Release Of Danger-associated Molecular Patterns Research Articles

Abstract LB275: Reactivation of mitotic arrest upon targeting TACC3 restores immunogenic cell death and T-DM1 response in HER2-positive breast cancer

Abstract Immunogenic cell death (ICD) is a specific form of cell death that is characterized by the release of danger-associated molecular patterns (DAMPs), such as cell surface exposure of calreticulin, ATP and HMGB1 release, and can be induced by several different anti-cancer therapies. This, in turn, evokes a strong anti-tumor immune response, leading to reduced tumor growth. Despite being the first and one of the most successful antibody-drug conjugates (ADCs) approved for refractory HER2-positive breast cancer, little is known if response and resistance to trastuzumab emtansine (T-DM1) involves ICD modulation that can be leveraged to enhance T-DM1 response. Here we report that T-DM1 induces spindle assembly checkpoint (SAC)-dependent ICD in sensitive cells, mediated by its payload by inducing eIF2α phosphorylation, surface exposure of calreticulin, ATP and HMGB1 release, and secretion of ICD-related cytokines, all of which are lost in resistance. Accordingly, ICD-related gene signatures correlate with clinical response to T-DM1-containing therapy in pre-treatment samples, and increased infiltration of anti-tumor CD8+ T cells is correlated with better T-DM1 response in post-treatment samples. We found that transforming acidic coiled-coil containing 3 (TACC3) is overexpressed in T-DM1 resistant cells, and that T-DM1 responsive patients have reduced TACC3 protein while the non-responders exhibited increased TACC3 expression during T-DM1 treatment. Notably, genetic or pharmacological inhibition of TACC3 revives T-DM1-induced SAC activation and induction of ICD markers in vitro. TACC3 inhibition elicits ICD in vivo as shown by vaccination assay. It also potentiates T-DM1 in the human HER2-overexpressing MMTV-huHER2#5 (Fo5) transgenic model by activating ICD markers, release of cytokines and inducing dendritic cell (DC) maturation and enhancing infiltration of cytotoxic T cells. Together, our results show that ICD is a key mechanism of action of T-DM1 which is lost in resistance, and that targeting TACC3 restores T-DM1-mediated ICD and overcomes resistance. Citation Format: Ozge Saatci, Mustafa Emre Gedik, Nathaniel Oberholtzer, Meral Uner, Ozge Akbulut, Metin Cetin, Mertkaya Aras, Kubra Ibis, Burcu Caliskan, Erden Banoglu, Stefan Wiemann, Aysegul Uner, Sercan Aksoy, Shikhar Mehrotra, Ozgur Sahin. Reactivation of mitotic arrest upon targeting TACC3 restores immunogenic cell death and T-DM1 response in HER2-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB275.

Abstract 3740: Tusamitamab ravtansine induces immunogenic cell death and synergizes with anti-PD-1 or anti-PD-L1 antibody combination in solid tumor

Abstract Tusamitamab ravtansine (SAR408701) is an antibody-drug conjugate (ADC) targeted to carcino-embryonic antigen-related cell adhesion molecule 5 (CEACAM5) and delivering DM4, a maytansinoid cytotoxic. CEACAM5 is highly expressed in several tumor types, including non-squamous non-small cell lung cancer (NSQ NSCLC). It is currently being evaluated in patients with advanced CEACAM5-positive NSQ NSCLC, in a phase II study in combination with pembrolizumab, and a phase III trial as single agent in comparison with docetaxel. Preclinical investigations were performed to determine whether SAR408701 could be beneficially combined with immune checkpoint inhibitors. We, first, demonstrated that the tusamitamab ravtansine payload, DM4, is able to induce robust immunogenic cell death (ICD) using an in vivo vaccination assay. In this assay, immunocompetent mice were “vaccinated” by DM4-treated CT26 cells and then later rechallenged with CT26 tumor cells. Tumor rejection was observed in more than 50% of “vaccinated” mice. The ICD was characterized by the expression or release of danger-associated molecular patterns (DAMPs), and we observed DAMP modulation such as calreticulin (CRT) exposure at the cell surface of dying tumor cells, and the release of the High-Mobility Group Box 1 (HMGB1) in the extracellular space by CT26 cells in response to DM4 treatment. We also showed that DM4-treated CT26 tumor cells implanted into mice or SAR408701 treatment of MC38 tumor-bearing mice induced an elevation of plasmatic level of murine IL-6 and INFγ, revealing potential antitumor immunity modulation by the ADC and its payload. Immunohistochemistry analysis of MC38 tumors also revealed that tusamitamab ravtansine treatment induced a dose-dependent increase of CD8 T-cell infiltration in tumors correlating to dose-dependent efficacy. Finally, tusamitamab ravtansine was combined with immune checkpoint inhibitors (ICIs) in immunocompetent mice bearing MC38 tumor model. In this model, the combination of tusamitamab ravtansine with anti-PD-1 or anti-PD-L1 antibody led to complete response and, even, tumor-free survivors at 120 days post tumor implantation while single agents were inactive or transiently active. In summary, these preclinical data show that tusamitamab ravtansine could be beneficially combined with immune checkpoint and strongly support their evaluation in clinical development. Citation Format: Celine Nicolazzi, Anne-Marie Lefebvre, Nicolas Moindrot, Christelle Larois, Marion Classe, Sukhvinder Sidhu. Tusamitamab ravtansine induces immunogenic cell death and synergizes with anti-PD-1 or anti-PD-L1 antibody combination in solid tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3740.

