Phorbol 12-myristate 13-acetate Research Articles

Naked Antibody-Based Conditioning Targeting CD47 and CD117 Plus Janus Kinase Inhibition Enables Toxin-Free, Radiation-Free, and Chemotherapy-Free Fully Mismatched Allogeneic Hematopoietic Stem Cell Transplantation

INTRODUCTION: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for numerous malignant and non-malignant hematologic diseases. To enable engraftment of donor hematopoietic stem cells (HSCs), recipients undergo conditioning with chemotherapy and irradiation to make marrow space for incoming donor HSCs and suppress host immune responses that may lead to graft rejection. These cytotoxic conditioning regimens carry risks of severe toxicities which prevents many patients from accessing the lifesaving benefit of allo-HSCT. Previously, we developed fully ablative CD45- and cKit-targeted antibody-drug conjugates (ADC) which, when combined with Janus kinase 1/2 (JAK1/2) inhibitors, enabled fully mismatched allo-HSCT with full donor chimerism and minimal toxicity while providing durable antileukemia activity (Persaud et al (2022), ASH Abstract). However, even ADCs carry potential risks of acute and chronic toxicities that may preclude their common use as HSCT conditioning agents. Moreover, unlike malignant diseases, allo-HSCT for non-malignant diseases does not require the conditioning regimen to provide antitumor benefit and partial donor chimerism may be sufficient for cure, strongly favoring the use of less intense conditioning strategies with the fewest possible toxicities. We hypothesized that naked antibody-based conditioning strategies can achieve the therapeutic benefits of allo-HSCT for non-malignant hematologic diseases, obviating exposure to toxic ADC payloads. METHODS: We combined unconjugated antibodies targeting CD47 and cKit with JAK1/2 inhibition to perform minor histocompatibility antigen (miHA)-mismatched (129/Sv→B6-Ly5.1) and fully mismatched (BALB/c→B6) murine allo-HSCT. In some full mismatch HSCT experiments, recipients included B6-gp91phox -/- mice, which serve as a model of chronic granulomatous disease (CGD) due to their deficiency in a catalytic subunit of NADPH oxidase. Mice received 100 µg anti-CD47 (clone mIAP410; BioXcell) 8 days before HSCT (d-8) as a priming dose to reduce the severity of CD47 blockade-induced anemia. Next, mice received 500 µg anti-cKit (clone ACK2; BioLegend) on d-6 and daily 500 µg doses of anti-CD47 from d-6 through d-2. For JAK1/2 inhibition, mice received oral ruxolitinib (Incyte; 2 mg drug/g chow) starting d-3 or d-1 and continuing until d+28. On d0, recipients were transplanted with 20 x 10 6 whole bone marrow cells from 129/Sv (miHA) or BALB/c (fully mismatched) mice. Peripheral blood donor chimerism was followed monthly by flow cytometry for CD45.2 + (miHA) or H-2D d+ (fully mismatched) cells. Oxidative burst among granulocytes in B6-gp91phox -/- recipients was assessed by dihydrorhodamine 123 (DHR123) staining after phorbol 12-myristate 13-acetate (PMA) stimulation. RESULTS: CD47 and cKit antibodies plus ruxolitinib enabled multilineage engraftment in both miHA and fully mismatched allo-HSCT, including myeloid lineage donor chimerism of 95-99% (Figure 1A). As expected, CD47 blockade induced significant but transient morbidity from anemia during the peritransplant period; notably, a shorter duration of ruxolitinib treatment before HSCT (starting d-1 instead of d-3) improved the tolerability of the regimen without impacting engraftment success. Allo-HSCT of B6-gp91phox -/- mice led to near complete replacement of host granulocytes in peripheral blood with donor granulocytes, whose DHR123 positivity upon PMA stimulation indicated an intact oxidative burst capacity (Figure 1B). CONCLUSIONS: Combination of antibodies targeting CD47 and cKit with JAK1/2 inhibition provides a novel conditioning strategy for allo-HSCT in mouse models, achieving high-level, stable multilineage engraftment without radiation, chemotherapy, or exposure to toxic ADC payloads. The near complete donor chimerism among granulocytes made it particularly well suited to correcting phagocyte oxidase function in the gp91phox -/- model of CGD. Future studies seek to 1) mitigate the peritransplant anemia caused by CD47 blockade using Fc-silenced anti-CD47 reagents, 2) determine the proportions of donor- versus recipient-derived myeloid cells in non-hematopoietic tissues of allo-HSCT recipients, and 3) use murine infection models to evaluate the impact of allo-HSCT on host defense in transplanted B6-gp91phox -/- recipients.

Alpha-secretase dependent nuclear localization of the amyloid-β precursor protein-binding protein Fe65 promotes DNA repair

Fe65 is a brain enriched adaptor protein involved in various cellular processes, including actin cytoskeleton regulation, DNA repair and transcription. A well-studied interacting partner of Fe65 is the transmembrane amyloid-β precursor protein (APP), which can undergo regulated intramembrane proteolysis (RIP). Following β- and γ-secretase-mediated RIP, the released APP intracellular domain (AICD) together with Fe65 can translocate to the nucleus and regulate transcription. In this study, we investigated if Fe65 nuclear localization can also be regulated by different α-secretases, also known to participate in RIP of APP and other transmembrane proteins. We found that in both Phorbol 12-myristate 13-acetate and all-trans retinoic acid differentiated neuroblastoma cells a strong negative impact on Fe65 nuclear localization, equal to the effect observed upon γ-secretase inhibition, could be detected following inhibition of all three (ADAM9, ADAM10 and ADAM17) α-secretases. Moreover, using the comet assay and analysis of Fe65 dependent DNA repair associated posttranslational modifications of histones, we could show that inhibition of α-secretase-mediated Fe65 nuclear translocation resulted in impaired capacity of the cells to repair DNA damage. Taken together this suggests that α-secretase processing of APP and/or other Fe65 interacting transmembrane proteins play an important role in regulating Fe65 nuclear translocation and DNA repair.

