Levels Of Damage-associated Molecular Patterns Research Articles

Berberine sensitizes immune checkpoint blockade therapy in melanoma by NQO1 inhibition and ROS activation

Unprecedented progress in immune checkpoint blockade (ICB) therapy has been made in cancer treatment. However, the response to ICB therapy is limited to a small subset of patients. The development of ICB sensitizers to improve cancer immunotherapy outcomes is urgently needed. Berberine (BBR), a well-known phytochemical compound isolated from many kinds of medicinal plants such as Berberis aristata, Coptis chinensis, and Phellondendron chinense Schneid, has shown the ability to inhibit the proliferation, invasion and metastasis of cancer cells. In this study, we investigated whether BBR can enhance the therapeutic benefit of ICB for melanoma, and explored the underlying mechanisms involved. The results showed that BBR could sensitize ICB to inhibit tumor growth and increased the survival rate of mice. Moreover, BBR stimulated intracellular ROS production partially by inhibiting NQO1 activity, which induced immunogenic cell death (ICD) in melanoma, elevated the levels of damage-associated molecular patterns (DAMPs), and subsequently activated DC cells and CD8 + T cells in vitro and in vivo. In conclusion, BBR is a novel ICD inducer. BBR could enhance the therapeutic benefit of ICB for melanoma. These effects were partially mediated through the inhibition of NQO1 and ROS activation.

Immune markers of severe acute pancreatitis.

Acute pancreatitis is a common acute inflammatory disorder of the pancreas, and its incidence has been increasing worldwide. Approximately 10% of acute pancreatitis progresses to severe acute pancreatitis (SAP), which carries significant morbidity and mortality. Disordered immune response to pancreatic injury is regarded as a key event that mediates systemic injury in SAP. In this article, we review recent developments in immune biomarkers of SAP and future directions for research. Given the importance of the NLRP3-inflammasome pathway in mediating systemic inflammatory response syndrome and systemic injury, recent studies have investigated associations of SAP with systemic levels of activators of NLRP3, such as the damage associated molecular patterns (DAMPs) for the first time in human SAP. For example, circulating levels of histones, mitochondrial DNAs, and cell free DNAs have been associated with SAP. A panel of mechanistically relevant immune markers (e.g., panel of Angiopoeitin-2, hepatocyte growth factor, interleukin-8 (IL-8), resistin and sTNF-α R1) carried higher predictive accuracies than existing clinical scores and individual immune markers. Of the cytokines with established relevance to SAP pathogenesis, phase 2 trials of immunotherapies, including tumor necrosis factor (TNF)-alpha inhibition and stimulation of IL-10 production, are underway to determine if altering the immunologic response can reduce the severity of acute pancreatitis (AP). Circulating systemic levels of various DAMPs and a panel of immune markers that possibly reflect activities of different pathways that drive SAP appear promising as predictive biomarkers for SAP. But larger multicenter studies are needed for external validation. Studies investigating immune cellular pathways driving SAP using immunophenotyping techniques are scarce. Interdisciplinary efforts are also needed to bring some of the promising biomarkers to the bedside for validation and testing for clinical utility. Studies investigating the role of and characterization of altered gut-lymph and gut-microbiota in severe AP are needed.

NIR-responsive CN-Pt-GEM hydrogel induces necroptosis and immunotherapeutic responses prevent postoperative recurrence and wound infection in lung carcinoma

BackgroundCancer recurrence following surgical resection is a major cause of treatment failure. Finding effective methods to prevent postoperative recurrence and wound infection is an important component of successful surgery. With the development of new nanotechnology, more treatment options have been provided for postoperative adjuvant therapy. This study presents an innovative hydrogel system that stimulates tumoricidal immunity after surgical resection of non-small cell lung cancer (NSCLC) and prevents cancer relapse.ResultsThe hydrogel system is based on the excellent photothermal conversion performance of single-atom platinum (CN-Pt) along with the delivery and release of the chemotherapy drug, gemcitabine (GEM). The system is coated onto the wound surface after tumor removal with subsequent near-infrared (NIR) photothermal therapy, which efficiently induces necroptosis of residual cancer cells, amplifies the levels of damage-associated molecular patterns (DAMPs), and increases the number of M1 macrophages. The significantly higher levels of phagocytic macrophages enhance tumor immunogenicity and sensitize cancer cells to CD8 + T-cell immunity to control postoperative recurrence, which has been verified using an animal model of postoperative lung cancer recurrence. The CN-Pt-GEM-hydrogel with NIR can also inhibit postoperative wound infection.ConclusionsThese findings introduce an alternative strategy for supplementing antitumor immunity in patients undergoing resection of NSCLC tumors. The CN-Pt-GEM-hydrogel with the NIR system also exhibits good biosafety and may be adaptable for clinical application in relation to tumor resection surgery, wound tissue filling, infection prevention, and recurrence prevention.

