Myeloid cells play a critical role in perpetuating inflammation during various chronic diseases. Recently the death of macrophages through programmed necrosis (necroptosis) has emerged as an important mechanism in inflammation and pathology. We evaluated the mechanisms that lead to the induction of necrotic cell death in macrophages. Our results indicate that type I IFN (IFN-I) signaling is a predominant mechanism of necroptosis, because macrophages deficient in IFN-α receptor type I (IFNAR1) are highly resistant to necroptosis after stimulation with LPS, polyinosinicpolycytidylic acid, TNF-α, or IFN-β in the presence of caspase inhibitors. IFN-I-induced necroptosis occurred through both mechanisms dependent on and independent of Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF) and led to persistent phosphorylation of receptor-interacting protein 3 (Rip3) kinase, which resulted in potent necroptosis. Although various IFN-regulatory factors (IRFs) facilitated the induction of necroptosis in response to IFN−β, IRF-9-STAT1-or -STAT2-deficient macrophages were highly resistant to necroptosis. Our results indicate that IFN-β-induced necroptosis of macrophages proceeds through tonic IFN-stimulated gene factor 3 (ISGF3) signaling, which leads to persistent expression of STAT1, STAT2, and IRF9. Induction of IFNAR1/ Rip3-dependent necroptosis also resulted in potent inflammatory pathology in vivo. These results reveal how IFN-I mediates acute inflammation through macrophage necroptosis.

Receptor interacting protein kinase 1 (RIPK1) participates in several cell signaling complexes that promote cell activation and cell death. Stimulation of RIPK1 in the absence of caspase signaling induces regulated necrosis (necroptosis), which promotes an inflammatory response. Understanding of the mechanisms through which RIPK1 promotes inflammation has been unclear. Herein we have evaluated the impact of a K45A mutation of RIPK1 on necroptosis of macrophages and the activation of inflammatory response. We show that K45A mutation of RIPK1 results in attenuated necroptosis of macrophages in response to stimulation with LPS, TNFα and IFNβ in the absence of caspase signaling. Impairment in necroptosis correlated with poor phosphorylation of RIPK1, RIPK3 and reduced trimerization of MLKL. Furthermore, K45A mutation of RIPK1 resulted in poor STAT1 phosphorylation (at S727) and expression of RANTES and MIP-1α following TNF-R engagement in the absence of caspase activation. Our results further indicate that in the absence of stimulation by pathogen-associated molecular patterns (PAMPs), cellular inhibitors of apoptotic proteins (cIAPs) prevent the K45-dependent auto-phosphorylation of RIPK1, leading to resistance against necroptosis. Finally, RIPK1K45A mice displayed attenuated inflammatory response in vivo as they were significantly resistant against endotoxin shock, but highly susceptible against a challenge with Salmonella typhimurium. This correlated with reduced expression of IL-1β and ROS, and poor processing of caspase 8 by RIPK1K45A macrophages. Overall, these results indicate that K45 mediated kinase activity of RIPK1 is not only important for necroptosis but it also has a key role in promoting cytokine signaling and host response to inflammatory stimuli. Apoptosis is considered as the predominant, programmed pathway of cell death; however, recently several pathways of regulated cell death have been shown to operate in cells that are considered highly inflammatory.1,2 Although apoptosis has a major role during fetal development, 3 regulated necrotic cell death does not appear to influence fetal development. 4 Interestingly, receptor interacting protein kinase 1 (RIPK1) deficiency cause embryonic lethality, suggesting a key role of RIPK1 in host survival. 5 Besides the kinase activity, RIPK1 appears to have a scaffolding function, which may influence immune homeostasis. 6,7 Depending on the interacting partners and posttranslational modifications, RIPK1 has a multifaceted role in cell signaling and cell survival.8 Following TNF-R1 signaling, RIPK1 transitions between pro-survival and pro-cell death signaling complexes. 9,10 Necroptosis is a form of regulated necrosis of cells that operates in the absence of caspase activity 9 and is initiated by the engagement of various TLRs or cytokine receptors.11 The first cardinal signaling step in necrosome signaling is the phosphorylation of RIPK1, which leads to RIPK1-RIPK3 interaction and phosphorylation of RIPK3.12 Although necroptotic signaling has been shown to be...

Background The coronavirus disease 2019 (COVID-19) pandemic has negatively affected many aspects of daily life. In Saudi Arabia, many studies, using a range of assessment approaches, have examined how the pandemic has affected the mental health of both the general public and healthcare workers. However, to develop effective public-health initiatives for such crisis events, it would also be relevant to determine the pandemic’s impact on the behavioral, emotional, and social lives of Saudi children. Objective To assess, among Saudi children aged 3-15 years, the behavioral, emotional, and social changes that have occurred in their daily lives due to the COVID-19 pandemic. Materials and methods This study featured a cross-sectional design. Potential participants were approached through the most popular social media in Saudi Arabia, and the final sample size was 651 parents. As, at the time of data collection, the members of the Saudi public were requested to avoid face-to-face meetings where possible, a well-designed electronic questionnaire featuring closed-ended questions was used. Results Descriptive statistics showed that the mean age of the parents was 29±7 years (range: 20-60 years); over half (58%) were female. During the COVID-19 outbreak, one-third of children had asked to sleep in their parents’ beds. Furthermore, approximately 30% of children demonstrated increased irritability and mood swings when compared with the period before the pandemic. Concerning adaptive social behaviors, during the pandemic, 22% of children appeared calmer and 14% of children seemed more thoughtful. Our study reports more screen time, less physical activity, and reduced sleep time among children compared with the pre-pandemic period. Conclusion The COVID-19 pandemic has psychologically affected children. The present results highlight the need to reduce this psychological burden by enhancing children’s emotional resilience and involving parents in health-promotion programs aimed at mitigating the negative impacts of such public-health crises.

Understanding the molecular signaling in programmed cell death is vital to a practical understanding of inflammation and immune cell function. Here we identify a previously unrecognized mechanism that functions to downregulate the necrosome, a central signaling complex involved in inflammation and necroptosis. We show that RipK1 associates with RipK3 in an early necrosome, independent of RipK3 phosphorylation and MLKL-induced necroptotic death. We find that formation of the early necrosome activates K48-ubiquitin-dependent proteasomal degradation of RipK1, Caspase-8, and other necrosomal proteins. Our results reveal that the E3-ubiquitin ligase Triad3a promotes this negative feedback loop independently of typical RipK1 ubiquitin editing enzymes, cIAPs, A20, or CYLD. Finally, we show that Triad3a-dependent necrosomal degradation limits necroptosis and production of inflammatory cytokines. These results reveal a new mechanism of shutting off necrosome signaling and may pave the way to new strategies for therapeutic manipulation of inflammatory responses.