Human Neutrophil Cell Research Articles

Dual protection of aqueous garlic extract biomolecules against hemolysis and its oxidation products in preventing inflammation.

Garlic (Allium sativum) is recognized as functional food, rich in bioactive compounds that can combat diseases associated with oxidative stress. This study aims to investigate the protective potential of aqueous garlic extract against hemolysis and oxidation. Despite being caused by membrane fragility, hemolysis can lead to inflammation through the oxidation of its products, and in some cases, even exacerbate it in certain pathological contexts. Supplementation with antioxidant molecules can improves oxidative status, in this study, we selected garlic, an excellent functional food, and targeted its effects using aqueous extract and pure molecules. The aqueous garlic extract was prepared under safe conditions and subjected to toxicity on human neutrophils and red blood cells before experimentation. The results indicate that aqueous garlic extract significantly reduces hemolysis with a maximum protection of 98. 74 ± 1. 08 % at a concentration of 5μg/ml. Additionally, experiments were conducted with pure compounds found in garlic such as quercetin, gallic acid, and caffeic acid. The outcomes show that quercetin reduces hemolysis of RBC with a maximum protection of 88. 8 ± 2. 89 % at 20 µM followed by caffeic acid and gallic acid. The action mechanism of the extract was tested on human neutrophil cells, the extract significantly reduced luminol-amplified chemiluminescence of PMA-stimulated neutrophils up to 50 % at 10 µg/ml in addition to its ability to directly scavenge hydrogen peroxide. Our results suggest that aqueous garlic extract exerts promising anti-inflammatory activity in vitro. Through its dual protection against hemolysis and Ros production, garlic may indirectly prevent inflammation reducing the oxidation of hemolysis products. These abilities make garlic aqueous extract promising candidate for improving cardiovascular health, reducing oxidative stress and modulating immunity.

Plasma-derived exosomal protein SHP2 deficiency induces neutrophil hyperactivation in Behcet's uveitis

To investigate the effect of plasma-derived exosomal proteins on neutrophil hyperactivation in Behcet's uveitis (BU), we treated neutrophils from healthy controls with plasma-derived exosomes from active BU patients, and determined the level of neutrophil activation by real-time quantitative PCR (RT-qPCR) and cytokine detection assay. The results revealed that exosomes from active BU patients could activate neutrophils as shown by increasing the expression levels of pro-inflammatory cytokines (IL-17 and IL-6), chemokines (IL-8 and MCP-1), and NETs (MPO and ELANE). Label-free quantitative proteomic analysis of plasma-derived exosomes from patients and healthy controls found a remarkably distinct protein profile and identified differentially expressed proteins (DEPs) between the two groups. The results of GO, KEGG, and GSEA enrichment analysis showed that DEPs were enriched in innate immune-mediated and neutrophil hyperactivation-related signaling pathways. The protein-protein interaction (PPI) analysis determined that SHP2 was a downregulated key hub protein in the exosomes of active BU patients. Knockdown of SHP2 in human neutrophil cell lines (NB4 cells) was shown to promote the secretion of pro-inflammatory cytokines, chemokines, and NETs. The converse effects were observed following SHP2 overexpression. In conclusion, we highlighted a pathogenic role of plasma-derived exosomal SHP2 deficiency in facilitating neutrophil activation and suggested that SHP2 might be an immunoprotective factor in BU pathologic process.

Innate activation of human neutrophils and neutrophil-like cells by the pro-inflammatory bacterial metabolite ADP-heptose and Helicobacter pylori

Lipopolysaccharide inner core heptose metabolites, including ADP-heptose, play a substantial role in the activation of cell-autonomous innate immune responses in eukaryotic cells, via the ALPK1-TIFA signaling pathway, as demonstrated for various pathogenic bacteria. The important role of LPS heptose metabolites during Helicobacter pylori infection of the human gastric niche has been demonstrated for gastric epithelial cells and macrophages, while the role of heptose metabolites on human neutrophils has not been investigated. In this study, we aimed to gain a better understanding of the activation potential of bacterial heptose metabolites for human neutrophil cells. To do so, we used pure ADP-heptose and, as a bacterial model, H. pylori, which can transport heptose metabolites into the human host cell via the Cag Type 4 Secretion System (CagT4SS). Main questions were how bacterial heptose metabolites impact on the pro-inflammatory activation, alone and in the bacterial context, and how they influence maturation of human neutrophils. Results of the present study demonstrated that neutrophils respond with high sensitivity to pure heptose metabolites, and that global regulation networks and neutrophil maturation are influenced by heptose exposure. Furthermore, activation of human neutrophils by live H. pylori is strongly impacted by the presence of LPS heptose metabolites and the functionality of its CagT4SS. Similar activities were determined in cell culture neutrophils of different maturation states and in human primary neutrophils. In conclusion, we demonstrated that specific heptose metabolites or bacteria producing heptoses exhibit a strong activity on cell-autonomous innate responses of human neutrophils.