Low serum double-stranded DNA levels are associated with higher survival rates in severe COPD patients.

Damage-associated molecular patterns (DAMPs) are endogenous danger signals that alert and activate the immune system upon cellular damage or death. It has previously been shown that DAMP release is increased in patients with COPD, leading to higher levels in extracellular fluids such as serum. In the current study we investigated whether the serum levels of DAMPs were associated with survival rates in COPD patients. A panel of seven DAMPs, consisting of HMGB1, fibrinogen, α-defensin, heat shock protein 70, S100A8, galectin-9 and double-stranded DNA (dsDNA), was measured in serum of 949 severe COPD patients. Maximally selected rank statistics was used to define cut-off values and a Cox proportional hazards model was used to evaluate the effect of high or low DAMP levels on 4-year survival. For DAMPs that were found to affect survival significantly, baseline characteristics were compared between the two DAMP groups. Out of the seven DAMPs, only dsDNA was significantly associated with 4-year survival. Patients with elevated serum level of dsDNA had higher 4-year mortality rates, lower FEV1 % predicted values and higher emphysema scores. In conclusion, in a clinical cohort of 949 patients with moderate-to-severe COPD, elevated serum levels of dsDNA were associated with a higher risk of death. This study further illustrates the potential role of circulating DAMPs, such as dsDNA, in the progression of COPD. Together, the results of this study suggest that levels of circulating dsDNA might serve as an additional prognostic biomarker for survival in COPD patients.

Investigation of folate-modified EGCG-loaded thermosensitive nanospheres inducing immunogenic cell death and damage-associated molecular patterns in hepatocellular carcinoma

BackgroundThe systemic treatment of advanced hepatocellular carcinoma is currently facing a bottleneck. EGCG, the primary active compound in green tea, exhibits anti-tumor effects through various pathways. However, there is a lack of study on EGCG-induced immunogenic cell death (ICD) in hepatocellular carcinoma. MethodsIn a previous study, we successfully synthesized folate-modified thermosensitive nano-materials, encapsulated EGCG within nanoparticles using a hydration method, and established the EGCG nano-drug delivery system. The viability of HepG2 cells post-EGCG treatment was assessed via the MTT and EdU assays. Cell migration and invasion were evaluated through wound healing experiments, Transwell assays, and Annexin V-FITC/PI assay for apoptosis detection. Additionally, the expression levels of damage-associated molecular patterns (DAMPs) were determined using immunofluorescence, ATP measurement, RT-qPCR, and Western Blot. ResultsThe drug sensitivity test revealed an IC50 value of 96.94 μg/mL for EGCG in HepG2 cells after 48 h. EGCG at a low concentration (50 μg/mL) significantly impeded the migration and invasion of HepG2 cells, showing a clear dose-dependent response. Moreover, medium to high EGCG concentrations induced cell apoptosis in a dose-dependent manner and upregulated DAMPs expression. Immunofluorescence analysis demonstrated a notable increase in CRT expression following low-concentration EGCG treatment. As EGCG concentration increased, cell viability decreased, leading to CRT exposure on the cell membrane. EGCG also notably elevated ATP levels. RT-qPCR and Western Blot analyses indicated elevated expression levels of HGMB1, HSP70, and HSP90 following EGCG intervention. ConclusionEGCG not only hinders the proliferation, migration, and invasion of hepatocellular carcinoma cells and induces apoptosis, but also holds significant clinical promise in the treatment of malignant tumors by promoting ICD and DAMPs secretion.

