Autophagy Impairment through Lysosome Dysfunction by Brucine Induces Immunogenic Cell Death (ICD).

Autophagy is an important tightly controlled cellular process that regulates cellular homeostasis and is involved in deciding cell fate such as cell survival and death. The role of autophagy in many intracellular signaling pathways explains its interaction with other different types of cell death, including apoptosis and immunogenic cell death (ICD). The reports showed the complex and intriguing relationship existing between autophagy and immune system signaling pathways. However, the role of autophagy in ICD remains to be clearly elucidated. In this study, we demonstrated that Brucine, a clinically-used small molecule in traditional Chinese medicine, elicited autophagy inhibition. Brucine also triggered cell stress and induced features of ICD, including calreticulin (CRT) exposure and high-mobility group box 1 (HMGB1) release in MDA-MB-231 and CT26 cancer cells. Brucine impaired autolysosomal degradation and exerted a feedback regulation of ERK1/2-mTOR-p70S6K signaling cascade. Brucine-elicited ICD was confirmed by the rejection of CT26 tumor cells, implanted in the mice after vaccination with Brucine-treated CT26 cells. The impaired autophagy contributed to Brucine-induced ICD, as knock-down of Atg5 significantly reduced Brucine-elicited CRT exposure and HMGB1 release. Our results revealed Brucine as a novel autophagy regulator, ICD inducer and hitherto undocumented role of autophagy in ICD. Thus, these results imply the importance of Brucine in cancer immunotherapy. Therefore, Brucine may be used as an ICD inducer and improve its application in cancer treatment with minimized toxicity.

NIR-Triggered Sequentially Responsive Nanocarriers Amplified Cascade Synergistic Effect of Chemo-Photodynamic Therapy with Inspired Antitumor Immunity.

A desirable cancer therapeutic strategy is supposed to have effective ability to not only exert maximum anticancer ability but also inspire antitumor immunity for preventing tumor relapse and metastasis. During this research, multifunctional upconversion nanoparticles (UCNPs) coated by ROS-responsive micelles are prepared for tumor targeting and near-infrared (NIR)-triggered photodynamic therapy (PDT)-combined synergistic effect of chemotherapy. Moreover, both PDT and chemotherapy agents could activate antitumor immunity via inducing immunogenic cell death with CD8+ and CD4+ T cells infiltrating in tumors. Through the experiments, intravenous administration of multifunctional nanocarriers with noninvasive NIR irradiation destroys the orthotopic tumors and efficiently suppresses lung metastasis in a metastatic triple-negative breast cancer model by cascade-amplifying chemo-PDT and systemic antitumor immunity. In conclusion, this study provides prospective chemo-PDT with inspired antitumor immunity for metastatic cancer treatment.

Selective Photokilling of Colorectal Tumors by Near-Infrared Photoimmunotherapy with a GPA33-Targeted Single-Chain Antibody Variable Fragment Conjugate.

Antibody-based near-infrared photoimmunotherapy (NIR-PIT) is an attractive strategy for cancer treatment. Tumor cells can be selectively and efficiently killed by the targeted delivery of an antibody-photoabsorber complex followed by exposure to NIR light. Glycoprotein A33 antigen (GPA33) is highly expressed in most human colorectal cancers (CRCs) and is an ideal diagnostic and therapeutic target. We previously produced a single-chain fragment of a variable antibody against GPA33 (A33scFv antibody). Here, we investigate the efficacy of NIR-PIT by combining A33scFv with the NIR photoabsorber IR700 (A33scFv-IR700). In vitro, recombinant A33scFv displayed specific binding and delivery of an NIR dye to GPA33-positive tumor cells. Furthermore, A33scFv-IR700-mediated NIR-PIT was successful in rapidly and specifically killing GPA33-positive colorectal tumor cells. NIR-PIT treatment induced the release of lactate dehydrogenase from tumor cells, followed by cell necrosis, rather than apoptosis, through the promotion of reactive oxygen species accumulation in tumor cells. In mice bearing LS174T tumor grafts, A33scFv selectively accumulated in GPA33-positive tumors. Following only a single injection of the conjugate and subsequent illumination, A33scFv-IR700-mediated NIR-PIT induced a significant increase in therapeutic response in LS174T-tumor mice compared with that in the non-NIR-PIT groups (p < 0.001). Because the GPA33 antigen is specifically expressed in CRC tumors, A33scFv-IR700 might be a promising antibody fragment-photoabsorber conjugate for NIR-PIT of CRC.