Multiomic Characterization of Myelodysplastic Neoplasms (MDS) with Micromegakaryocytes Highlights the Role of EZH2-RUNX1 deregulation in Disease Physiopathology and Response to Targeted Therapies

Micromegakaryocytes (micro-MKs), an unequivocal myeloid dysplastic feature in adults, are present in 15-20% of myelodysplastic neoplasms (MDS). We recently associated the detection of these small megakaryocytes with a mono- or bi-lobated nucleus in bone marrow (BM) smears of MDS patients with an unfavorable prognosis. The aim of the present study was to elucidate the molecular bases of micro-MK formation and its impact on MDS response to targeted therapies. We selected retrospectively a cohort of 40 MDS patient samples subjected to NGS myeloid panel and RNA sequencing and carried out a morphological re-evaluation that included micro-MKs recounting. Cases with isolated del(5q) or SF3B1 mutation were excluded. MDS cases were selected according to the following morphological findings: no dismegakaryopoiesis, dismegakaryopoiesis without micro-MK, low rate of micro-MK (<10%; micro-MK low) and high rate of micro-MK (>40%; micro-MK high). No patient harbored an intermediate (10-40%) rate of micro-MK. A comparative characterization of 8 representative patients was carried out subjecting diagnosis BM samples to Tapestri and Chromium single cell proteogenomic and transcriptomic platforms, gene set enrichment analysis, and deep cytogenetic characterization by optical genome mapping. Functional validation studies were performed using the megakaryoblastic cell line MEG01 differentiated to a mature MK phenotype by exposure to phorbol myristate acetate (PMA), as well as using the CRISPR-Cas9 tool for gene editing. Finally, activity of investigational drugs including 5-azacytidine, lenalidomide, venetoclax, rigosertib and pevonedistat, was evaluated in MEG01-activated cells and in vivo using a chicken embryo chorioallantoic membrane (CAM) model of MDS patient-derived xenograft (PDX). The most frequently mutated genes across all cohorts were ASXL1 (58%), SRSF2 (38%), RUNX1 (30%), TET2 (30%), STAG2 (20%), EZH2 (18%), DDX41 (10%), U2AF1 (10%), and ZRSR2 (10%) . Interestingly, EZH2 and RUNX1 mutations were enriched in the micro-MK high group ( EZH2: 0%, 10%, 14.3% vs. 66.7%, p 0.006; RUNX1: 0%, 20%, 35.7% vs. 83.3%, p 0.003), and both mutations co-occurred in 85.7% (6/7) of the EZH2 mutated cases. Single cell analyses revealed an enrichment of RUNX1 alterations in three populations within micro-MK high BM samples, including hematopoietic stem and progenitor cells, non-classical monocytes and immature erythroid cells. These samples were also characterized by a 5-fold increase in MK progenitor abundance and by the upregulation of a set of 10 EZH2-repressed genes in neutrophil-like cells and myeloid progenitors, with 7q loss presumably affecting the EZH2 locus in 50% of the cases with micro-MK. Using the in vitro MK differentiation model, we further demonstrated that EZH2 downregulation hampered the acquisition of MK surface markers CD41 and CD61, and that venetoclax was the sole agent retaining notable cytotoxic activity in differentiated cells when compared to undifferentiated megakaryoblasts. Accordingly, in a first-in-kind in vivo CAM-MDS transplant model, venetoclax was able to impair BM infiltration by human cells in a PDX model of micro-MK+ MDS. In summary, supporting the notion that EZH2 and RUNX1 exert a crucial role in the regulation of megakaryopoiesis, our present findings suggest that deregulation of EZH2-RUNX1 activity by loss-of-function mutation and/or genetic deletion affecting myeloid precursors, impairs a proper megakaryocytic differentiation process in MDS. Thus, this phenomenon may be considered as a characteristic feature of MDS cases with an elevated proportion of micro-MK. Of note, preliminary in vitro and in vivo results support the use of venetoclax for the treatment of this subgroup of patients with dismal outcome.

The PMA phorbol ester tumor promoter increases canonical Wnt signaling via macropinocytosis

Activation of the Wnt pathway lies at the core of many human cancers. Wnt and macropinocytosis are often active in the same processes, and understanding how Wnt signaling and membrane trafficking cooperate should improve our understanding of embryonic development and cancer. Here, we show that a macropinocytosis activator, the tumor promoter phorbol 12-myristate 13-acetate (PMA), enhances Wnt signaling. Experiments using the Xenopus embryo as an in vivo model showed marked cooperation between the PMA phorbol ester and Wnt signaling, which was blocked by inhibitors of macropinocytosis, Rac1 activity, and lysosome acidification. Human colorectal cancer tissue arrays and xenografts in mice showed a correlation of cancer progression with increased macropinocytosis/multivesicular body/lysosome markers and decreased GSK3 levels. The crosstalk between canonical Wnt, focal adhesions, lysosomes, and macropinocytosis suggests possible therapeutic targets for cancer progression in Wnt-driven cancers.