Toll-like receptor expression and functional behavior in platelets from patients with systemic lupus erythematosus

BackgroundMultiple blood cell abnormalities participate in the development of inflammation in systemic lupus erythematosus (SLE). Although platelets have been suggested as one of these contributors through the release of their content during activation, there are limited specific data about their role as immune players in SLE. Materials and MethodsThirteen SLE patients were included. Flow cytometry was used to measure Toll-like receptors (TLR) 2, 4, and 9 in resting platelets, platelet-activation markers (PAC-1 binding, P-selectin, CD63, and CD40 ligand -L) and platelet-leukocyte aggregates before and after specific TLR stimulation. Soluble CD40L and von Willebrand factor (vWf) release from stimulated platelets was measured using ELISA. ResultsIn resting conditions, SLE platelets showed normal expression levels of TLR 2, 4 and 9. Platelet surface activation markers, soluble CD40L, and vWf release were normal at baseline and after TLR stimulation. Platelet-monocyte aggregates were elevated in resting conditions in SLE samples and showed only a marginal increase after TLR stimulation, while baseline and stimulated platelet-neutrophil and platelet-lymphocyte aggregates were normal. C-reactive protein levels positively correlated with platelet-monocyte aggregates both at baseline and after stimulation with the TLR-2 agonist PAM3CSK4, suggesting these complexes could reflect the inflammatory activity in SLE. In our cohort, 12 of 13 patients received treatment with hydroxychloroquine (HCQ), a known inhibitor of endosomal activity and a potential inhibitor of platelet activation. The fact that SLE platelets showed an adequate response to TLR agonists suggests that, despite this treatment, they retain the ability to respond to the increased levels of damage-associated molecular patterns (DAMPs), which represent known TLR ligands, present in the circulation of SLE patients. Interestingly, elevated plasma levels of high mobility group box 1 (HMGB1), a classical DAMP, correlated with vWf release from TLR-stimulated platelets, suggesting that HMGB1 may also be released by platelets, thereby creating a positive feedback loop for platelet activation that contributes to inflammation. ConclusionOur study demonstrates normal platelet TLR expression and function together with increased circulating platelet-monocyte aggregates. In addition, a direct correlation was observed between plasma HMGB1 levels and platelet vWf release following TLR2 stimulation. This platelet behavior in a group of patients undergoing HCQ treatment suggests that platelets could play a role in the inflammatory state of SLE.

Stimuli-Responsive Nanoadjuvant Rejuvenates Robust Immune Responses to Sensitize Cancer Immunotherapy.

Despite their immense therapeutic potential, cancer immunotherapies such as immune checkpoint blockers (ICBs) benefit only a small subset of patients. Toll-like receptor agonists reverse the immunosuppressive tumor microenvironment (TME) to enhance antitumor immunity, but their systemic administration induces side effects. This work describes a TME-responsive nanotherapeutic platform for the site-specific release of drug candidates in tumors with a significant antitumor efficacy. Imidazoquinoline (IMQ)-derived liposomal nanovesicles (LN-IMQ) triggered the antitumor ability of macrophages, mobilized T-cell immunity, and promoted the secretion of antitumor cytokines, explaining the synergistic effect of LN-IMQ with ICBs. LN-IMQ monotherapy observed complete tumor regression in 6/8 of 4T1-bearing mouse, and cured mice resisted secondary tumor challenge. Besides, LN-IMQ decreased the occurrence of lung metastases, being effective against advanced metastases. On the other hand, neoantigen-based cancer vaccine has very low immune responses. Here, we also verified that LN-IMQ can serve as an ideal tumor antigen delivery vector. Cancer cells in vitro treated with chemotherapeutic drugs included multiple neoantigens and high levels of damage-associated molecular patterns, which were then successfully encapsulated in LN-IMQ to obtain a "personalized nanovaccine" with artificially amplified antigenicity and adjuvant properties. This study developed an attractive potential personalized nanovaccine for chemotherapeutic-drug-induced tumor neoantigens and immunotherapy.

Potential of damage associated molecular patterns in synergising radiation and the immune response in oesophageal cancer.

