Stereotactic body radiation combined with oncolytic vaccinia virus induces potent anti-tumor effect by triggering tumor cell necroptosis and DAMPs.

Radiation is an integral part of cancer therapy. With the emergence of oncolytic vaccinia virus immunotherapy, it is important to study the combination of radiation and vaccinia virus in cancer therapy. In this study, we investigated the anti-tumor effect of and immune mechanisms underlying the combination of high-dose hypofractionated stereotactic body radiotherapy (SBRT) and oncolytic vaccinia virus in preclinical murine models. The combination enhanced the in vivo anti-tumor effect and increased the numbers of splenic CD4+Ki-67+ helper T lymphocytes and CD8+Ki-67+ cytotoxic T lymphocytes. Combinational therapy also increased tumor-infiltrating CD3+CD4+ helper T lymphocytes and CD3+CD8+ cytotoxic T lymphocytes, but decreased tumor-infiltrating regulatory T cells. In addition, SBRT combined with oncolytic vaccinia virus enhanced in vitro cell death, partly through necroptosis, and subsequent release of damage-associated molecular patterns (DAMPs), and shifted the macrophage M1/M2 ratio. We concluded that SBRT combined with oncolytic vaccinia virus can trigger tumor cell necroptosis and modify macrophages through the release of DAMPs, and then generate potent anti-tumor immunity and effects. Thus, combined therapy is potentially an important strategy for clinical cancer therapy.

Immunogenic Cell Death Induction by Ionizing Radiation.

Immunogenic cell death (ICD) is a form of regulated cell death (RCD) induced by various stresses and produces antitumor immunity via damage-associated molecular patterns (DAMPs) release or exposure, mainly including high mobility group box 1 (HMGB1), calreticulin (CRT), adenosine triphosphate (ATP), and heat shock proteins (HSPs). Emerging evidence has suggested that ionizing radiation (IR) can induce ICD, and the dose, type, and fractionation of irradiation influence the induction of ICD. At present, IR-induced ICD is mainly verified in vitro in mice and there is few clinical evidence about it. To boost the induction of ICD by IR, some strategies have shown synergy with IR to enhance antitumor immune response, such as hyperthermia, nanoparticles, and chemotherapy. In this review, we focus on the molecular mechanisms of ICD, ICD-promoting factors associated with irradiation, the clinical evidence of ICD, and immunogenic forms of cell death. Finally, we summarize various methods of improving ICD induced by IR.

Basal epithelial stem cells cross an alarmin checkpoint for postviral lung disease.

Epithelial cells are charged with protection at barrier sites, but whether this normally beneficial response might sometimes become dysfunctional still needs definition. Here, we recognized a pattern of imbalance marked by basal epithelial cell growth and differentiation that replaced normal airspaces in a mouse model of progressive postviral lung disease due to the Sendai virus. Single-cell and lineage-tracing technologies identified a distinct subset of basal epithelial stem cells (basal ESCs) that extended into gas-exchange tissue to form long-term bronchiolar-alveolar remodeling regions. Moreover, this cell subset was selectively expanded by crossing a cell-growth and survival checkpoint linked to the nuclear-localized alarmin IL-33 that was independent of IL-33 receptor signaling and instead connected to autocrine chromatin accessibility. This mechanism creates an activated stem-progenitor cell lineage with potential for physiological or pathological function. Thus, conditional loss of Il33 gene function in basal epithelial cells disrupted the homeostasis of the epithelial barrier at skin and gut sites but also markedly attenuated postviral disease in the lung based on the downregulation of remodeling and inflammation. Thus, we define a basal ESC strategy to deploy innate immune machinery that appears to overshoot the primordial goal of self-defense. Our findings reveal new targets to stratify and correct chronic and often deadly postviral disease.

Ferroptosis, necroptosis, and pyroptosis in anticancer immunity.

