Phorbol 12-myristate 13-acetate Research Articles

Anti-Inflammatory Effect of Protopine through MAPK and NF-κB Signaling Regulation in HepG2 Cell.

Protopine is a substance used for hemostasis with an anti-inflammatory action and is one of the substances that are actively undergoing experiments to confirm their utility as anticancer agents. This study examined the molecular changes in the cellular signaling pathways associated with inflammatory responses in phorbol 12-myristate 13 acetate (PMA)-induced human hepatocellular carcinoma cell line (Hep G2). The inhibition of PMA-induced phosphorylation of I-κB in HepG2, the effect of protopine on the MAPK signals, the inhibition of COX-2 activity, and the inhibition of MMP-9 as a medium of inflammatory response were evaluated by Western blot and qPCR. The effect of protopine on the survival rates in HepG2 cells was evaluated and found to be stable to a processing concentration of up to 40μM. Subsequent Western blot analyses showed that protopine blocks the transfer of the MAPKs cell signals induced by PMA and the transfer of the subunit of the nuclear factor-kappa B (NF-κB) to the nucleolus. Protopine inhibited the kappa alpha (I-κBα) phosphorylation in the cytosol and blocked PMA-induced inflammation via COX-2 activity inhibition. The expression of MMP-9 at the gene and protein levels, which is associated with cell migration and metastasis, was reduced by protopine.

Oxygen-dependent regulation of permeability in low resistance intestinal epithelial cells infected with Giardia lamblia

Intestinal epithelial cells (IECs) reside in a highly anaerobic environment that is subject to daily fluctuations in partial oxygen pressure (pO2), depending on intestinal tissue perfusion. This condition, known as physiological hypoxia, has a major impact on the maintenance of gut homeostasis, such as effects on the integrity and function of the intestinal epithelial barrier. Giardia lamblia is a microaerophilic protozoan parasite that infects and colonizes the small intestine of its host, causing watery diarrhea. The disease, known as giardiasis, is associated with enhanced intestinal permeability and disruption or reorganization of tight junction (TJ) proteins between IECs. Given the central role of oxygen in gut homeostasis, in this study, we aimed to evaluate whether pO2 affects intestinal permeability (flux of ions and macromolecules) and TJ protein expression in human IECs during G. lamblia infection. Using human cell lines HuTu-80 and Caco-2 as models of “loose” (low resistance) and “tight” (high resistance) intestines, respectively, we elucidated that low pO2 drives intestinal barrier dysfunction in IECs infected with trophozoites through dephosphorylation of protein kinase C (PKC α/β II). Additionally, we demonstrated that IECs infected with trophozoites in the presence of a pharmacological PKC activator (phorbol 12-myristate 13-acetate) partially restored the barrier function, which was correlated with increased protein expression levels of zonula occludens (ZO)-2 and occludin. Collectively, these results support the emerging theory that molecular oxygen impacts gut homeostasis during Giardia infection via direct host signaling pathways. These findings further our knowledge regarding Giardia-host interactions and the pathophysiological mechanisms of human giardiasis.

Defective RAB31-mediated megakaryocytic early endosomal trafficking of VWF, EGFR, and M6PR in RUNX1 deficiency

AbstractTranscription factor RUNX1 is a master regulator of hematopoiesis and megakaryopoiesis. RUNX1 haplodeficiency (RHD) is associated with thrombocytopenia and platelet granule deficiencies and dysfunction. Platelet profiling of our study patient with RHD showed decreased expression of RAB31, a small GTPase whose cell biology in megakaryocytes (MKs)/platelets is unknown. Platelet RAB31 messenger RNA was decreased in the index patient and in 2 additional patients with RHD. Promoter-reporter studies using phorbol 12-myristate 13-acetate–treated megakaryocytic human erythroleukemia cells revealed that RUNX1 regulates RAB31 via binding to its promoter. We investigated RUNX1 and RAB31 roles in endosomal dynamics using immunofluorescence staining for markers of early endosomes (EEs; early endosomal autoantigen 1) and late endosomes (CD63)/multivesicular bodies. Downregulation of RUNX1 or RAB31 (by small interfering RNA or CRISPR/Cas9) showed a striking enlargement of EEs, partially reversed by RAB31 reconstitution. This EE defect was observed in MKs differentiated from a patient-derived induced pluripotent stem cell line (RHD-iMKs). Studies using immunofluorescence staining showed that trafficking of 3 proteins with distinct roles (von Willebrand factor [VWF], a protein trafficked to α-granules; epidermal growth factor receptor; and mannose-6-phosphate) was impaired at the level of EE on downregulation of RAB31 or RUNX1. There was loss of plasma membrane VWF in RUNX1- and RAB31-deficient megakaryocytic human erythroleukemia cells and RHD-iMKs. These studies provide evidence that RAB31 is downregulated in RHD and regulates megakaryocytic vesicle trafficking of 3 major proteins with diverse biological roles. EE defect and impaired vesicle trafficking is a potential mechanism for the α-granule defects observed in RUNX1 deficiency.

