You had me at PELO: a “Ribosome Rescuer” induces NLR inflammasome assembly

Pyroptosis, an inflammatory form of programmed cell death, eliminates a niche where pathogens replicate, providing overall benefits to a host. When left uncontrolled, pyroptosis can lead to severe pathology. Overactive pyroptotic activation has been implicated in neuroinflammation, cardiovascular disease, acute respiratory distress syndrome (ARDS), and many other diseases [1–3]. Because of its multiple and permanent consequences, pyroptosis is a highly regulated signaling pathway. Major regulators of pyroptosis are NLR inflammasomes, which are activated by various “danger” signals such as ATP. Notably, high NLR levels are associated with cell death. Once assembled, this inflammasome promotes pyroptotic cell death by activating the pore-forming protein gasdermin D (GSDMD) protein. GSDMD oligomerizes, is inserted into the cell membrane, where it forms a pore, leading to lytic cell death and release of biologically active interleukin 1β (IL-1β). The activation of IL-1β and GSDMD depend on caspase 1, which is also activated after inflammasome assembly. Despite being a major regulatory mechanistic factor in pyroptotic cell death and release of biologically active IL-1β, the conformational changes required for inflammasome activation and assembly are poorly understood. In this regard, a recent study from Wu et al. shed some light on the requirements for NLR inflammasome assembly by introducing a previously unknown contributor to NLR inflammasome assembly [4].

The authors used an unbiased approach based on mass spectrometry to observe the association of the inflammasome with PELO, which is known for reactivating ribosomes and releasing those that are ectopically attached to mRNAs. In addition to NLRP3, PELO interacts with several cytosolic NLRs but not with other pathogen recognition receptors (PRRs), suggesting that PELO is a protein unique to inflammasomes (Fig. 1)

Fig. 1.

One gene- many functions. In addition to recently discovered role of PELO in the inflammasome assembly, this gene product regulates translation and ribosome disassembly, stem cells population, genome instability and several other biological functions. While most of these functions are waiting to be mapped to specific domains of PELO, the staggering complexity of PELO biology exemplifies that perhaps thousands genes encode proteins with multiple functions thus explaining surprisingly small number of genes that organisms are made of

Inflammasome-mediated pyroptotic cell death is essential to a robust immune response to many pathogens. For example, L. monocytogenes, an intracellular gram-positive pathogen, has been shown to activate many cytosolic NLR inflammasomes by using bacterial toxins that dissolve the phagosome, enabling pathogen entry into the intracellular space [5]. These toxins, together with bacterial flagellin, activate multiple NLR inflammasomes, inducing the cleavage of both GSDMD and pro-IL-1β into their active forms. Active GSDMD oligomerizes and is inserted into the host cell membrane, forming pores that release IL-1β and induce lytic cell death. S. typhimurium, a gram-negative intracellular pathogen, also activates NLR inflammasomes via the action of LPS, bacterial toxins, and flagellin [6]. Inducing pyroptotic death ensures the elimination of the niches where these pathogens replicate, trapping them in cellular debris and inducing an antibacterial inflammatory response. When pyroptotic death is inhibited, pathogens replicate easily, making them detrimental to a host.

The authors used L. monocytogenes and S. typhimurium to evaluate the action of PELO during infection. They hypothesized that if PELO exerts any impact on inflammasome function, mice with myeloid-specific PELO knockout will present with impaired NLR activation and inhibited pyroptotic cell death, and therefore, these mice will be more susceptible to infection. Indeed, these myeloid cell PELO-knockout mice exhibited reduced survival times when infected with these bacterial infections. In support of the hypothesis, BMDMs from these mice did not express the hallmarks of pyroptosis, including the activation of neither caspase 1 nor IL-1β or the release of LDH. Myeloid cell-specific PELO-knockout mice were similarly protected from the effects of FlaTox (flagellin), indicating that NLRC4 activity also relies greatly on PELO.

