Nemo-like kinase orchestrates the PAMP and circumstance of macrophage death in sepsis

Abstract

Sepsis is a life-threatening acute inflammatory condition characterized by dysfunctional immune responses to infection, including dangerous cytokine storms.¹ While sepsis can cause death within hours, the underlying risk can also extend years beyond the initial infection.² Macrophages are tissue immune sentinels that integrate external stimuli, such as pathogen-associated molecular patterns (PAMPs) found on invading microbes via pattern recognition receptors (PRRs), and dictate tissue-level and systemic inflammatory outcomes.³ Therefore, it is unsurprising that macrophages strongly contribute to the overall inflammatory milieu and pathology of sepsis. Key to this network of inflammatory processes in sepsis is programmed cell death (PCD), including the recently discovered PANoptosis with features of Pyroptosis, Apoptosis and Necroptosis (Figure 1).⁴ Unlike apoptosis, which is part of homeostasis and resolves inflammation, pyroptosis and necroptosis are lytic forms of cell death that release intracellular contents, which in turn heighten inflammatory responses, such as in sepsis.⁵

PANoptotic cell death is characterised by the PANoptosome, a complex that coordinates PCD fate decisions, which consists of signalling partners and effector proteins of each of these PCD pathways.⁴ Although there are different flavours of PANoptosomes, dictated by the incorporation of varying cell death effector proteins, a common and critical feature in dictating PCD is caspase-8. Upon recruitment to the PANoptosome, caspase-8 homodimerizes for its auto-cleavage and active functions. Here active caspase-8 cleaves caspase-3/7 and gasdermin-D (GSDM-D) to activate apoptotic and pyroptotic cell death, respectively (Figure 1). Inactivation or absence of caspase-8 shifts PANoptosome signalling towards necroptosis. Therefore, caspase-8 represents a critical PCD checkpoint, and a better understanding of its regulation could support therapeutic opportunities to leverage inflammatory processes and improve overall survival after infection.

A recent study by Xia et al. employed multidisciplinary approaches using mouse models and clinical samples to identify Nemo-like kinase (NLK) as a regulator of PANoptosis and inflammatory outcomes in sepsis.⁶ NLK is a conserved kinase belonging to the mitogen-activated protein kinase (MAPK) family and has been implicated in a variety of processes including embryonic and nervous system development, regulation of cell signaling cascades such as by transcription factor phosphorylation, and in an array of diseases including cancer and neurodegenerative diseases.⁷ Using independent transcriptomic datasets, the authors identify increased NLK in peripheral blood mononuclear cells (PBMCs) from sepsis patients, where higher NLK levels are associated with poorer survival outcomes.⁶ In particular, monocytes, the precursors of tissue-infiltrating macrophages, show significant and positive correlations between NLK and PANoptosis-related effectors (CASP8, GSDMD, NLRP3, IL-18, ASC, CASP3, RIPK3 and MLKL), suggesting a relationship between NLK, PCD, and sepsis.

To investigate the role of NLK in macrophage-mediated inflammation in sepsis, the authors utilised conditional (Csf1r-iCre) Nlk knockout mice subjected to cecal ligation and puncture (CLP) to induce polymicrobial infection characteristic of sepsis.⁶ In agreement with data obtained from sepsis patient PBMCs, Nlk deletion in macrophages blunts multiorgan injury after CLP. Notably, in the lung, a primary site of tissue injury in sepsis, Nlk deletion polarises macrophages towards M2 or pro-resolving subsets, accompanied by reductions in circulating cytokines such as tumour necrosis factor α (TNFα) and pro-inflammatory interleukins (IL)-6, -1β and -18. Analysis of splenic macrophages derived from these mice reveals engagement of PANoptosis in response to CLP, combining both morphologic and activated signalling features of pyroptosis (cleaved GSDM-D & caspase-1), apoptosis (cleaved caspase-3), and necroptosis (phosphorylated mixed lineage kinase domain-like protein [pMLKL]). Interestingly, deletion of Nlk shifted PANoptosis towards necroptosis compared to pyroptosis and apoptosis, suggesting a potential NLK-mediated regulation of caspase-8 and the PANoptosome. Indeed, Nlk deletion reduces CLP-induced caspase-8 incorporation and activation in the PANoptosome, correlating with the aforementioned shifts in necroptosis. These data are also recapitulated in macrophages treated ex vivo with lipopolysaccharide (LPS). The authors further define this cooperativity of NLK and caspase-8 using molecular docking and immunoprecipitation studies, demonstrating hydrophobic and hydrogen bond interactions, which were surprisingly independent of NLK's canonical kinase function.

Given the urgency and challenges in treating septic patients, exploring additional avenues to modulate inflammatory outcomes is crucial. Here the authors demonstrate that NLK promotes PANoptosis via its support of caspase-8 in the PANoptosome, leading to widespread inflammation, cytokine storm, and multiorgan injury. The authors propose that loss of NLK shifts PCD towards necroptosis, dampens cytokine release, while concomitantly promoting the release of chemotactic factors such as CXCL10, and the shift towards pro-resolving macrophage subsets that together may function to resolve the microbial challenge of sepsis, although this remains to be formally tested. Through the identification of interfaces that mediate NLK and caspase-8 interactions, the authors provide the potential for development of small molecule inhibitors that can augment this non-canonical function of NLK, without compromising its other effects and ultimately increasing survival in septic patients. Although it is likely that NLK is induced by invading pathogens, as its expression is stimulated by LPS, whether these mechanisms in NLK regulation of PANoptosis also extend to the setting of sterile inflammation remains to be elucidated. Further investigation of the consequences of perturbed PANoptosis in other diseases also requires evaluation, as septic patients also present with co-morbidities from chronic diseases such as atherosclerosis, where necroptosis promotes plaque vulnerability, and apoptosis resolves inflammation.⁸ Nevertheless, this study provides an exciting new area that moves beyond targeting PANoptosome components individually and reveals a novel mediator of the pathway that may serve to calibrate and leverage PCD pathways to yield inflammatory-mediated benefits in the setting of infection.

Julia E. Steinbeck reviewed the manuscript and generated the figure. Adil Rasheed drafted the manuscript and generated the figure. All authors approved the final manuscript for submission.

The authors declare no conflicts of interest.

Not applicable.

No new data were generated for this review.