Identification and Functional Evaluation of Polyphenols That Induce Regulatory T Cells.

Regulatory T cells (Tregs) and CD4+/CD25+ T cells play an important role in the suppression of excessive immune responses, homeostasis of immune function, and oral tolerance. In this study, we screened for food-derived polyphenols that induce Tregs in response to retinaldehyde dehydrogenase (RALDH2) activation using macrophage-like THP-1 cells. THP-1 cells were transfected with an EGFP reporter vector whose expression is regulated under the control of mouse Raldh2 promoter and named THP-1 (Raldh2p-EGFP) cells. The THP-1 (Raldh2p-EGFP) cells were treated with 33 polyphenols after inducing their differentiation into macrophage-like cells using phorbol 12-myristate 13-acetate. Of the 33 polyphenols, five (kaempferol, quercetin, morin, luteolin and fisetin) activated Raldh2 promoter activity, and both quercetin and luteolin activated the endogenous Raldh2 mRNA expression and enzymatic activity. Furthermore, these two polyphenols increased transforming growth factor beta 1 and forkhead box P3 mRNA expression, suggesting that they have Treg-inducing ability. Finally, we verified that these polyphenols could induce Tregs in vivo and consequently induce IgA production. Oral administration of quercetin and luteolin increased IgA production in feces of mice. Therefore, quercetin and luteolin can induce Tregs via RALDH2 activation and consequently increase IgA production, suggesting that they can enhance intestinal barrier function.

The overexpressed transcription factor RGPR-p117 suppresses the proliferation of normal rat kidney proximal tubular epithelial NRK-52E cells: Involvement of diverse signaling pathways

AimsRGPR-p117 was originally discovered as a novel transcription factor, which specifically binds to a nuclear factor I (NFI) consensus motif TTGGC(N)6CC in the promoter region of the regucalcin gene. RGPR-p117 is also called as Lztr2 and SEC16B. The role of RGPR-p117 in cell regulation is poorly understood. This study was undertaken to determine whether the overexpression of RGPR-p117 impacts the proliferation of normal rat kidney proximal tubular epithelial NRK-52E cells in vitro. Main methodsThe NRK-52E wild-type cells and RGPR-p117-overexpressing NRK-52E cells were cultured in DMEM containing fetal bovine serum. Key findingsThe overexpression of RGPR-p117 repressed colony formation and proliferation of NRK-52E cells. Interestingly, RGPR-p117 overexpression blocked cell proliferation promoted by culturing with Bay K 8644, a calcium-entry agonist, and phorbol 12-myristate 13-acetate, an activator of protein kinase C. The depressive effects of RGPR-p117 overexpression on cell proliferation were not occurred by culturing with various inhibitors of cell cycle and intracellular signaling processes. RGPR-p117 overexpression increased the translocation of RGPR-p117 into the nucleus of NRK-52E cells. Mechanistically, RGPR-p117 overexpression diminished the levels of Ras, PI3 kinase, Akt, mitogen-activated protein kinase, and mTOR, while it raised the levels of p53, Rb, p21, and regucalcin. Furthermore, RGPR-p117 overexpression protected cell death caused by apoptosis-inducing factors, suggesting that the suppressive effects of RGPR-p117 on cell growth are independent of cell death. SignificanceThe present study demonstrates that the overexpressed transcription factor RGPR-p117 suppresses cell proliferation via targeting diverse signaling processes, suggesting a role of RGPR-p117 in cell regulation.

Propofol enhances the sensitivity of glioblastoma cells to temozolomide by inhibiting macrophage activation in tumor microenvironment to down-regulate HIF-1α expression