Oncolytic vaccinia virus immunotherapy antagonizes image-guided radiotherapy in mouse mammary tumor models.

Ionizing radiation (IR) and oncolytic viruses are both used to treat cancer, and the effectiveness of both agents depends upon stimulating an immune response against the tumor. In this study we tested whether combining image guided ionizing radiation (IG-IR) with an oncolytic vaccinia virus (VACV) could yield a better therapeutic response than either treatment alone. ΔF4LΔJ2R VACV grew well on irradiated human and mouse breast cancer cells, and the virus can be combined with 4 or 8 Gy of IR to kill cells in an additive or weakly synergistic manner. To test efficacy in vivo we used immune competent mice bearing orthotopic TUBO mammary tumors. IG-IR worked well with 10 Gy producing 80% complete responses, but this was halved when the tumors were treated with VACV starting 2 days after IG-IR. VACV monotherapy was ineffective in this model. The antagonism was time dependent as waiting for 21 days after IG-IR eliminated the inhibitory effect but without yielding any further benefits over IR alone. In irradiated tumors, VACV replication was also lower, suggesting that irradiation created an environment that did not support infection as well in vivo as in vitro. A study of how four different treatment regimens affected the immune composition of the tumor microenvironment showed that treating irradiated tumors with VACV altered the immunological profiles in tumors exposed to IR or VACV alone. We detected more PD-1 and PD-L1 expression in tumors exposed to IR+VACV but adding an αPD-1 antibody to the protocol did not change the way VACV interferes with IG-IR therapy. VACV encodes many immunosuppressive gene products that may interfere with the ability of radiotherapy to induce an effective anti-tumor immune response through the release of danger-associated molecular patterns. These data suggest that infecting irradiated tumors with VACV, too soon after exposure, may interfere in the innate and linked adaptive immune responses that are triggered by radiotherapy to achieve a beneficial impact.

Co-delivery of doxorubicin and STING agonist cGAMP for enhanced antitumor immunity

Many chemotherapeutic agents can induce immunogenic cell death (ICD), which leads to the release of danger-associated molecular patterns (DAMPs) and tumor-associated antigens. This process promotes dendritic cells (DCs) maturation and cytotoxic T lymphocyte (CTL) infiltration. However, cancer cells can employ diverse mechanisms to evade the host immune system. Recent studies have shown that stimulator of interferon genes (STING) agonists, such as cGAMP, can amplify ICD-triggered immune responses and enhance the infiltration of immune cells into the tumor microenvironment (TME). Building upon these findings, we constructed a doxorubicin (DOX) and cGAMP co-delivery system (DOX/cGAMP@NPs) for melanoma and triple-negative breast cancer (TNBC) therapy. The results demonstrated that DOX could effectively destroy tumors and induce the release of DAMPs by ICD. Furthermore, in orthotopic 4T1 tumors mice model and subcutaneous B16 tumor mice model, cGAMP could promote the maturation of DCs and CD8+ T cell activation and infiltration by inducing the secretion of type I interferons and pro-inflammation cytokine, which amplified the antitumor immune response induced by DOX. This strategy also promoted the depletion of immunosuppressive cells, potentially alleviating the immunosuppressive TME. In conclusion, our study highlights the combination of DOX-induced ICD and the immune-enhancing properties of cGAMP holds significant implications for future research and clinical applications.

