TNF-alpha Secretion Research Articles

The association of TNF-alpha secretion and mtDNA copy number in CD14+ monocytes of patients with obesity and CHD.

Mitochondrial dysfunction may be one of the causes of inflammatory activation of monocytes and macrophages, which leads to excessive secretion of inflammatory mediators and the development of chronic inflammation. The study was aimed to evaluate the secretion of inflammatory cytokine tumor necrosis factor-α (TNF-α) in the primary culture of monocytes, and to analyze its relationship with the number of mitochondrial DNA (mtDNA) copies in the blood of patients with coronary heart disease (CHD) and obesity. 108 patients with obesity and concomitant CHD and a control group of 25 participants were included in the study. CD14+ monocytes were isolated by a standard method in a ficoll-urographin gradient, followed by separation using magnetic particles. The number of mtDNA copies was estimated using qPCR. It was demonstrated that the number of mtDNA copies was significantly increased in groups of patients with CHD and obesity + CHD in comparison with control group. mtDNA copy number positively correlated with basal and LPS-stimulated TNF-α secretion, the most significant correlation was found in the group of patients with CHD and obesity. Thus, the change in mtDNA copy number in CD14+ monocytes which indicates the presence of mitochondrial dysfunction, confirm the direct involvement of mitochondria in the violation of the inflammatory response of monocytes revealed in this study as an increased secretion of inflammatory cytokine TNF-α.

EFFECT OF ARTIFICIAL MITOCHONDRIAL TRANSFER FROM LINE CELLULAR HSC-3 TO LYMPHOCYTE TCD4+, AS A MECHANISM INDUCTOR OF EXHAUSTED PHENOTYPE.

Abstract Background: Oral squamous cell carcinoma (OSCC) is the most frequent type of oral cancer. The tumor microenvironment of OSCC induces an alteration of the T lymphocyte, promoting an exhausted phenotype. The main organelle of metabolism is the mitochondrion and in recent years it has been indicated that several cells have the capacity to transfer mitochondria, including cancer cells. However, it has not been evaluated whether mitochondria transfer from cancer cells to T lymphocytes induces an exhausted phenotype in T lymphocyte. The aim of this work was to analyze the exhausted phenotype in TCD4+ lymphocytes after artificial transfer of mitochondria (MitoCeption) obtained from the oral cancer cell line HSC-3. Methods: Isolated MitoTracker-labeled mitochondria from oral cancer tumor cells were Mitocepted into CD4+ T lymphocytes. Then, surface molecule expression, proliferation and cytokine secretion mediated by tumor mitochondrial transfer were analyzed by flow cytometry. Results: The results showed that TCD4+ lymphocytes that acquired mitochondria had increased expression of 2 inhibitory proteins (TIGIT and CTLA4) and 3 proteins associated with exhausted phenotype (PD-1, PLD-1 and LAG3), compared to the control group. In addition, the mitocepted lymphocytes exhibited a significant decrease in proliferation compared to the control. For cytokine analysis, a significant decrease was observed in the mitocepted group in the secretion of IFN-gamma, TNF-alpha, IL-10 and IL-4, but not IL-17, compared to the control. Conclusions: Therefore, we concluded that the acquisition of isolated mitochondria from HSC-3 cancer cells by CD4+ T lymphocyte induces an exhausted phenotype in the TCD4+ lymphocyte. Regular Fondecyt Project 1211480

Role of macrophage AHR/TLR4/STAT3 signaling axis in the colitis induced by non-canonical AHR ligand aflatoxin B1

Here we report that macrophage AHR/TLR/STAT signaling axis is implicated in the colon colitis induced by non-canonical AHR ligand aflatoxin B1 (AFB1). In BALB/c mice gavaged with 5, 25 and 50 µg/kg body weight/day AFB1, we observed severe colitis featured by over-recruitment of myeloid lineage immune cells such as monocytes/macrophage in colon lamina propria. Stressed and damaged colon epithelial cells were observed in low-dose group, while twisted and shortened intestinal crypts being found in middle dose group. Severe tissue damage was induced in the high-dose group. Dose-dependent increases of ROS, NO, and decrease of mitochondrial ROS-suppressor STAT3 were observed in the exposure groups. Further investigation in AFB1-treated human macrophage model found: (1) functional adaptations such as elevation of TNF-alpha and IL-6 secretion, stimulation of phagocytosis, elevation of LTE4 level; (2) overall inflammatory status confirmed by RNA-sequence analysis, in line with up-regulation of immune functional proteins such as ICAM-1, IDO-1, NF-kB-p65, NLRP3, COX-2 and iNOS; (3) mRNA disruption of mitochondrial oxidative phosphorylation complex I units and STATs; (4) perturbation of AHR/TLR/STAT3 signaling axis, including elevated AHR, TLR2, TLR4, and decreased STAT3, p-STAT3 Ser727. Mechanism investigation revealed regulatory links of ligand-dependent AHR/TLR4/STAT3. AHR-TLR4 together regulate MyD88, and STAT3 may be directly regulated by MyD88 (TLR4 downstream molecule) upon AHR/TLR4 binding with ligands. Solely protein level changes of AHR/TLR4 cannot regulate STAT3. Our study suggests that macrophage AHR/TLR4/STAT3 is involved with the colitis induced by sub-acute exposure to AFB1. Future follow-up study will focus on the intervention of the colitis using AHR-anti-inflammatory ligands.