Radiation-Induced Cell Death: Signaling and Pharmacological Modulation.

Currently, more than half of newly diagnosed cancer patients receive radiation treatment. However, the radioresistance of tumor cells as well as the early and late side effects limit the beneficial outcome of radiotherapy. Accordingly, the innovative approaches to maximize tumor killing and/or minimize radiation toxicity remain a major focus of interest. In the past decade, several pieces of evidence have shown the importance of different modes of regulated cell death (RCD) in the radioresponse of malignant and normal tissues. Furthermore, the biological modulation of radiation-induced RCDs has come to attention as a novel therapeutic means. Here, we review the major signaling pathways that orchestrate all types of RCD initiated by exposure to ionizing radiation. The latest advances in the development of small-molecule RCD modulators (both natural and synthetic) that are intended for widening the therapeutic window of radiotherapy are also discussed.

Nano-Pulse Stimulation is a physical modality that can trigger immunogenic tumor cell death.

BACKGROUND: We have been developing a non-thermal, drug-free tumor therapy called Nano-Pulse Stimulation (NPS) that delivers ultrashort electric pulses to tumor cells which eliminates the tumor and inhibits secondary tumor growth. We hypothesized that the mechanism for inhibiting secondary tumor growth involves stimulating an adaptive immune response via an immunogenic form of apoptosis, commonly known as immunogenic cell death (ICD). ICD is characterized by the emission of danger-associated molecular patterns (DAMPs) that serve to recruit immune cells to the site of the tumor. Here we present evidence that NPS stimulates both caspase 3/7 activation indicative of apoptosis, as well as the emission of three critical DAMPs: ecto-calreticulin (CRT), ATP and HMGB1. METHODS: After treating three separate cancer cell lines (MCA205, McA-RH7777, Jurkat E6-1) with NPS, cells were incubated at 37 °C. Cell-culture supernatants were collected after three-hours to measure for activated caspases 3/7 and after 24 h to measure CRT, ATP and HMGB1 levels. We measured the changes in caspase-3 activation with Caspase-Glo® by Promega, ecto-CRT with anti-CRT antibody and flow cytometry, ATP by luciferase light generation and HMGB1 by ELISA. RESULTS: The initiation of apoptosis in cultured cells is greatest at 15 kV/cm and requires 50 A/cm2. Reducing this current inhibits cell death. Activated caspase-3 increases 8-fold in Jurkat E6-1 cells and 40% in rat hepatocellular carcinoma and mouse fibrosarcoma cells by 3 h post treatment. This increase is non-linear and peaks at 15-20 J/mL for all field strengths. 10 and 30 kV/cm fields exhibited the lowest response and the 12 and 15 kV/cm fields stimulated the largest amount of caspase activation. We measured the three DAMPs 24 h after treatment. The expression of cell surface CRT increased in an energy-dependent manner in the NPS treated samples. Expression levels reached or exceeded the expression levels in the majority of the anthracycline-treated samples at energies between 25 and 50 J/mL. Similar to the caspase response at 3 h, secreted ATP peaked at 15 J/mL and then rapidly declined at 25 J/mL. HMGB1 release increased as treatment energy increased and reached levels comparable to the anthracycline-treated groups between 10 and 25 J/mL. CONCLUSION: Nano-Pulse Stimulation treatment at specific energies was able to trigger the emission of three key DAMPs at levels comparable to Doxorubicin and Mitoxantrone, two known inducers of immunogenic cell death (ICD). Therefore NPS is a physical modality that can trigger immunogenic cell death in tumor cells.

Colon cancer cell treatment with rose bengal generates a protective immune response via immunogenic cell death.