There is an intimate crosstalk between cancer formation, dissemination, treatment response and the host immune system, with inducing tumour cell death the ultimate therapeutic goal for most anti-cancer treatments. However, inducing a purposeful synergistic response between conventional therapies and the immune system remains evasive. The release of damage associated molecular patterns (DAMPs) is indicative of immunogenic cell death and propagation of established immune responses. However, there is a gap in the literature regarding the importance of DAMP expression in oesophageal adenocarcinoma (OAC) or by immune cells themselves. To investigate the effects of conventional therapies on DAMP expression and to determine whether OAC is an immunogenic cancer. We investigated the levels of immunogenic cell death-associated DAMPs, calreticulin (CRT) and HMGB1 using an OAC isogenic model of radioresistance. DAMP expression was also assessed directly using ex vivo cancer patient T cells (n = 10) and within tumour biopsies (n = 9) both pre and post-treatment with clinically relevant chemo(radio)therapeutics. Hypoxia in combination with nutrient deprivation significantly reduces DAMP expression by OAC cells in vitro. Significantly increased frequencies of T cell DAMP expression in OAC patients were observed following chemo(radio)therapy, which was significantly higher in tumour tissue compared with peripheral blood. Patients with high expression of HMGB1 had a significantly better tumour regression grade (TRG 1-2) compared to low expressors. In conclusion, OAC expresses an immunogenic phenotype with two distinct subgroups of high and low DAMP expressors, which correlated with tumour regression grade and lymphatic invasion. It also identifies DAMPs namely CRT and HMGB1 as potential promising biomarkers in predicting good pathological responses to conventional chemo(radio)therapies currently used in the multimodal management of locally advanced disease.

Abstract P2-20-09: Enhancement of anti-tumor immunity in ER+ breast cancer through dual inhibition of MCL1 and caspases

Abstract Breast cancer is one of the most commonly diagnosed cancer types and a leading cause of cancer death in women in the United States. However, it is difficult to treat with immunotherapy because of its immunological “cold” status. This underscores the importance of a novel clinical approach to increase efficacy of immunotherapy including acute upregulation of Type I Interferon (IFN) response. One strategy to enhance Type I IFN response is by release of damage-associated molecular patterns (DAMPs) from mitochondria, including mitochondrial DNA, RNA and oxidized proteins that are recognized by pattern recognition receptors (PRRs) in cytoplasm and activate downstream IFN signaling. Because many cancers including breast cancer experience increased and dysregulated mitochondrial biogenesis at baseline, they contain significantly higher levels of DAMPs compared to normal cells, but these substrates are sequestered in mitochondria and not accessible to PRRs. We set out to develop a strategy to exploit increased levels of DAMPs in cancer cell mitochondria to enhance Type I IFN response. While DAMPs are released from mitochondria upon apoptosis caused by mitochondrial outer membrane permeabilization (MOMP), Type I IFN response is still silenced due to cleavage of PRRs by activated caspases. One way to induce MOMP is by inhibition of Myeloid Cell Leukemia-1 (Mcl-1), an anti-apoptotic family protein that is frequently upregulated in breast cancer. Interestingly, breast cancer cell lines showed the highest dependency on Mcl-1 among solid cancers and inhibitors of Mcl-1 have shown efficacy in breast cancer early clinical trials. Based on our preliminary observations showing that Mcl-1 inhibition activates a robust Type I IFN response in MCF7 breast cancer cells deficient for caspase 3, we hypothesized that combining Mcl-1 inhibitors with caspase inhibitors would greatly enhance Type I IFN response in a robust and specific manner by facilitating release on DAMPs into the cytoplasm while preventing inactivation of PRRs. Strikingly, in contrast with Mcl-1 inhibitor treatment alone, combination treatment led to dramatic increase in expression levels of IFN-β and interferon-stimulated genes (ISGs) in MCF7 and ZR-75-1 ER+ breast cancer cell lines with minimal effects on cytotoxicity. In addition, T47d, a breast cancer line highly resistant to Mcl-1 inhibition due to compensation by other Bcl-2 family proteins, showed greatly enhanced Type I IFN response upon combined Mcl-1, Bcl-2 and caspase inhibition. Because reduced expression of MHC class I molecules on cell surface is one of the most important immune escape mechanisms in tumors including breast cancer, we examined whether the proposed combinatorial treatment would overcome this by enhancing expression of MHC class I. Immunofluorescence (IF) staining confirmed that the combination treatment dramatically increased total HLA-ABC protein expression on plasma membrane of ZR-75-1. Furthermore, combinatorial treatment upregulated transcription of most genes responsible for antigen presentation, a multistep process consisting of antigen peptide generation and loading of MHC class I molecules. Lastly, we sought to examine how elevated chemokine expression may impact patients with breast cancer. We examined association of cytokines highly upregulated by the combination treatment with immune cell infiltration using a TCGA dataset that included over a thousand breast cancer samples. This analysis was performed by xCell algorithm and demonstrated strong association of that cytokine expression with CD8+ T, B and DC infiltrate. Our results demonstrate that the combination of Mcl-1 and caspase inhibitors greatly enhances Type I IFN response and may improve anti-tumor immunity. We plan to further test this drug combination in immunocompetent mouse models, laying the foundation for a clinical trial assessing the combination treatment in patients with metastatic ER+ breast cancer. Citation Format: Jae Kyo Yi, Alexander Spektor. Enhancement of anti-tumor immunity in ER+ breast cancer through dual inhibition of MCL1 and caspases. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-20-09.