In recent years, cancer immunotherapy based on immune checkpoint inhibitors (ICIs) has achieved considerable success in the clinic. However, ICIs are significantly limited by the fact that only one third of patients with most types of cancer respond to these agents. The induction of cell death mechanisms other than apoptosis has gradually emerged as a new cancer treatment strategy because most tumors harbor innate resistance to apoptosis. However, to date, the possibility of combining these two modalities has not been discussed systematically. Recently, a few studies revealed crosstalk between distinct cell death mechanisms and antitumor immunity. The induction of pyroptosis, ferroptosis, and necroptosis combined with ICIs showed synergistically enhanced antitumor activity, even in ICI-resistant tumors. Immunotherapy-activated CD8+ T cells are traditionally believed to induce tumor cell death via the following two main pathways: (i) perforin-granzyme and (ii) Fas-FasL. However, recent studies identified a new mechanism by which CD8+ T cells suppress tumor growth by inducing ferroptosis and pyroptosis, which provoked a review of the relationship between tumor cell death mechanisms and immune system activation. Hence, in this review, we summarize knowledge of the reciprocal interaction between antitumor immunity and distinct cell death mechanisms, particularly necroptosis, ferroptosis, and pyroptosis, which are the three potentially novel mechanisms of immunogenic cell death. Because most evidence is derived from studies using animal and cell models, we also reviewed related bioinformatics data available for human tissues in public databases, which partially confirmed the presence of interactions between tumor cell death and the activation of antitumor immunity.

Enhanced Wound Healing- and Inflammasome-Associated Gene Expression in TNFAIP3-Interacting Protein 1- (TNIP1-) Deficient HaCaT Keratinocytes Parallels Reduced Reepithelialization.

TNIP1 protein is a widely expressed, cytoplasmic inhibitor of inflammatory signaling initiated by membrane receptors such as TLRs which recognize pathogen-associated and damage-associated molecular patterns (PAMPs and DAMPs). Keratinocyte TNIP1 deficiency sensitizes cells to PAMPs and DAMPs promoting hyperresponsive expression and secretion of cytokine markers (e.g., IL-8 and IL-6) relevant to cases of chronic inflammation, like psoriasis, where TNIP1 deficiency has been reported. Here, we examined the impact of TNIP1 deficiency on gene expression and cellular responses (migration and viability) relevant to acute inflammation as typically occurs in wound healing. Using siRNA-mediated TNIP1 expression knockdown in cultured HaCaT keratinocytes, we investigated TNIP1 deficiency effects on signaling downstream of TLR3 agonism with low-concentration poly (I:C), a representative PAMP/DAMP. The combination of TNIP1 knockdown and PAMP/DAMP signaling disrupted expression of specific keratinocyte differentiation markers (e.g., transglutaminase 1 and involucrin). These same conditions promoted synergistically increased expression of wound-associated markers (e.g., S100A8, TGFß, and CCN2) suggesting potential benefit of increased inflammatory response from reduced TNIP1 protein. Unexpectedly, poly (I:C) challenge of TNIP1-deficient cells restricted reepithelialization and reduced cell viability. In these cells, there was not only increased expression for genes associated with inflammasome assembly (e.g., ASC, procaspase 1) but also for A20, a TNIP1 partner protein that represses cell-death signaling. Despite this possibly compensatory increase in A20 mRNA, there was a decrease in phospho-A20 protein, the form necessary for quenching inflammation. Hyperresponsiveness to poly (I:C) in TNIP1-deficient keratinocytes was in part mediated through p38 and JNK pathways. Taken together, we conclude that TNIP1 deficiency promotes enhanced expression of factors associated with promoting wound healing. However, the coupled, increased potential priming of the inflammasome and reduced compensatory activity of A20 has a net negative effect on overall cell recovery potential manifested by poor reepithelialization and viability. These findings suggest a previously unrecognized role for TNIP1 protein in limiting inflammation during successful progression through early wound healing stages.

S100-Alarmins Are Essential Pilots of Postnatal Innate Immune Adaptation.

The restricted capacity of newborn infants to mount inflammatory responses toward microbial challenges has traditionally been linked to the high risk of septic diseases during the neonatal period. In recent years, substantial evidence has been provided that this characteristic of the neonatal immune system is actually a meaningful physiologic state that is based on specific transiently active cellular and molecular mechanisms and required for a favorable course of postnatal immune adaptation. The identification of physiologically high amounts of S100-alarmins in neonates has been one of the crucial pieces in the puzzle that contributed to the change of concept. In this context, innate immune immaturity could be redefined and assigned to the epigenetic silence of adult-like cell-autonomous regulation at the beginning of life. S100-alarmins represent an alternative age-specific mechanism of immune regulation that protects neonates from hyperinflammatory immune responses. Here, we summarize how infants are provided with S100-alarmins and why these allow an uneventful clash between the innate immune system and the extrauterine world. The mode of action of S100-alarmins is highlighted including their tuning functions at multiple levels for establishing a state of homeostasis with the environment in the newborn individual.