Functionality of a Peripheral Blood Derived Microglia-like Cell System for Drug Discovery to Treat Sickle Cell Related Chronic Pain

Background: Abnormal microglial activation is emerging as a clinically promising drug target to treat chronic pain in sickle cell disease (SCD). But research involving microglia has been stymied by difficulty in obtaining human CNS microglia and differences between human and animal neurobiology. To address such limitations, we have developed an in vitro culture system to induce human microglia-like cells (MLC) from peripheral blood mononuclear cells. In this study, we further validated the functionality of the cultured peripheral blood derived microglia-like cell (PB-MLC) and evaluate the pathological characteristics captured in the PB-MLC model system, which is an important requirement for disease model systems used in drug discovery. Methods: Human peripheral blood mononuclear cells were cultured with human GM-CSF (10 ng/ml), human IL-34 (100 ng/ml), TGF-β2 (2 ng/mL) and cholesterol (1.5 µg/mL) for 7 days to induce Human peripheral blood derived microglia-like cells (HPB-MLC). HPB-MLC were morphologically analyzed by phase contrast microscopy, phenotyped by immunofluorescence staining with anti-TMEM119, P2RY12, and Iba1 antibodies. To validate that the HPB-MLC culture system recapitulates important pathological features, we compared the microglial morphology, activation states and phagocytic activity of HPB-MLC from individuals with SCD with or without chronic pain, individuals with headache and normal donors without pain: Cells were stimulated with 100 ng/ml LPS for 24 hours, and secretion of TNF-alpha, IL-1beta, and IL-6 was measured by ELISA. The microglial phagocytic function of HPB-MLC was determined by assessing the uptake of fluorescent-labeled Zymosan particles with fluorescent microscopy and imaging flow cytometry. The model system was also validated by comparing cultured PB-MLC to CNS derived microglia cells using Sprague-Dawley rats: Rat brain derived microglia (BDM) and rat peripheral blood monocytes were isolated and then cultured with murine IL-34 (100 ng/ml), murine GM-CSF (10 ng/ml). TGF-β2 (2 ng/mL), and cholesterol (1.5 µg/mL) for 7 days. The cultured cells were morphologically analyzed as above; phenotyped with anti-TMEM119, P2RY12, and Iba1 antibodies. Rat BDM, rat PB-MLC and HPB-MLC were treated with 100 ng/ml LPS with or without minocycline (2.5, 5, 10, 25, 50 μg/mL) for 24 hours; TNF-alpha secretion was measured by ELISA. Results: Rat PB-MLC resemble BDM morphologically and express microglia specific markers. HPB-MLC express microglia specific markers (TMEM119, P2RY12, Iba1), exhibit microglial phagocytic activity. HPB-MLC retain the patient pain phenotype: Cultures from donors without chronic pain show more ramified morphology and lower phagocytic activity than cultures from individuals with chronic pain (Figure 1); HPB-MLC can be activated by LPS, causing microglial deramification and increased the expression of microglial activation marker CD68. HPB-MLC from patients with pain are more easily activated and secrete more inflammatory cytokines in response to LPS; SCD pain and headache samples exhibit different pro-inflammatory cytokine profile. To evaluate the possibility of using the PB-MLC model system to screen compounds to inhibit microglia activation, we tested PB-MLC cells with the microglial inhibitor, minocycline. The compound significantly suppressed the release of proinflammatory cytokine TNF-alpha from LPS-induced activated PB-MLC in a dose-dependent manner (Figure 2). Conclusions: We have developed and validated a microglial-like cell culture system from the peripheral blood of patients with sickle cell disease that retains the patient pain phenotype. HPB-MLC are physiologically functional and exhibit mature microglial features, including LPS-induced activation, microglial specific morphological transition, microglial phagocytic activity, and drug response to a well-studied microglia inhibitor, minocycline. As a cost-effective, efficient, and patient-specific microglia model, HPB-MLC culture system can be applied to identify target compounds that normalize microglia activation, and potentially treat chronic pain. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Bacteriocin BacSp222 and Its Succinylated Forms Exhibit Proinflammatory Activities Toward Innate Immune Cells .

PurposeThe zoonotic opportunistic pathogen Staphylococcus pseudintermedius 222 produces BacSp222 – an atypical peptide exhibiting the features of a bacteriocin, a virulence factor, and a molecule modulating the host inflammatory reaction. The peptide is secreted in an unmodified form and, additionally, two forms modified posttranslationally by succinylation. This study is a comprehensive report focusing on the proinflammatory properties of such molecules.MethodsThe study was performed on mouse monocyte/macrophage-like and endothelial cell lines as well as human neutrophils. The following peptides were studied: BacSp222, its succinylated forms, the form deprived of formylated methionine, and a reference bacteriocin – nisin. The measurements of the nitric oxide (NO) level, induced NO synthase (iNOS) expression, the profile of secreted cytokines, NF-kappa-B activation, reactive oxygen species (ROS) biosynthesis, and the formation of extracellular traps were conducted to evaluate the proinflammatory activity of the studied peptides.ResultsBacSp222 and its succinylated forms effectively induced NO production and iNOS expression when combined with IFN-gamma in macrophage-like cells. All natural BacSp222 forms used alone or with IFN-gamma stimulated the production of TNF-alpha, MCP-1, and IL-1-alpha, while the co-stimulation with IFN-gamma increased IL-10 and IL-27. Upregulated TNF-alpha secretion observed after BacSp222 exposition resulted from increased expression but not from membrane TNF-alpha proteolysis. In neutrophils, all forms of bacteriocin upregulated IL-8, but did not induce ROS production or NETs formation. In all experiments, the activities of deformylated bacteriocin were lower or unequivocal in comparison to other forms of the peptide.ConclusionAll naturally secreted forms of BacSp222 exhibit proinflammatory activity against monocyte-macrophage cells and neutrophils, confirming that the biological role of BacSp222 goes beyond bactericidal and cytotoxic effects. The atypical posttranslational modification (succinylation) does not diminish its immunomodulatory activity in contrast to the lower antibacterial potential or cytotoxicity of such modified form established in previous studies.