Inflammation and damage-associated molecular patterns in major psychiatric disorders.

Emerging evidence indicates that inflammation plays an important role as a mechanism underlying mental disorders. However, most of the research on inflammatory mechanisms focuses on serum levels of interleukins and very few studies have investigated molecules that initiate and expand innate immune pathways such as damage-associated molecular patterns (DAMPs). This study investigated the levels of DAMPs among patients diagnosed with major depressive disorder (MDD), bipolar disorder (BD) I and II, schizophrenia (SCZ), and generalized anxiety disorder (GAD). We quantified serum levels of heat shock proteins (HSPs) 70 and 60 and of S100 calcium-binding protein B (S100B). Serum levels of HSP70, HSP60, and S100B were assessed in a sample of participants with psychiatric disorders (n = 191) and a control group (CT) (n = 59) using enzyme-linked immunosorbent assay (ELISA). Serum HSP70 concentrations were significantly higher in the MDD group compared to the CT, SCZ, and BD groups. The GAD group had higher concentrations of HSP70 than the SCZ group. Exploring associations with medications, lithium (p = 0.003) and clozapine (p = 0.028) were associated with lower HSP70 levels. Approximately 64% of the sample had DAMPs levels below the limits of detection indicated by the respective ELISA kit. This was the first study to assess DAMPs levels in a transdiagnostic sample. Our preliminary findings suggest that HSP70 may be associated with MDD pathophysiology. Medications such as lithium and clozapine were associated with lower HSP70 levels in BD and SCZ groups, respectively. Therefore, it is worth mentioning that all participants were medicated and many psychotropic drugs exert an anti-inflammatory effect, possibly reducing the signs of inflammation.

Platelet dysfunction after trauma: From mechanisms to targeted treatment.

Platelet dysfunction after trauma: From mechanisms to targeted treatment.

Diagnostic and prognostic significance of cell death markers in patients with cirrhosis and acute decompensation.

BackgroundThe transition from compensated to decompensated liver cirrhosis is a hallmark of disease progression, however, reliable predictors to assess the risk of decompensation in individual patients from routine diagnostics are lacking. Here, we characterize serum levels of cell death-associated markers and routine biochemistry from patients with chronic liver disease with and without decompensation.MethodsA post-hoc analysis was based on prospectively collected clinical data from 160 patients with chronic liver disease, stably compensated or decompensated at baseline or during follow-up, over a median period of 721 days. Serum levels of damage-associated molecular patterns (DAMPs) and routine biochemistry are quantified at baseline (for all patients) and during follow-up (for patients with acute decompensation). The panel of DAMPs assessed in this study comprises high-mobility group-box protein 1 (HMGB1), cytochrome C (cyt C), soluble Fas-ligand (sFasL), interleukin 6 (IL-6), soluble cytokeratin-18 (CK18-M65) and its caspase‐cleaved fragment CK18-M30.ResultsIn this cohort study, 80 patients (50%) were diagnosed with alcoholic liver cirrhosis, 60 patients (37.5%) with hepatitis C virus- and 20 patients (13.5%) with hepatitis B virus-related liver cirrhosis. At baseline, 17 patients (10.6%) showed decompensated liver disease and another 28 patients (17.5%) developed acute decompensation during follow-up (within 24 months). One hundred fifteen patients showed stable liver disease (71.9%). We found DAMPs significantly elevated in patients with decompensated liver disease versus compensated liver disease. Patients with acute decompensation during follow-up showed higher baseline levels of IL-6, sFasL, CK18-M65 and–M30 (P<0.01) compared to patients with stably compensated liver disease. In multivariate analyses, we found an independent association of baseline serum levels of sFasL (P = 0.02; OR = 2.67) and gamma-glutamyl transferase (GGT) (P<0.001; OR = 2.1) with acute decompensation. Accuracy of the marker combination for predicting acute decompensation was high (AUC = 0.79). Elevated aminotransferase levels did not correlate with decompensated liver disease and acute decompensation.ConclusionsDAMPs are elevated in patients with decompensated liver disease and patients developing acute decompensation. The prognostic value of a marker combination with soluble Fas-ligand and GGT in patients with liver cirrhosis should be further evaluated.