Temozolomide (TMZ) is the first-line drug for the clinical treatment of glioblastoma (GBM), but drug resistance limits its treatment benefits. This study was intended to investigate whether propofol could restrict the resistance of GBM cells to TMZ and uncover the underlying mechanisms. Human GBM cell line U251 and TMZ-resistant U251/TMZ cell line were transplanted into mice to construct GBM and TMZ-resistant GBM xenograft tumors. Tumor growth in mice was monitored, and the tumor tissues were collected for biochemical analysis. THP-1 cell differentiated into M0 subtype macrophage using phorbol 12-myristate 13-acetate (PMA). The culture medium of M0 macrophage was collected for treating U251 cells with the presence or absence of propofol or propofol + DMOG (HIF-1α activator). Results showed that propofol significantly enhanced the inhibitory effect of TMZ on tumor growth, macrophage infiltration and inflammation in TMZ-resistant GBM xenograft tumors in vivo. Compared with GBM xenograft tumors, higher expression of HIF-1α, O6-methylguanine-DNA methyltransferase (MGMT), p-p65 and cyclooxygenase 2 (Cox2) was observed in TMZ-resistant GBM xenograft tumors, but propofol co-treatment markedly reduced the expression of these proteins. In in vitro experiments, culture medium from M0 macrophage promoted U251 cell survival, inflammation and expression of HIF-1α, MGMT, p65 and Cox2, whereas inhibited cell apoptosis. However, propofol suppressed the PMA-induced THP-1 M0 macrophage activation, and propofol-treated culture medium from M0 macrophage blocked all the effects of M0 medium on U251 cells. Additionally, DMOG reversed the effect of propofol-treated M0 medium on U251 cells. In conclusion, Propofol restricted TMZ resistance via inhibiting macrophage activation and down-regulating HIF-1α expression in GBM.

A systematic exploration of ginsenoside Rg5 reveals anti-inflammatory functions in airway mucosa cells

BackgroundHyperactivated airway mucosa cells overproduce mucin and cause severe breathing complications. Here, we aimed to identify the effects of saponins derived from Panax ginseng on inflammation and mucin overproduction. MethodsNCI–H292 cells were pre-incubated with 16 saponins derived from P. ginseng, and mucin overproduction was induced by treatment with phorbol 12-myristate 13-acetate (PMA). Mucin protein MUC5AC was quantified by enzyme-linked immunosorbent assay, and mRNA levels were analyzed using quantitative polymerase chain reaction (qPCR). Moreover, we performed a transcriptome analysis of PMA-treated NCI–H292 cells in the absence or presence of Rg5, and differential gene expression was confirmed using qPCR. Phosphorylation levels of signaling molecules, and the abundance of lipid droplets, were measured by western blotting, flow cytometry, and confocal microscopy. ResultsGinsenoside Rg5 effectively reduced MUC5AC secretion and decreased MUC5AC mRNA levels. A systematic functional network analysis revealed that Rg5 upregulated cholesterol and glycerolipid metabolism, resulting in the production of lipid droplets to clear reactive oxygen species (ROS), and modulated the mitogen-activated protein kinase and nuclear factor (NF)-κB signaling pathways to regulate inflammatory responses. Rg5 induced the accumulation of lipid droplets and decreased cellular ROS levels, and N-acetyl-l-cysteine, a ROS inhibitor, reduced MUC5AC secretion via Rg5. Furthermore, Rg5 hampered the phosphorylation of extracellular signal-regulated kinase and p38 proteins, affecting the NF-κB signaling pathway and pro-inflammatory responses. ConclusionRg5 alleviated inflammatory responses by reducing mucin secretion and promoting lipid droplet-mediated ROS clearance. Therefore, Rg5 may have potential as a therapeutic agent to alleviate respiratory disorders caused by hyperactivation of mucosa cells.

Comparison of Rapid Cytokine Immunoassays for Functional Immune Phenotyping.

BackgroundCell-based functional immune-assays may allow for risk stratification of patients with complex, heterogeneous immune disorders such as sepsis. Given the heterogeneity of patient responses and the uncertain immune pathogenesis of sepsis, these assays must first be defined and calibrated in the healthy population.ObjectiveOur objective was to compare the internal consistency and practicality of two immune assays that may provide data on surrogate markers of the innate and adaptive immune response. We hypothesized that a rapid turnaround, microfluidic-based immune assay (ELLA) would be comparable to a dual-color, enzyme-linked immunospot (ELISpot) assay in identifying tumor necrosis factor (TNF) and interferon (IFN)γ production following ex vivo whole blood stimulation.DesignThis was a prospective, observational cohort analysis. Whole blood samples from ten healthy, immune-competent volunteers were stimulated for either 4 hours or 18 hours with lipopolysaccharide, anti-CD3/anti-CD28 antibodies, or phorbol 12-myristate 13-acetate with ionomycin to interrogate innate and adaptive immune responses, respectively.Measurements and Main ResultsELLA analysis produced more precise measurement of TNF and IFNγ concentrations as compared with ELISpot, as well as a four- to five-log10 dynamic range for TNF and IFNγ concentrations, as compared with a two-log10 dynamic range with ELISpot. Unsupervised clustering accurately predicted the ex vivo immune stimulant used for 90% of samples analyzed via ELLA, as compared with 72% of samples analyzed via ELISpot.ConclusionsWe describe, for the first time, a rapid and precise assay for functional interrogation of the innate and adaptive arms of the immune system in healthy volunteers. The advantages of the ELLA microfluidic platform may represent a step forward in generating a point-of-care test with clinical utility, for identifying deranged immune phenotypes in septic patients.

