Cell death has a fundamental impact on the evolution of degenerative disorders, autoimmune processes, inflammatory diseases, tumor formation and immune surveillance. Over the past couple of decades extensive studies have uncovered novel cell death pathways, which are independent of apoptosis. Among these is necroptosis, a tightly regulated, inflammatory form of cell death. Necroptosis contribute to the pathogenesis of many diseases and in this review, we will focus exclusively on necroptosis in humans. Necroptosis is considered a backup mechanism of apoptosis, but the in vivo appearance of necroptosis indicates that both caspase-mediated and caspase-independent mechanisms control necroptosis. Necroptosis is regulated on multiple levels, from the transcription, to the stability and posttranslational modifications of the necrosome components, to the availability of molecular interaction partners and the localization of receptor-interacting serine/threonine-protein kinase 1 (RIPK1), receptor-interacting serine/threonineprotein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). Accordingly, we classified the role of more than seventy molecules in necroptotic signaling based on consistent in vitro or in vivo evidence to understand the molecular background of necroptosis and to find opportunities where regulating the intensity and the modality of cell death could be exploited in clinical interventions. Necroptosis specific inhibitors are under development, but >20 drugs, already used in the treatment of various diseases, have the potential to regulate necroptosis. By listing necroptosis-modulated human diseases and cataloging the currently available drug-repertoire to modify necroptosis intensity, we hope to kick-start approaches with immediate translational potential. We also indicate where necroptosis regulating capacity should be considered in the current applications of these drugs. Facts • Necroptosis is closely associated with the pathogenesis of many human diseases. How effective can the off-label use of already approved drugs in necroptosis-driven diseases be?

Distinct types of immune responses are activated by infections, which cause the development of type I, II, or III inflammation, regulated by Th1, Th2, Th17 helper T cells and ILC1, ILC2 and ILC3 cells, respectively. While the classification of immune responses to different groups of pathogens is widely accepted, subtypes of the immune response elicited by sterile inflammation have not yet been detailed. Necroinflammation is associated with the release of damage-associated molecular patterns (DAMP) from dying cells. In this review, we present that the distinct molecular mechanisms activated during apoptosis, necroptosis, pyroptosis, and ferroptosis lead to the release of different patterns of DAMPs and their suppressors, SAMPs. We summarize the currently available data on how regulated cell death pathways and released DAMPs and SAMPs direct the differentiation of T helper and ILC cells. Understanding the subtypes of necroinflammation can be crucial in developing strategies for the treatment of sterile inflammatory diseases caused by cell death processes.

Controlling the balance of pro-inflammatory M1 versus anti-inflammatory M2 macrophages may have paramount therapeutic benefit in cardiovascular diseases, infections, cancer and chronic inflammation. The targeted depletion of different macrophage populations provides a therapeutic option to regulate macrophage-mediated functions. Macrophages are highly sensitive to necroptosis, a newly described regulated cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1), RIPK3 and mixed lineage kinase domain like pseudokinase. Antagonists of inhibitors of apoptosis proteins (SMAC mimetics) block RIPK1 ubiquitination, while TGF-activated kinase 1 (TAK1) inhibitors prevent the phosphorylation of RIPK1, resulting in increased necroptosis. We compared the sensitivity of monocyte-derived human M1 and M2 cells to various apoptotic and necroptotic signals. The two cell types were equally sensitive to all investigated stimuli, but TAK1 inhibitor induced more intense necroptosis in M2 cells. Consequently, the treatment of co-cultured M1 and M2 cells with TAK1 inhibitor shifted the balance of the two populations toward M1 dominance. Blockage of either Aurora Kinase A or glycogen synthase kinase 3β, two newly described necroptosis inhibitors, increased the sensitivity of M1 cells to TAK1-inhibitor-induced cell death. Finally, we demonstrated that in vitro differentiated tumor-associated macrophages (TAM-like cells) were as highly sensitive to TAK1 inhibitor-induced necroptosis as M2 cells. Our results indicate that at least two different necroptotic pathways operate in macrophages and the targeted elimination of different macrophage populations by TAK1 inhibitor or SMAC mimetic may provide a therapeutic option to regulate the balance of inflammatory/anti-inflammatory macrophage functions.