Bis(4-glycosyloxybenzyl) 2-isobutyltartrate and dihydrophenanthrene derivatives from the pseudobulbs of Pholidota chinensis and their anti-inflammatory activity

Six previously undescribed components, bis(4-glycosyloxybenzyl) 2-isobutyltartrate derivatives (pholidotoside A-E) and phenolic glycoside (pholidotosin A), together with twenty known compounds were isolated from the pseudobulbs of Pholidota chinensis. Their structures and absolute configuration were elucidated and established through various spectroscopic and chemical methods. The anti-inflammatory potential of selected compounds was examined using a human neutrophil cell model activated by N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB). Among these, dihydrophenanthrenes exhibited potent inhibitory effect on both superoxide anion generation and elastase release assays with IC50 values ranging from 0.41 ± 0.05 to 7.14 ± 0.30 μM.

Human IL-17 and TNF-α Additively or Synergistically Regulate the Expression of Proinflammatory Genes, Coagulation-Related Genes, and Tight Junction Genes in Porcine Aortic Endothelial Cells.

Immune rejection is the major limitation for porcine xenograft survival in primate recipients. Proinflammatory cytokines play important roles in immune rejection and have been found to mediate the pathological effects in various clinical and experimental transplantation trials. IL-17 and TNF-α play critical pathological roles in immune disorders, such as psoriasis and rheumatoid arthritis. However, the pathological roles of human IL-17 (hIL-17) and human TNF-α (hTNF-α) in xenotransplantation remain unclear. Here we found that hIL-17 and hTNF-α additively or synergistically regulate the expression of 697 genes in porcine aortic endothelial cells (PAECs). Overall, 415 genes were found to be synergistically regulated, while 282 genes were found to be additively regulated. Among these, 315 genes were upregulated and 382 genes were downregulated in PAECs. Furthermore, we found that hIL-17 and hTNF-α additively or synergistically induced the expression of various proinflammatory cytokines and chemokines (e.g., IL1α, IL6, and CXCL8) and decreased the expression of certain anti-inflammatory genes (e.g., IL10). Moreover, hIL-17 plus hTNF-α increased the expression of IL1R1 and IL6ST, receptors for IL1 and IL6, respectively, and decreased anti-inflammatory gene receptor expression (IL10R). hIL-17 and hTNF-α synergistically or additively induced CXCL8 and CCL2 expression and consequently promoted primary human neutrophil and human leukemia monocytic cell migration, respectively. In addition, hIL-17 and hTNF-α induced pro-coagulation gene (SERPINB2 and F3) expression and decreased anti-coagulation gene (TFPI, THBS1, and THBD) expression. Additionally, hIL-17 and hTNF-α synergistically decreased occludin expression and consequently promoted human antibody-mediated complement-dependent cytotoxicity. Interestingly, hTNF-α increased swine leukocyte antigen (SLA) class I expression; however, hIL-17 decreased TNF-α-mediated SLA-I upregulation. We concluded that hIL-17 and hTNF-α likely promote the inflammatory response, coagulation cascade, and xenoantibody-mediated cell injury. Thus, blockade of hIL-17 and hTNF-α together might be beneficial for xenograft survival in recipients.