Location is the key to function: HMGB1 in sepsis and trauma-induced inflammation.

High mobility group box 1 (HMGB1) is a multifunctional nuclear protein, probably known best as a prototypical alarmin or damage-associated molecular pattern (DAMP) molecule when released from cells. However, HMGB1 has multiple functions that depend on its location in the nucleus, in the cytosol, or extracellularly after either active release from cells, or passive release upon lytic cell death. Movement of HMGB1 between cellular compartments is a dynamic process induced by a variety of cell stresses and disease processes, including sepsis, trauma, and hemorrhagic shock. Location of HMGB1 is intricately linked with its function and is regulated by a series of posttranslational modifications. HMGB1 function is also regulated by the redox status of critical cysteine residues within the protein, and is cell-type dependent. This review highlights some of the mechanisms that contribute to location and functions of HMGB1, and focuses on some recent insights on important intracellular effects of HMGB1 during sepsis and trauma.

Nanoparticles and danger signals: Oral delivery vehicles as potential disruptors of intestinal barrier homeostasis.

Gut immune system homeostasis involves diverse structural interactions among resident microbiota, the protective mucus layer, and a variety of cells (intestinal epithelial, lymphoid, and myeloid). Due to the substantial surface area in direct contact with an "external" environment and the diversity of xenobiotic, abiotic, and self-interactions coordinating to maintain gut homeostasis, there is enhanced potential for the generation of endogenous danger signals when this balance is lost. Here, we focus on the potential generation and reception of damage in the gut resulting from exposure to nanoparticles (NPs), common food and drug additives. Specifically, we describe recent evidence in the literature showing that certain NPs are potential generators of damage-associated molecular patterns, as well as potential immune-stimulating molecular patterns themselves.

High mobility group box-1 (HMGB1) is increased in injured mouse spinal cord and can elicit neurotoxic inflammation.

Inflammation is a ubiquitous but poorly understood consequence of spinal cord injury (SCI). The mechanisms controlling this response are unclear but culminate in the sequential activation of resident and recruited immune cells. Collectively, these cells can exert divergent effects on cell survival and tissue repair. HMGB1 is a ubiquitously expressed DNA binding protein and also a potent inflammatory stimulus. Necrotic cells release HGMB1, but HMGB1 also is actively secreted by inflammatory macrophages. A goal of this study was to quantify spatio-temporal patterns of cellular HMGB1 expression in a controlled mouse model of experimental SCI then determine the effects of HMGB1 on post-SCI neuroinflammation and recovery of function. We documented SCI-induced changes in nuclear and cytoplasmic distribution of HMGB1 in various cell types after SCI. The data reveal a time-dependent increase in HMGB1 mRNA and protein with protein reaching maximal levels 24-72 h post-injury then declining toward baseline 14-28 days post-SCI. Although most cells expressed nuclear HMGB1, reduced nuclear labeling with increased cytoplasmic expression was found in a subset of CNS macrophages suggesting that those cells begin to secrete HMGB1 at the injury site. In vitro data indicate that extracelluar HMGB1 helps promote the development of macrophages with a neurotoxic phenotype. The ability of HMGB1 to elicit neurotoxic macrophage functions was confirmed in vivo; 72 h after injecting 500 ng of recombinant HMGB1 into intact spinal cord ventral horn, inflammatory CNS macrophages co-localized with focal areas of neuronal killing. However, attempts to confer neuroprotection after SCI by blocking HMGB1 with a neutralizing antibody were unsuccessful. Collectively, these data implicate HMGB1 as a novel regulator of post-SCI inflammation and suggest that inhibition of HMGB1 could be a novel therapeutic target after SCI. Future studies will need to identify better methods to deliver optimal concentrations of HMGB1 antagonists to the injured spinal cord.

Polymorphisms in inflammasome genes and risk of asthma in Brazilian children.