Selected adipocytokine concentrations in patients hospitalized for exacerbated chronic heart failure

Introduction: The role of adipose tissue as energy storage and endocrine organ is an area of interest in the treatment of several diseases. This study aimed to to evaluate blood adiponectin (ADP), leptin, tumor necrosis factor type alpha (TNF-alpha) concentrations, and their prognostic value in patients with exacerbated chronic heart failure (CHF). Methods and results: The following were assessed in 64 consecutive patients hospitalized for exacerbated CHF and a control group of 32 age and sex-matched individuals admitted due to life-limiting symptoms of peripheral artery disease: serum leptin, ADP, TNF-alpha concentrations, and body composition determined by bioelectrical impedance analysis. Compared to the control group, CHF patients had significantly higher blood ADP concentrations, a higher ratio of ADP to fat mass expressed as a percentage of body mass, and lower blood TNF-alpha concentrations and ratios of TNF-alpha to the visceral fat level (VFL). Compared to patients who survived, patients with CHF who died during the one-year follow-up had significantly higher values of ADP and higher ratios of ADP to body surface area. In the Cox regression model, blood ADP concentration was the only independent risk factor in respect of all-cause mortality during the one-year follow-up (HR; 95% CI: 1.16; 1.03-1.31). Conclusions: Patients with CHF present dysregulation in the secretion of ADP and TNF-alpha. Increased blood ADP concentration was associated with an increase in one-year all-cause mortality by 16%.

Abstract 5557: PMC-309, a highly selective anti-VISTA antibody reverses immunosuppressive TME to immune-supportive TME

Abstract Purpose: We investigated the pharmacology of PMC-309 in ex vivo or in vivo characterization for the development of the preclinical study. Experimental procedures: Target selectivity: it was measured by SPR assay. Human VISTA expressing CHO k1 cells or CHO k1 mock cells were used in cell binding assay. Target cell analysis: human peripheral blood mononuclear cells (PBMC) containing lymphocyte and myeloid lineage cells were used for target cells analysis with flow cytometry. In vitro T cell activity: human PBMC was employed for the evaluation of T cell activity (IRB #1041107-201703-BR-002-02) and was stimulated with or w/o PMC-309 in the presence of anti-CD3/28 Antibody. In vivo study: MC38 bearing human VISTA knock-in (KI) mice were employed for the assessment of anti-tumor activity of PMC-309. The tumor infiltrated immune cells: Immune cells in the TME were evaluated by immunohistochemistry (IHC) or flow cytometry (FACS) analysis. Statistics: All data were presented as the mean ± SEM and analyzed using one-way ANOVA followed by Dunnett’s multiple comparison tests in Graph pad prism®. ***p<0.001, **p<0.01 and *p<0.05 compared to Control group. Summary of data: PMC-309 binding to VISTA expressing cells is highly selective and the selectivity is maintained even in the low pH conditions that mimic TME. PMC-309 enhances the secretion of IFN-gamma, TNF-alpha, and IL-2 in Mixed Lymphocyte Reaction (MLR) settings. In addition, PMC-309 promoted monocyte differentiation into M1 macrophage that stimulates proinflammatory cytokine secretion of T cells. For the in vivo study, PMC-309 was intravenously administrated in VISTA-KI mice. The tumor growth rate was suppressed in accompanied by a synergistic effect with an anti-PD1 antibody. The anti-tumor activity was associated with enhanced T cell activation, increased secretion of pro-inflammatory cytokines, and increased penetration of cytotoxic T cells, but lowing immune-suppressive MDSC cells into TME as demonstrated with IHC analysis. Conclusions: PMC-309 is a fully human anti-VISTA antibody that targets human VISTA specifically. PMC-309 enhanced both T cell activation and monocyte activation when monocytes were cultured with or without T cells by targeting VISTA. PMC-309 increased the number of T cell infiltration while a decrease of MDSCs in the TME. PMC-309 in combination with chemotherapy or other IO drugs could address highly medical unmet needs from patients with drug resistance to currently available IO treatment options. Citation Format: Cheon Ho Park, Sang Soon Byun, Jin Yong An, Hyerim Han, Weon Sup Lee. PMC-309, a highly selective anti-VISTA antibody reverses immunosuppressive TME to immune-supportive TME [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 5557.