Necroptosis and Its Involvement in Various Diseases.

Necroptosis is a regulated form of cell death involved in the development of various pathological conditions. In contrast to apoptosis, plasma membrane rupture (PMR) occurs in cells in the relatively early stage of necroptosis; therefore, necroptosis induces a strong inflammatory response. Stimuli, including tumor necrosis factor (TNF), interferon (IFN)α/β, lipopolysaccharide, polyI:C, and viral infection, induce the formation of necrosomes that lead to membrane rupture and the release of intracellular contents, termed danger-associated molecular patterns (DAMPs). DAMPs are the collective term for molecules that normally reside in the cytoplasm or nucleus in living cells without inducing inflammation but induce strong inflammatory responses when released outside cells. Recent studies have provided a better understanding of the mechanisms underlying PMR and the release of DAMPs. Moreover, necroptosis is involved in various pathological conditions, and mutations in necroptosis-related genes can cause hereditary autoinflammatory syndromes. Thus, manipulating necroptosis signaling pathways may be useful for treating diseases involving necroptosis.

Glycyrrhizic Acid Inhibits High-Mobility Group Box-1 and Homocysteine-Induced Vascular Dysfunction.

Hyperhomocysteinemia (HHcy) worsens cardiovascular outcomes by impairing vascular function and promoting chronic inflammation via release of danger-associated molecular patterns, such as high-mobility group box-1 (HMGB-1). Elevated levels of HMGB-1 have recently been reported in patients with HHcy. Therefore, targeting HMGB-1 may be a potential therapy to improve HHcy-induced cardiovascular pathologies. This study aimed to further elucidate HMGB-1's role during acute HHcy and HHcy-induced atherogenesis and to determine if inhibiting HMGB-1 with glycyrrhizic acid (Glyz) improved vascular function. Male New Zealand White rabbits (n = 25) were placed on either a standard control chow (CD; n = 15) or atherogenic diet (AD; n = 10) for 4 weeks. Rabbit serum and Krebs taken from organ bath studies were collected to quantify HMGB-1 levels. Isometric tension analysis was performed on abdominal aorta (AA) rings from CD and AD rabbits. Rings were incubated with homocysteine (Hcy) [3 mM] for 60 min to induce acute HHcy or rhHMGB-1 [100 nM]. Vascular function was assessed by relaxation to cumulative doses of acetylcholine. Markers of vascular dysfunction and inflammation were quantified in the endothelium, media, and adventitia of AA rings. HMGB-1 was significantly upregulated in serum (p < 0.0001) and Krebs (p < 0.0001) after Hcy exposure or an AD. Incubation with Hcy (p < 0.0001) or rhHMGB-1 (p < 0.0001) and an AD (p < 0.0001) significantly reduced relaxation to acetylcholine, which was markedly improved by Glyz. HMGB-1 expression was elevated (p < 0.0001) after Hcy exposure and AD (p < 0.0001) and was normalized after Glyz treatment. Moreover, markers of vascular function, cell stress and inflammation were also reduced after Glyz. These results demonstrate that HMGB-1 has a central role during HHcy-induced vascular dysfunction and inhibiting it with Glyz could be a potential treatment option for cardiovascular diseases.

The APC-EPCR-PAR1 axis in sickle cell disease.

Sickle Cell Disease (SCD) is a group of inherited hemoglobinopathies. Sickle cell anemia (SCA) is caused by a homozygous mutation in the β-globin generating sickle hemoglobin (HbS). Deoxygenation leads to pathologic polymerization of HbS and sickling of erythrocytes. The two predominant pathologies of SCD are hemolytic anemia and vaso-occlusive episodes (VOE), along with sequelae of complications including acute chest syndrome, hepatopathy, nephropathy, pulmonary hypertension, venous thromboembolism, and stroke. SCD is associated with endothelial activation due to the release of danger-associated molecular patterns (DAMPs) such as heme, recurrent ischemia-reperfusion injury, and chronic thrombin generation and inflammation. Endothelial cell activation is mediated, in part, by thrombin-dependent activation of protease-activated receptor 1 (PAR1), a G protein coupled receptor that plays a role in platelet activation, endothelial permeability, inflammation, and cytotoxicity. PAR1 can also be activated by activated protein C (APC), which promotes endothelial barrier protection and cytoprotective signaling. Notably, the APC system is dysregulated in SCD. This mini-review will discuss activation of PAR1 by APC and thrombin, the APC-EPCR-PAR1 axis, and their potential roles in SCD.

