Regulated necrosis and innate immunity
Unlike apoptosis, which is considered a less immunogenic variant of PCD, all forms of regulated necrosis are highly inflammatory (Pasparakis & Vandenabeele, 2015; Vanden Berghe et al., 2015). This type of PCD involves the massive release of intracellular molecules which act as danger associated molecular patterns (DAMPs), activating the immune system and amplifying the inflammatory response (D’Arcy, 2019; Linkermann et al., 2014). The same process is triggered during innate immune response to microbes, inducing the release of pathogen associated molecular patterns (PAMPs) from infected cells, leading to immune cell recruitment and activation (Kaczmarek, Vandenabeele, & Krysko, 2013). This concept was introduced by certain studies that demonstrated that unlike apoptotic cells, necrotic cells are able to release specific molecules that activate immune cells like macrophages and antigen presenting cells. For example the research of Basu and colleges in 2000 who demonstrate that necrotic but no apoptotic E.G7 cells release HSPs such as hsp90, hsp70 and gp96 which can activate macrophages and dendritic cells through the stimulation of the NF-κB pathway. Although in this work they do not induce an specific necroptotic process (just a mimic or a regular necrosis), this was a first approach to the idea that during oncosis and non-apoptotic cell death the release of these intracellular factors is a crucial internal signaling to activate the immune response (Basu, Binder, Suto, Anderson, & Srivastava, 2000). This has been experimentally corroborated in the context of inflammatory necroptosis. For example, in spite of their pleiotropic role in apoptosis-necrosis transition, inflammosome activation and TNF signaling, the kinases RIPK1 and RIPK3 are actively involved in antiviral and antibacterial response in a necroptotic-dependent way (reviewed in (Humphries et al., 2015; Silke et al., 2015)). It has been demonstrated in vitro and in vivo that certain viral entities like cytomegalovirus or herpes simplex virus regulate the necroptotic response in murine infected cells through the interaction between viral encoded proteins and the RIP homotypic interaction motif (RHIM) domains of RIPK1 and RIPK3 (Huang et al., 2015; Upton, Kaiser, & Mocarski, 2008, 2010). This has also been observed in human infected cells (Omoto et al., 2015). However, necroptosis can be also manipulated as a microbial strategy to impair immune function. In line with this, intracellular bacteria such as Salmonella enterica are also strong inducers of RIPK3-dependent necroptosis in infected macrophages (Robinson et al., 2012). The same mechanism is used by other pathogenic entities, which induce necroptosis in immune cells to interfere with its defensive function and amplify the inflammatory loop (Weinlich, Oberst, Beere, & Green, 2017). In spite of that, necroptosis is still an efficient mechanism to eliminate and expose intracellular pathogens. Some bacteria have even evolved to suppress necroptosis as an infective mechanism. That is the case of Porphyromonas gingivalis , which is able to cleave RIPK1 in human infected endothelial cells via a lysine-specific (Kgp) protease (Madrigal, Barth, Papadopoulos, & Genco, 2012). The key role of RIP kinases and necroptosis in the maintenance of an immunocompetent status has been confirmed in vivo , in RIPK3-knockout mice which are more susceptible to viral and bacterial infections (Jorgensen, Rayamajhi, & Miao, 2017; Kaiser, Upton, & Mocarski, 2013). Moreover, the role of RIP kinases in innate immunity is beyond necroptosis induction. RIPK1 and RIPK3 are key regulators of cell final destination during an infection or immune challenge, but they can also participate in signal transduction for the downstream activation of NF-κB acting as adaptor proteins in the signaling cascade of nucleic acid sensing proteins like the DNA-dependent activator of IFN regulatory factors (DAI) or Toll-like receptors such as TLR3 and TLR4 (Humphries et al., 2015).
Not only necroptosis but other forms of regulated necrosis are also implicated in particular and interesting mechanisms of innate immunity. One of the clearest examples is the form of PCD called Pyroptosis/Pyronecrosis, which receives this denomination due to its final and distinctive molecular event: the releasing of pyrogenic cytokines like IL-1β and IL-1α (Κεππ ετ αλ., 2010). This special form of PCD occurs in different cell types but primary in immune cells, mainly macrophages, as a direct response to pathogens leading to the Caspase 1-dependent processing and release of IL-1β, a potent inflammation mediator and IL18, which induces Interferon-γ (IFN-γ) production in TH1 cells, NK cells and cytotoxic T lymphocytes (CTLs), and also promotes TH2 cell development (Dinarello, 2009; B. Liu et al., 2004; Miao et al., 2011). However, besides of its function in cytokine maturation and release, pyroptosis itself seems to be an efficient mechanism to eliminate and release intracellular bacteria from infected macrophages, exposing them to neutrophil-mediated clearance trough a molecular mechanism that relies on Caspase 1 activity. This has been demonstrated in vivo for Salmonella typhimurium, Legionlla pneumophila and Burkholderia thailandensis in Casp1 knockout mice (Miao et al., 2011). This form of programed necrosis has been recently recognized by its role in the immune response against bacterial infections of the gastrointestinal tract and even in the control of transformed cells in the context of gastrointestinal cancer (Zhou & Fang, 2019).
On the other hand, NETosis is another important example of how regulated necrosis actively mediates microbe killing and immune enhancing. This cell death modality is known to be induced in specialized immune cells like neutrophils, leading to the release of the so called “extracellular traps”, complex structures composed of chromatin fragments and histones which opsonize and enclose circling pathogens, limiting their spreading within the body and facilitating their clearance by phagocytocis (Allam et al., 2014; Andrade & Darrah, 2013). These extracellular traps also contain antimicrobial proteins such as, MPO, NE, Proteinase 3 (PR3), Cathepsin G, Lysozyme and α-defensins representing a non-phagocytic mode of neutralize and degrade invading pathogens (Branzk & Papayannopoulos, 2013; Urban et al., 2009) and are able to stimulate the production of Interferon-α (IFN-α), a strong antiviral cytokine (Garcia-Romo et al., 2011). In the past 10 years, this form of PCD has been extensively studied to elucidate the sequence of its molecular events and its role in immune response, demonstrating that is actually one of the most specialized immune mechanism, which can be activated depending on microbe size and location, and as a strategy to control pathogens which are able to escape from neutrophil phagocytosis (reviewed in (Burgener & Schroder, 2020)). The effectiveness of this antimicrobial defense is evident in some pathogens such as Group A Streptococcus and Staphylococcus aureus , that produce nucleases and DNA binding proteins to impair NET formation and block NETosis as an active evasion mechanism (Storisteanu et al., 2017). In the same way, it has been reported that chronic granulomatouse disease (CGD) patients are more susceptible to Aspergillus nidulans growth, due to their deficiency in NADPH oxidase function and consequently in NET formation (Bianchi et al., 2009). Invasive aspergillosis is a leading cause of death in CGD patients, suggesting the importance of NETosis in pathogen control and immunocompetent status.