Exploration of Deformation of F-Actin during Macropinocytosis by Confocal Microscopy and 3D-Structured Illumination Microscopy

Since their invention, confocal microscopy and super-resolution microscopy have become important choices in cell biology research. Macropinocytosis is a critical form of endocytosis. Deformation of the cell membrane is thought to be closely related to the movement of F-actin during macropinocytosis. However, it is still unclear how the morphology of F-actin and the membrane change during this process. In this study, confocal microscopy was utilized for macroscopic time-series imaging of the cell membranes and F-actin in cells induced by phorbol 12-myristate 13-acetate (PMA). Super-resolution structured illumination microscopy (SIM), which can overcome the diffraction limit, was used to demonstrate the morphological characteristics of F-actin filaments. Benefiting from the advantages of SIM in terms of resolution and 3D imaging, we speculated on the regular pattern of the deformation of F-actin during macropinocytosis. The detailed visualization of structures also helped to validate the speculation regarding the role of F-actin filaments in macropinocytosis in previous studies. The results obtained in this study will provide a better understanding of the mechanisms underlying macropinocytosis and endocytosis.

Identification of IL-10 competent B cells in swine

Progress in the phenotypic characterisation of porcine B cells is ongoing, with recent advances in the identification of B1 cell subsets and plasma cells. However, regulatory B cells, commonly identified by interleukin (IL)-10 production, have not been studied in pigs so far. Here we investigate IL-10 expression in B cell subsets in response to CpG-oligodeoxynucleotides, phorbol 12-myristate 13-acetate and ionomycin stimulation in vitro. Our results reflect similar findings in human and mice. We identify a small subset of IL-10 competent B cells, present within both porcine B1 and B2 cell subsets across blood, spleen, mediastinal lymph nodes and lung tissue, with varied differentiation statuses. The capacity for IL-10 production coincided with CD95 expression, suggesting an activated phenotype of IL-10 competent B cells. These findings support the emerging paradigm that B cell IL-10 production is a function of various B cell subsets influenced by activation history and microenvironmental factors.

Effects of neuronal cell adhesion molecule L1 and nanoparticle surface modification on microglia

Microelectrode arrays for neural recording suffer from low yield and stability partly due to the inflammatory host responses. A neuronal cell adhesion molecule L1 coating has been shown to promote electrode-neuron integration, reduce microglia activation and improve recording. Coupling L1 to surface via a nanoparticle (NP) base layer further increased the protein surface density and stability. However, the exact L1-microglia interaction in these coatings has not been studied. Here we cultured primary microglia on L1 modified surfaces (with and without NP) and characterized microglia activation upon phorbol myristate acetate (PMA) and lipopolysaccharide (LPS) stimulation. Results showed L1 coatings reduced microglia's superoxide production in response to PMA and presented intrinsic antioxidant properties. Meanwhile, L1 decreased iNOS, NO, and pro-inflammatory cytokines (TNF alpha, IL-6, IL-1 beta), while increased anti-inflammatory cytokines (TGF beta 1, IL-10) in LPS stimulated microglia. Furthermore, L1 increased Arg-1 expression and phagocytosis upon LPS stimulation. Rougher NP surface showed lower number of microglia attached per area than their smooth counterpart, lower IL-6 release and superoxide production, and higher intrinsic reducing potential. Finally, we examined the effect of L1 and nanoparticle modifications on microglia response in vivo over 8 weeks with 2-photon imaging. Microglial coverage on the implant surface was found to be lower on the L1 modified substrates relative to unmodified, consistent with the in vitro observation. Our results indicate L1 significantly reduces superoxide production and inflammatory response of microglia and promotes wound healing, while L1 immobilization via a nanoparticle base layer brings added benefit without adverse effects. Statement of significanceSurface modification of microelectrode arrays with L1 has been shown to reduce microglia coverage on neural probe surface in vivo and improves neural recording, but the specific mechanism of action is not fully understood. The results in this study show that surface bound L1 reduces superoxide production from cultured microglia via direct reduction reaction and signaling pathways, increases anti-inflammatory cytokine release and phagocytosis in response to PMA or LPS stimulation. Additionally, roughening the surface with nanoparticles prior to L1 immobilization further increased the benefit of L1 in reducing microglia activation and oxidative stress. Together, our findings shed light on the mechanisms of action of nanotextured and neuroadhesive neural implant coatings and guide future development of seamless tissue interface.

MiR-let-7c-3p targeting on Egr-1 contributes to the committed differentiation of leukemia cells into monocyte/macrophages.