Abstract 3196: Soluble factors released by pancreatic cancer cells enhance neutrophil survival

Abstract Pancreatic cancer (PC) remains a challenge to modern-day cancer therapeutics, with a dismal five-year survival rate of 10%. Due to the pancreas' tricky location and the dense stroma surrounding it, patients have late diagnosis and fail to respond to chemotherapy regimens. The genetic stability of the tumor microenvironment (TME) in comparison with rapidly evolving tumor cells makes it an attractive target for the development of cancer therapeutics. Neutrophils, the most abundant immune cell, is an essential player in the TME. These neutrophils can become pro-tumorigenic based on signals they receive in the TME by regulating immune suppression, tumor metastasis, and angiogenesis. Previous studies from our laboratory demonstrate an increase in the number of neutrophils in the pancreatic tumor with the PC disease's progression in the KPC Model (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre). Based on our previous observation and accumulating literature, we hypothesize that pancreatic tumor cells can increase neutrophil proliferation and/or survival in circulation and tumor in situ. We investigated the effect of soluble factors released by pancreatic tumor cells on neutrophil proliferation/survival with this rationale. For this objective, we utilized murine promyelocytes cells (MPRO), a mouse neutrophil cell line, and a human leukemia cell line (HL60), a human neutrophil cell line. We treated MPRO and HL60 cells with a gradient of supernatants collected from T3M-4- and CD18/HPAF-parent and gemcitabine-resistant cells. By utilizing the WST assay, we observed an increase in MRPO and HL60 cells' viability with an increasing gradient of supernatant collected from both T3M-4, CD18/HPAF-parent gemcitabine-resistant cells with serum-free media as a control. We also observed a slight increase in neutrophil viability when treated with the supernatant collected from parent cell-lines supernatant compared to supernatant collected from the gemcitabine resistant cell-lines. Next, to delineate the effect of direct interaction between pancreatic tumor cells and neutrophils, we utilized a co-culture assay between MPRO cells and CD18/HPAF cells. We observed an increase in the viability of CD18/HPAF cells when placed in contact with the neutrophils; however, we observed inhibition in the viability of neutrophils placed in contact with the tumor cells. Our results showing positive viability of neutrophils with the treatment of soluble tumor factors explain higher neutrophil to lymphocyte ratio in PC patients' blood circulation. Neutrophils in the TME release pro-tumorigenic cytokines that increase tumor cell proliferation, albeit resulting in neutrophil death as observed by neutrophil growth inhibition. Along with neutrophil survival, a more mechanistic understanding of neutrophil recruitment and polarization in TME will facilitate designing novel therapeutics for PC. Citation Format: Paran Goel, Sugandha Saxena, Caitlin Molczyk, Rakesh Singh. Soluble factors released by pancreatic cancer cells enhance neutrophil survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3196.

Human neutrophil membrane-derived nanovesicles as a drug delivery platform for improved therapy of infectious diseases

Resolvins are a group of specialized proresolving lipid mediators (SPMs) enzymatically produced from omega-3 fatty acids during acute inflammation response to infections or tissue injury. Resolvin D1 (RvD1) is one of resolvins and is well studied in resolution of inflammation to treat inflammatory diseases. Resolution of inflammation includes the inhibition of polymorphonuclear leukocyte recruitment and reduced cytokine production. However, effective delivery of RvD1 to inflammatory tissues is challenging because of its lack of tissue targeting and poor physicochemical properties. Here, we proposed nanovesicles made from human neutrophil membrane which can specifically target inflamed tissues, and we loaded RvD1 on the surface of nanovesicles and antibiotic (ceftazidime, CEF) inside nanovesicles for improved treatment of bacterial infections. In a mouse model of bacterium-induced peritonitis, we demonstrated that human neutrophil cell membrane-formed vesicles (NMVs) enhanced inflammation resolution and bacterial killing after co-delivery of RvD1 and CEF. Our studies reveal that neutrophil nanovesicles may be critical for enhanced therapy to infectious diseases.

An Anti-Inflammatory 2,4-Cyclized-3,4-Secospongian Diterpenoid and Furanoterpene-Related Metabolites of a Marine Sponge Spongia sp. from the Red Sea.

Chemical investigation of a Red Sea Spongia sp. led to the isolation of four new compounds, i.e., 17-dehydroxysponalactone (1), a carboxylic acid, spongiafuranic acid A (2), one hydroxamic acid, spongiafuranohydroxamic acid A (3), and a furanyl trinorsesterpenoid 16-epi-irciformonin G (4), along with three known metabolites (−)-sponalisolide B (5), 18-nor- 3,17-dihydroxy-spongia-3,13(16),14-trien-2-one (6), and cholesta-7-ene-3β,5α-diol-6-one (7). The biosynthetic pathway for the molecular skeleton of 1 and related compounds was postulated for the first time. Anti-inflammatory activity of these metabolites to inhibit superoxide anion generation and elastase release in N-formyl-methionyl-leucyl phenylalanine/cytochalasin B (fMLF/CB)-induced human neutrophil cells and cytotoxicity of these compounds toward three cancer cell lines and one human dermal fibroblast cell line were assayed. Compound 1 was found to significantly reduce the superoxide anion generation and elastase release at a concentration of 10 μM, and compound 5 was also found to display strong inhibitory activity against superoxide anion generation at the same concentration. Due to the noncytotoxic activity and the potent inhibitory effect toward the superoxide anion generation and elastase release, 1 and 5 can be considered to be promising anti-inflammatory agents.