Immunotherapeutic approaches to manage patients with advanced gastrointestinal malignancies are desired; however, mechanisms to incite tumor-specific immune responses remain to be elucidated. Rose bengal (RB) is toxic at low concentrations to malignant cells and may induce damage-associated molecular patterns; therefore, we investigated its potential as an immunomodulator in colon cancer. Murine and human colon cancer lines were treated with RB (10% in saline/PV-10) for cell cycle, cell death, and apoptosis assays. Damage-associated molecular patterns were assessed with western blot, ELISA, and flow cytometry. In an immunocompetent murine model of colon cancer, we demonstrate that tumors regress upon RB treatment, and that RB induces cell death in colon cancer cells through G2/M growth arrest and predominantly necrosis. RB-treated colon cancer cells expressed distinct hallmarks of immunogenic cell death (ICD), including enhanced expression of calreticulin and heat-shock protein 90 on the cell surface, a decrease in intracellular ATP, and the release of HMGB1. To confirm the ICD phenotype, we vaccinated immunocompetent animals with syngeneic colon cancer cells treated with RB. RB-treated tumors served as a vaccine against subsequent challenge with the same CT26 colon cancer tumor cells, and vaccination with in vitro RB-treated cells resulted in slower tumor growth following inoculation with colon cancer cells, but not with syngeneic non-CT26 cancer cells, suggesting a specific antitumor immune response. In conclusion, RB serves as an inducer of ICD that contributes to enhanced specific antitumor immunity in colorectal cancer.

Inflammasomes Coordinate Pyroptosis and Natural Killer Cell Cytotoxicity to Clear Infection by a Ubiquitous Environmental Bacterium.

Defective neutrophils in patients with chronic granulomatous disease (CGD) cause susceptibility to extracellular and intracellular infections. Microbes must first be ejected from intracellular niches to expose them to neutrophil attack, so we hypothesized that inflammasomes detect certain CGD pathogens upstream of neutrophil killing. Here, we identified one such ubiquitous environmental bacterium, Chromobacterium violaceum, whose extreme virulence was fully counteracted by the NLRC4 inflammasome. Caspase-1 protected via two parallel pathways that eliminated intracellular replication niches. Pyroptosis was the primary bacterial clearance mechanism in the spleen, but both pyroptosis and interleukin-18 (IL-18)-driven natural killer (NK) cell responses were required for liver defense. NK cells cleared hepatocyte replication niches via perforin-dependent cytotoxicity, whereas interferon-γ was not required. These insights suggested a therapeutic approach: exogenous IL-18 restored perforin-dependent cytotoxicity during infection by the inflammasome-evasive bacterium Listeria monocytogenes. Therefore, inflammasomes can trigger complementary programmed cell death mechanisms, directing sterilizing immunity against intracellular bacterial pathogens.

T cell lipid peroxidation induces ferroptosis and prevents immunity to infection.

The selenoenzyme glutathione peroxidase 4 (Gpx4) is a major scavenger of phospholipid hydroperoxides. Although Gpx4 represents a key component of the reactive oxygen species-scavenging network, its relevance in the immune system is yet to be defined. Here, we investigated the importance of Gpx4 for physiological T cell responses by using T cell-specific Gpx4-deficient mice. Our results revealed that, despite normal thymic T cell development, CD8(+) T cells from T(ΔGpx4/ΔGpx4) mice had an intrinsic defect in maintaining homeostatic balance in the periphery. Moreover, both antigen-specific CD8(+) and CD4(+) T cells lacking Gpx4 failed to expand and to protect from acute lymphocytic choriomeningitis virus and Leishmania major parasite infections, which were rescued with diet supplementation of high dosage of vitamin E. Notably, depletion of the Gpx4 gene in the memory phase of viral infection did not affect T cell recall responses upon secondary infection. Ex vivo, Gpx4-deficient T cells rapidly accumulated membrane lipid peroxides and concomitantly underwent cell death driven by ferroptosis but not necroptosis. These studies unveil an essential role of Gpx4 for T cell immunity.

Differential adjuvant activities of TLR7 and TLR9 agonists inversely correlate with nitric oxide and PGE2 production.