In preliminary experiments, it was found that the expression of early growth response-1 (Egr-1) was upregulated during the committed differentiation of leukemia cells into monocytes/macrophages. The cross-analysis of gene chip detection and database prediction indicated that Egr-1 was associated with upstream microRNA (miR)-let-7c-3p, thus the present study focused on the role of the miR-let-7c-3p/Egr-1 signaling axis in the committed differentiation of leukemia cells into monocytes/macrophages. Phorbol 12-myristate 13-acetate (PMA) was used to induce the directed differentiation of human K562 leukemia cells into monocytes/macrophages and the differentiation of K562 leukemia cells was determined by cell morphology observation and expression of differentiation antigens CD11b and CD14 by flow cytometry. The expression levels of Egr-1 and miR-let-7c-3p were detected by reverse transcription-quantitative PCR and the protein expression of Egr-1 was detected by western blotting. The effect of Egr-1 on the differentiation of K562 cells was detected by short interfering (si)RNA interference assay. A dual-luciferase reporter assay was used to detect target binding of miR-let-7c-3p on the 3′UTR of Egr-1. Cell transfection of miR-let-7c-3p mimics and inhibitors was used to modulate the expression of miR-let-7c-3p, as indicated by RT-qPCR assays. Western blotting was also used to examine the effect of miR-let-7c-3p on Egr-1 expression. The PMA-induced differentiation of K562 cells was transfected with miR-let-7c-3p and the expression of differentiation antigen was detected by flow cytometry. A differentiation model of K562 leukemia cells into monocytes/macrophages was induced by PMA, which was indicated by morphological observations and upregulation of CD11b and CD14 antigens. The gene or protein expression of Egr-1 was significantly higher compared with that of the control group, while the expression of miR-let-7c-3p was significantly lower compared with that of the control group. siRNA interference experiments showed that the expression of cell differentiation antigen CD14 in the 100 µg/ml PMA + si-Egr-1 group was significantly lower compared with that in the 100 µg/ml PMA + si-ctrl group. The dual luciferase reporter gene results showed that the luciferase activity of the co-transfected mimic and Egr-1 WT groups was significantly lower than that of the NC control group, while the luciferase activity of the co-transfected mimic and Egr-1 MUT groups was comparable to that of the NC control group. Therefore, the dual-luciferase reporter gene assay confirmed that miR-let-7c-3p can target Egr-1. Western blotting showed that the expression of Egr-1 following transfection with miR-let-7c-3p inhibitor was significantly higher compared with that of the negative control and the expression of Egr-1 after transfection with miR-let-7c-3p mimic was significantly lower than that of the negative control. Following exposure to PMA, the expressions of CD11b and CD14 in the miR-let-7c-3p inhibitor group were significantly higher than those in the miR-let-7c-3p NC group, as indicated by CD11b and CD14 respectively. In conclusion, miR-let-7c-3p could bind to the 3′UTR of Egr-1 and negatively regulated Egr-1 expression. The miR-let-7c-3p/Egr-1 signaling axis was closely associated with the committed differentiation of K562 cells from leukemia cells to monocytes/macrophages.

Repression of HIV-1 reactivation mediated by CRISPR/dCas9-KRAB in lymphoid and myeloid cell models

BackgroundDespite antiretroviral treatment efficacy, it does not lead to the complete eradication of HIV infection. Consequently, reactivation of the virus from latently infected cell reservoirs is a major challenge toward cure efforts. Two strategies targeting viral latency are currently under investigation: the “shock and kill” and the “block and lock.” The “Block and Lock” methodology aims to control HIV-1 latency reactivation, promoting a functional cure. We utilized the CRISPR/dCas9-KRAB platform, which was initially developed to suppress cellular genes transcription, to block drug-induced HIV-1 reactivation in latently infected T cells and myeloid cells.ResultsWe identified a set of five sgRNAs targeting the HIV-1 proviral genome (LTR1-LTR5), having the lowest nominated off-target activity, and transduced them into the latently infected lymphoid (J-Lat 10.6) and myeloid (U1) cell lines. One of the sgRNAs (LTR5), which binds specifically in the HIV-1 LTR NFκB binding site, was able to promote robust repression of HIV-1 reactivation in latently infected T cells stimulated with Phorbol 12-Myristate 13-Acetate (PMA) and Ingenol B (IngB), both potent protein kinase C (PKC) stimulators. Reactivation with HDAC inhibitors, such as SAHA and Panobinostat, showed the same strong inhibition of reactivation. Additionally, we observed a hundred times reduction of HIV-1 RNA expression levels in the latently infected myeloid cell line, U1 induced with IngB.ConclusionTaken together, our results show that the KRAB fused CRISPR/dCas9 system can robustly prevent the HIV-1 latency reactivation process, mediated by PMA or IngB and SAHA or Panobinostat, both in myeloid and lymphoid HIV-1 latently infected cells. In addition, we demonstrated that KRAB repressor protein is crucial to reactivation resistance phenotype, and we have identified some useful hotspots sequences in HIV-1 LTR for the design sgRNAs.