Carbonic anhydrase IX inhibitor S4 triggers release of DAMPs related to immunogenic cell death in glioma cells via endoplasmic reticulum stress pathway

BackgroundImmunogenic cell death (ICD), which releases danger-associated molecular patterns (DAMP) that induce potent anticancer immune response, has emerged as a key component of therapy-induced anti-tumor immunity. The aim of this work was to analyze whether the carbonic anhydrase IX inhibitor S4 can elicit ICD in glioma cells.MethodsThe effects of S4 on glioma cell growth were evaluated using the CCK-8, clonogenic and sphere assays. Glioma cell apoptosis was determined by flow cytometry. Surface-exposed calreticulin (CRT) was inspected by confocal imaging. The supernatants of S4-treated cells were concentrated for the determination of HMGB1and HSP70/90 expression by immunoblotting. RNA-seq was performed to compare gene expression profiles between S4-treated and control cells. Pharmacological inhibition of apoptosis, autophagy, necroptosis and endoplasmic reticulum (ER) stress was achieved by inhibitors. In vivo effects of S4 were evaluated in glioma xenografts. Immunohistochemistry (IHC) was performed to stain Ki67 and CRT.ResultsS4 significantly decreased the viability of glioma cells and induced apoptosis and autophagy. Moreover, S4 triggered CRT exposure and the release of HMGB1 and HSP70/90. Inhibition of either apoptosis or autophagy significantly reversed S4-induced release of DAMP molecules. RNA-seq analysis indicated that the ER stress pathway was deregulated upon exposure to S4. Both PERK-eIF2α and IRE1α- XBP1 axes were activated in S4-treated cells. Furthermore, pharmacological inhibition of PERK significantly suppressed S4-triggered ICD markers and autophagy. In glioma xenografts, S4 significantly reduced tumor growth.ConclusionsAltogether, these findings suggest S4 as a novel ICD inducer in glioma and might have implications for S4-based immunotherapy.Avm3R8fPrFVn1VZUdJULS6Video

230-LB: A Composite Capsule Strategy Support Longevity of Microencapsulated Pancreatic ß Cells

Microencapsulation of islets in immunoprotective membranes offers an alternative treatment for type 1 diabetes in absence of systemic immunosuppression. However, periods of survival of transplanted islets are still limited. A factor that might be involved in a limited duration of survival is permanent damage to the extracellular matrix (ECM) during enzymatic islet-isolation which enhances susceptibility of islets for inflammatory stress. Here we investigated whether a combination of inclusion of ECM components (collagen IV and RGD), necroptosis inhibitor necrostatin-1 (Nec-1), amino acids (AA), and inclusion of the immunomodulatory polymer pectin in microcapsules can enhance the functionality of β-cells. The impact of these factors individually or combined was tested on MIN6 β-cells and human islet-cells. Viability, function, and immune activation via danger-associated molecular patterns (DAMPs) release and NF-κB activation in THP1-reporter cells were determined. Also, impact on mitochondrial dynamics was tested. Our data show that composite capsules of pectin/ECM/Nec-1/AA improve insulin secretion and enhance the mitochondrial activity of β-cells in vitro till day 7 after encapsulation. The alginate-pectin based microcapsules also showed immunomodulatory effects with decreased DAMPs release and lower NF-κB activation. After cytokine exposure, pectin/ECM/Nec-1/AA prevented the cytokine-induced decrease of mitochondrial activity and prevented viability loss until day 5. The rescuing effects were accompanied by modulation of mitochondrial fusion and fission gene expression, similar to that of untreated controls. Our data suggest that this composite capsule strategy using pectin/ECM/Nec-1/AA supports survival of microencapsulated β-cells and lowers susceptibility of β-cells for inflammatory stress. This strategy to create a better intracapsular microenvironment is a potentially promising way to preserve islet function and enhance graft survival time. Disclosure T. Qin: None. A. M. Smink: None. P. De vos: None. Funding JDRF (2-SRA-2018-523-S-B); China Scholarship Council (201906230339)

Induction of immunogenic cell death effect of licoricidin in cervical cancer cells by enhancing endoplasmic reticulum stress-mediated high mobility group box 1 expression.

