Agnieszka Kaczmarek

Immunogenic cell death and DAMPs in cancer therapy

Although it was thought that apoptotic cells, when rapidly phagocytosed, underwent a silent death that did not trigger an immune response, in recent years a new concept of immunogenic cell death (ICD) has emerged. The immunogenic characteristics of ICD are mainly mediated by damage-associated molecular patterns (DAMPs), which include surface-exposed calreticulin (CRT), secreted ATP and released high mobility group protein B1 (HMGB1). Most DAMPs can be recognized by pattern recognition receptors (PRRs). In this Review, we discuss the role of endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) in regulating the immunogenicity of dying cancer cells and the effect of therapy-resistant cancer microevolution on ICD.

A novel pathway combining calreticulin exposure and ATP secretion in immunogenic cancer cell death

Surface‐exposed calreticulin (ecto‐CRT) and secreted ATP are crucial damage‐associated molecular patterns (DAMPs) for immunogenic apoptosis. Inducers of immunogenic apoptosis rely on an endoplasmic reticulum (ER)‐based (reactive oxygen species (ROS)‐regulated) pathway for ecto‐CRT induction, but the ATP secretion pathway is unknown. We found that after photodynamic therapy (PDT), which generates ROS‐mediated ER stress, dying cancer cells undergo immunogenic apoptosis characterized by phenotypic maturation (CD80high, CD83high, CD86high, MHC‐IIhigh) and functional stimulation (NOhigh, IL‐10absent, IL‐1βhigh) of dendritic cells as well as induction of a protective antitumour immune response. Intriguingly, early after PDT the cancer cells displayed ecto‐CRT and secreted ATP before exhibiting biochemical signatures of apoptosis, through overlapping PERK‐orchestrated pathways that require a functional secretory pathway and phosphoinositide 3‐kinase (PI3K)‐mediated plasma membrane/extracellular trafficking. Interestingly, eIF2α phosphorylation and caspase‐8 signalling are dispensable for this ecto‐CRT exposure. We also identified LRP1/CD91 as the surface docking site for ecto‐CRT and found that depletion of PERK, PI3K p110α and LRP1 but not caspase‐8 reduced the immunogenicity of the cancer cells. These results unravel a novel PERK‐dependent subroutine for the early and simultaneous emission of two critical DAMPs following ROS‐mediated ER stress.

ER stress-induced inflammation: does it aid or impede disease progression?

Different lines of research have revealed that pathways activated by the endoplasmic reticulum (ER) stress response induce sterile inflammation. When activated, all three sensors of the unfolded protein response (UPR), PERK, IRE1, and ATF6, participate in upregulating inflammatory processes. ER stress in various cells plays an important role in the pathogenesis of several diseases, including obesity, type 2 diabetes, cancer, and intestinal bowel and airway diseases. Moreover, it has been suggested that ER stress-induced inflammation contributes substantially to disease progression. However, this generalization can be challenged at least in the case of cancer. In this review, we emphasize that ER stress can either aid or impede disease progression via inflammatory pathways depending on the cell type, disease stage, and type of ER stressor.

ATP release from dying autophagic cells and their phagocytosis are crucial for inflammasome activation in macrophages

Pathogen-activated and damage-associated molecular patterns activate the inflammasome in macrophages. We report that mouse macrophages release IL-1β while co-incubated with pro-B (Ba/F3) cells dying, as a result of IL-3 withdrawal, by apoptosis with autophagy, but not when they are co-incubated with living, apoptotic, necrotic or necrostatin-1 treated cells. NALP3-deficient macrophages display reduced IL-1β secretion, which is also inhibited in macrophages deficient in caspase-1 or pre-treated with its inhibitor. This finding demonstrates that the inflammasome is activated during phagocytosis of dying autophagic cells. We show that activation of NALP3 depends on phagocytosis of dying cells, ATP release through pannexin-1 channels of dying autophagic cells, P2X7 purinergic receptor activation, and on consequent potassium efflux. Dying autophagic Ba/F3 cells injected intraperitoneally in mice recruit neutrophils and thereby induce acute inflammation. These findings demonstrate that NALP3 performs key upstream functions in inflammasome activation in mouse macrophages engulfing dying autophagic cells, and that these functions lead to pro-inflammatory responses.