Differences in H3K4me3 and chromatin accessibility contribute to altered T-cell receptor signaling in neonatal naïve CD4 T cells.

Neonatal CD4+ T cells have reduced or delayed T-cell receptor (TCR) signaling responses compared with adult cells, but the mechanisms underlying this are poorly understood. This study tested the hypothesis that human neonatal naïve CD4+ TCR signaling and activation deficits are related to differences in H3K4me3 patterning and chromatin accessibility. Following initiation of TCR signaling using anti-CD3/anti-CD28 beads, adult naïve CD4+ T cells demonstrated increased CD69, phospho-CD3ε and interleukin (IL)-2, tumor necrosis factor-α (TNF-α), interferon-γ and IL-17A compared with neonatal cells. By contrast, following TCR-independent activation using phorbol myristate acetate (PMA)/ionomycin, neonatal cells demonstrated increased expression of CD69, IL-2 and TNF-α and equivalent phospho-ERK compared with adult cells. H3K4me3 chromatin immunoprecipitation-sequencing (ChIP-seq) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) were performed on separate cohorts of naïve CD4+ T cells from term neonates and adults, and RNA-seq data from neonatal and adult naïve CD4+ T cells were obtained from the Blueprint Consortium. Adult cells demonstrated overall increased chromatin accessibility and a higher proportion of H3K4me3 sites associated with open chromatin and active gene transcription compared with neonatal cells. Adult cells demonstrated increased mRNA expression of the TCR-associated genes FYN, ITK, CD4, LCK and LAT, which was associated with increased H3K4me3 at the FYN and ITK gene loci and increased chromatin accessibility at the CD4, LCK and LAT loci. These findings indicate that neonatal TCR-dependent defects in activation are epigenetically regulated and provide a potentially targetable mechanism to enhance neonatal CD4+ T-cell responses.

Abstract 2455: Development of a bioluminescence reporter mouse model for tracking and quantifying Ly6G+ neutrophils in vivo

Abstract Immunotherapy has revolutionized the way cancer is treated; however, despite tremendous success, not all patients benefit from immunotherapy. Identifying biomarkers predictive of effective treatment response to current and novel immunotherapies is of utmost importance. Multiple studies have indicated neutrophil-lymphocyte ratio (NLR) may be a predictive biomarker of immunotherapy response. We analyzed hematological parameters across multiple preclinical murine tumors to determine if NLR predicts tumor growth and response to treatment. The hematological survey identified NLR to be strongly correlated to tumor weights, with larger tumors exhibiting higher NLRs. In addition, mouse tumors that have been characterized as resistant or partially responsive to immunotherapy had a higher NLR than tumors responsive to immunotherapy. To track neutrophils in vivo, we generated a BAC transgenic mouse wherein firefly luciferase (Luc2), diphtheria toxin receptor (DTR), and enhanced green fluorescence protein (eGFP) genes were expressed under the endogenous Ly6G promoter. The Ly6G promoter enables tracking of Ly6G+ neutrophils using luciferase-based bioluminescence imaging (BLI), depletion of Ly6G+ neutrophils by diphtheria toxin treatment, and the ability to perform flow analyses using the eGFP marker. In vivo and ex vivo BLI demonstrated that the Ly6G+ cells were primarily expressed in bone marrow, blood, spleen, and the digestive tract. To ensure the specificity of the bioluminescence signal, we isolated Ly6G+ and Ly6G- immune cells and performed in vitro BLI. In vitro BLI confirmed that bioluminescence was exclusive to Ly6G+ cells. To confirm that the Luc2-DTR-eGFP (LDG) reporter cassette expression did not affect neutrophil activity and function, we quantified the level of myeloperoxidase (MPO), a marker of neutrophil activation, in the phorbol myristate acetate (PMA)-induced ear edema mouse model. Neutrophil activity was measured using luminol enabled BLI of MPO. Luminol-BLI following PMA-induced ear edema demonstrated similar MPO activity between wildtype and transgenic mice. Finally, we investigated if the LDG reporter cassette impacted tumor growth. Measures of tumor burden in this transgenic line recapitulated our hematological findings. Tumors with higher NLR showed higher tumor BLI signal when compared to tumors with lower NLR ratio, demonstrating that the LDG reporter cassette did not impact tumor growth in the transgenic mice and importantly, providing a tool to track Ly6G+ cells in tumors. Our results demonstrate that NLR in tumor correlates with response to immunotherapy across multiple tumor types. Here we describe the development of a transgenic mouse to track intratumoral Ly6G+ cells. These transgenic mice provide a valuable tool for profiling oncology compounds, including those targeting immune cells, and for understanding the mechanism of action of such agents. Citation Format: Milind D. Chalishazar, Nicolas Solban, Johnny Kopinja, Doug Linn, Razvan Cristescu, Heather Zhou, Thomas Rosahl, Brian Long, Weisheng Zhang, Eric Hostetler. Development of a bioluminescence reporter mouse model for tracking and quantifying Ly6G+ neutrophils in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2455.