Licoricidin (LCD) is an activity compound of the roots of Glycyrrhiza uralensis, which has therapeutic efficacy, including anti-virus, anti-cancer, and enhanced immunity in Traditional Chinese Medicine. Herein, this study aimed to clarify the effect of LCD on cervical cancer cells. In the present study, we found that LCD significantly inhibited cell viability via inducing cell apoptosis and companies with cleaved-PARP protein expression and caspase-3/-9 activity. Cell viability was markedly reversed these effects by pan-caspase inhibitor Z-VAD-FMK treatment. Furthermore, we showed that LCD-induced ER (endoplasmic reticulum) stress triggers upregulating the protein level of GRP78 (Bip), CHOP, and IRE1α, and subsequently confirmed the mRNA level by quantitative real-time polymerase chain reaction. In addition, LCD exhibited the release of danger-associated molecular patterns from cervical cancer cells, such as the release of high-mobility group box 1 (HMGB1), secretion of ATP, and exposure of calreticulin (CRT) on the cell surface, which led to immunogenic cell death (ICD). These results provide a novel foundation that LCD induces ICD via triggering ER stress in human cervical cancer cells. LCD might be an ICD inducer of immunotherapy in progressive cervical cancer.

Early DNase-I therapy delays secondary brain damage after traumatic brain injury in adult mice

Traumatic brain injury (TBI) causes the release of danger-associated molecular patterns (DAMP) from damaged or dead cells, which contribute to secondary brain damage after TBI. Cell-free DNA (cfDNA) is a DAMP known to cause disruption of the blood–brain barrier (BBB), promote procoagulant processes, brain edema, and neuroinflammation. This study tested the hypothesis that administration of deoxyribonuclease-I (DNase-I) has a beneficial effect after TBI. Mice (n = 84) were subjected to controlled cortical impact (CCI) and posttraumatic intraperitoneal injections of low dose (LD) or high dose (HD) of DNase-I or vehicle solution at 30 min and 12 h after CCI. LD was most effective to reduce lesion volume (p = 0.003), brain water content (p < 0.0001) and to stabilize BBB integrity (p = 0.019) 1 day post-injury (dpi). At 6 h post injury LD-treated animals showed less cleavage of fibrin (p = 0.0014), and enhanced perfusion as assessed by micro-computer-tomography (p = 0.027). At 5 dpi the number of Iba1-positive cells (p = 0.037) were reduced, but the number of CD45-positive cells, motoric function and brain lesion volume was not different. Posttraumatic-treatment with DNase-I therefore stabilizes the BBB, reduces the formation of brain edema, immune response, and delays secondary brain damage. DNase-I might be a new approach to extend the treatment window after TBI.

Cisplatin and Albumin-Based Gold–Cisplatin Nanoparticles Enhance Ablative Radiation Therapy–Induced Antitumor Immunity in Local and Distant Tumor Microenvironment

Ablative radiation therapy (RT) is an important strategy to eliminate primary tumor and can potentially induce the abscopal effect. Human serum albumin nanoparticle (NP) was used for controlled release of cisplatin to decrease cisplatin's systemic toxicity, and gold (Au) was added to increase RT-induced immunogenic cell death and potentiate the abscopal antitumor immunity. The designed albumin-based cisplatin-conjugated AuNPs were administered concurrently with ablative RT. C57BL/6 mice implanted with syngeneic murine Lewis lung carcinoma or murine MB49 tumor models were treated with ablative RT (12 Gy per fraction for 2 fractions, total 24 Gy), cisplatin, or Au-cisplatin NPs. Combining ablative RT with cisplatin or Au-cisplatin NPs both destroyed the primary tumor effectively and elicited immunogenic cell death accompanied by release of danger-associated molecular patterns. This enhanced recruitment of effector tumor-infiltrating immune cells, including natural killer T cells and CD8+ T cells, and elicited an increased percentage of professional antigen-presenting CD11c+ dendritic cells. Transient weight loss, accompanying hepatotoxicity, nephrotoxicity, and hematopoietic suppression, was observed as a systemic adverse event in the cisplatin but not the Au-cisplatin NPs group. Cisplatin and Au-cisplatin NPs both showed equivalent ability to reduce metastatic potential when combined with ablative RT, confirmed by suppressed unirradiated flank tumor growth and decreased metastatic lung tumor burden, which translated to improved survival. Mobilization and abundance of effector tumor-infiltrating immune cells including CD8+ T cells and dendritic cells were observed in the distant lung tumor microenvironment after ablative RT with cisplatin or Au-cisplatin NPs, demonstrating increased antitumor immunotherapeutic activity as an abscopal effect. Compared with cisplatin, the albumin-based Au-cisplatin NPs exhibited equivalent but no superior antitumor immunotherapeutic activity while reducing systemic adverse events and can be safely administered concurrently with ablative RT. Alternative NP formulations may be designed to further improve anticancer outcomes.