Activation of different pattern recognition receptors causes distinct profiles of innate immune responses, which in turn dictate the adaptive immune response. We found that mice had higher CD4+ T cell expansion to an immunogen, ovalbumin, when coadministered with CpG than with CL097 in vivo. To account for this differential adjuvanticity, we assessed the activities of CpG and CL097 on antigen-specific CD4+ T cell expansion in vitro using an OT-II CD4+ T cell/bone marrow-derived dendritic cell (DC) co-culture system. Unexpectedly, ovalbumin-stimulated expansion of OT-II CD4+ T cells in vitro was potently suppressed by both TLR agonists, with CL097 being stronger than CpG. The suppression was synergistically reversed by co-inhibition of cyclooxygenases 1 and 2, and inducible nitric oxide (NO) synthase. In addition, stimulation of OT-II CD4+ T cell/DC cultures with CL097 induced higher levels of CD4+ T cell death than stimulation with CpG, and this CD4+ T cell turnover was reversed by NO and PGE2 inhibition. Consistently, the co-cultures stimulated with CL097 produced higher levels of prostaglandin E2 (PGE2) and NO than stimulation with CpG. CL097 induced higher PGE2 production in DC cultures and higher IFN-γ in the OT-II CD4+ T cell/DC cultures, accounting for the high levels of PGE2 and NO. This study demonstrates that the adjuvant activities of immunostimulatory molecules may be determined by differential induction of negative regulators, including NO and PGE2 suppressing clonal expansion and promoting cell death of CD4+ T cells.

Targeting the heat shock response in combination with radiotherapy: Sensitizing cancer cells to irradiation-induced cell death and heating up their immunogenicity.

Radiotherapy represents an essential treatment option for the majority of cancer patients in different stages of their disease. Physical achievements of the recent years led to the implementation of high precision treatment planning procedures, and image-guided dose delivery is current state of the art. Yet, radiotherapy still faces several limitations with cancer intrinsic radioresistance being a key driver of therapeutic failure. Accordingly, the mechanisms orchestrating radioresistance and their therapeutic targeting by combined modality approaches are in the center of attention of numerous radiation oncologists. In the present review, we summarize and discuss therapeutic approaches that exploit the heat shock response, either by hyperthermia or by pharmacological heat shock protein inhibition, in combination with radiotherapy. These strategies appear particularly promising, since they sensitize cancer cells to irradiation-induced cell death and at the same time have proven the potential to promote systemic anti-tumor immune mechanisms, which may target not only locally surviving tumor cells, but also distant out-of-field metastases.

Triggering cell death by nanographene oxide mediated hyperthermia.

Graphene oxide (GO) has been proposed as an hyperthermia agent for anticancer therapies due to its near-infrared (NIR) optical absorption ability which, with its small two-dimensional size, could have a unique performance when compared to that of any other nanoparticle. Nevertheless, attention should be given to the hyperthermia route and the kind of GO-cell interactions induced in the process. The hyperthermia laser irradiation parameters, such as exposure time and laser power, were investigated to control the temperature rise and consequent damage in the GOs containing cell culture medium. The type of cell damage produced was evaluated as a function of these parameters. The results showed that cell culture temperature (after irradiating cells with internalized GO) increases preferentially with laser power rather than with exposure time. Moreover, when laser power is increased, necrosis is the preferential cell death leading to an increase of cytokine release to the medium.

Insulin treatment modulates the host immune system to enhance Pseudomonas aeruginosa wound biofilms.

Diabetes affects 25.8 million people in the United States, or 8.3% of the population, and these numbers are even higher in developing countries. Diabetic patients are more susceptible to the development of chronic wounds with debilitating bacterial infections than nondiabetics. Previously, we compared the ability of the opportunistic pathogen Pseudomonas aeruginosa to cause biofilm-associated infections in chronic wounds of diabetic and nondiabetic mice (C. Watters, K. DeLeon, U. Trivedi, J. A. Griswold, M. Lyte, K. J. Hampel, M. J. Wargo, and K. P. Rumbaugh, Med. Microbiol. Immunol. 202:131-141, 2013). Unexpectedly, we observed that insulin-treated diabetic mice had significantly more biofilm in their wounds, which correlated with higher antibiotic tolerance. Here, we investigated whether insulin treatment modulates the diabetic immune system to favor P. aeruginosa biofilm formation. Utilizing a murine chronic wound model, we found that DNA protected P. aeruginosa in the wounds of insulin-treated diabetic mice from antibiotic treatment. We also observed increased numbers of neutrophils, reduced numbers of macrophages, and increased cell death in the wounds of diabetic mice on insulin therapy. Taken together, these data suggest that high levels of lysed neutrophils in the wounds of diabetic mice on insulin, combined with fewer macrophages to remove the cellular debris, contribute to increased DNA levels, which enhance P. aeruginosa biofilms.