TLR-2 and TLR-9 are sensors of apoptosis in a mouse model of doxorubicin-induced acute inflammation

Anthracycline antibiotics are inducers of an immunogenic form of apoptosis that has immunostimulatory properties because of the release of damage-associated molecular patterns. To study the mechanisms used by the innate immune system to sense this immunogenic form of cell death, we established an in vivo model of cell death induced by intraperitoneal injection of doxorubicin, a prototype of anthracyclines. The acute sterile inflammation in this model is characterized by rapid influx of neutrophils and increased levels of IL-6 and monocyte chemotactic protein-1. We demonstrate that acute inflammation induced by doxorubicin is associated with apoptosis of monocytes/macrophages and that it is specific for doxorubicin, an immunogenic chemotherapeutic. Further, the inflammatory response is significantly reduced in mice deficient in myeloid differentiation primary response gene 88 (MyD88), TLR-2 or TLR-9. Importantly, a TLR-9 antagonist reduces the recruitment of neutrophils induced by doxorubicin. By contrast, the acute inflammatory response is not affected in TRIFLps2 mutant mice and in TLR-3, TLR-4 and caspase-1 knockout mice, which shows that the inflammasome does not have a major role in doxorubicin-induced acute inflammation. Our findings provide important new insights into how the innate immune system senses immunogenic apoptotic cells and clearly demonstrate that the TLR-2/TLR-9-MyD88 signaling pathways have a central role in initiating the acute inflammatory response to this immunogenic form of apoptosis.

Severity of doxorubicin‐induced small intestinal mucositis is regulated by the TLR‐2 and TLR‐9 pathways

Intestinal mucositis is a serious complication of cancer chemotherapy and radiotherapy; it frequently compromises treatment and dramatically reduces the quality of life of patients. Different approaches to limit the damage to the intestine during anti‐cancer therapy have been largely ineffective due to insufficient knowledge of the mechanism of mucositis development. This study aimed to define the role of TLR‐2 and TLR‐9 in the modulation of small intestinal damage in a model of doxorubicin‐induced mucositis. Doxorubicin‐induced intestinal damage was verified by a histological score (HS), analysis of leukocyte influx into the lamina propria, and determination of the number of apoptotic cells. Additionally, the activation status of glycogen synthase kinase 3β (GSK‐3β) was assessed. Wild‐type (WT) mice injected with doxorubicin demonstrated severe intestinal damage (HS 8.0 ± 0.81), reduction of villus length to 43.9% ± 13.7% of original length, and increased influx of leukocytes as compared to vehicle‐injected mice (HS 1.33 ± 1.15). The protective effect of TLR‐2 or TLR‐9 deficiency was associated with a significant decrease of the HS as compared to WT mice. In the ileum, a minor reduction of villus length and a decreased number of infiltrating leukocytes and TUNEL‐positive cells was observed. We demonstrate that the TLR‐9 antagonist ODN2088 reduces doxorubicin‐induced intestinal damage. Furthermore, we show that GSK‐3β activity is inhibited in the absence of TLR‐2. The protective capacity of GSK‐3β suppression was observed in WT mice by inhibiting it with the specific inhibitor SB216763. Overall, our findings demonstrate that the TLR‐2/GSK‐3β and TLR‐9 signalling pathways play a central role in the development of intestinal mucositis and we suggest a new therapeutic strategy for limiting doxorubicin‐induced intestinal inflammation. Copyright

TNF/TNF-R1 pathway is involved in doxorubicin-induced acute sterile inflammation.