DAMPs Released from Proinflammatory Macrophages Induce Inflammation in Cardiomyocytes via Activation of TLR4 and TNFR.

Cardiac dysfunction is a life-threatening complication in sepsis. Upon infection and cardiac stress, the cardiac macrophage population expands. Recruited macrophages exhibit a predominantly proinflammatory phenotype and release danger-associated molecular patterns (DAMPs) that contribute to cardiac dysfunction. However, the underlying pathomechanisms are highly complex and not fully understood. Here, we utilized an indirect macrophage-cardiomyocyte co-culture model to study the effects of proinflammatory macrophages on the activation of different cardiac receptors (TLR3, TLR4, and TNFR) and their role in cardiac inflammation and caspase-3/7 activation. The stimulation of cardiomyocytes with conditioned medium of LPS-stimulated macrophages resulted in elevated IL-6 protein concentrations and relative IL-6 and TNFα mRNA levels. Conditioned medium from LPS-stimulated macrophages also induced NFκB translocation and increased caspase-3/7 activation in cardiomyocytes. Analyzing the role of different cardiac receptors, we found that TLR4 and TNFR inhibition reduces cardiac inflammation and that the inhibition of TNFR prevents NFκB translocation into the nuclei of cardiomyocytes, induced by exposure to conditioned medium of proinflammatory macrophages. Moreover, we demonstrated that TLR3 inhibition reduces macrophage-mediated caspase-3/7 activation. Our results suggest that the immune response of macrophages under inflammatory conditions leads to the release of DAMPs, such as eRNA and cytokines, which in turn induce cardiomyocyte dysfunction. Thus, the data obtained in this study contribute to a better understanding of the pathophysiological mechanisms of cardiac dysfunction.

Adenosine metabolized from extracellular ATP promotes type 2 immunity through triggering A2BAR signaling in intestinal epithelial cells.

SUMMARYIntestinal nematode parasites can cross the epithelial barrier, causing tissue damage and release of danger-associated molecular patterns (DAMPs) that may promote host protective type 2 immunity. We investigate whether adenosine binding to the A2B adenosine receptor (A2BAR) on intestinal epithelial cells (IECs) plays an important role. Specific blockade of IEC A2BAR inhibits the host protective memory response to the enteric helminth, Heligmosomoides polygyrus bakeri (Hpb), including disruption of granuloma development at the host-parasite interface. Memory T cell development is blocked during the primary response, and transcriptional analyses reveal profound impairment of IEC activation. Extracellular ATP is visualized 24 h after inoculation and is shown in CD39-deficient mice to be critical for the adenosine production mediating the initiation of type 2 immunity. Our studies indicate a potent adenosine-mediated IEC pathway that, along with the tuft cell circuit, is critical for the activation of type 2 immunity.

Immunogenic Cell Death, DAMPs and Prothymosin α as a Putative Anticancer Immune Response Biomarker.

The new and increasingly studied concept of immunogenic cell death (ICD) revealed a previously unknown perspective of the various regulated cell death (RCD) modalities, elucidating their immunogenic properties and rendering obsolete the notion that immune stimulation is solely the outcome of necrosis. A distinct characteristic of ICD is the release of danger-associated molecular patterns (DAMPs) by dying and/or dead cells. Thus, several members of the DAMP family, such as the well-characterized heat shock proteins (HSPs) HSP70 and HSP90, the high-mobility group box 1 protein and calreticulin, and the thymic polypeptide prothymosin α (proTα) and its immunoreactive fragment proTα(100–109), are being studied as potential diagnostic tools and/or possible therapeutic agents. Here, we present the basic aspects and mechanisms of both ICD and other immunogenic RCD forms; denote the role of DAMPs in ICD; and further exploit the relevance of human proTα and proTα(100–109) in ICD, highlighting their possible clinical applications. Furthermore, we present the preliminary results of our in vitro studies, which show a direct correlation between the concentration of proTα/proTα(100–109) and the levels of cancer cell apoptosis, induced by anticancer agents and γ-radiation.

