levels of lactate dehydrogenase released by cells were determined in the supernatant using the CytoTox 96 non-radioactive cytotoxicity assay (Promega, G1780) according to the manufacturer’s instructions.

GraphPad Prism v8.0 software was used for data analysis. Data are shown as mean ± SEM. Statistical significance was determined by two-way ANOVA. A p value <0.05 was considered statistically significant.

To characterize P. aeruginosa–induced cell death, we first examined the activation of cell death signaling molecules in infected WT BMDMs. We observed robust cell death in these cells (Fig. 1A, 1B), along with activation of caspase-1, GSDMD, caspase-8, caspase-3, and caspase-7 (Fig. 1C). Additionally, pMLKL, pRIPK1, and an increase in total RIPK1 cleavage were observed in response to infection (Fig. 1D). Under these conditions, these data show that P. aeruginosa activates a combination of molecules indicative of PANoptosis. The activation of a molecular signature consistent with PANoptosis led us to hypothesize that inhibiting PANoptosis, by deleting key components of pyroptosis, apoptosis, and necroptosis, would provide complete protection against cell death. When PANoptosis-deficient BMDMs (Casp1/11^−/−Ripk3^−/−Casp8^−/−; referred to as QKO) were challenged with P. aeruginosa infection, QKO BMDMs were protected from cell death (Fig. 2A–C) and showed impaired activation of caspase-1, GSDMD, caspase-8, and caspase-3 (Fig. 2D). Activation of pMLKL was also impaired in QKO BMDMs in response to infection (Fig. 2E). This suggests that PANoptosis is involved in mediating the cell death in response to P. aeruginosa infection.

Because of the flexibility built into the PANoptotic cell death pathway, with multiple cell death effectors and executioners being activated, modifying it through a combination of gene deletions may result in a compensatory response of other key cell death signaling components. This phenomenon has previously been shown in Salmonella and Bacillus anthracis lethal toxin-treated cells void of pyroptosis (Casp1^−/−, Gsdmd^−/−, or Casp1/11^−/− cells) where, instead, caspase-8–mediated apoptosis executes a cell death response (20, 31, 35). However, the contribution of necroptosis, in the absence of pyroptosis, is less characterized in infections. To gain insight into the interconnectivity of these key cell death pathways in response to P. aeruginosa infection, we examined necroptotic markers in infected BMDMs deficient in the upstream pyroptotic molecules, NAIP5 and NLRC4. We observed reduced cell death in Naip5^−/− and Nlrc4^−/− BMDMs compared with WT BMDMs after P. aeruginosa infection (Fig. 3A, 3B). As infection progressed, the percentage of cells dying increased in Naip5^−/− and Nlrc4^−/− BMDMs, suggesting that the loss of pyroptotic sensors cannot completely protect against cell death (Fig. 3B). We also monitored the cleavage of caspase-1 and GSDMD, and, consistent with the observed partial protection from cell death, Naip5^−/− and Nlrc4^−/− BMDMs showed impaired activation of these molecules (Fig. 3C). We also observed decreased activation of caspase-8, caspase-3, and caspase-7 in Naip5^−/− and Nlrc4^−/− BMDMs (Fig. 3C). However, the loss of Naip5 or Nlrc4 resulted in an increase in the activation of pMLKL (Fig. 3D). Consistent with MLKL activation, there was increased pRIPK1 and total RIPK1 cleavage (Fig. 3D). Components of necroptosis can be activated by TNF-α (36). To determine whether NAIP5 or NLRC4 deficiency triggered enhanced production of TNF-α upon Pseudomonas infection, we measured TNF-α release in the supernatant of WT, Naip5^−/−, and Nlrc4^−/− BMDMs. All released similar amounts of TNF-α upon Pseudomonas infection (Fig. 3E). Altogether, these data suggest that in response to P. aeruginosa infection, cells deficient in inflammasome sensors modulate their overall cell death response and enhance activation of some PANoptotic effectors when others are impaired.