Influence of the autotaxin-lysophosphatidic acid axis on cellular function and cytokine expression in different breast cancer cell lines

Previous studies provide high evidence that autotaxin (ATX)-lysophosphatidic acid (LPA) signaling through LPA receptors (LPAR) plays an important role in breast cancer initiation, progression, and invasion. However, its specific role in different breast cancer cell lines remains to be fully elucidated to offer improvements in targeted therapies. Within this study, we analyzed in vitro the effect of LPA 18:1 and the LPAR1, LPAR3 (and LPAR2) inhibitor Ki16425 on cellular functions of different human breast cancer cell lines (MDA-MB-231, MDA-MB-468, MCF-7, BT-474, SKBR-3) and the human breast epithelial cell line MCF-10A, as well as Interleukin 8 (IL-8), Interleukin 6 (IL-6) and tumor necrosis factor (TNF)-alpha cytokine secretion after LPA-incubation. ATX-LPA signaling showed a dose-dependent stimulatory effect especially on cellular functions of triple-negative and luminal A breast cancer cell lines. Ki16425 inhibited the LPA-induced stimulation of triple-negative breast cancer and luminal A cell lines in variable intensity depending on the functional assay, indicating the interplay of different LPAR in those assays. IL-8, IL-6 and TNF-alpha secretion was induced by LPA in MDA-MB-468 cells. This study provides further evidence about the role of the ATX-LPA axis in different breast cancer cell lines and might contribute to identify subtypes suitable for a future targeted therapy of the ATX-LPA axis.

Abstract P035: A promising cancer immunotherapy target: Novel fully human agonist antibodies against the human T-cell costimulatory receptor CD27

Abstract CD27 is a member of the TNF receptor superfamily and plays a critical role in T-cell activation by providing a costimulatory signal. CD27 signaling enhances T-cell proliferation, activation and differentiation of effector and memory T cells and therefore promotes cytotoxic T cell (CTL)-based anti-tumor immunity. Agonistic stimulation of CD27 is a promising cancer immunotherapy approach to boost specific T cell driven anti-tumor responses. In this study, we generated a series of 147 fully human monoclonal anti-CD27 antibodies and tested their agonist properties to stimulate T cell activation. Using a NF-κB reporter Jurkat cell line, we evaluated in vitro the ability of anti-CD27 antibodies to induce CD27 receptor activation. With this assay, five antibodies have been selected for their agonist properties. When combined with suboptimal T cell receptor (TCR) stimulation, agonist antibodies induced CD27 receptor activation with an EC50 of 1-5 ug/mL. We also used human peripheral blood T cells to characterize the CD27-mediated costimulatory effects of agonist antibodies in combination with TCR stimulation. Our anti-CD27 monoclonal antibodies boosted T cell proliferation and induced IL-2 and TNFalpha secretion only in a presence of TCR engagement. Moreover, CD27 agonists induce strong T cell proliferation in a Mixed Lymphocyte Reaction. CD27 antibodies were shown to bind human and cynomolgus monkey CD27 with a KD value of 5-20 nM as determined by BioLayer Interferometry, but do not bind to mouse CD27. In vivo experiments are currently ongoing to demonstrate the efficient anti-tumor activity of the selected CD27 agonist antibodies in different mice tumor models. In conclusion, we have developed and successfully selected efficient fully human immunostimulatory agonist CD27 mAbs as a promising cancer immunotherapy. Citation Format: Yulia Ovechkina, Shaarwari Sridhar, David Jurchen, David Peckham, Eric Tarcha, Shawn Iadonato, Thierry Guillaudeux. A promising cancer immunotherapy target: Novel fully human agonist antibodies against the human T-cell costimulatory receptor CD27 [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P035.

Hepatitis B virus genomic nucleic acid in the activation and maturation of bone marrow-derived dendritic cells

Hepatitis B virus (HBV) is the etiological agent that causes a self-limiting or chronic infection in the hepatocytes of about 250 million people worldwide. The role of adaptive immune system during HBV infection has been well studied. However, the innate immune system's responses against HBV during the early stage of infection largely remain unclear. In this study, we found that HBV genomic DNA or Salmon Sperm DNA (SSD) was able to induce the innate immune response in the macrophages cell line RAW264.7 but not the hepatocyte cell line, HepG2, indicating that hepatocytes may lack of a functional DNA-sensing pathway and hence are unable to respond to the presence of foreign DNA in the cytosol with type 1 IFN response. Thus, we hypothesized that non-parenchymal cells like the Antigen Presenting Cells (APC) might be crucial in triggering the initial immune response to suppress the virus replication and link the innate and adaptive responses. Using bone marrow-derived DCs (BMDC) as a model, this study demonstrated that HBV genomic DNA is able to induce cytokines like TNF-alpha, IL-6, and IL-12p40 secretion. We also examined the activation and maturation of BMDCs when exposed to the HBV genomic DNA intracellularly and extracellularly. A significant shift of CD86+ and CD40+ cell populations was observed during extracellular exposure of BMDC to Poly I:C and HBV genomic DNA, indicating that TLRs may be vital in the uptake of the extracellular viral DNA to activate the BMDCs. Moreover, transfection of intracellular nucleic acid stimuli, including HBV genomic DNA as well induced BMDCs maturation. Our findings highlight the critical function of DCs in antiviral response as a potential connection between the innate and adaptive immune systems during HBV pathogenesis. Nevertheless, further study is required to determine the role of cytosol DNA sensing pathway in DCs during HBV infection.

Characterization of Iturin V, a Novel Antimicrobial Lipopeptide from a Potential Probiotic Strain Lactobacillus sp. M31

Members of lactic acid bacteria group are known to produce various antimicrobial substances. Cyclic lipopeptides are one such potent class of amphipathic natural biosurfactants that exhibit bactericidal and immunomodulatory properties. In this study, we aimed to investigate antimicrobial and immunomodulatory activities of a lipopeptide secreted by a LAB isolate strain M31 identified as a member of the genus Lactobacillus. The lipopeptide that was purified using a combination of chromatographic techniques and matrix-assisted laser desorption/ionization-time of flight of pure lipopeptide displayed a molecular weight of 1002Da. MS/MS analysis confirmed the presence of 7 amino acids (Asp-Tyr-Asp-Val-Pro-Asp-Ser) and a C13 beta-hydroxy fatty acid. The amino acid composition assigned lipopeptide to iturin class. However, the replacement of Gln with Val revealed it to represent a novel iturin named as iturin V. Iturin V showed antibacterial activity and did not cause hemolysis or cytotoxicity upto 125µg/mL. It induced secretion of pro-inflammatory cytokines TNF-alpha and IL-12 in murine dendritic cells. Probiotic features of strain M31 coupled with notable activity of iturin V against species of the genera Pseudomonas and Vibrio suggest that strain M31 has potential application for pathogen intervention treatments in processing of aquatic food products.