The PMA phorbol ester tumor promoter increases canonical Wnt signaling via macropinocytosis.

Activation of the Wnt pathway lies at the core of many human cancers. Wnt and macropinocytosis are often active in the same processes, and understanding how Wnt signaling and membrane trafficking cooperate should improve our understanding of embryonic development and cancer. Here, we show that a macropinocytosis activator, the tumor promoter phorbol 12-myristate 13-acetate (PMA), enhances Wnt signaling. Experiments using the Xenopus embryo as an in vivo model showed marked cooperation between the PMA phorbol ester and Wnt signaling, which was blocked by inhibitors of macropinocytosis, Rac1 activity, and lysosome acidification. Human colorectal cancer tissue arrays and xenografts in mice showed a correlation of cancer progression with increased macropinocytosis/multivesicular body/lysosome markers and decreased GSK3 levels. The crosstalk between canonical Wnt, focal adhesions, lysosomes, and macropinocytosis suggests possible therapeutic targets for cancer progression in Wnt-driven cancers.

CD138 promotes the accumulation and activation of autoreactive T cells in autoimmune MRL/lpr mice.

Autoreactive T cells, specifically CD138+ (syndecan-1) T cells produced in Fas-deficient systemic lupus erythematosus (SLE) mouse models, were shown to significantly promote the generation of autoantibodies. In the present study, Murphy Roths Large lymphoproliferative (MRL/lpr) lupus mice were used to investigate the role of CD138 protein expression in T cells in the progression of SLE. Measurement of flow cytometry, immunofluorescence and Luminex were performed to determine the effect of CD138 on T cells in MRL/lpr mice. The results demonstrate that CD138+ T cells induce apoptosis via a Fas-dependent pathway. CD138 protein expression in T cells of MRL/lpr mice significantly reduced T cell apoptosis and contributed to the accumulation of T cells and double negative (DN) T cells, whilst simultaneously promoting T cell activation in Fas-deficient lupus mice. CD138 protein expression in DN T cells also significantly increased the protein expression of Fas ligand to enhance the cytotoxicity of DN T cells. Furthermore, phorbol 12-myristate 13-acetate and ionomycin (PI) stimulation reduced CD138 protein expression in CD3+ T cells and prevented CD138+ T cell accumulation by inducing specific apoptosis. PI stimulation also activated T cells in MRL/lpr mice to increase CD69 protein expression. CD69 protein expression in CD138+ T cells significantly increased the frequency of apoptotic CD138+ T cells. In addition, results from the present study demonstrated that CD138- T cells of MRL/lpr lupus mice had an activation defect. CD138 protein expression in T cells significantly reversed the defective activation and activating T cells could significantly reduce CD138 protein expression in CD3+ T cells of MRL/lpr mice. This suggests that CD138 protein expression in CD3+CD138- T cells of MRL/lpr mice may be a consequence of the impaired activation in autoreactive T cells prior to exposure to self-antigens by the immune system. CD138 expression in autoreactive T cells has a central role in promoting the progression and development of autoimmune response in MRL/lpr mice.

A truncated HIV Tat demonstrates potent and specific latency reversal activity.

A major barrier to HIV-1 cure is caused by the pool of latently infected CD4 T-cells that persist under combination antiretroviral therapy (cART). This latent reservoir is capable of producing replication-competent infectious viruses once prolonged suppressive cART is withdrawn. Inducing the reactivation of HIV-1 gene expression in T-cells harboring a latent provirus in people living with HIV-1 under cART may result in depletion of this latent reservoir due to cytopathic effects or immune clearance. Studies have investigated molecules that reactivate HIV-1 gene expression, but to date, no latency reversal agent has been identified to eliminate latently infected cells harboring replication-competent HIV in cART-treated individuals. Stochastic fluctuations in HIV-1 tat gene expression have been described and hypothesized to allow the progression into proviral latency. We hypothesized that exposing latently infected CD4+ T-cells to Tat would result in effective latency reversal. Our results indicate the capacity of a truncated Tat protein and mRNA to reactivate HIV-1 in latently infected T-cells ex vivo to a similar degree as the protein kinase C agonist: phorbol 12-myristate 13-acetate, without T-cell activation or any significant transcriptome perturbation.

Akt1 players promote PMA U937 cell line differentiation into macrophage-like cells

PurposeMonocytes are a leukocytes’ subset that plays an important role in immunity. Protein kinase B (AKT) is involved in monocytes' survival, proliferation and differentiation. Using phorbol 12-myristate 13-acetate (PMA) as an inducer for cell line U937 differentiation into macrophage-like cells may be used as a model for cancer cell therapy or other biomedical research studies. The authors investigated the Akt1 signaling pathway's involvement with PMA as a differentiating agent and survival in the U937 cell line.Design/methodology/approachPMA was utilized to stimulate the differentiation of the U937 cell line into macrophage-like cells at a concentration of 10 nM. Akt1-phosphorylated Serine 473, Bad-phosphorylated Serine 136 and Caspase9-phosphorylated Serine 196 were tested by flow cytometry for the involvement of the Akt1 signaling pathway during differentiation in addition to the expression of CD14, CD206 and CD83. DNA cell cycle variation analysis was done using PI staining and cell viability and apoptosis detection using Annexin V and PI flow cytometry.FindingsThere was a decrease in phosphorylated Akt1 and Bad activation and an increase in Caspase9 activation, with an increase in surface markers CD14, CD206 and CD83 acquired by PMA-differentiated cells. DNA cell cycle analysis revealed cell accumulation in the G2/M phase and fewer cells in the S phase of PMA-induced U937. Apoptosis induction for Ly294002 or Wortmannin-inhibited cells and part of PMA-induced cells were detected.Originality/valueThese results may be used to create a model for biomedical research studies and advance the understanding of the mechanism involving differentiation of the U937 cell line.