Platelet Toll-like Receptor 4-Related Innate Immunity Potentially Participates in Transfusion Reactions Independent of ABO Compatibility: An Ex Vivo Study.

The role of platelet TLR4 in transfusion reactions remains unclear. This study analyzed platelet TLR4 and certain damage-associated molecular patterns (DAMPs) and evaluated how ABO compatibility affected TLR4 expression after a simulated ex vivo transfusion. A blood bank was the source of donor red blood cells. Blood from patients undergoing cardiac surgery was processed to generate a washed platelet suspension to which the donor blood was added in concentrations 1, 5, and 10% (v/v). Blood-mixing experiments were performed on four groups: a 0.9% saline control group (n = 31); a matched-blood-type mixing group (group M, n = 20); an uncross-matched ABO-specific mixing group (group S, n = 20); and an ABO-incompatible blood mixing group (group I, n = 20). TLR4 expression in the platelets was determined after blood mixing. We evaluated levels of TLR4-binding DAMPs (HMGB1, S100A8, S100A9, and SAA), lipopolysaccharide-binding protein, and endpoint proteins in the TLR4 signaling pathway. In the M, S, and I groups, 1, 5, and 10% blood mixtures significantly increased TLR4 expression (all p < 0.001) in a concentration-dependent manner. Groups M, S, and I were not discovered to have significantly differing TLR4 expression (p = 0.148). HMGB1, S100A8, and S100A9 levels were elevated in response to blood mixing, but SAA, lipopolysaccharide-binding protein, TNF-α, IL-1β, and IL-6 levels were not. Blood mixing may elicit innate immune responses by upregulating platelet TLR4 and DAMPs unassociated with ABO compatibility, suggesting that innate immunity through TLR4-mediated signaling may induce transfusion reactions.

P12.02 Systemic Anticancer Therapy Upregulate Plasma Levels of Damage-Associated Molecular Patterns in Patients With Advanced Lung Cancer

Immunogenic cell death (ICD) characterized by the release of damage-associated molecular patterns (DAMPs) from dying cancer cells may contribute to the synergistic antitumor effect of cytotoxic chemotherapy combined with an immune checkpoint inhibitor. The kinetics of circulating DAMP levels in cancer patients have remained largely uncharacterized, however.

AIM/CD5L attenuates DAMPs in the injured brain and thereby ameliorates ischemic stroke.

The sterile inflammation caused by damage-associated molecular patterns (DAMPs) worsens the prognosis following primary injury such as ischemic stroke. However, there are no effective treatments to regulate DAMPs. Here, we report that AIM (or CD5L) protein reduces sterile inflammation by attenuating DAMPs and that AIM administration ameliorates the deleterious effects of ischemic stroke. AIM binds to DAMPs via charge-based interactions and disulfide bond formation. This AIM association promotes the phagocytic removal of DAMPs and neutralizes DAMPs by impeding their binding to inflammatory receptors. In experimental stroke, AIM-deficient mice exhibit severe neurological damage and higher mortality with greater levels of DAMPs and associated inflammation in the brain than wild-type mice, in which brain AIM levels increase following stroke onset. Recombinant AIM administration reduces sterile inflammation in the infarcted region, leading to a profound reduction of animal mortality. Our findings provide a basis for the therapies targeting DAMPs to improve ischemic stroke.