Abstract PO-036: Immunological characterization of mouse HR+ mammary tumors relapsing after radiation therapy

Abstract Background. Hormone receptor (HR)+ breast cancer (BC) causes the majority of BC-related deaths in the US (Siegel, Miller et al. 2020). Standard treatment includes surgery, endocrine therapy +/- chemotherapy or radiation therapy (RT), depending on surgical procedure and risk assessment). However, approximately 50% of patients with HR+ do not experience disease eradication after standard-of-care therapy, ultimately relapse and succumb to the disease. It has been postulated that such cases of relapse reflect situations in which treatment is unable to elicit a strong immune response that would enable long-term disease control. Objective and procedures. To obtain insights into the immunological alterations accompanying disease relapse in HR+ BC exposed to RT, we harnessed an endogenous model of BC driven in immunocompetent mice by the implantation of a slow-release medroxyprogesterone acetate (MPA, M) pellet and oral administration of the carcinogen DMBA (D). This model recapitulates multiple key aspects of human luminal B BC, including a relatively ´cold´ microenvironment in basal conditions and hence limited sensitivity to PD-1 blockage (Buque, Bloy et al. 2020). To identify immunological alterations associated with disease relapse after RT, we undertook an in-depth characterization of the tumor (by DNAseq and RNAseq) and systemic (by flow cytometry on the splenic compartment) microenvironment of C57BL/6 female mice bearing tumors that recovered normal growth after either 21 Gy in a single fraction, or 3 consecutive doses of 10 Gy each (10 Gy X 3, total dose 30 Gy), which we previously demonstrated to mediate differential local control of the disease, with the latter approach being more efficient. Results. We observed a trend for reduction in splenic macrophage activation and abundance of B cells, T cells and NKT cells amongst tumors relapsing after single-dose vs fractionated RT, alongside an increased abundance of immunosuppressive TREG cells, elevated markers of exhaustion in both CD4+ and CD8+ cells and limited responsiveness to ex vivo stimulation in terms of TNFalpha secretion. Moreover, mice with M/D-driven tumors relapsing after single-dose RT exhibited a more pronounced secretion of IL17 by splenic CD8+ cells, which has been previously associated with immunosuppression in the BC microenvironment (Chabab, Barjon et al. 2020). As compared to control tumor-naïve mice, the splenocytes of mice with M/D-driven tumors relapsing after fractionated RT were sub-efficient at responding to ex vivo stimulation with cytokine and effector molecules, potentially linked to disease progression. DNAseq and RNAseq data are being analyzed and results will be available shortly. Impact: Identifying immunological correlates of relapse after RT can direct strategies to overcome resistance in HR+ BC patients, the majority of BC patients. If successful, this can inform therapeutic approaches to enable superior therapeutic responses in patients with HR+ BC, hence significantly reducing BC-related deaths. Citation Format: Norma Bloy, Aitziber Buque, Giulia Petroni, Takahiro Yamazaki, Ai Sato, Bhavneet Bhinder, Olivier Elemento, Silvia C. Formenti, Lorenzo Galluzzi. Immunological characterization of mouse HR+ mammary tumors relapsing after radiation therapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-036.

CTLA-2 Alpha Is a Potent Inhibitor of Angiogenesis in Murine Ocular Tissue.

Cytotoxic T lymphocyte antigen-2 (CTLA-2) alpha has been reported to suppress the activities of cathepsin L (Cath L), which is deeply involved in angiogenesis. Therefore, we assessed whether CTLA-2 alpha plays a role in angiogenesis in ocular tissue. To establish models of corneal inflammation and experimental choroidal neovascularization (CNV), male C57BL/6J mice (n = 5) underwent corneal suture placement or laser-induced CNV, respectively. Mice were then injected with recombinant CTLA-2 alpha (1 µg) into the peritoneal cavity at day 0 and every 2 days after operation. In vitro experiments were performed to assess the inflammatory response by measuring TNF-alpha secretion in peritoneal cavity exudate cells (PECs) or the proliferation of mouse vascular endothelial cells (mVECs). CTLA-2 alpha treatment dramatically suppressed corneal angiogenesis, as well as laser-induced CNV. Moreover, CTLA-2 alpha inhibited the proliferation of mVECs in vitro, while CTLA-2 alpha abolishment was able to rescue proliferation. However, CTLA-2 alpha could not suppress cytokine secretion from inflammatory cells such as PECs. In summary, CTLA-2 alpha was able to suppress angiogenesis by suppressing endothelial cell proliferation. Further studies are needed to investigate its usefulness as a new antiangiogenic treatment for a variety of conditions, including age-related macular degeneration.