Dear Editor, Doxorubicin is an anthracycline antibiotic, which is used in treatment of cancer and it works by induction of apoptosis in cancerous cells.1 In vivo, apoptotic cells are rapidly cleared preferentially by monocytes and neutrophil migration is inhibited2 to limit tissue injury and inflammation. More recently, few anti-cancer drugs, including anthracyclines (doxorubicin) were shown to evoke apoptosis in cancerous cells, which is associated with immune system activation (i.e. immunogenic apoptosis).3, 4 In our previous study we reported that doxorubicin can induce acute inflammation in peritoneum, which is associated with apoptosis.5 Doxorubicin-killed cells could be also a source of damage-associated molecular patterns (DAMPs) leading to inflammation and tumor necrosis factor (TNF) production, which would amplify the inflammatory response triggered by DAMPs. TNF is a pleiotropic cytokine that binds to receptors TNF-R1 and TNF-R2 and, depending on cell type, triggers different signaling pathways, including cell death and inflammation.6 This study aimed to examine whether TNF contributes to the doxorubicin-induced acute sterile inflammation. Intraperitoneal injection of doxorubicin provoked an acute inflammatory response accompanied by the influx of neutrophils.5 We observed an increased level of LDH, a marker of tissue damage and secondary necrotic cells, already after 6 h with further increase after 16 h post injection of doxorubicin (Supplementary Figure 1a). Lavage fluid collected 16 h after doxorubicin injection contained increased levels of TNF as compared to vehicle-injected group (0.09±0.14 versus 2.11±0.84 pg/ml, P<0.0001). We next tested the involvement of TNF-R1 and TNF-R2 in the sterile inflammation in response to doxorubicin. Wild-type mice had abundant neutrophils in their abdominal cavities, but this response was markedly decreased in TNF-R1/2 double knockout mice (by 2.4-fold, Supplementary Figure 1b). The number of neutrophils attracted in response to doxorubicin-induced acute inflammation was 3.4-fold lower in TNF-R1-deficient mice than in controls (Figure 1a), but in TNF-R2-deficient mice, there was no difference as compared to wild-type mice (Figure 1b). These results show that TNF-R1 was involved in mediating the inflammatory response but TNF-R2 was not. Importantly, cell death in TNF-R1/2 double knockout mice was not affected in comparison to wild-type mice (data not shown) indicating that TNF does not contribute to the cell death process itself. Figure 1 Role of TNF-R1 and TNF-R2 and the effect of inhibiting soluble TNF on doxorubicin-induced acute inflammatory response. The number of neutrophils in peritoneal lavage was decreased in TNF-R1−/− (a) but not in TNF-R2−/− ( ... Further, we investigated whether acute inflammation can be reduced in wild-type mice by the administration of Etanercept, a recombinant dimeric soluble form of TNF-R2 that blocks the interaction of TNF with its cell surface receptors.7 Wild-type mice were injected intraperitoneally with Etanercept together with doxorubicin and 16 h later the recruited cells were phenotyped. Etanercept significantly reduced the recruitment of neutrophils (Figure 1c). This further confirms the importance of TNF in doxorubicin-induced acute inflammation. In conclusion, we report that intraperitoneal injection of doxorubicin in mice leads to increase in TNF levels in the lavage fluid. It has been also reported that patients undergoing chemotherapy with doxorubicin had elevated levels of TNF in their plasma.8 Similarly, mouse in vitro and in vivo studies demonstrated up-regulation of TNF after doxorubicin administration both on mRNA and protein levels.9, 10 Previously, we found that the majority of cells that died apoptotically due to doxorubicin treatment were monocytes/macrophages with some minor neutrophils.5 Therefore, it is possible that either dying macrophages or attracted neutrophils might be a source of TNF, and that by binding to TNF-R1 it could amplify the inflammation. We demonstrated that the acute inflammatory response to doxorubicin was reduced in TNF-R1- but not TNF-R2-deficient mice. In addition, Etanercept decreases the attraction of neutrophils after doxorubicin administration. These studies show that TNF and the TNF-R1 signaling pathway are key elements in the acute sterile inflammatory response to doxorubicin.

Molecular Pathways of Different Types of Cell Death: Many Roads to Death

Cell death is a fundamental cellular response that has a crucial role in shaping our bodies during development and in regulating tissue homeostasis by eliminating unwanted cells. Three major morphologies of cell death have been described: apoptosis (type I), cell death associated with autophagy (type II) and necrosis (type III). In mammalian cells, the apoptotic response is mediated by either an intrinsic or an extrinsic pathway, depending on the origin of the death stimuli, and is almost always caspase-dependent. For a long time necrosis has been considered to be an accidental and uncontrolled form of cell death. However, evidence is accumulating that necrotic cell death in some cases can be as well controlled and programmed as caspase-dependent apoptosis. Autophagy is foremost a survival mechanism that is activated in cells subjected to nutrient or obligate growth factor deprivation. When cellular stress continues, cell death may continue by autophagy alone, or else it often becomes associated with features of apoptotic or necrotic cell death, depending on the stimulus and cell type. It is debatable whether autophagic cell death is an alternative way of dying, different from apoptotic and necrotic cell death, or whether failure of autophagy to rescue the cell can lead to cell death by either pathway. The aim of this chapter is to provide a general overview of current knowledge on signalling events that result in apoptosis, necrosis and cell death associated with autophagy.

ER Stress and Inflammation

Accumulating evidence indicates that ER-stress-activated pathways, e.g. the unfolded protein response (UPR), lead to activation of various inflammatory processes, such as the acute-phase response (APR) and those instigated by transcriptional factors like NF-κB and AP-1. ER stress-mediated inflammation has been found to be associated with several diseases, such as obesity, type 2 diabetes, intestinal bowel disease and cancer. The role of ER stress-mediated inflammation is not straightforward as it can vary from disease-promoting/supporting to disease-controlling depending upon the cell type or disease in focus. This makes therapeutic targeting of ER stress-mediated inflammation very challenging and tough. In this chapter, we discuss the biology of ER stress-induced inflammation, its role in various diseases, and the possibility of targeting it for therapeutic purposes.