Caspase-1 and GSDMD activation were not completely abolished in Naip5^−/− and Nlrc4^−/− BMDMs (Fig. 3C, 3D), most likely due to the fact that P. aeruginosa has been shown to activate the NLRP3 inflammasome sensor to induce pyroptosis in response to a bacterial infection (37–41). To determine the contribution of NLRP3 in response to infection, we used Nlrc4^−/−Nlrp3^−/− BMDMs. Although Nlrc4^−/−Nlrp3^−/− and Nlrc4^−/− BMDMs both had less cell death than did WT BMDMs, the loss of both inflammasome sensors resulted in significantly less cell death than for the single Nlrc4^−/− knockout (Fig. 4A, 4B). Additionally, there was further reduction in the activation of caspase-1 and GSDMD when both inflammasome sensors were deleted (Fig. 4C). However, Nlrc4^−/−Nlrp3^−/− BMDMs were still not fully protected from cell death, indicating that other signaling molecules may be responsible for cell death in the absence of canonical P. aeruginosa–induced inflammasome sensors. Notably, activation of molecules involved in necroptosis was markedly enhanced in pyroptosis-deficient BMDMs compared with WT BMDMs (Fig. 4D). There was a complete absence of active caspase-1 in Nlrc4^−/−Nlrp3^−/− BMDMs, although GSDMD was cleaved (Fig. 4C). This could possibly be due to caspase-11–mediated GSDMD activation (29), and, indeed, we observed activation of caspase-11 in Nlrc4^−/−Nlrp3^−/− BMDMs (Fig. 4C) upon P. aeruginosa infection. Overall, these results suggest that upon infection, the activation of MLKL-dependent cell death is enhanced in the absence of key P. aeruginosa inflammasome sensors. P. aeruginosa likely induces a PANoptosis response that activates MLKL in a compensatory manner when cells are unable to activate inflammasomes.

The critical role of cross-talk between various cell death signaling molecules in the host innate immune response has been increasingly recognized, particularly cross-talk resulting in functional redundancy between pyroptotic and apoptotic molecules. Previous studies have shown an increase in caspase-8–mediated cell death in response to caspase-1, caspase-1/11, or Gsdmd deletions (20, 31, 35). Cross-talk is further highlighted by the pyroptotic caspase-1, which can cleave caspase-7 (10), and inflammasome activation, which can result in the cleavage of the apoptotic substrate poly(ADP-ribose) polymerase 1 (6). In the context of osteomyelitis, the NLRP3 inflammasome or caspase-1 can play a redundant role with caspase-8 in promoting IL-1β–mediated disease (5, 19). Additionally, in GSDMD-deficient macrophages, caspase-3 and caspase-7 are activated (42), showcasing the interplay between pyroptosis and apoptosis to achieve cell death. Enabling apoptosis in lieu of pyroptosis may prevent the host from initiating a hyper-inflammatory response to pathogens.

Cross-talk between apoptosis and necroptosis is also well understood, as necroptosis functions as the backup cell death pathway when the apoptotic caspase-8 is inhibited (36). Although suspected, few studies have aimed at discovering cross-talk between pyroptosis and molecules associated with necroptosis. In this study, we provide evidence of cross-talk between components involved in pyroptosis and necroptosis in response to P. aeruginosa infection. The NAIP5-NLRC4 cytosolic bacterial sensor complex modulated the biochemical features of cell death induced by P. aeruginosa; its absence activated RIPK1 and pMLKL, suggesting that RIPK1 and MLKL activation can serve as a backup for not only the loss of caspase-8 activation but also caspase-1 activation. Non-cleavable caspase-8 (Casp8^DA/DA) can facilitate cleavage of RIPK1 in response to TNF and cycloheximide treatment (43). Similarly, we also observed reduced cleavage of caspase-8 and increased cleavage of total RIPK1 in Naip5^−/− or Nlrc4^−/− BMDMs, suggesting that non-cleaved caspase-8 might still regulate RIPK1 cleavage in Naip5^−/− or Nlrc4^−/− BMDMs upon Pseudomonas infection. Although we observed no difference in the amounts of TNF released between WT and NAIP5- or NLRC4-deficient cells, future studies should be aimed at determining the activation of components of necroptosis in Nlrc4^−/−Tnf^−/− macrophages to mechanistically define the contribution of TNF in Nlrc4^−/− BMDMs upon Pseudomonas infection.

Overall, our results highlight that P. aeruginosa–induced cell death is characterized by a compensatory activation of MLKL in response to a deficiency in inflammasome sensing. Not only do our data lead to a more comprehensive understanding of PCD responses, but they provide more evidence of a unified PCD response, PANoptosis, and showcase the ability of P. aeruginosa to induce a finely tuned cell death response that specifically modulates PANoptosis executioners in a way that dampens components of apoptosis and activates components of necroptosis in BMDMs deficient in inflammasome sensors. In the context of infection, it would benefit the host to respond to pathogens by utilizing a myriad of cell death molecules; pathogens carrying specific inhibitors would be less successful at colonization due to the ability of the host immune system to reroute PCD in real time. PANoptosis is a strategy built on flexible redundancy, allowing for the use of multiple paths to achieve cell death. The connection between components of pyroptosis and necroptosis presented in the present study expands our knowledge of the intricacies of a Gram-negative bacteria–induced PANoptosis cell death response.

We thank all members of the Kanneganti laboratory for comments and suggestions during the development of this manuscript. We also thank Dr. J. Gullett and Dr. R. Tweedell for scientific editing and writing support.

The authors have no financial conflicts of interest.