Yin and Yang of innate immune cell death

Lytic innate immune cell death releases a variety of pathogen-associated and damage-associated molecular patterns, cytokines, metabolites and other molecules into the extracellular environment, where they can drive further physiological responses. In a recent study in Nature, Mehrotra et al. studied the secretome released during NLRP1B inflammasome-driven pyroptosis and identified that metabolites released by dying cells can promote wound healing.

Dead cells are well-known for their homeostatic and immunomodulatory functions during development and pathophysiology. The molecular mechanism that drives cell death often determines the effect of dead and dying cells on neighboring cells and the local tissue environment.¹ For instance, non-lytic cell death, apoptosis, is historically considered to be immunologically silent or even immune suppressive.² In contrast, lytic cell death pathways driven by innate immune activation, such as pyroptosis³ and PANoptosis,^4–6 are inflammatory due to the production and release of pro-inflammatory cytokines and damage-associated molecular patterns (DAMPs). These molecules can synergize with the local or systemic inflammatory milieu to further induce PANoptosis and tissue damage^4–6 (Fig. 1). Additionally, the production of inflammasome-dependent cytokines IL-1β and IL-18 during cell death is a well-known pro-inflammatory signature of pyroptosis. However, little is known about effector functions of other components in the “secretome”, or the set of molecules released, during lytic cell death.

Fig. 1. Differential release of molecules during innate immune cell death can drive pro-tissue repair or pro-inflammatory effects.

Activation of the NLRP1B inflammasome with LeTox in unprimed macrophages induces production of a pyroptotic “secretome” (i.e., a set of molecules released during the cell lysis) that lacks the major inflammatory factors IL-1α or IL-1β (termed “Pyro^–1”; left side). The Pyro^–1 secretome contains PGE₂, and this secretome induces a pro-repair phenotype in macrophages, leading to IL-27 production and improved wound healing. In contrast, induction of other forms of lytic innate immune cell death can drive a pro-inflammatory phenotype. This can occur in primed macrophages (right side), where IL-1α, IL-1β, and additional pro-inflammatory cytokines, DAMPs, and other molecules are released to drive a pro-inflammatory amplification loop, leading to PANoptosis and contributing to tissue damage, organ failure, and pathology in disease.

During pyroptosis, the physiological outcome is generally dominated by the pro-inflammatory effects of the IL-1 family cytokines released. However, anthrax lethal toxin (LeTox) can induce pyroptosis in unprimed mouse macrophages through activation of the NLRP1B inflammasome without releasing IL-1α or IL-1β⁷ (Fig. 1). Using this system, new research now suggests an unexpected effector function for the secretome from these pyroptotic cells that lacks the major inflammatory factors IL-1α or IL-1β (termed “Pyro^–1”) in promoting tissue repair.⁸ When cultured macrophages were treated with the Pyro^–1 secretome, instead of inducing pro-inflammatory responses (e.g., TNF or IL-6 production), the cells exhibited an increase in proliferation and migration characteristics,⁸ mirroring the well-documented “compensatory proliferation” induced by apoptotic cells during development.⁹ In vivo, the Pyro^–1 secretome promoted wound healing in colonic and cutaneous punch wound models.⁸ Furthermore, application of the Pyro^–1 secretome on full-thickness skin wounds induced expansion of the CD301⁺ macrophage population, which produced IL-27 to promote skin wound healing⁸ (Fig. 1).

Metabolomic analysis of the Pyro^–1 secretome identified prostaglandin E₂ (PGE₂) as a key metabolite that is present in the Pyro^–1 secretome and absent in the apoptotic secretome.⁸ Furthermore, PGE₂ contributed to the tissue repair function of the Pyro^–1 secretome.⁸ PGE₂ is one of the key effectors released by efferocytes after apoptotic cell engulfment,² and it can also be induced by particulate matter (e.g., silica and alum) after LPS priming in an NLRP3 inflammasome-independent manner through lysosome rupture.¹⁰ A set of washout experiments using several covalent and noncovalent inhibitors of COX1/2, the enzymes involved in prostaglandin synthesis, showed that PGE₂ was synthesized in pyroptotic cells during the process of pyroptosis.⁸ In addition, the pyroptotic executioner GSDMD was required for PGE₂ to be present in the Pyro^–1 secretome,⁸ suggesting that GSDMD is needed for PGE₂ synthesis or release during LeTox-induced pyroptosis, possibly through GSDMD-dependent lysosomal permeablization.¹⁰ Together, these findings suggest that PGE₂ production is coupled with NLRP1B inflammasome-induced pyroptosis. This raises the question of whether PGE₂ can be produced and released during other cell death modalities, such as during pyroptosis in response to other inflammasomes or during PANoptosis, where inflammasomes can act as integral components of the PANoptosome that drives lytic cell death [4, 6]. Further work to discover bioactive molecules released during different forms of lytic cell death and to understand the specificity and physiological impact of these molecules can define the effector functions of different cell death modalities, which can be leveraged for therapeutic intervention.

These new findings suggest a “Yin-Yang” feature of the innate immune cell death pathway, pyroptosis: the pyroptotic secretome, or the pathogen-associated molecular patterns (PAMPs), DAMPs, cytokines, metabolites, and other molecules released during pyroptosis, acts in both pro-inflammatory and pro-wound healing capacities. The presence of PGE₂ and other metabolites with “masked” pro-repair functions provides a potential mechanism of action for the previously identified efficacy of IL-1-blocking therapies to facilitate wound healing in vivo, suggesting that these treatments could hold promise for improving patient outcomes. Additionally, while the Pyro^–1 secretome specifically may be difficult to induce therapeutically or in physiological conditions, the concept of a balanced secretome opens new avenues in the field for identifying novel bioactive molecules released by cells dying through lytic cell death that can be therapeutically valuable. Current literature suggests that the release of PAMPs, DAMPs, and cytokines from lytic cells often induces an inflammation amplification loop, which can drive PANoptosis and contribute to tissue damage, organ failure, and lethality in a variety of infections and inflammatory conditions.^4–6 The identification of strategies to counteract these pro-inflammatory effects, potentially by harnessing the untapped potential of the other molecules in the secretome of innate immune cell death, as suggested by this study, will provide exciting therapeutic opportunities.