Cytosolic DNA Sensors as a New Pharmacological Target in EVLP

<h3>Purpose</h3> Innate immunity is a key component of the inflammatory responses ensuing ischemia/reperfusion (I/R) injury. Cell damage and death caused by I/R promotes the release of danger-associated molecular patterns (DAMPs) recognized by specific pattern recognition receptors (PRRs). DNA released from nucleus and mitochondria of injured cells is a known DAMP, which sustains inflammation and cell death when recognized by specific PRRs like cytosolic DNA (cDNA) sensors cGAS and AIM2. The cGAS/STING pathway induces the production of type I interferons (IFNs) and IFN stimulated genes (ISGs). The role of cDNA sensors in reperfused lung is currently not characterized. Thus, using the rat <i>ex vivo</i> lung perfusion (EVLP) and lung transplantation (LTx) models, we begin to investigate the role of cGAS/STING in the early inflammatory events of lung I/R. <h3>Methods</h3> Heart-Lung blocks from male Sprague-Dawley rats were cannulated in the pulmonary artery and left atrium and assigned to four groups (n=5/6 rats/group). Cold ischemia (CI): under mechanical ventilation, the lungs were flushed with 4°C Perfadex. The heart-lung block was stored inflated (FiO2 0.5) for 7h in 4°C Perfadex. Warm ischemia (WI): After cannulation, the lungs were deflated for 1h at room temperature, flushed and stored as above for 6h. CI+EVLP: lungs were harvested as above, stored at 4°C for 2h, followed by 3-hour EVLP, and stored again (2h) in 4°C Perfadex. WI+EVLP: after 1 hour WI, lungs were flushed and stored at 4°C for 1 hour, followed by 3h EVLP, and stored again (2h) in 4°C Perfadex. Right lungs were used as controls and left lungs were transplanted and sampled 2h after LTx. We analyzed by RT-PCR and western blot the expression of the cytosolic DNA sensors cGAS, STING and AIM2 and by RT-PCR the expression of the downstream gene <i>Ifnb</i>, and the ISGs <i>Ifit1</i> and <i>Isg15</i> in whole lung tissue<i>.</i> <h3>Results</h3> cGAS, STING and AIM2 proteins are expressed in lung after the ischemic period. Their mRNA expression is strongly induced by the reperfusion (EVLP, LTx or EVLP+LTx) regardless of the conditions of preservation (CI or WI). We observed a similar behavior for the mRNA expression of <i>Ifnb</i> and the ISGs <i>Ifit1</i> and <i>Isg15</i>. <h3>Conclusion</h3> These preliminary data provide evidence that cytosolic DNA sensors, especially the cGAS-STING axis, may play a prominent role in the early inflammation of the reperfused lung regardless of the preservation conditions.

Redox Activation of Mitochondrial DAMPs and the Metabolic Consequences for Development of Autoimmunity.

Significance: Reactive oxygen species (ROS) are well known to promote innate immune responses during and in the absence of microbial infections. However, excessive or prolonged exposure to ROS provokes innate immune signaling dysfunction and contributes to the pathogenesis of many autoimmune diseases. The relatively high basal expression of pattern recognition receptors (PRRs) in innate immune cells renders them prone to activation in response to minor intrinsic or extrinsic ROS misbalances in the absence of pathogens. Critical Issues: A prominent source of ROS are mitochondria, which are also major inter-organelle hubs for innate immunity activation, since most PRRs and downstream receptor molecules are directly located either at mitochondria or at mitochondria-associated membranes. Due to their ancestral bacterial origin, mitochondria can also act as quasi-intrinsic self-microbes that mimic a pathogen invasion and become a source of danger-associated molecular patterns (DAMPs) that triggers innate immunity from within. Recent Advances: The release of mitochondrial DAMPs correlates with mitochondrial metabolism changes and increased generation of ROS, which can lead to the oxidative modification of DAMPs. Recent studies suggest that ROS-modified mitochondrial DAMPs possess increased, persistent immunogenicity. Future Directions: Herein, we discuss how mitochondrial DAMP release and oxidation activates PRRs, changes cellular metabolism, and causes innate immune response dysfunction by promoting systemic inflammation, thereby contributing to the onset or progression of autoimmune diseases. The future goal is to understand what the tipping point for DAMPs is to become oxidized, and whether this is a road without return. Antioxid. Redox Signal. 36, 441-461.