Uridine Diphosphate Glucose (UDP-G) Activates Oxidative Stress and Respiratory Burst in Isolated Neutrophils.

The extracellular purinergic agonist uridine diphosphate glucose (UDP-G) activates chemotaxis of human neutrophils (PMN) and the recruitment of PMN at the lung level, via P2Y14 purinergic receptor signaling. This effect is similar to the activation of PMN with N-formyl-methionyl-leucyl-phenylalanine (fMLP), a mechanism that also triggers the production of superoxide anion and hydrogen peroxide via the NADPH oxidase system. However, the effects of UDP-G on this system have not been studied. Defects in the intracellular phagocyte respiratory burst (RB) cause recurrent infections, immunodeficiency, and chronic and severe diseases in affected patients, often with sepsis and hypoxia. The extracellular activation of PMN by UDP-G could affect the RB and oxidative stress (OS) in situations of inflammation, infection and/or sepsis. The association of PMNs activation by UDP-G with OS and RB was studied. OS was evaluated by measuring spontaneous chemiluminescence (CL) of PMNs with a scintillation photon counter, and RB by measuring oxygen consumption with an oxygen Clark electrode at 37 °C, in non-stimulated cells and after activation (15 min) with lipopolysaccharides (LPS, 2 µg/mL), phorbol myristate acetate (PMA, 20 ng/mL), or UDP-G (100 μM). The stimulation index (SI) was calculated in order to establish the activation effect of the three agonists. After stimulation with LPS or PMA, the activated PMNs (0.1 × 106 cells/mL) showed an increase in CL (35%, p < 0.05 and 56%, p < 0.01, SI of 1.56 and 2.20, respectively). Contrariwise, the stimulation with UDP-G led to a decreased CL in a dose-dependent manner (60%, 25 μM, p < 0.05; 90%, 50-150 μM, p < 0.001). Nonetheless, despite the lack of oxidative damage, UDP-G triggered RB (SI 1.8) in a dose-dependent manner (38-50%, 100-200 μM, p < 0.0001). UDP-G is able to trigger NADPH oxidase activation in PMNs. Therefore, the prevention of OS and oxidative damage observed upon PMN stimulation with UDP-G indicates an antioxidant property of this molecule which is likely due to the activation of antioxidant defenses. Altogether, LPS and UDP-G have a synergistic effect, suggesting a key role in infection and/or sepsis.

Trichinella spiralis excretory/secretory products from adult worms inhibit NETosis and regulate the production of cytokines from neutrophils

Upon encountering exogenous pathogens, polymorphonucleocytes (PMNs) engage in various processes to destroy them, including releasing neutrophil extracellular traps (NETs) that trap pathogens and induce phagocytosis and cytokine production. Parasites have unique strategies with which to evade the host's immune response. However, the strategy employed by Trichinella spiralis in response to the reaction of PMNs has yet to be elucidated. This study explored the effect of excretory/secretory products (ESP) on three major functions: NETs, phagocytosis, and cytokine production. Specifically, PMNs were pre-treated with the ESP of 3-day-old adults and then stimulated with phorbol 12-myristate 13-acetate (PMA). We found that in PMNs pretreated with ESP, PMA-induced NET generation was suppressed by ESP. ROS production is a hallmark of PMA-induced NETosis. The LDH assay results showed that ESP inhibits NETs by suppressing ROS rather than promoting PMN death. Furthermore, ESP enhanced Escherichia coli engulfment by PMNs, improving overall phagocytic function. Finally, cytokine analysis revealed an increase in pro-inflammatory cytokine IL-1β, and other cytokines (IL-10, TNF-α), while IL-4 displayed a significant reduction. In conclusion, this study has unraveled T. spiralis' evasion and regulation mechanisms against innate immune cells, providing insights into parasite strategies to manipulate host immunity, potentially informing new treatments for NET-related autoimmune diseases.Graphical

Cortical spheroid on perfusable microvascular network in a microfluidic device.

Human induced pluripotent stem cell (hiPSC)-derived brain spheroids can recapitulate the complex cytoarchitecture of the brain, as well as the genetic/epigenetic footprint of human brain development. However, hiPSC-derived 3D models such as spheroid and organoids does not have a perfusable microvascular network, which plays a vital role in maintaining homeostasis in vivo. With the critical balance of positive and negative angiogenic modulators, 3D microvascular network can be achieved by angiogenesis. This paper reports on a microfluidic-based three-dimensional, cortical spheroid grafted on the vascular-network. Vascular network was formed by inducing angiogenic sprouting using concentration gradient-driven angiogenic factors in the microfluidic device. We investigate critical factors for angiogenic vascular network formation with spheroid placement, including 1) a PKCα activator, phorbol-12-myristate-13-acetate (PMA); 2) orientation of endothelial cells under perfusion and permeability of vascular network; 3) effect of extracellular matrix (ECM) types and their densities on angiogenesis; and 4) integration with cortical spheroid on vascular network. This paper demonstrates proof of concept for the potential utility of a membrane-free in vitro cortical spheroid tissue construct with perfusable microvascular network that can be scaled up to a high throughput platform. It can provide a cost-effective alternative platform to animal testing by modeling brain diseases and disorders, and screening drugs.

