Constitutive interferon signaling maintains critical threshold of MLKL expression to license necroptosis

Abstract

Interferons (IFNs) are critical determinants in immune-competence and autoimmunity, and are endogenously regulated by a low-level constitutive feedback loop. However, little is known about the functions and origins of constitutive IFN. Recently, lipopolysaccharide (LPS)-induced IFN was implicated as a driver of necroptosis, a necrotic form of cell death downstream of receptor-interacting protein (RIP) kinase activation and executed by mixed lineage kinase like-domain (MLKL) protein. We found that the pre-established IFN status of the cell, instead of LPS-induced IFN, is critical for the early initiation of necroptosis in macrophages. This pre-established IFN signature stems from cytosolic DNA sensing via cGAS/STING, and maintains the expression of MLKL and one or more unknown effectors above a critical threshold to allow for MLKL oligomerization and cell death. Finally, we found that elevated IFN-signaling in systemic lupus erythematosus (SLE) augments necroptosis, providing a link between pathological IFN and tissue damage during autoimmunity.

Hierarchy of clinical manifestations in SAVI N153S and V154M mouse models

“…Besides type I interferons, several studies have linked cytosolic DNA-sensing pathways to necroptosis downstream of STING activation. IFN production upon STING activation was reported to engage RIPK3 and MLKL, leading to oligomerization of MLKL and MLKL-dependent necroptotic cell death (22)(23)(24). Because we observed significant loss of lymphoid cells in these SAVI strains, we considered the possibility that necroptosis led to cell death in SAVI mice.…”

Constitutive Interferon Attenuates RIPK1/3-Mediated Cytokine Translation

“…We have previously taken advantage of the genetic diversity of wild-derived MOLF/Ei (MOLF) mice for mapping and identification of genes conferring resistance to necroptosis in MOLF macrophages (Schworer et al, 2014;Sarhan et al, 2018). Here, to extend our line of inquiry to the genetic level, we compared LZ-driven inflammatory responses in BMDMs from MOLF and B6 mice using TNF-a and CXCL-1 as a readout.…”

“…We have termed this MOLF-specific increase in cytokine and chemokine production upon LZ treatment ''RIPK-kinase-dependent inflammation'' or the ''RIPK-kinase-dependent inflammatory phenotype.'' MOLF BMDMs are resistant to LZ-induced necroptosis compared with B6 ( Figure 1C), because of a hypomorphic allele of Sting (stimulator of IFN genes), which regulates MLKL, an important effector of necroptosis, as well as a number of other ISGs (Surpris et al, 2015;Sarhan et al, 2018). Although total MLKL levels were reduced in MOLF macrophages, MLKL phosphorylation and upstream RIPK1 phosphorylation were uninhibited, indicating that RIPK1 kinase activity was functional in MOLF macrophages ( Figure 1D) (Sarhan et al, 2018).…”

“…MOLF BMDMs are resistant to LZ-induced necroptosis compared with B6 ( Figure 1C), because of a hypomorphic allele of Sting (stimulator of IFN genes), which regulates MLKL, an important effector of necroptosis, as well as a number of other ISGs (Surpris et al, 2015;Sarhan et al, 2018). Although total MLKL levels were reduced in MOLF macrophages, MLKL phosphorylation and upstream RIPK1 phosphorylation were uninhibited, indicating that RIPK1 kinase activity was functional in MOLF macrophages ( Figure 1D) (Sarhan et al, 2018). To determine if resistance to necroptosis in MOLF permitted increased cytokine In all panels, BMDMs were stimulated with LPS, LPS/zVAD (LZ), LPS/zVAD/Nec-1 s (LZNs), zVAD (Z), or Nec-1 s (Ns) as indicated.…”

“…Stimulation of IFNAR leads to activation of Janus kinase (JAK) and the transcription factors STAT1/2, which regulate a class of IFN-stimulated genes (ISGs) that serve many antiviral and antibacterial functions (Battistini, 2009;Schneider et al, 2014;Mostafavi et al, 2016). IFN signaling has been shown to regulate necroptosis and regulate the levels and activation of the key necroptotic effectors MLKL, RIPK3, and ZBP1 (Fu et al, 1999;Thapa et al, 2013;McComb et al, 2014;Sarhan et al, 2018;Ingram et al, 2019). In the absence of activation or pathogen infection, sufficient levels of these effectors are sustained by so-called constitutive (or tonic) IFN signaling, the origins of which remain poorly understood (Vogel et al, 1979;Yoshida et al, 2005;Abt et al, 2012;Ahn et al, 2012).…”