Abstract 1933: Improved DAMP assays for the in vitro assessment of immunogenic cell death

Abstract The extracellular levels of damage-associated molecular patterns (DAMPs) released during immunogenic cell death (ICD) are positively correlated with the magnitude and efficacy of the resulting in vivo immune response. Therefore, extracellular ATP (eATP) and high mobility group box 1 (HMGB1) have been identified as key biomarkers for their predictive capacity during in vitro ICD screening activities. Current methods for identifying eATP and HMGB1 inducers are laborious, costly, and hampered by poor throughput. To overcome these challenges, we developed easy-to-use, homogeneous, bioluminescent assays that measure dose-dependent release of these immunostimulatory agents directly in cell culture. The eATP assay utilizes an optimized ATP detection chemistry that can be employed directly to assess live-cell kinetic responses for up to 24 h, with even longer exposures supported by a staggered reagent addition approach. The HMGB1 assay measures the protein's concentration at exposure endpoint in the same sample well using complementary luciferase fragment-labelled monoclonal antibodies. We tested the utility of the assays using U20S, Jurkat and U937 cells dosed with serial dilutions of known ICD inducers (doxorubicin, idarubicin, mitoxantrone, and bortezomib). The resulting data suggest the potency of eATP and HMGB1 release is dependent upon cell model and agent but can be reproducibly and robustly measured in 96 and 384 well environments. Further, the eATP assay produced remarkable early dose-dependent response resolution whereas the HMGB1 data provided a confirmatory post-mortem ICD parameter. This new ICD biomarker detection workflow may help to efficiently define and rank-order the capacity of new chemical entities to induce apoptosis and immunogenic cell death. Citation Format: Andrew L. Niles, Kevin R. Kupcho, Dan F. Lazar, James J. Cali. Improved DAMP assays for the in vitro assessment of immunogenic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1933.

Increased plasma levels of damage-associated molecular patterns during systemic anticancer therapy in patients with advanced lung cancer.

BackgroundImmunogenic cell death (ICD) characterized by the release of damage-associated molecular patterns (DAMPs) from dying cancer cells may contribute to the synergistic antitumor effect of cytotoxic chemotherapy combined with an immune checkpoint inhibitor. The kinetics of circulating DAMP levels in cancer patients have remained largely uncharacterized, however.MethodsWe evaluated the possible effects of various systemic anticancer therapy modalities on the kinetics of plasma DAMP concentrations in a prospective observational study of patients with advanced lung cancer. The plasma concentrations of high-mobility group box 1 (HMGB1), calreticulin (CRT), heat shock protein 70 (HSP70), annexin A1, and histone H3 were thus determined in 121 such patients at four time points during the first cycle of treatment.ResultsThe mean of the maximum fold change in HMGB1, HSP70, or annexin A1 concentration observed during treatment was significantly greater than the corresponding baseline value (P<0.005). The maximum fold changes in HMGB1 and CRT concentrations tended to be associated with clinical response as evaluated by RECIST criteria, although the changes in the levels of these two DAMPs were not correlated, suggestive of differential induction mechanisms. Among the various treatment modalities administered, platinum-based combination or single-agent chemotherapy tended to elicit robust increases in the concentrations of HMGB1 and CRT.ConclusionsSerial monitoring of plasma revealed that systemic anticancer therapy increased the circulating levels of HMGB1 and CRT and that these changes tended to be associated with clinical response, suggesting that agents capable of releasing these DAMPs into plasma might induce ICD in advanced lung cancer patients.

Role of Mitochondria-Derived Danger Signals Released After Injury in Systemic Inflammation and Sepsis.