Formononetin ameliorates the LPS-induced inflammatory response and apoptosis of neuronal cells via NF-κB/NLRP3 signaling pathway.

The objective of this study was to investigate the impact of formononetin on cellular apoptosis and inflammatory responses following spinal cord injury (SCI), as well as the underlying mechanisms involved. In this study, PC12 cells were treated with lipopolysaccharide (LPS) and different concentrations of Formononetin (FT) (50μM, 100μM, 200μM). To confirm the effect of nuclear factor-κB (NF-κB)/NLR family pyrin domain containing 3 (NLRP3) signaling pathways, the cells in the phorbol-12-myristate-13-acetate (PMA) group were treated with 0.1μmol/L PMA (NF-κB/NLRP3 signaling pathway activators). The lactate dehydrogenase (LDH) concentration and cell viability, proliferating cell nuclear antigen (PCNA) fluorescence intensity, and cell apoptosis were determined using an LDH kit, Cell Counting Kit-8 (CCK-8), immunofluorescence, and flow cytometry assays, respectively. Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-16 (IL-6) expression levels were detected by quantitative ELISA assay. The expression of proteins related to the NF-κB/NLRP3 signaling pathway was detected by western blotting. Our results showed that LPS increased LDH levels in PC12 cells, suggesting that inflammation caused PC12 cell damage. However, the PC12 cell damage was decreased by methylprednisolone. Formononetin promotes cell survival and proliferation, and prevents apoptosis in a concentration-dependent manner. Formononetin reduced the TNF-α, IL-1β, and IL-6 levels in the LPS-treated model. Moreover, formononetin decreased the levels of p-p65 NF-κB and NLRP3 in PC12 cells. We conclude that formononetin ameliorated the inflammatory response and apoptosis in LPS-induced inflammatory injury in neuronal cells via the NF-κB/NLRP3 signaling pathway.

Comparison of whole blood cytokine immunoassays for rapid, functional immune phenotyping in critically ill patients with sepsis

BackgroundSepsis is characterized by highly heterogeneous immune responses associated with a spectrum of disease severity. Methods that rapidly and sensitively profile these immune responses can potentially personalize immune-adjuvant therapies for sepsis. We hypothesized that the ELLA microfluidic approach to measure cytokine production from the whole blood of septic and critically ill patients would deliver faster, more precise results than the existing optic-driven ELISpot quantification. We tested our hypothesis by measuring ex vivo-stimulated production of TNF and IFNγ in critically ill and septic patients (n = 22), critically ill and non-septic patients (n = 10), and healthy volunteers (n = 10) through both ELLA and ELISpot immunoassays. Blood samples were subjected to one of three stimulants for 4 h or 18 h durations during days 1, 7–10, and 14 of critical illness. Stimulants for lymphocytes included anti-CD3/anti-CD28 and phorbol 12-myristate 13-acetate (PMA), whereas LPS was used for monocytes. Stimulated TNF and IFNγ concentrations were then associated with 30-day mortality.ResultsBoth ELISpot and ELLA immunoassays showed substantial agreement in TNF concentrations post 4 h and 18 h LPS stimulation, with concordance correlation coefficients at 0.62 and 0.60, respectively. ELLA had a broad dynamic measurement range and provided accurate TNF and IFNγ readings at both minimal and elevated cytokine concentrations (with mean coefficients of variation between triplicate readings at 2.1 ± 1.4% and 4.9 ± 7.2%, respectively). However, there was no association between the ELLA-determined cytokine concentrations on the first day of critical illness and 30-day mortality rate. In contrast, using the ELISpot for cytokine quantification revealed that non-survivors had reduced baseline TNF levels at 18 h, decreased LPS-induced TNF levels at 18 h, and diminished TNF levels post 4 h/18 h anti-CD3/28 stimulation.ConclusionsOur study affirms the feasibility of obtaining dependable immune phenotyping data within 6 h of blood collection from critically ill patients, both septic and non-septic, using the ELLA immunoassay. Both ELLA and ELISpot can offer valuable insights into prognosis, therapeutic strategies, and the underlying mechanisms of sepsis development.

The mechanism of acrolein exposure inhibited the release of neutrophil extracellular traps: By reducing respiratory burst and Raf/MEK/ERK pathway and promote cell apoptosis