Validation of a Peripheral Blood-Derived Microglia-like Cell System

Background: Individuals with chronic pain may have hyperactive microglia, which stimulate neurons to send a pain signal with little or no stimuli. Microglia are therefore a potential drug target to treat chronic pain, but drug discovery has been stymied by differences between human and animal neurobiology, and lack of healthy human CNS microglia. We cultured peripheral blood derived monocytes to develop characteristics of CNS derived microglia, termed peripheral blood derived microglia like cells (PB-MLC). We found that lipopolysaccharide (LPS) treated PB-MLCs from patients with chronic pain, from sickle cell disease (SCD) or chronic headaches, secreted more pro-inflammatory cytokines (TNF-alpha, IL-1beta, and IL-6) than PB-MLCs from normal donors, suggesting that patient pain phenotype was preserved in culture; PB-MLC from individuals with chronic pain were hyperactive in vitro as their microglia are in vivo. We hypothesize that PB-MLCs can be developed as a cell-based assay to screen compounds to treat chronic pain. To validate our model system, we compared cultured PB-MLCs to CNS derived microglia cells, using Sprague-Dawley rats, and treated human PB-MLC with microglia activation inhibitors shown to work in vivo in murine models. Methods: We isolated rat brain derived microglia (BDM) and rat peripheral blood monocytes; both were cultured with murine IL-34 (100 ng/ml) and GM-CSF (10 ng/ml). BDM and rPB-MLC were morphologically analyzed by fluorescence imaging microscopy, combined with machine learning, phenotyped by RT-qPCR and indirect immunofluorescence with anti-TMEM119, CD68, and Iba1 antibodies. Cells were treated with LPS for 24 hours, and TNF-alpha, IL-1beta, and IL-6 secretion measured by ELISA. For human PB-MLC studies, monocytes were cultured with GM-CSF (10 ng/ml) and IL-34 (100 ng/ml) for 7 days. PB-MLC morphology was analyzed as above; phenotyped with anti-CX3CR1, TMEM119, CD68, and Iba1 antibodies. PB-MLCs were treated with 100 ng/ml LPS with or without minocycline (2.5, 5, 10, 25 μg/mL), clopidogrel (1, 2, 4 μM) and MRS2395 (1, 5, 10 μM), for 24 h; TNF-alpha and IL-1beta secretion measured by ELISA. Results: We found that rPB-MLC resemble BDM morphologically, express the same microglia specific markers (TMEM119, P2RY12) and can be activated by LPS (Figure 1). Monocytes not cultured with IL-34 and GM-CSF did not express microglia specific genes (Figure 2A). To evaluate the possibility of using the PB-MLC model system to screen compounds to inhibit microglia activation, we tested PB-MLC cells with the following microglial inhibitors shown to be active in murine models in vivo: minocycline, MRS2395, and clopidogrel. MRS2395 and clopidogrel significantly suppressed the release of proinflammatory cytokine TNF-alpha from LPS-induced activated PB-MLCs in a dose-dependent manner (Figure 2B); minocycline did not. Conclusions: We validated our model system by comparing CNS derived microglia to rPB-MLCs and found they share morphology, similar cytokine secretion in response to LPS, and expression of microglia-specific genes. We confirmed that human PB-MLC expressed microglia specific genes while the original monocytes did not. Since P2Y12 is implicated in chronic pain, we tested two P2Y12 receptor agonists, clopidogrel and MRS2395, in our human PB-MLC system. When challenged with LPS, clopidogrel and MRS2395 inhibited LPS-induced PB-MLC activation in vitro as it had in vivo in a murine chronic pain model. We propose to use our human PB-MLC to screen for compounds that reduce microglia hyperactivity, to identify pharmacologic agents to treat chronic pain. Disclosures No relevant conflicts of interest to declare.

Exclusive enteral nutrition mediates gut microbial and metabolic changes that are associated with remission in children with Crohn\u2019s disease

A nutritional intervention, exclusive enteral nutrition (EEN) can induce remission in patients with pediatric Crohn’s disease (CD). We characterized changes in the fecal microbiota and metabolome to identify the mechanism of EEN. Feces of 43 children were collected prior, during and after EEN. Microbiota and metabolites were analyzed by 16S rRNA gene amplicon sequencing and NMR. Selected metabolites were evaluated in relevant model systems. Microbiota and metabolome of patients with CD and controls were different at all time points. Amino acids, primary bile salts, trimethylamine and cadaverine were elevated in patients with CD. Microbiota and metabolome differed between responders and non-responders prior to EEN. EEN decreased microbiota diversity and reduced amino acids, trimethylamine and cadaverine towards control levels. Patients with CD had reduced microbial metabolism of bile acids that partially normalized during EEN. Trimethylamine and cadaverine inhibited intestinal cell growth. TMA and cadaverine inhibited LPS-stimulated TNF-alpha and IL-6 secretion by primary human monocytes. A diet rich in free amino acids worsened inflammation in the DSS model of intestinal inflammation. Trimethylamine, cadaverine, bile salts and amino acids could play a role in the mechanism by which EEN induces remission. Prior to EEN, microbiota and metabolome are different between responders and non-responders.