Considering its role in inflammation and recently described "alternative" roles in epithelial homeostasis and Th1/Th2 balance, we hypothesize that inflammasome genetics could contribute to the development of asthma. Selected functional polymorphisms in inflammasome genes are evaluated in a cohort of asthmatic children and their families. Gain-of-function NLRP1 variants rs11651270, rs12150220 and rs2670660 resulted significantly associated to asthma in trios (TDT) analysis; and rs11651270 and rs2670660 also with asthma severity and total IgE level in asthmatic children. NLRP1 activators in humans are still unknown, however we hypothesized that individuals with gain-of-function SNPs in NLRP1 could be more prone in activating inflammasome in the presence of asthma-related cell stressors (i.e. ER stress or ROS), and this activation contribute to exacerbate inflammatory response and asthma development. Gain-of-function IL1A rs17561 resulted significantly associated with a reduced pulmonary capacity in asthmatic children. IL18 rs5744256 which lead to lower serum level of IL-18 appeared to be associated to a worse response to bronchodilators. Concluding, this work provides evidences about the contribution of inflammasome genetics in the development of paediatric asthma, both considering its inflammatory role in alveolar macrophages (i.e.: NLRP1) or its homeostatic role in lung epithelial cells (i.e.: IL1A, IL18).

Alarmins in chronic noncommunicable diseases: Atherosclerosis, diabetes and cancer.

There is a wide range of pathological conditions proved to be associated with inflammation. The inflammatory process offers protection against harmful stimuli such as induced cell injury and tissues damage by means of specialized mediators and cells. Alarmins, also known as endogenous danger signals or damage-associated molecular patterns (DAMPs) molecules, are critical players of immune response to tissue suffering. In many inflammatory and autoimmune conditions, alarmins are released into the extracellular milieu and bind to specific receptors to stimulate and promote activation of innate immune cells, cell differentiation, cell death or secretion of inflammatory mediators. This paper, based on biochemical and mass spectrometry proteomic data, highlights the role of heat shock proteins (HSPs), high-mobility group box 1 (HMGB1) protein and S100 proteins as main alarmins involved in the maintaining and amplifying inflammation in atherosclerosis, diabetes and cancer. BIOLOGICAL SIGNIFICANCE: This paper, based on biochemical and mass spectrometry proteomic data, highlights the role of the heat shock proteins (HSPs), high-mobility group box 1 (HMGB1) protein and S100 proteins as main alarmins involved in maintaining and amplifying atherosclerosis, diabetes and cancer inflammation.

Immunogenic Apoptotic Cell Death and Anticancer Immunity.

For many years it has been thought that apoptotic cells rapidly cleared by phagocytic cells do not trigger an immune response but rather have anti-inflammatory properties. However, accumulating experimental data indicate that certain anticancer therapies can induce an immunogenic form of apoptosis associated with the emission of damage-associated molecular patterns (DAMPs), which function as adjuvants to activate host antitumor immune responses. In this review, we will first discuss recent advances and the significance of danger signaling pathways involved in the emission of DAMPs, including calreticulin, ATP, and HMGB1. We will also emphasize that switching on a particular signaling pathway depends on the immunogenic cell death stimulus. Further, we address the role of ER stress in danger signaling and the classification of immunogenic cell death inducers in relation to how ER stress is triggered. In the final part, we discuss the role of radiotherapy-induced immunogenic apoptosis and the relationship of its immunogenicity to the fraction dose and concomitant chemotherapy.

Cancer Cell Death-Inducing Radiotherapy: Impact on Local Tumour Control, Tumour Cell Proliferation and Induction of Systemic Anti-tumour Immunity.

Radiotherapy (RT) predominantly is aimed to induce DNA damage in tumour cells that results in reduction of their clonogenicity and finally in tumour cell death. Adaptation of RT with higher single doses has become necessary and led to a more detailed view on what kind of tumour cell death is induced and which immunological consequences result from it. RT is capable of rendering tumour cells immunogenic by modifying the tumour cell phenotype and the microenvironment. Danger signals are released as well as the senescence-associated secretory phenotype. This results in maturation of dendritic cells and priming of cytotoxic T cells as well as in activation of natural killer cells. However, RT on the other hand can also result in immune suppressive events including apoptosis induction and foster tumour cell proliferation. That's why RT is nowadays increasingly combined with selected immunotherapies.

Killing Is Not Enough: How Apoptosis Hijacks Tumor-Associated Macrophages to Promote Cancer Progression.

