Equally potent: Nlrp3 mutation in macrophages or neutrophils is sufficient to drive autoinflammation

Gain‐of‐function mutation in NLRP3 is associated with a spectrum of autoinflammatory disorders including familial cold autoinflammatory syndrome, Muckle–Wells syndrome, and neonatal onset multisystem inflammatory disease, collectively known as cryopyrin‐associated periodic syndrome (CAPS). However, the cell types mediating the pathogenesis of CAPS are not completely understood. Two studies in EMBO Reports now demonstrate that gain‐of‐function Nlrp3 mutation in either macrophages or neutrophils alone is sufficient to trigger systemic autoinflammation and lethality in mice.

Gain‐of‐function mutations in Nlrp3 are associated with a wide spectrum of inflammatory diseases. Two recent studies demonstrate that Nlrp3 mutations in either macrophages or neutrophils alone are sufficient to trigger autoinflammation and lethality.

NLPR3 is a sensor of microbial infection and cellular stress. Upon activation, NLRP3 assembles into a large multiprotein inflammasome complex comprising NLRP3 itself, an adaptor protein ASC, and the protease caspase‐1. Active caspase‐1 cleaves the pore‐forming protein, gasdermin D (GSDMD) to liberate the cytotoxic N‐terminal fragment, which inserts into the plasma membrane to mediate pyroptotic cell death in most cell types. Caspase‐1 also cleave the pro‐inflammatory cytokines, pro‐IL‐1β and pro‐IL‐18 into its mature active form where it is secreted out of the cells through GSDMD pores. Collectively, these processes mount a potent inflammatory response that ensures effective clearance of the invading pathogen. As such, dysregulated NLRP3 activity is associated with several heritable and acquired inflammatory diseases (Schroder & Tschopp, ²⁰¹⁰). For example, gain‐of‐function NLRP3 mutation causes a spectrum of rare autoinflammatory diseases including familial cold autoinflammatory syndrome, Muckle–Wells syndrome, and neonatal onset multisystem inflammatory disease that are collectively termed cryopyrin‐associated periodic syndrome (CAPS). Although murine models have established that gain‐of‐function Nlrp3 mutation in myeloid cells is sufficient to recapitulate most of the pathogenesis observed in human CAPS (Brydges et al, ²⁰⁰⁹), the specific contribution of each myeloid cell subset to systemic autoinflammation remains unclear.

To investigate the role of macrophages in CAPS, Frising et al (2022) generated a mouse model in which the murine gain‐of‐function Nlrp3 mutation is selectively expressed in macrophages. These mice were born at expected Mendelian ration; however, they display severe weight loss, high serum cytokines, multi‐organ failure, and perinatal lethality, indicating that activating‐Nlrp3 mutation in macrophages is sufficient to drive CAPS (Fig 1). In a complementary study published in this issue of EMBO Reports, Kaufmann et al (2022) found that selective expression of mutant Nlrp3 in neutrophils is also sufficient to drive CAPS in mice, a finding that is independently confirmed by another group (Stackowicz et al, ²⁰²¹). These findings are significant, as they provide genetic evidence that neutrophil inflammasome are as potent as macrophages in driving inflammatory response in vivo, thus refuting the common perception that neutrophils are merely effector cells that are recruited after inflammasome activation (Fig 1).

Figure 1. Gain‐of‐function NLRP3 mutation in macrophage or neutrophils alone is sufficient to drive systemic inflammation.

Gain‐of‐function Nlrp3 mutation in (A) macrophages or (B) neutrophils results in constitutive caspase‐1 activation, leading to enhanced cytokine processing (e.g., IL‐1β and IL‐18) and GSDMD cleavage. Constitutive NLRP3 activation in (A) macrophages or (B) neutrophils results in multi‐organ failure, defective hematopoiesis, systemic inflammation, and delayed growth and early lethality. These pathological effects can be partially suppressed by neutralizing IL‐1β and IL‐18. Global deletion of GSDMD rescues the auto‐inflammatory phenotype seen in in neutrophil‐restricted Nlrp3 mutant mice, while contribution of GSDMD in in macrophage‐restricted Nlrp3 mutant mice was not investigated.

To better understand the pathogenesis of macrophage‐ or neutrophil‐specific Nlrp3 mutation, the authors performed histological analysis and immune cell profiling in various organs such as the liver and spleen. Both studies found evidence of inflammatory foci and necrotic lesions in the tissues that are accompanied with increased macrophage and monocyte numbers. By contrast, neutrophil abundance, quantified using anti‐Ly6G staining, appears to be significantly decreased in the liver of mutant mice compared with healthy controls (Frising et al, ²⁰²²; Kaufmann et al, ²⁰²²). These observations are surprising, since neutrophilic inflammation is a hallmark of inflammasome activation and CAPS (Brydges et al, ²⁰⁰⁹). Subsequent in‐depth analysis revealed that gain‐of‐function Nlrp3 mutation in macrophages or neutrophils disrupted granulopoiesis and neutrophil maturation, as these mice display an accumulation of precursor neutrophils, immature neutrophils, or an unique population of Ly6G⁻CD101⁺ cells in the liver, blood, and bone marrow (Frising et al, ²⁰²²; Kaufmann et al, ²⁰²²). Blockade of IL‐1/18 signaling in macrophage‐restricted Nlrp3 mutant animals only provided a slight improvement in weight loss and survival, while global deletion of Gsdmd completely reversed disease onset in neutrophil‐restricted Nlrp3 mutant animals, suggesting that both arms of caspase‐1‐mediated inflammation—pyroptosis and cytokine secretion is required for CAPS (Brydges et al, ²⁰¹³). Although in vitro studies revealed that GSDMD promotes cytokine secretion but not pyroptosis in caspase‐1‐activated neutrophils (Chen et al, ²⁰¹⁸; Karmakar et al, ²⁰²⁰), it is possible that constitutive NLRP3 activity sensitizes CAPS neutrophils to pyroptosis in vivo. Since Ninj1 was recently identified to mediate terminal lysis in GSDMD‐perforated cells (Kayagaki et al, ²⁰²¹), future studies using Ninj1‐deficient animals will be useful to discriminate the contribution of GSDMD pores and cellular lysis to the pathogenesis of CAPS.

In summary, these two studies demonstrated that gain‐of‐function Nlrp3 mutation in macrophage or neutrophil alone is sufficient to recapitulate most of the autoinflammatory phenotype observed in CAPS mice and humans. Understanding the contribution of Ninj1 to the pathogenesis of CAPS will be important, as it may offer an alternative therapeutic option for patients that are unresponsive to anti‐IL‐1/18 treatment.

Disclosure and competing interests statement

The author declares that he has no conflict of interest.

EMBO reports (2022) 23: e56091