Acrolein (AC) is a highly toxic volatile substance in the environment, and studies have found that excessive AC had a toxic effect on the immune system. Neutrophils are the first line of defense against pathogen invasion. The release of neutrophil extracellular traps (NETs) is a protective mechanism for neutrophils, and its release is affected by environmental pollutants. However, the effect of AC on NETs release and its mechanism remains unclear. In this study, chicken peripheral blood neutrophils were pretreated with 20 μM AC and treated with 5 μM Phorbol 12-myristate 13-acetate (PMA) to stimulate the release of NETs. The results showed that AC exposure significantly inhibited the release of NETs induced by PMA, respiratory burst, and the expression levels of phospho-rapidly accelerated fibrosarcoma (p-Raf), phospho-mitogen-activated extracellular signal-regulated kinase (p-MEK) and phospho-extracellular regulated protein kinases (p-ERK). In addition, AC exposure significantly inhibited the expression of B-cell lymphoma-2 (Bcl-2) and promoted the expression of apoptotic factors Bcl2-Associated X (Bax), cytochrome c (Cyt C), cysteinyl aspartate specific proteinase 9 (Casp 9) and cysteinyl aspartate specific proteinase 3 (Casp 3). Further inhibition of neutrophil apoptosis significantly improved the release of NETs. The above results indicated that AC exposure led to a decrease in the formation of NETs, which is caused by excessive AC-induced neutrophil apoptosis. Our study clarified the immune toxicity mechanism of AC on chickens, which is of great significance and reference value for protecting the ecological environment and poultry health.

SAT297 Major Immunophenotypic Abnormalities In Patients With Primary Adrenal Insufficiency Of Different Etiology

Abstract Disclosure: H.F. Nowotny: None. T. Marchant Seiter: None. J. Ju: None. A. Gottschlich: None. H. Schneider: None. S. Zopp: None. F. Vogel: None. L. Tschaidse: None. M.K. Auer: None. C. Lottspeich: None. S. Kobold: None. S. Rothenfusser: None. F. Beuschlein: None. M. Reincke: None. L. Braun: None. N. Reisch: None. Background: Increased risk of infection, adrenal crises and a higher mortality rate have been reported for patients with primary adrenal insufficiency (PAI). Such dismal outcomes have been inferred to immune cell dysregulation as a consequence of unphysiological cortisol replacement. As the immune landscape of patients with different types of PAI has not been systematically explored, we set out to immunophenotype PAI patients with different causes of glucocorticoid deficiency. Methods: The present cross-sectional single center study includes 28 patients with congenital adrenal hyperplasia (CAH), 27 patients after bilateral adrenalectomy due to Cushing’s syndrome (BADx), 21 patients with Addison’s disease (AD) and 52 healthy controls. All patients with PAI were on a stable glucocorticoid replacement regimen using a median dose of 25 mg hydrocortisone (IQR 5 mg for BADx and AD and IQR 10 mg for CAH) per day. Heparinized blood samples were processed to isolate peripheral blood mononuclear cells. All samples were processed for analysis of immune cell subsets using multicolor flow cytometry after four-hour stimulation with phorbol myristate acetate (PMA)/Ionomycin, as well as analysis of natural killer (NK-) cell cytotoxicity and clock gene expression. Results: Analysis of immune cell profiles revealed reduced percentages of IFNγ-secreting T helper (Th1) in AD (p = 0.0024) and cytotoxic (Tc1) cells in CAH (p = 0.0055) and AD patients (p = 0.0075) compared to controls. Moreover, we observed a downregulation of the percentage of IL-4+ Th2 cells in AD patients (p = 0.0157) and Tc2 cells in AD (p = 0.0154) and CAH patients (p = 0.0012) compared to controls. Th17 and Tc17 cells were also downregulated in patients with AD compared to the other patient groups and controls (p &amp;lt; 0.0001). NK-cell cytotoxicity (NKCC) using bioluminescence measurements was reduced in all subsets of PAI patients compared to controls with the lowest change in patients with CAH (mean specific lysis 57.5% in controls, 21.7% in CAH, -0.5% in AD and -28.7% in BADx). The percentage of activated NK-cells was upregulated in BADx (p = 0.0008) and AD patients (p = 0.0348) compared to controls, also expressed as an increase in CD107a expression as a marker of NK-cell degranulation (BADx p &amp;lt; 0.0001; AD p = 0.0002). While the percentage of NK-cell activating receptor expressing NK-cells did not differ from controls, expression percentage of NK-cell inhibiting receptor CD94 was upregulated in both BADx and AD patients (p &amp;lt; 0.0001). Conclusion: In patients with three different etiologies of PAI, distinct differences in T- and NK-cell-phenotypes became apparent despite the use of same GC preparation and dose. Our results highlight so far unsuspected differences in immune cell composition and NK-cell function in PAI patients of different causes and suggest disease-specific alterations that might necessitate disease-specific treatment. Presentation: Saturday, June 17, 2023

Ursolic Acid Alleviates Neuroinflammation after Intracerebral Hemorrhage by Mediating Microglial Pyroptosis via the NF-κB/NLRP3/GSDMD Pathway.

The neuroinflammatory response after intracerebral hemorrhage (ICH) causes a large amount of neuronal loss, and inhibiting the inflammatory response can improve the prognosis. In previous laboratory studies and clinical trials, ursolic acid (UA) inhibited the inflammatory response, but whether it can be administered to inhibit the neuroinflammatory response after cerebral hemorrhage is unknown. The aim of this study was to investigate the effects of ursolic acid after cerebral hemorrhage. Online databases were used to obtain potential therapeutic targets of ursolic acid for the treatment of cerebral hemorrhage, and possible mechanisms were analyzed by KEGG, GO, and molecular docking. A rat model of cerebral hemorrhage was established using collagenase, and an in vitro cerebral hemorrhage model was constructed by adding hemin to BV2 cell culture medium. Enzyme-linked immunosorbent assay (ELISA), Western blotting (WB), immunofluorescence, TUNEL staining, and calcein/PI staining were used to investigate the degree of microglial M1 polarization, changes in the levels of inflammatory factors, activation of the NF-κB pathway, and changes in the indicators of cellular death after ursolic acid treatment. In addition, phorbol 12-myristate 13-acetate (PMA) was used to activate the NF-κB pathway to verify that ursolic acid exerts its anti-neuroinflammatory effects by regulating the NF-κB/NLRP3/GSDMD pathway. Network pharmacology and bioinformatics analyses revealed that ursolic acid may exert its therapeutic effects on cerebral hemorrhage through multiple pathways. Together, in vivo and in vitro experiments showed that ursolic acid inhibited microglial M1 polarization and significantly reduced the levels of p-NF-κB, GSDMD-N, cleaved caspase-1, TNF-α, IL-6, and IL-1β, which were significantly inhibited by the use of PMA. Ursolic acid inhibits microglial pyroptosis via the NF-κB/NLRP3/GSDMD pathway to alleviate neuroinflammatory responses after cerebral hemorrhage.