Abstract 3014: PHGDH- A therapeutic vulnerability in melanoma

Abstract Recurrently amplified oncogenes may represent melanoma vulnerabilities that can be exploited for therapy. The genomic locus containing the Phosphoglycerate Dehydrogenase (PHGDH) gene at chromosome 1p12 is frequently amplified in melanoma. PHGDH is the rate limiting enzyme that converts the glycolytic intermediate 3-Phospho Glycerate (3-PG) to serine through the serine synthesis pathway (SSP). Serine is utilized to make glycine, glutathione, one carbon metabolites, proteins and lipids. PHGDH inhibition suppresses proliferation of 1p12 amplified melanoma cells. However, the role of PHGDH in chromosome 1p12 non-amplified melanoma is poorly understood. We observed that 1p12 non-amplified melanoma cell lines have enhanced expression of the SSP enzymes as compared to melanocytes cultured in tetradecanoyl-phorbol acetate (TPA, a growth factor that stimulates MAPK pathway). Moreover, TPA starvation in melanocytes increased PHGDH expression. This led us to hypothesize that activation of oncogenic MAPK signaling leads to high expression of PHGDH which can serve as therapeutic vulnerability in melanomagenesis. Acute expression of oncogenic BRAFV600E in melanocytes resulted in upregulation of SSP enzymes, while treatment of melanoma cells with either BRAF or MEK inhibitor resulted in downregulation of SSP enzymes. We confirmed that PHGDH is regulated by BRAF-ERK-mTOR axis. Downregulation of PHGDH in 1p12 non-amplified melanoma cell lines decreased their cell proliferation and colony formation ability in vitro. We also observed that Phgdh knockdown in a BrafV600E/WT and Pten-/- (BPP) melanoma mouse model significantly increased survival. Furthermore, serine-glycine starvation (external source of Serine-glycine) in BPP chimeras did not affect the survival of these mice while on the other end, it suppressed the growth of the xenograft tumors injected in the NSG mice, which warrants further investigation. These results suggest the importance of PHGDH for melanoma cell proliferation and tumor growth irrespective of its amplification status. The exact mechanism of PHGDH regulation by BRAFV600E and the therapeutic effect of PHGDH inhibition in melanoma initiation and progression are the focus of our studies. Citation Format: Neel N. Jasani, Florian Karreth. PHGDH- A therapeutic vulnerability in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3014.

Abstract 2488: PET imaging of innate immunity activation In Vivo with redox-tuned 4-[18F]fluoro-1-naphthol

Abstract High redox potential reactive oxygen and nitrogen species (ROS/RNS), such as O2 free radicals, superoxide, and hypochlorous acid, generated by activities of the NADPH oxidase-2 (NOX2)/myeloperoxidase (MPO) axis and related enzymes, are key effector molecules of innate immunity in physiological and diseased inflammatory states. Other lower energy species (H2O2, NO) provide adjuvant signaling functions. NOX2- and MPO-derived high energy radicals are known to oxidize naphthol species, wherein the naphthol products bind to proximate proteins and activated myeloid cells. Herein, we present 4-[18F]fluoro-1-naphthol ([18F]4FN), a novel redox-tuned radiopharmaceutical that selectively detects by positron emission tomography (PET) high energy radicals produced by activated innate immunity. The products of human MPO plus H2O2, but not H2O2 alone, rapidly and completely oxidized [18F]4FN. All-trans-retinoic acid-differentiated HL-60 "neutrophil-like" human cells activated with phorbol-12-myristate-13-acetate (PMA) retained [18F]4FN 5-fold over unstimulated cells. 4-ABAH, an MPO-specific inhibitor, or DPI, a broad oxidase inhibitor, blocked cellular retention by >95%. [18F]4FN PET/CT imaging readily discriminated foci of inflammation in vivo in three distinct murine models of acute inflammation: endotoxin-induced whole-body toxic shock, PMA-induced mild contact dermatitis of the ear, and lipopolysaccharide (LPS)-induced ankle arthritis. Mechanistically, in mice in vivo, 4-ABAH reduced inflammation-induced [18F]4FN retention, and Cybb-/- (Nox2-/-) gene-deletion strongly and significantly abrogated PMA-induced [18F]4FN retention. Thus, [18F]4FN shows promise as a robust redox-tuned reporter for imaging activation states of innate immunity by PET/CT, is ready for translation. [18F]4FN PET imaging may find application in a variety of inflammatory states associated with cancer therapy, immunotherapy-related adverse events, as well as other diseases, including arthritis, hepatitis, atherosclerosis, COVID-19, as well as up-staging and monitoring multi-organ inflammation. Citation Format: Seth T. Gammon, Federica Pisaneschi, Vincenzo Paolillo, Sarah Qureshy, David Piwnica-Worms. PET imaging of innate immunity activation In Vivo with redox-tuned 4-[18F]fluoro-1-naphthol [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2488.

