Abstract
The NLRC4 inflammasome recognizes bacterial flagellin and components of the type III secretion apparatus. NLRC4 stimulation leads to caspase-1 activation followed by a rapid lytic cell death known as pyroptosis. NLRC4 is linked to pathogen-free auto-inflammatory diseases, suggesting a role for NLRC4 in sterile inflammation. Here, we show that NLRC4 activates an alternative cell death program morphologically similar to apoptosis in caspase-1-deficient BMDMs. By performing an unbiased genome-wide CRISPR/Cas9 screen with subsequent validation studies in gene-targeted mice, we highlight a critical role for caspase-8 and ASC adaptor in an alternative apoptotic pathway downstream of NLRC4. Furthermore, caspase-1 catalytically dead knock-in (Casp1 C284A KI) BMDMs genetically segregate pyroptosis and apoptosis, and confirm that caspase-1 does not functionally compete with ASC for NLRC4 interactions. We show that NLRC4/caspase-8-mediated apoptotic cells eventually undergo plasma cell membrane damage in vitro, suggesting that this pathway can lead to secondary necrosis. Unexpectedly, we found that DFNA5/GSDME, a member of the pore-forming gasdermin family, is dispensable for the secondary necrosis that follows NLRC4-mediated apoptosis in macrophages. Together, our data confirm the existence of an alternative caspase-8 activation pathway diverging from the NLRC4 inflammasome in primary macrophages.
Highlights
Inflammation is a beneficial host mechanism that protects against injury caused by harmful agents such as pathogens or environmental irritants, excessive or chronic inflammation can be detrimental and lead to a myriad of inflammatory diseases[1]
Consistent with previous reports[10,11,12], flagellin-triggered cell death measured by lactate dehydrogenase (LDH) release was fully dependent on NLRC4 and NAIP5 (Fig. 1a no pre-stimulation)
When bone marrow derived macrophages (BMDMs) were pre-stimulated with a TLR2-agonist (Pam3CSK4) as a method to mimic the presence of bacteria, Casp1−/−Casp11−/− BMDMs became resistant to NAIP5/NLRC4-mediated cell death (Fig. 1a,b Pam3CSK4 pre-stimulation)
Summary
Inflammation is a beneficial host mechanism that protects against injury caused by harmful agents such as pathogens or environmental irritants, excessive or chronic inflammation can be detrimental and lead to a myriad of inflammatory diseases[1]. Macrophages are vital sentinels of the immune system, surveying their environment via innate immune receptors, which include membrane-bound Toll-like receptors (TLRs) and cytosolic NOD-like receptors (NLRs). These receptors function in tightly regulated responses against pathogen-associated molecular patterns (PAMPs) derived from microbes or damage associated molecular patterns (DAMPs) released as a result of cellular injury[1,2,3,4]. We sought to identify mechanisms of NLRC4-mediated cell death in sterile bacteria-free conditions to gain invaluable insights into the etiology of NLRC4 mediated auto-inflammatory diseases. Genetic evidence of an alternative NLRC4 mediated caspase-8 pathway in primary macrophages with bacterial infection free conditions has not been clearly studied
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