In Vitro Generation of Human Neutrophilic Myeloid-Derived Suppressor Cells.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells of myeloid origin. MDSC are functionally defined by their capacity to suppress T, NK, and B cell responses and henceforth altering the disease outcome in various pathological conditions. MDSC are further subdivided into three distinct subsets: monocytic (M-) MDSC, neutrophilic or polymorphonuclear (PMN-) MDSC, and early-stage (e-) MDSC. However, since surface markers utilized to define MDSC are expressed on other myeloid cells too, it is mandatory to functionally assess the suppressive activity for characterizing these cells. Here, we provide a protocol for generation of PMN-MDSC in vitro from freshly isolated human peripheral blood mononuclear cells. These MDSC can be used further to perform functional assays to determine their immunosuppressive potential or test their activities in various biological conditions, for instance in infection and cancer.

The Role of Zinc in Gliotoxin Biosynthesis of Aspergillus fumigatus.

Zinc performs diverse physiological functions, and virtually all living organisms require zinc as an essential trace element. To identify the detailed function of zinc in fungal pathogenicity, we carried out cDNA microarray analysis using the model system of Aspergillus fumigatus, a fungal pathogen. From microarray analysis, we found that the genes involved in gliotoxin biosynthesis were upregulated when zinc was depleted, and the microarray data were confirmed by northern blot analysis. In particular, zinc deficiency upregulated the expression of GliZ, which encodes a Zn2-Cys6 binuclear transcription factor that regulates the expression of the genes required for gliotoxin biosynthesis. The production of gliotoxin was decreased in a manner inversely proportional to the zinc concentration, and the same result was investigated in the absence of ZafA, which is a zinc-dependent transcription activator. Interestingly, we found two conserved ZafA-binding motifs, 5′-CAAGGT-3′, in the upstream region of GliZ on the genome and discovered that deletion of the ZafA-binding motifs resulted in loss of ZafA-binding activity; gliotoxin production was decreased dramatically, as demonstrated with a GliZ deletion mutant. Furthermore, mutation of the ZafA-binding motifs resulted in an increase in the conidial killing activity of human macrophage and neutrophil cells, and virulence was decreased in a murine model. Finally, transcriptomic analysis revealed that the expression of ZafA and GliZ was upregulated during phagocytosis by macrophages. Taken together, these results suggest that zinc plays an important role in the pathogenicity of A. fumigatus by regulating gliotoxin production during the phagocytosis pathway to overcome the host defense system.

OR04-5 Stimulation of Protein Disulfide Isomerase Activity by Activation of The Renin-Angiotensin System