PKC regulates αKlotho gene expression in MDCK and NRK-52E cells

Particularly expressed in the kidney, αKlotho is a transmembrane protein that acts together with bone hormone fibroblast growth factor 23 (FGF23) to regulate renal phosphate and vitamin D homeostasis. Soluble Klotho (sKL) is released from the transmembrane form and controls various cellular functions as a paracrine and endocrine factor. αKlotho deficiency accelerates aging, whereas its overexpression favors longevity. Higher αKlotho abundance confers a better prognosis in cardiovascular and renal disease owing to anti-inflammatory, antifibrotic, or antioxidant effects and tumor suppression. Serine/threonine protein kinase C (PKC) is ubiquitously expressed, affects several cellular responses, and is also implicated in heart or kidney disease as well as cancer. We explored whether PKC is a regulator of αKlotho. Experiments were performed in renal MDCK or NRK-52E cells and PKC isoform and αKlotho expression determined by qRT-PCR and Western Blotting. In both cell lines, PKC activation with phorbol ester phorbol-12-myristate-13-acetate (PMA) downregulated, while PKC inhibitor staurosporine enhanced αKlotho mRNA abundance. Further experiments with PKC inhibitor Gö6976 and RNA interference suggested that PKCγ is the major isoform for the regulation of αKlotho gene expression in the two cell lines. In conclusion, PKC is a negative regulator of αKlotho gene expression, an effect which may be relevant for the unfavorable effect of PKC on heart or kidney disease and tumorigenesis.

The state of mitochondrial membrane potential in peripheral blood leukocytes of patients with chronic obstructive pulmonary disease

Introduction. Mitochondria provide energy homeostasis of the cell by maintaining an optimal transmembrane electrochemical gradient (ΔΨm), which does not allow excessive formation of reactive oxygen species (ROS). However, under conditions of pathology, the normal functioning of mitochondria is disrupted, which can lead to ATP deficiency and/or increased production of ROS.Aim. The aim of this study was to investigate the ΔΨm parameters and their relationship with the expression of TRP channels in peripheral blood leukocytes of patients with chronic obstructive pulmonary disease (COPD).Materials and methods. The study included 23 patients with COPD of varying severity, 8 smokers without signs of bronchial obstruction and 9 healthy volunteers who had never smoked. All subjects underwent spirometry to assess the lung function. ΔΨm was determined by staining the cells with tetramethylrhodamine ethyl ester (TMRE) and measuring the fluorescent signal by flow cytometry, under basal conditions and pro-inflammatory stimulation with phorbol-12-myristate-13-acetate (PMA).Results. We found that COPD patients were characterized by a significant increase in basal ΔΨm of monocytes (161.8 [153.8; 206.8] vs. 129.3 [75.5; 161.8], p=0.03) and lymphocytes (209,7 [184.7; 257.8] vs. 122.5 [67.9; 164.3], p=0.003) as compared with the control group. Stimulation of cells with PMA led to multidirectional changes in ΔΨm, while its increased level was still preserved in COPD. In monocytes of COPD patients, a decrease in ΔΨm in response to PMA stimulation was prevalent (75%), while in the majority (53.9%) of individuals in the control group ΔΨm, on the contrary, increased (p=0.08). In addition, among COPD patients, an increase in ΔΨm in monocytes was accompanied by an enhanced expression of TRPV4, while in the control group, among individuals with positive dynamics of ΔΨm, TRPV4 expression was, on the contrary, reduced.Conclusion. The increased level of ΔΨm in the mononuclears of COPD patients is consistent with previously detected enhanced ROS production, but does not support the assumption about energy deficit in the cells. The revealed differences in the relationship between TRPV4 expression and ΔΨm dynamics may indicate the presence of pathological features in TRP signaling in COPD patients.