Necroptosis is a regulated necrotic-like cell death modality which has come into the focus of attention since it is known to contribute to the pathogenesis of many inflammatory and degenerative diseases as well as to tumor regulation. Based on current data, necroptosis serves as a backup mechanism when death receptor-induced apoptosis is inhibited or absent. However, the necroptotic role of the proteins involved in mitochondrial apoptosis has not been investigated. Here, we demonstrated that the stimulation of several death and pattern recognition receptors induced necroptosis under caspase-compromised conditions in wild-type, but not in caspase-9-negative human Jurkat and murine MEF cells. Cerulein-induced pancreatitis was significantly reduced in mice with acinar cell-restricted caspase-9 gene knockout. The absence of caspase-9 led to impaired association of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3 and resulted in decreased phosphorylation of RIP kinases, but the overexpression of RIPK1 or RIPK3 rescued the effect of caspase-9 deficiency. Inhibition of either Aurora kinase A (AURKA) or its known substrate, glycogen synthase kinase 3b (GSK3ß) restored necroptosis sensitivity of caspase-9-deficient cells, indicating an interplay between caspase-9 and AURKA-mediated pathways to regulate necroptosis. Our findings suggest that caspase-9 acts as a newly identified regulator of necroptosis, and thus, caspase-9 provides a promising therapeutic target to manipulate the immunological outcome of cell death.

Efficient adjuvants have the potential to trigger both innate and adaptive immune responses simultaneously. Flagellin is a unique pathogen-derived protein, which is recognized by pattern recognition receptors (PRRs) as well as by B-cell and T cell receptors thus providing an important link between innate and adaptive immunity. The aforementioned properties define flagellin as an optimal adjuvant. The induction of immunogenic cell death could be an additional expectation for adjuvants in the context of cancer immunotherapy due to their ability to activate dendritic cells (DC) to present tumor antigens through the engulfment of dying cells. The immunostimulatory potential of flagellin in the course of DC and lymphocyte activation is well documented, however the exact mechanism is not fully explored. Based on this limitation we sought to investigate the potential modulatory effects of flagellin on various cell death processes knowing that it plays detrimental roles in regulating the final outcome of various types of immune responses. Here we provide evidence that the pre-treatment of Jurkat T-cells with recombinant flagellin is able to increase the degree of cell death provoked by FasL or TNF-α, and concomitantly increases the cytotoxic potential of phytohemagglutinin activated T-lymphocytes in a TLR5 dependent way. In contrast to these flagellin-mediated effects on the death receptor-induced signaling events, the mitochondrial apoptotic pathway remained unaffected. Furthermore, the cell culture supernatant of wild type Salmonella enteritidis bacteria, but not their flagellin deficient variant, was able to enhance the Fas-induced cell death process. To define the molecular mechanisms of flagellin-mediated elevated levels of cell death we were able to detect the upregulation of RIP1-dependent signaling events. These findings demonstrate that the cooperative actions of pattern recognition and different death receptors are able to initiate the cell death process with the mobilization of RIP-dependent cell death modalities. This finding highlights the capability of flagellin to act as a potential adjuvant which is relevant for tumor immunotherapy.

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Macrophages and dendritic cells (DCs) are important contributors to anti-tumor immune responses. However, these highly plastic cells are also the primary targets of tumor manipulation, which may result in the development of tumor-promoting subtypes. The effect of chemotherapeutic agents on tumor cells is an area of intense study, but little is known about their effects on innate immune cells.We investigated the effects of four chemotherapeutic drugs (two platinum-based agents; oxaliplatin and cisplatin, and two anthracyclines; doxorubicin and epirubicin) on the differentiation, function, and viability of macrophages and DCs. Macrophages and DCs were differentiated from monocytes in the presence of these chemotherapeutic drugs and we compared their cell surface receptor expression, cytokine production, and chemotactic- and T-cell-polarizing ability.We have shown that differentiation in the presence of anthracyclines dose-dependently increases CTLA-4 expression in DCs. Antineoplastic agent-driven differentiation strongly modified the CCL2- or CCL5-induced chemotactic activity of both macrophages and DCs. DCs differentiated in the presence of high-dose cisplatin and a low dose of epirubicin promoted regulatory T-cell development, whereas oxaliplatin at specific doses induced both DCs and macrophages to enhance cytotoxic T-cell responses. Furthermore, we found that inflammatory macrophages are more sensitive to doxorubicin-induced cell death than their counterparts.In summary, our results confirm that chemotherapeutic agents acting on a similar basis may have different effects on the anti-tumor immune response. Treatment with optimal dose, combinations, and timing of chemotherapy may determine tumor immunity and the metastatic potential of tumors.

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