Understanding the molecular and cellular mechanisms that are affected by activation of the renin-angiotensin system (RAS) are important for development of novel therapeutic strategies to more specifically treat hypertension and reduce cardiovascular risk. RAS regulates blood pressure (BP) in part via its effector molecule, angiotensin II (AngII), a potent vasopressor that stimulates the production of reactive oxygen species (ROS). Protein disulfide isomerase (PDI) is a multifunctional oxidoreductase that catalyzes thiol/disulfide interchange reactions within the endoplasmic reticulum and regulates ROS production. Also, PDI is present in circulation and at the cell surface of platelets, lymphocytes, erythrocytes and endothelial cells and plays a critical role in initiating thrombus formation. However, the interaction of RAS and PDI remain unclear. We hypothesized that RAS activation would lead to increased PDI levels thus contributing to oxidative stress. First, we studied the in vitro effects of AngII on EA.hy926, a human endothelial cell line, and measured PDI activity. Our results show that AngII dose and time dependently increased extracellular PDI activity (p<0.05, n=6); an event that was reduced to baseline levels by preincubation with 0.5 uM losartan, an AngII type 1 receptor antagonist (ARB). Consistent with these results, AngII stimulated: 1) a 3-fold rise of extracellular PDI levels as determined by ELISA (p<0.05, n=4); 2) a 2.5-fold increase in PDI mRNA levels by RT-PCR (p<0.05, n=4); and 3) increased PDI protein levels by western blot analyses that were blocked by losartan. We also studied the effect of AngII on extracellular PDI activity in differentiated HL-60 cells, a human polymorphonuclear neutrophil cell line, and observed significantly increased extracellular PDI activity following incubation with AngII (p<0.05, n=4). We then characterized the in vivo effects of exogenous AngII infusion on PDI in Sprague-Dawley rats, a model of increased BP, inflammation and target organ damage. We studied three conditions: (1) control; (2) AngII infused (80 ng/min x 28 days); (3) AngII + ARB (10 mg losartan/kg/day x 21 days). AngII infusion led to significant increases in plasma PDI levels that were reduced by 20% following ARB treatment (p<0.05, n=5/group). We then studied a model of naturally increased AngII and hypertension, the Otsuka Long Evans Tokushima Fatty rats (OLETF) and their strain controls that were randomly assigned to the following groups: (1) untreated strain controls; (2) untreated OLETF; (3) OLETF + ARB. OLETF rats had increased BP and significantly greater circulating PDI activity than control rats that was likewise blocked by ARB treatment (p<0.05; n=6/group). Thus, RAS activation represents a novel mechanism for increased PDI levels and activity that may contribute to the deleterious effects of disordered RAS in cardiovascular disease.

The effect of cassava (Manihot esculenta) leaf extract on COX-2 expression in the neutrophil cell culture exposed to the lipopolysaccharide of Escherichia coli (in-vitro study)

Introduction: Neutrophils are cells which played an initial role in the inflammation area and can be induced by lipopolysaccharide (LPS). Activated neutrophils will secrete the COX-2 enzyme which is involved in arachidonic acid synthesis to produce inflammatory mediators. However, excessive inflammation can cause tissue destruction; thus, the body needs anti-inflammation agents. Cassava leaf contains biochemical component which has an anti-inflammation effect. This study was aimed to determine the expression of COX-2 in neutrophils administered with cassava leaf extract and the LPS of E. coli. Methods: This study was experimental laboratories (in vitro study) using human neutrophil cells culture. The samples of this study were divided into 4 groups, administered with 12.5% cassava leaf extract (T1); 25% cassava leaf extract (T2); control group which contained only neutrophils cells (C1); and control group contained neutrophils and LPS (C2). Human neutrophil cells culture was incubated in the 12.5% and 25% cassava leaf extract before exposed with the LPS of E. coli. COX-2 expression was observed with immunochemistry methods. Results: The COX-2 expression in the 12.5% cassava leaf extract group was significantly higher than the 25% cassava leaf extract group (p &lt; 0.05). Conclusion: Cassava leaf extract can inhibit the COX-2 expression and can be used as an alternative anti-inflammation agent.Keywords: Anti-inflammation, cassava leaf extract, COX-2 expression, neutrophils.

A Genome-Wide Screen Identifies Factors Involved in S. aureus-Induced Human Neutrophil Cell Death and Pathogenesis.

Staphylococcus aureus is a commensal organism in approximately 30% of the human population and colonization is a significant risk factor for invasive infection. As a result of this, there is a great need to better understand how S. aureus overcomes human immunity. Neutrophils are essential during the innate immune response to S. aureus, yet this microorganism uses multiple evasion strategies to avoid killing by these immune cells, perhaps the most catastrophic of which is the rapid induction of neutrophil cell death. The aim of this study was to better understand the mechanisms underpinning S. aureus-induced neutrophil lysis, and how this contributes to pathogenesis in a whole organism model of infection. To do this we screened the genome-wide Nebraska Transposon Mutant Library (NTML) in the community acquired methicillin resistant S. aureus strain, USA300, for decreased ability to induce neutrophil cell lysis. Out of 1,920 S. aureus mutants, a number of known regulators of cell lysis (including the master regulators accessory gene regulator A, agrA and Staphylococcus exoprotein expression protein S, saeS) were identified in this blinded screen, providing validity to the experimental system. Three gene mutations not previously associated with cell death: purB, lspA, and clpP were found to be significantly attenuated in their ability to induce neutrophil lysis. These phenotypes were verified by genetic transductants and complemented strains. purB and clpP were subsequently found to be necessary for bacterial replication and pathogenesis in a zebrafish embryo infection model. The virulence of the clpP mutant was restored in a neutrophil-depleted zebrafish model, suggesting the importance of ClpP in mechanisms underpinning neutrophil immunity to S. aureus. In conclusion, our work identifies genetic components underpinning S. aureus pathogenesis, and may provide insight into how this commensal organism breaches innate immune barriers during infection.