Impact of calcium and reactive oxygen species on human sperm function: Role of NOX5

NOX5 is introduced as a new therapeutic target for infertility treatment. This study aimed to compare the basal and stimulated reactive oxygen species (ROS) production and sperm function in human teratozoospermic (n= 15) and normozoospermic (n= 17) semen samples following calcium overload and NOX5 activation. Washed spermatozoa incubated for 1h under five various conditions: control group, adding a calcium ionophore A23187, phorbol myristate acetate (PMA), A23187 + PMA, and diphenylene iodonium (DPI) + A23187 + PMA. ROS generation was measured immediately after treatment for 30 min. Motility, viability, acrosome reaction, and apoptosis were evaluated after 1-h incubation. ROS production significantly increased when A23187 or PMA was added to the sperm medium. DPI had suppressive effects on ROS generation. Progressive and total motility significantly decreased following calcium elevation and NOX5 activation, which was somewhat returned by DPI. Necrotic and live cells in teratozoospermia was, respectively, higher and lower than normozoospermia samples. Incubation with A23187 significantly increased the percentage of early and late apoptosis. Teratozoosperm are more vulnerable than normal spermatozoa, and produce more basal and stimulated ROS. It seems that calcium overload induces apoptosis in spermatozoa and loss of viability through MPT pore opening and increased intracellular ROS.

Quantitative proteomics and in-cell cross-linking reveal cellular reorganisation during early neuronal differentiation of SH-SY5Y cells

The neuroblastoma cell line SH-SY5Y is commonly employed to study neuronal function and disease. This includes cells grown under standard conditions or differentiated to neuron-like cells by administration of chemical reagents such as retinoic acid (RA) or phorbol-12-myristate-13-acetate (PMA). Even though SH-SY5Y cells are widely explored, a complete description of the resulting proteomes and cellular reorganisation during differentiation is still missing. Here, we relatively quantify the proteomes of cells grown under standard conditions and obtained from two differentiation protocols employing RA or a combination of RA and PMA. Relative quantification and KEGG pathway analysis of the proteins reveals the presence of early differentiating cells and provides a list of marker proteins for undifferentiated and differentiated cells. For characterisation of neuronal sub-types, we analyse expression of marker genes and find that RA-differentiated cells are acetylcholinergic and cholinergic, while RA/PMA-differentiated cells show high expression of acetylcholinergic and dopaminergic marker genes. In-cell cross-linking further allows capturing protein interactions in different cellular organelles. Specifically, we observe structural reorganisation upon differentiation involving regulating protein factors of the actin cytoskeleton.

Tryptanthrin attenuates TLR3-mediated STAT1 activation in THP-1 cells.

Upon viral infection, dysregulated immune responses are associated with the disease exacerbation and poor prognosis. The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway are essential for the innate immune responses against invading viruses as well as for sustained activation of macrophages. Tryptanthrin, a natural alkaloid, exhibits various bioactivities, including anti-microbial and anti-inflammatory effects. The aim of this study was to elucidate the effects of tryptanthrin on toll-like receptor 3 (TLR3)–mediated STAT1 activation in macrophages in vitro. Using phorbol myristate acetate (PMA)–differentiated THP-1 cells, we analyzed the protein level of phosphorylated-STAT1 (p-STAT1) upon stimulation with polyinosinic-polycytidylic acid (poly IC), a well-known TLR3 ligand, with and without tryptanthrin. We found that tryptanthrin decreased the protein level of p-STAT1 in a concentration-dependent manner after poly IC stimulation. On the other hand, tryptanthrin did not affect the levels of p-STAT1 upon stimulation with lipopolysaccharide from Escherichia coli. Consistently, tryptanthrin suppressed poly IC–induced mRNA expression of interferon (IFN)–stimulated genes which are regulated by STAT1. Moreover, tryptanthrin decreased the protein level of phosphorylated-IFN regulatory factor 3 and the subsequent IFN-β mRNA induction after poly IC stimulation. Tryptanthrin is a promising therapeutic agent for the aberrant activation of macrophages caused by viral infection.