Glatiramer acetate attenuates pro-inflammatory T cell responses but does not directly protect neurons from inflammatory cell death.

Glatiramer acetate (GA) is a synthetic, random, basic copolymer capable of modulating adaptive T cell responses. In animal models of various inflammatory and degenerative central nervous system disorders, GA-induced T cells cross the blood-brain barrier, secrete high levels of anti-inflammatory cytokines and neurotrophins, and thus both reduce neuronal damage and promote neurogenesis. Recently, it has been suggested that GA itself may permeate the (impaired) blood-brain-barrier and directly protect neurons under conditions of inflammation-mediated neurodegeneration. To test this hypothesis, we examined the direct effects of GA on neuronal functionality and T cell-mediated neuronal apoptosis in culture, acute brain slices, and focal experimental autoimmune encephalomyelitis. GA caused a depolarization of the resting membrane potential and led to an immediate impairment of action potential generation in neurons. Moreover, GA-incubated neurons underwent dose-dependent apoptosis. Apoptosis of ovalbumin peptide-loaded major histocompatibility complex class I-expressing neurons induced by ovalbumin-specific effector T cells could be reduced by pre-incubation of T cells, but not neurons with GA. Similar results could be found using acute brain slices. In focal experimental autoimmune encephalomyelitis, lesion size and neuronal apoptosis could be limited by pretreating rats with GA, whereas intracerebral GA application into the inflammatory lesion had no effect on neuronal survival. Our data suggest that GA attenuates adaptive pro-inflammatory T cell responses, but does not exert direct neuroprotective effects.

Complement-dependent tumor cell lysis triggered by combinations of epidermal growth factor receptor antibodies.

Therapeutic monoclonal antibodies against the epidermal growth factor receptor (EGFR) have advanced the treatment of colon and head and neck cancer, and show great promise for the development of treatments for other solid cancers. Antibodies against EGFR have been shown to act via inhibition of receptor signaling and induction of antibody-dependent cellular cytoxicity. However, complement-dependent cytotoxicity, which is considered one of the most powerful cell killing mechanisms of antibodies, seems inactive for such antibodies. Here, we show a remarkable synergy for EGFR antibodies. Combinations of antibodies against EGFR were identified, which resulted in potent complement activation via the classic pathway and effective lysis of tumor cells. Studies on a large panel of antibodies indicated that the observed synergy is a general mechanism, which can be activated by combining human IgG1 antibodies recognizing different, nonoverlapping epitopes. Our findings show an unexpected quality of therapeutic EGFR antibodies, which may be exploited to develop novel and more effective treatments for solid cancers.

The role of annexin A5 in the modulation of the immune response against dying and dead cells.

Annexins are characterized by the ability to bind phospholipids of membranes in the presence of Ca2+. Annexin A5 represents a typical member of this protein family and is a natural occurring highly specific ligand for phosphatidylserine (PS). The exposure of PS is one major "eat me" signal for phagocytes of apoptotic and necrotic cells. Apoptotic cells are normally cleared via an anti-inflammatory pathway. In contrast, the uptake and removal of necrotic cells normally involves inflammation and an immune response. Interestingly, the lack of endogenous annexin A5 also leads to a reduced inflammatory potential of necrotic cells. Annexin A5 may interfere in vivo with the immunosuppressive effects of apoptotic cells since it preferentially binds PS with high affinity and inhibits apoptotic cell uptake by macrophages. In this review we focus on how defects in the clearance process can lead to chronic autoimmunity. Furthermore, the role of annexin A5 as important adjuvant for apoptotic cell-based tumour vaccines is discussed. The mechanism of how the immunogenicity of apoptotic cells can be restored by blocking their PS-dependent clearance is outlined in detail. Taken together, annexin A5 is an important modulator of the immune response against PS-exposing particles like apoptotic cells, necrotic cells, and certain viruses.