Significance: Sepsis is a major public health concern, with high mortality and morbidity, especially among patients undergoing trauma. It is characterized by a systemic inflammatory response syndrome (SIRS) occurring in response to infection. Although classically associated with pathogens, many patients with SIRS do not have infection. The variability of the disease course cannot be fully explained by our current understanding of its pathogenesis. Thus, other factors are likely to play key roles in the development and progression of SIRS/sepsis. Recent Advances: Circulating levels of damage-associated molecular patterns (DAMPs) seem to correlate with SIRS/sepsis morbidity and mortality. Of the known DAMPs, those of mitochondrial (mt) origin have been of particular interest, since their DNA (mtDNA) and formyl peptides (mtFPs) resemble bacterial DNA and peptides, and hence, when released, may be recognized as "danger signals." Critical Issues: mtDAMPs released after tissue injury trigger immune responses similar to those induced by pathogens. Thus, they can result in systemic inflammation and organ damage, similar to that observed in SIRS/sepsis. We will discuss recent findings on the roles of mtDAMPs, particularly regarding the less recognized mtFPs, in the activation of inflammatory responses and development of SIRS/sepsis. Future Directions: There are no established methods to predict the course of SIRS/sepsis, but clinical studies reveal that plasma levels of mtDAMPs may correlate with the outcome of the disease. We propose that non-pathogen-initiated, mtDAMPs-induced SIRS/sepsis events need further studies aimed at early clinical recognition and better treatment of this disease.

The up-to-date pathophysiology of Kawasaki disease.

Kawasaki disease (KD) is an acute systemic vasculitis of an unknown aetiology. A small proportion of children exposed to severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) or infected by Yersinia reproducibly develop principal symptoms of KD in various ethnic areas, but not in all studies. These microbes provoke a rapid cell‐damaging process, called ‘pyroptosis’, which is characterised by a subsequent release of proinflammatory cellular components from damaged endothelial and innate immune cells. In agreement with these molecular events, patients with KD show elevated levels of damage‐associated molecular patterns derived from cell death. In addition, an overwhelming amount of oxidative stress‐associated molecules, including oxidised phospholipids or low‐density lipoproteins, are generated as by‐products of inflammation during the acute phase of the disease. These molecules induce abnormalities in the acquired immune system and activate innate immune and vascular cells to produce a range of proinflammatory molecules such as cytokines, chemokines, proteases and reactive oxygen species. These responses further recruit immune cells to the arterial wall, wherein inflammation and oxidative stress closely interact and mutually amplify each other. The inflammasome, a key component of the innate immune system, plays an essential role in the development of vasculitis in KD. Thus, innate immune memory, or ‘trained immunity’, may promote vasculitis in KD. Hence, this review will be helpful in understanding the pathophysiologic pathways leading to the development of principal KD symptoms and coronary artery lesions in patients with KD, as well as in subsets of patients with SARS‐CoV‐2 and Yersinia infections.

Induction of NF-κB inflammatory pathway in monocytes by microparticles from patients with systemic lupus erythematosus

BackgroundElevated levels of circulating microparticles (MPs) and molecules of the complement system have been reported in patients with systemic lupus erythematosus (SLE). Moreover, microparticles isolated from patients with SLE (SLE-MPs) contain higher levels of damage-associated molecular patterns (DAMPs) than MPs from healthy controls (CMPs). We hypothesize that the uptake of MPs by monocytes could contribute to the chronic inflammatory processes observed in patients with SLE. Therefore, the aim of this study was to evaluate the expression of activation markers, production of proinflammatory mediators, and activation of the NF-κB signaling pathway in monocytes treated with CMPs and SLE-MPs. MethodologyMonocytes isolated from healthy individuals were pretreated or not with pyrrolidine dithiocarbamate (PDTC) and cultured with CMPs and SLE-MPs. The cell surface expression of CD69 and HLA-DR were evaluated by flow cytometry; cytokine and eicosanoid levels were quantified in culture supernatants by Cytokine Bead Array and ELISA, respectively; and the NF-κB activation was evaluated by Western blot and epifluorescence microscopy. ResultsThe cell surface expression of HLA-DR and CD69, and the supernatant levels of IL-6, IL-1β, PGE2, and LTB4 were higher in cultures of monocytes treated with SLE-MPs than CMPs. These responses were blocked in the presence of PDTC, a pharmacological inhibitor of the NF-κB pathway, with concomitant reduction of IκBα and cytoplasmic p65, and increased nuclear translocation of p65. ConclusionsThe present findings indicate that significant uptake of SLE-MPs by monocytes results in activation, production of inflammatory mediators, and triggering of the NF-κB signaling pathway.