Macrophages are a group of heterogeneous cells of the innate immune system that are crucial to the initiation, progression, and resolution of inflammation. Moreover, they control tissue homeostasis in healthy tissue and command a broad sensory arsenal to detect disturbances in tissue integrity. Macrophages possess a remarkable functional plasticity to respond to irregularities and to initiate programs that allow overcoming them in order to return back to normal. Thus, macrophages kill malignant or transformed cells, rearrange extracellular matrix, take up and recycle cellular as well as molecular debris, initiate cellular growth cascades, and favor directed migration of cells. As an example, apoptotic death of bystander cells is sensed by macrophages, initiating functional responses that support all hallmarks of cancer. In this chapter, we describe how tumor cell apoptosis hijacks tumor-associated macrophages to promote tumor growth. We propose that tumor therapy should not only kill malignant cells but also target the interaction of the host with apoptotic cancer cells, as this might be efficient to limit the protumor action of apoptotic cells and boost the antitumor potential of macrophages. Leaving the apoptotic cell/macrophage interaction untouched might also limit the benefit of conventional tumor cell apoptosis-focused therapy since surviving tumor cells might receive overwhelming support by the wound healing response that apoptotic tumor cells will trigger in local macrophages, thereby enhancing tumor recurrence.

Death in the fast lane: what's next for necroptosis?

Necroptosis is a form of programmed cell death that is both mechanistically and morphologically distinct from apoptosis, the canonical mechanism of cell suicide. Although early descriptions of necroptosis date back decades, the last 5 years have seen a proliferation of studies of this process. This surge in interest has included the recent publication of several excellent, in-depth reviews of the literature [Chan FK-M et al. (2014) Annu Rev Immunol 33, 141210135520002; Weinlich R & Green DR (2014) Mol Cell 56, 469-480; Silke J et al. (2015) Nat Immunol 16, 689-697; Linkermann A & Green DR (2014) N Engl J Med 370, 455-465]. Rather than contribute another summary to this well-summarized field, in this Minireview I will briefly discuss key recent findings, then touch on some of the major outstanding questions - the known unknowns - that remain.

Immunopathogenesis of IBD: current state of the art.

IBD is a chronic inflammatory condition of the gastrointestinal tract encompassing two main clinical entities: Crohn's disease and ulcerative colitis. Although Crohn's disease and ulcerative colitis have historically been studied together because they share common features (such as symptoms, structural damage and therapy), it is now clear that they represent two distinct pathophysiological entities. Both Crohn's disease and ulcerative colitis are associated with multiple pathogenic factors including environmental changes, an array of susceptibility gene variants, a qualitatively and quantitatively abnormal gut microbiota and a broadly dysregulated immune response. In spite of this realization and the identification of seemingly pertinent environmental, genetic, microbial and immune factors, a full understanding of IBD pathogenesis is still out of reach and, consequently, treatment is far from optimal. An important reason for this unsatisfactory situation is the currently limited comprehension of what are the truly relevant components of IBD immunopathogenesis. This article will comprehensively review current knowledge of the classic immune components and will expand the concept of IBD immunopathogenesis to include various cells, mediators and pathways that have not been traditionally associated with disease mechanisms, but that profoundly affect the overall intestinal inflammatory process.

Stress-associated erythropoiesis initiation is regulated by type 1 conventional dendritic cells.

Erythropoiesis is an important response to certain types of stress, including hypoxia, hemorrhage, bone marrow suppression, and anemia, that result in inadequate tissue oxygenation. This stress-induced erythropoiesis is distinct from basal red blood cell generation; however, neither the cellular nor the molecular factors that regulate this process are fully understood. Here, we report that type 1 conventional dendritic cells (cDC1s), which are defined by expression of CD8α in the mouse and XCR1 and CLEC9 in humans, are critical for induction of erythropoiesis in response to stress. Specifically, using murine models, we determined that engagement of a stress sensor, CD24, on cDC1s upregulates expression of the Kit ligand stem cell factor on these cells. The increased expression of stem cell factor resulted in Kit-mediated proliferative expansion of early erythroid progenitors and, ultimately, transient reticulocytosis in the circulation. Moreover, this stress response was triggered in part by alarmin recognition and was blunted in CD24 sensor- and CD8α+ DC-deficient animals. The contribution of the cDC1 subset to the initiation of stress erythropoiesis was distinct from the well-recognized role of macrophages in supporting late erythroid maturation. Together, these findings offer insight into the mechanism of stress erythropoiesis and into disorders of erythrocyte generation associated with stress.