B-075 Biophysical Changes of Leukocyte Activation (and NETosis) in the Cellular Host Response to Sepsis

Abstract Background During sepsis, neutrophils, upon activation, undergo biophysical and biochemical changes that may terminate in the release of neutrophil extracellular traps (NETs) (1–3). The IntelliSep test is a rapid sepsis diagnostic that assesses immune activation by quantifying biophysical properties of leukocytes from whole blood in &amp;lt;10 minutes. The test results in the IntelliSep Index (ISI), ranging between 0.1–10.0, stratified into three interpretation bands (Band 1, Band 2, and Band 3) based on the probability of the clinical syndrome of sepsis (4, 5). We investigated the correlation between the IntelliSep result and immunological changes observed during leukocyte activation through in-vitro studies and using clinical samples from non-septic and septic patients. Methods In-vitro Studies: Healthy blood samples were collected between April - August 2022 from 18 volunteers at a blood donor center. Aliquots of each blood sample were incubated for 10 minutes with phorbol myristate acetate (PMA) in phosphate-buffered saline at one of 3 concentrations: 0, 200, and 400 nM prior to the IntelliSep test (3 repeats per donor per concentration). Clinical Studies: Subjects with signs or symptoms of infection were enrolled from Emergency Departments (EDs) in three similar but distinct prospective cohort studies (February 2016—September 2019) (1, 2). The IntelliSep test was performed on a aliquot of fresh whole blood from each subject, and remnant plasma was prepared and frozen for later analysis. Plasma levels of neutrophil elastase-DNA (NE DNA) and citrullinated histone H3-DNA (Cit-H3 DNA) complexes were quantified using previously reported custom ELISAs 3,6. Subjects were retrospectively adjudicated as sick (adjudicated as septic with SOFA scores peaking on the day of enrollment compared to two subsequent days) and “healthy” (SOFA for day of enrollment &amp;lt;2, hospital length of stay &amp;lt;3 days). Results Significant increases in ISI scores were observed with increasing concentration of PMA in healthy blood samples (0 and 200: P &amp;lt; 10−10; 0 and 400: P &amp;lt; 10−10). The clinical analysis cohort consisted of 39 “sick” and 42 “healthy” subjects. Linear correlation was observed between the ISI and NE DNA and Cit-H3 DNA quantities with significant increases across ISI Interpretation Bands (Band 1 to Band 3: P &amp;lt; 0.001). Conclusion Correlation of increasing ISI and PMA concentration supports the hypothesis that the biophysical changes measured by the IntelliSep test result from leukocyte activation. Observed correlation between the ISI and NET quantities suggest that NET formation is one of the activation pathways that result in the biophysical changes measured in the ISI. Together these experiments support that biophysical changes of leukocyte activation measured using a cellular host response test could provide a window into a patient’s state of dysregulated immunity and may have the potential to aid ED physicians in timely diagnosis of sepsis.

B-144 A Comparative Analysis of Unsupervised Machine Learning Algorithms for Polyploidy Analysis Using Flow Cytometry

Abstract Background Recently, there has been a growing interest in using cell differentiation inducers to produce platelets in vitro by inducing megakaryocytic differentiation in cell lines. During this process, cells undergo polyploidization through endomitosis. To measure the degree of polyploidy, cell cycle analysis by flow cytometry is commonly used. However, manual gating can be time-consuming and prone to human error, especially when analyzing large datasets. To address this issue, this study aimed to compare the performance of unsupervised machine learning algorithms for polyploidy analysis of flow cytometry data. Methods The K562 cell line (Korea Cell Line Bank, Korea) was cultured under four conditions, including 1 nM and 2.5 nM of PMA (phorbol 12-myristate 13-acetate; Sigma Aldrich, USA) and 5 μg/mL of phytosphingosine (TCI, Japan), for four days. Propidium iodide (Sigma Aldrich, USA) was used to stain the cells for ploidy analysis, and flow cytometry (Navios, Beckman Coulter Inc, USA) was used to obtain the intensity values. Twelve flow cytometry measurements were obtained for K562 cells cultured under four different conditions, with each condition tested in triplicate. Unsupervised machine learning algorithms, including K-Means, Bisecting K-Means (BKM), Mini Batch K-Means (MBKM), Agglomerative Clustering (AC), Gaussian Mixture Models (GMM), K-Medoids, and Partitioning Around Medoids (PAM), were used to generate boundaries among three clusters (2n, 4n, and ≥8n). The boundary values generated by each algorithm were comparatively analyzed by calculating the standard derivation index (SDI) on each boundary. The concordance of each algorithm with manual gating of flow cytometry data was evaluated by calculating residual errors. Results A total of 168 SDI values were obtained from seven different unsupervised machine learning algorithms for two boundaries among 2n, 4n, and ≥8n clusters of four culture conditions in triplicate. When comparing the average (±95% confidence interval) of SDI for each algorithm for the entire dataset, K-Means showed the most acceptable average SDI value of −0.07 (±0.17) with the narrowest confidence interval followed by −0.13 (±0.54) of AC, −0.26 (±0.18) of MBKM, −0.53 (±0.2) of K-Medoids, −0.58 (±0.2) of PAM, 0.74 (±0.3) of GMM, and 0.84 (±0.35) of BKM. In addition, the average (±95% confidence interval) of the absolute value of the residual errors of each algorithm was calculated, with K-Means showing the lowest value of 6.67 (±2.3), indicating higher concordance with manual gating. AC, MBKM, K-Medoids, PAM, BKM, and GMM had higher average residual errors of 10.04 (±2.5), 10.46 (±4.07), 11.13 (±4.65), 12.04 (±4.83), 12.54 (±3.0), and 12.75 (±3.5), respectively. Conclusion K-means demonstrated the best performance for clustering each ploidy among the seven unsupervised machine learning algorithms tested for the flow cytometry data. In addition, the algorithms achieved acceptable concordance with manual gating in most cases in this study. Therefore, unsupervised machine learning algorithms show promise in automating the gating of flow cytometry data for polyploidy analysis and measuring megakaryocytic differentiation in cell lines.