As a known “ribosome rescuer”, PELO is an unlikely hero in the story of pyroptosis. PELO binds stalled ribosomes and catalyzes the separation of ribosomal subunits, ensuring that functioning ribosomal subunits are recycled. Mouse models have demonstrated the importance of PELO ribosomal rescue for epidermal homeostasis, wound healing, and neurogenesis [7, 8]. In these critical activities, PELO relies on HBS1L, a GTPase elongation factor. To decouple the role of PELO in inflammasome activation from its ribosomal function, the authors demonstrated that HBS1L-deficient BMDMs underwent pyroptotic cell death. Specifically, Hbs1l−/− BMDMs stimulated by classic inflammasome activators demonstrated a complete pyroptotic response, producing IL-1β and releasing substantial quantities of LDH. In fact, the ribosomal activities of PELO seemed to compete with the inflammasome incorporation of PELO, indicating that these two processes are unrelated. When 4-NQO was used to generate NOS and induce downstream ribosome stalling, inflammasome activation was abrogated.

The authors demonstrated that PELO plays a unique role in NLR inflammasome activation that is independent of ribosomal rescue activity, but this result begs the question, how does PELO contribute to inflammasome activation? The authors ruled out a role for PELO in ASC oligomerization, as deletion of PELO exerted a substantial effect on NLRC4 activation, which does not require ASC activity. However, a final and essential step of NLR inflammasome activation is the oligomerization of NLR components. Oligomerization is thought to be induced by an ATPase-dependent conformational change that exposes binding sites, and the authors questioned whether this step relies on PELO [9, 10]. Using Blue Native (BN)–PAGE for identification of multiprotein complexes, the authors visualized large oligomers of NLRs by Western blotting. Indeed, the oligomerization of NLRP3 and NLRC4 stimulated by classic inflammasome activators was completely abrogated in PELO-KO BMDMs and HEK293T cells. These results suggested a potential role for PELO in the ATPase activity needed for NLR inflammasome assembly and activation.

To evaluate the ATPase-promoting effect of PELO, the authors reconstituted inflammasome activity in vitro. That is, they incubated ATP with various recombinant NLRs and PELO and measured the amount of ATP converted into ADP. All NLRs, including NLRP3 and NLRC4, converted ATP to ADP when incubated with PELO. HEK293Ts coexpressing NLRC4 inflammasome components and PELO were stimulated with flagellin to activate the NLRC4 inflammasome, and the authors observed coimmunoprecipitation of PELO with flagellin and NLRC4. The quantity of PELO that was immunoprecipitated largely depended on the quantity of NLRC4, and when lysates were fractionated, PELO seemed to move between light and heavy fractions independent of the fractional NLRC4 content, suggesting the possible release of PELO after initial NLR oligomerization.

This elegant and extensive study points to PELO as a novel regulator of NLR inflammasome assembly that promotes the ATPase activity required for NLR oligomerization. This function is entirely independent of the previously characterized ribosome-rescuing function of PELO, providing another example of the multiple functions of a gene. Thus, this study supports further unbiased inquiries into the discovery of gene functions. The authors propose a model in which transient binding of PELO to the flagellin–NLRC4 complex activates ATPase activity to induce the conformational change required to bind NLRC4 unit sequentially. PELO likely falls away and promotes the ATPase activity needed to attach the next subunit to the complex until the NLR inflammasome is fully oligomerized and activated. As noted in the study, the activity of PELO plays a significant role in pyroptotic activity in myeloid cells and intracellular pathogen defense at the organismal level. The identification of PELO not only adds to our understanding of inflammasome activation, a process involved in many pathologies, but also introduces a novel connection of inflammasome assembly to ribosomal rescue. As the authors clearly show, ribosomal distress pulls PELO away from stabilized inflammasomes, inhibiting pyroptotic cell death. As we continue to study the powerful NLR inflammasome complex, we need to consider if we oversimplify the effect of cellular stress on the pyroptotic response and resistance to infection. Additionally, this study proposes intriguing and concerning implications regarding the many cancer drugs designed to induce ROS production or cellular stress and how these outcomes may impact patient susceptibility to infection.

Author contributions

ZM and AP wrote the manuscript. AP designed and generated the figure.

Competing interests

The authors declare no competing interests.