Murine transfer of a human gene variant associated with exceptional longevity displays senolytic effects both in immune compartment and endothelium of aged mice

Abstract The persistence and accumulation of senescent cells has been shown to potentially play a role in the pathophysiology of age-related cardiovascular diseases. Indeed with time, a decline in immune efficacy, termed immunesenescence, and a deleterious secretory phenotype of senescent cells (SASP) generate that inflammatory background mainly mediating the elderly cardiovascular phenotype. Long Living Individuals (LLI) which delay aging, represent a model of positive biology and an exceptional resource to study and find a way to improve general public health. Previous studies from our group have shown that a human gene associated with exceptional longevity (LAV-BPIFB4) was able to block the atherosclerotic process in ApoE−/− mice by conferring the animals with a pro-resolving M2 macrophages profile. Furthermore, LAV-BPIFB4 promotes the recruitment of hematopoietic stem cells, reparative vascularization and frailty reduction. Here we sought to underpinn the role of LAV-BPIFB4 in counteracting the age-related remodeling of the immune responses. The effects of systemic adeno-associated viral vector-mediated LAV-BPIFB4 gene transfer on the immune dynamics in old mice have been investigated by an extensive flow cytometric approach in lymphoid tissues (bone marrow, spleen and peripheral blood). C57BL/6J mice were assigned to two age-matched experimental groups: a treatment group (AAV-LAV-BPIFB4; N=6 mice, aged 18–23 months and a control group (AAV-GFP; N=6 mice, aged 18–23 months. 30th and 60th day since the beginning of the infection, SA-beta Gal substrate has been used to identify CD45+ senescent cells in freshly isolated PBMC, splenocytes, bone marrow (BM)-derived cells. As expected, we monitored an increase in SA-betaGal activity in blood. This increase is most significant in CD11b+ myeloid cells, whithout affecting neither CD3+T neither NK1.1+Natural Killer (NK) cell compartment. Notably 30 days AAV-LAV-BPIFB4 infection and to a more the 60 days-treatment, resulted in a significant decrease in senescent pool of peripheral immune cells and a concomitant enrichment of senescent cells in spleen. Concomitantly, aorta from AAV-LAV treated mice showed significant reduction in SA-beta Gal positive area. Furthermore a LAV-BPIFB4 induction of pro-resolving M2 macrophages compared to control group was documented in the main haemocateretic organ. As consequence the senolytic effect of LAV-BPIFB4 gene-therapy well correlated with the rescue of proliferative index of splenocytes upon PHA stimulation, and their functional protective response to lipopolysaccharide (LPS) in term of IL-6 and TNF-alpha secretion. The restoration of a protective and balanced immune response finally reflected the reduction of senescent phenopype acquired by mouse aortic endothelial cells during the aging process in vivo. A better underpinning of the senolytic action of LAV-BPIFB4 may offer a valuable therapeutic tool to reverse aging phenotype causing most of cardiac diseases Funding Acknowledgement Type of funding source: Private company. Main funding source(s): Cariplo Foundation (n.2016-0874) to AAP and CV

As a biopatch, fibrin and bone marrow-derived cells modulate macrophage polarization and cardiomyobasts proliferation

Abstract Introduction Repair of the cardiac structure and function is the holy grail for curative treatment of myocardial infarction (MI). Clinical trials for cardiac cell-based therapies have revealed numerous challenges and led to a shift in the initial paradigms. The current concept is that cell/matrix-based therapy modifies the local immune response, releases factors involved in tissue repair, mitigates the progression of ventricular remodelling, and rescues the cardiac function. Macrophages are critical players in cardiac remodelling post-MI with a complex role yet not fully elucidated in cell-based therapy. Purpose We, first, investigated in an MI animal model, the myocardial macrophage content following an epicardial application of a biopatch composed of bone marrow-derived cells (BMDCs) and fibrin-based matrix. Second, we analyzed in vitro the cell interactions between the biopath, macrophages and cardiomyoblasts. Methods Two weeks post-infarct, performed on female rats, a Cell and Matrix BioPatch (CMBP) composed of fibrin and BMDCs was administered. Heart function and infarct were evaluated by high-resolution echocardiography and histology. Macrophages present 4 weeks post-MI were quantified by immunostaining for CD86 and CD206. In vitro, macrophages (undifferentiated (M0), triggered by IL4 (M2) or LPS/IFN-d (M1)), or cardiomyoblasts (H9C2) were cultured with conditioned media from CMBP. Their proliferation and plasticity were assessed by EDU incorporation and real-time cell analyzer and RT-PCR. Results Our data showed a significant improvement in ejection fraction after treatment and a reduced infarct expansion index (IEI MI/untreated= 0.08±0.05 vs. IEI MI/CMBP_treated =0.22±0.09, p=0.002). The ratio of CD86+/CD206+ cells was also significantly reduced in treated compared to untreated groups (ratioMI/untreated=0.96±0.82 vs ratioMI/fibrin_treated=0.51±0.12 and ratioMI/CMBP_treated=0.17±0.13, p<0.05) suggesting an alteration of the macrophage polarization. In vitro assays with CMBP-conditioned medium revealed a significant increase in the proliferation of M0 and M2. Also, M0 were polarized towards an M2 profile. A switch of M1 toward M2 was revealed by Gene expression profiles with a reduction of pro-inflammatory markers (CD86, MCP-1, IL-1beta, CD80, RANTES and iNOS) and an increase in the anti-inflammatory ones (Arginase 1, CD163, CD106). The secretion profile of the macrophages was also modified with a lower secretion of IL-6 and TNF-alpha and increased IL-10. Interestingly, when cardiomyoblasts are cultured with the medium conditioned of the macrophages triggered by CMBP, their proliferation was significantly increased by 16%±1 with M0 or M2 and 18%±1 with M1. Conclusion Our data point to a new paracrine communication from a biopatch that triggered macrophages and indirectly cardiac cells. Cell cross-talks promoted an anti-inflammatory response that promoted cardiac cells proliferation. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): University of Fribourg