The Role of Extracellular HSP70 in the Function of Tumor-Associated Immune Cells

Simple SummaryThe intracellular heat shock protein 70 (HSP70) is essential for cells to respond to stress, for instance, by refolding damaged proteins or inhibiting apoptosis. However, in cancer, HSP70 is overexpressed and can translocate to the extracellular milieu, where it emerged as an important modulator of tumor-associated immune cells. By targeting the tumor microenvironment (TME) through different mechanisms, extracellular HSP70 can trigger pro- or anti-tumorigenic responses. Therefore, understanding the pathways and their consequences is crucial for therapeutically targeting cancer and its surrounding microenvironment. In this review, we summarize current knowledge on the translocation of extracellular HSP70. We further elucidate its functions within the TME and provide an overview of potential therapeutic options.Extracellular vesicles released by tumor cells (T-EVs) are known to contain danger-associated molecular patterns (DAMPs), which are released in response to cellular stress to alert the immune system to the dangerous cell. Part of this defense mechanism is the heat shock protein 70 (HSP70), and HSP70-positive T-EVs are known to trigger anti-tumor immune responses. Moreover, extracellular HSP70 acts as an immunogen that contributes to the cross-presentation of major histocompatibility complex (MHC) class I molecules. However, the release of DAMPs, including HSP70, may also induce chronic inflammation or suppress immune cell activity, promoting tumor growth. Here, we summarize the current knowledge on soluble, membrane-bound, and EV-associated HSP70 regarding their functions in regulating tumor-associated immune cells in the tumor microenvironment. The molecular mechanisms involved in the translocation of HSP70 to the plasma membrane of tumor cells and its release via exosomes or soluble proteins are summarized. Furthermore, perspectives for immunotherapies aimed to target HSP70 and its receptors for cancer treatment are discussed and presented.

New Insights in Mechanisms and Therapeutics for Short- and Long-Term Impacts of Hepatic Ischemia Reperfusion Injury Post Liver Transplantation.

Liver transplantation has been identified as the most effective treatment for patients with end-stage liver diseases. However, hepatic ischemia reperfusion injury (IRI) is associated with poor graft function and poses a risk of adverse clinical outcomes post transplantation. Cell death, including apoptosis, necrosis, ferroptosis and pyroptosis, is induced during the acute phase of liver IRI. The release of danger-associated molecular patterns (DAPMs) and mitochondrial dysfunction resulting from the disturbance of metabolic homeostasis initiates graft inflammation. The inflammation in the short term exacerbates hepatic damage, leading to graft dysfunction and a higher incidence of acute rejection. The subsequent changes in the graft immune environment due to hepatic IRI may result in chronic rejection, cancer recurrence and fibrogenesis in the long term. In this review, we mainly focus on new mechanisms of inflammation initiated by immune activation related to metabolic alteration in the short term during liver IRI. The latest mechanisms of cancer recurrence and fibrogenesis due to the long-term impact of inflammation in hepatic IRI is also discussed. Furthermore, the development of therapeutic strategies, including ischemia preconditioning, pharmacological inhibitors and machine perfusion, for both attenuating acute inflammatory injury and preventing late-phase disease recurrence, will be summarized in the context of clinical, translational and basic research.

Enzyme-instructed and mitochondria-targeting peptide self-assembly to efficiently induce immunogenic cell death

Immunogenic cell death (ICD) plays a major role in cancer immunotherapy by stimulating specific T cell responses and restoring the antitumor immune system. However, effective type II ICD inducers without biotoxicity are still very limited. Herein, a tentative drug- or photosensitizer-free strategy was developed by employing enzymatic self-assembly of the peptide F-pY-T to induce mitochondrial oxidative stress in cancer cells. Upon dephosphorylation catalyzed by alkaline phosphatase overexpressed on cancer cells, the peptide F-pY-T self-assembled to form nanoparticles, which were subsequently internalized. These affected the morphology of mitochondria and induced serious reactive oxygen species production, causing the ICD characterized by the release of danger-associated molecular patterns (DAMPs). DAMPs enhanced specific immune responses by promoting the maturation of DCs and the intratumoral infiltration of tumor-specific T cells to eradicate tumor cells. The dramatic immunotherapeutic capacity could be enhanced further by combination therapy of F-pY-T and anti-PD-L1 agents without visible biotoxicity in the main organs. Thus, our results revealed an alternative strategy to induce efficient ICD by physically promoting mitochondrial oxidative stress.