Psychoneuroimmunology of stroke.

There is now considerable evidence from both experimental and clinical studies that immune and inflammatory processes can contribute to the onset of stroke and the neurologic and psychologic outcomes. Several specific therapeutic targets have been identified that may significantly improve the devastating impact of stroke.

Selective leukemic-cell killing by a novel functional class of thalidomide analogs.

Using a novel cell-based assay to profile transcriptional pathway targeting, we have identified a new functional class of thalidomide analogs with distinct and selective antileukemic activity. These agents activate nuclear factor of activated T cells (NFAT) transcriptional pathways while simultaneously repressing nuclear factor-kappaB (NF-kappaB) via a rapid intracellular amplification of reactive oxygen species (ROS). The elevated ROS is associated with increased intracellular free calcium, rapid dissipation of the mitochondrial membrane potential, disrupted mitochondrial structure, and caspase-independent cell death. This cytotoxicity is highly selective for transformed lymphoid cells, is reversed by free radical scavengers, synergizes with the antileukemic activity of other redox-directed compounds, and preferentially targets cells in the S phase of the cell cycle. Live-cell imaging reveals a rapid drug-induced burst of ROS originating in the endoplasmic reticulum and associated mitochondria just prior to spreading throughout the cell. As members of a novel functional class of "redoxreactive" thalidomides, these compounds provide a new tool through which selective cellular properties of redox status and intracellular bioactivation can be leveraged by rational combinatorial therapeutic strategies and appropriate drug design to exploit cell-specific vulnerabilities for maximum drug efficacy.

Transcriptional profiling of gamma delta T cells identifies a role for vitamin D in the immunoregulation of the V gamma 9V delta 2 response to phosphate-containing ligands.

Vitamin D is a steroid hormone that, in addition to its well-characterized role in calcium/phosphate metabolism, has been found to have regulatory properties for immune system function. The nuclear vitamin D receptor is widely expressed in tissues, but has also been shown to be regulated by hormones, growth factors, and cytokines. In this study we show that activation of human Vdelta2Vgamma9 T cells by nonpeptidic monoalkyl phosphates such as isopentenyl pyrophosphate leads to the up-regulation of the vitamin D receptor via a pathway that involves the classical isoforms of protein kinase C. We further show that this receptor is active by demonstrating that the ligand 1alpha,25-dihydroxyvitamin D3 (vitD3) significantly inhibits in a dose-dependent fashion phospholigand-induced gammadelta T cell expansion, IFN-gamma production, and CD25 expression. We also show that vitD3 negatively regulates signaling via Akt and ERK and, at high concentrations, potentiates Ag-induced cell death. As such, these data provide further support for the immunoregulatory properties of vitamin D, and suggest that the ability of vitD3 to negatively regulate the proinflammatory activity of gammadelta T cells may contribute to the protection this vitamin affords against inflammatory and autoimmune disorders dependent upon Th1-type responses.

Evidence of an anti-inflammatory role for Vasogen's immune modulation therapy.

We have reported that Vasogen's immune modulation therapy (IMT), a procedure involving intramuscular administration of autologous/syngeneic blood, which has been exposed ex vivo to increased temperature, UVC light and oxidation, prevents several LPS-induced inflammatory changes in the hippocampus. Here, we investigated neuroprotective effects of IMT in cortical tissue, and report that the treatment acts as an anti-inflammatory and antioxidative agent, reducing the concentration of TNFalpha and the accumulation of reactive oxygen species. The data couple these changes with an increase in the concentration of the anti-inflammatory cytokine IL-10, and a decrease in activation of the stress-activated protein kinase, c-jun N-terminal kinase. Consistent with these putative protective effects of IMT, we report that the LPS-induced increase in TUNEL staining, which is indicative of cell death, is prevented by IMT.