Enrichment of apolipoprotein A-IV and apolipoprotein D in the HDL proteome is associated with HDL functions in diabetic kidney disease without dialysis

Background and aimsDiabetic kidney disease (DKD) is associated with lipid derangements that worsen kidney function and enhance cardiovascular (CVD) risk. The management of dyslipidemia, hypertension and other traditional risk factors does not completely prevent CVD complications, bringing up the participation of nontraditional risk factors such as advanced glycation end products (AGEs), carbamoylation and changes in the HDL proteome and functionality. The HDL composition, proteome, chemical modification and functionality were analyzed in nondialysis subjects with DKD categorized according to the estimated glomerular filtration rate (eGFR) and urinary albumin excretion rate (AER).MethodsIndividuals with DKD were divided into eGFR> 60 mL/min/1.73 m2 plus AER stages A1 and A2 (n = 10) and eGFR< 60 plus A3 (n = 25) and matched by age with control subjects (eGFR> 60; n = 8).ResultsTargeted proteomic analyses quantified 28 proteins associated with HDL in all groups, although only 2 were more highly expressed in the eGFR< 60 + A3 group than in the controls: apolipoprotein D (apoD) and apoA-IV. HDL from the eGFR< 60 + A3 group presented higher levels of total AGEs (20%), pentosidine (6.3%) and carbamoylation (4.2 x) and a reduced ability to remove 14C-cholesterol from macrophages (33%) in comparison to HDL from controls. The antioxidant role of HDL (lag time for LDL oxidation) was similar among groups, but HDL from the eGFR< 60 + A3 group presented a greater ability to inhibit the secretion of IL-6 and TNF-alpha (95%) in LPS-elicited macrophages in comparison to the control group.ConclusionThe increase in apoD and apoA-IV could contribute to counteracting the HDL chemical modification by AGEs and carbamoylation, which contributes to HDL loss of function in well-established DKD.

Modulating TNFα activity allows transgenic IL15-Expressing CLL-1 CAR T cells to safely eliminate acute myeloid leukemia

BackgroundC-type lectin-like molecule 1 (CLL-1) is highly expressed in acute myeloid leukemia (AML) but is absent in primitive hematopoietic progenitors, making it an attractive target for a chimeric antigen receptor...

Abstract 5571: Turning up the heat on pancreatic ductal adenocarcinoma via inhibiting vasoactive intestinal peptide signaling

Abstract Pancreatic ductal adenocarcinoma (PDAC) constitutes more than 90% of all pancreatic cancers and has a 5-year survival rate of only 5-7%. A dense stroma, an immunosuppressive tumor microenvironment (TME) and scarce tumor infiltrating T cells, contribute toward the poor responsiveness of PDAC to conventional and FDA-approved checkpoint therapies. In this study, we show that vasoactive intestinal peptide (VIP) signaling is a novel immune checkpoint pathway in PDAC, which when inhibited in combination with anti-PD1 checkpoint inhibitor, significantly decreases tumor burden and enhances T cell infiltration in mouse models of PDAC. VIP is a 28-amino acid neuropeptide secreted at the nerve terminals, pancreas and the GI tract, and is commonly associated with regulating gut motility and blood pressure. Interestingly, VIP is also secreted by immune cells and is known to have immunosuppressive properties such as decreasing T cell proliferation and polarizing T cells towards Th2 phenotype. Intriguingly, according to the cancer genome atlas (TCGA), human exocrine pancreatic cancers expresses the highest level of VIP mRNA expression when compared to other solid malignancies. Similarly, we have generated preliminary data that shows enhanced VIP levels both systemically and locally in mouse models of PDAC and in human PDAC patients. Thus, taking all this into consideration, we hypothesized that enhanced VIP signaling is a potential mechanism of immune escape in PDAC and that inhibiting VIP signaling either alone or in combination with checkpoint inhibitors would significantly enhance treatment response in mouse models of PDAC. We tested the effect of inhibiting VIP signaling by using a VIP receptor antagonist (VIPR antagonist) in orthotopic mouse models of PDAC, where luciferase transfected KPC cells (mouse PDAC cell line), were implanted in the tail of the pancreas. Mice were then treated with VIPR antagonist and/or anti-PD1 seven days after tumor implantation. Our results showed that the combination treatment significantly reduced tumor burden and growth rate, as well as, enhanced the infiltration of both CD4+ and CD8+ T cells into the tumor microenvironment (as per immunohistochemistry of tumor tissues). Furthermore, analysis of tumor infiltrating T cells via Nanostring analysis showed that the combination treatment also significantly enhanced levels of IFN gamma and TNF alpha secreting T cells and costimulatory and effector molecules, including CD27 and FasL. In summary, we have identified that VIP signaling is a novel and targetable immune checkpoint pathway in PDAC, which when inhibited in combination with checkpoint inhibitors, significantly improves treatment response in mouse PDAC models. As the VIP sequence is conserved across different species including mouse and human, and since we have observed VIPR antagonists stimulate proliferation of human T cells, there is an increased potential for clinical translation of VIPR antagonists in the treatment of human PDAC. Citation Format: Sruthi Ravindranathan, Brandon Ware, Mohammad Zaidi, Jingru Zhu, Rohan K. Dhamsania, Shuhua Wang, Yiwen Li, Ishani H. Rao, Susan N. Thomas, Gregory B. Lesinski, Bassel El-Rayes, Edmund K. Waller. Turning up the heat on pancreatic ductal adenocarcinoma via inhibiting vasoactive intestinal peptide signaling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5571.