Sex Steroids and the Induction of Human Neutrophil Cell Death by Monosodium Urate Crystals

Gout is characterized by the presence of monosodium urate (MSU) crystals in joints and surrounding tissues. When MSU is detected by tissue, it is treated as ＂danger/ damage＂, and there is rapid recruitment of neutrophils from the circulation into the affected area. Neutrophil death induced by MSU crystals has been found to be strongly related to gout flare-ups. Clinically, males are nearly three times more likely to develop gout than females, but the incidence of gout increases with age for both sex. Sex steroids have immunological properties and are able to modulate various immune factors and are able to relieve excessive inflammation. However, the effect of sex steroids on neutrophil cell death induced by MSU crystals remains unclear. We investigated whether progesterone (P4), estradiol (E2), testosterone (T), and dihydrotestosterone (DHT) individually are able to inhibit neutrophil cell death in the presence of MSU crystals. Neutrophils were isolated from the peripheral venous blood samples of male volunteers. Neutrophil cell death was detected using propidium iodide (PI) assays. The results confirmed that treatment of neutrophils with MSU crystals (600 μg/ml) induced significant cell death within 10 min. Furthermore, when neutrophils were pretreated with various sex steroids (P4, E2, T and DHT) individually for 15 min, the neutrophil cell death induced by MSU crystals was not affected. Sex steroids do not seem to be able to inhibit neutrophil cell lysis induced by MSU crystals and this implies that the rapid responses of neutrophils to sex steroids are not seem involved in gout flare-ups.

Alteration of microRNA 340-5p and Arginase-1 Expression in Peripheral Blood Cells during Acute Ischemic Stroke.

Acute stroke alters the systemic immune response as can be observed in peripheral blood; however, the molecular mechanism by which microRNA (miRNA) regulates target gene expression in response to acute stroke is unknown. We performed a miRNA microarray on the peripheral blood of 10 patients with acute ischemic stroke and 11 control subjects. Selected miRNAs were quantified using a TaqMan assay. After searching for putative targets from the selected miRNAs using bioinformatic analysis, functional studies including binding capacity and protein expression of the targets of the selected miRNAs were performed. The results reveal a total of 30 miRNAs that were differentially expressed (16 miRNAs were upregulated and 14 miRNAs were downregulated) during the acute phase of stroke. Using prediction analysis, we found that miR-340-5p was predicted to bind to the 3'-untranslated region of the arginase-1 (ARG1) gene; a luciferase reporter assay confirmed the binding of miR-340-5p to ARG1. miR-340-5p was downregulated whereas ARG1 mRNA was upregulated in peripheral blood in patients experiencing acute stroke. Overexpression of miR-340-5p in human neutrophil and mouse macrophage cell lines induced downregulation of the ARG1 protein. Transfection with miR-340-5p increased nitric oxide production after LPS treatment in a mouse macrophage cell line. Our results suggest that several miRNAs are dynamically altered in the peripheral blood during the acute phase of ischemic stroke, including miR-340-5p. Acute stroke induces the downregulation of miR-340-5p, which subsequently upregulates ARG1 protein expression.

Abstract 4706: Extracellular polysaccharides extracted from Parachlorella kessleri enhance host antitumor immune responses and inhibit colon carcinoma growth in mice

Abstract Natural products obtained from various resources are often found to be new sources of effective drugs for disease including cancer. The novel extracellular polysaccharides (exopolysaccharides) purified from Parachlorella kessleri (PCEPS), a generous gift by Panac ADVANCE Co. Ltd. (Tokyo, Japan), were evaluated on their antitumor and immunomodulatory effects in cell culture and mouse colon carcinoma peritoneal dissemination model. In two-dimensional cell culture, the PCEPS treatment inhibited cell growth of both murine and human colon carcinoma cells in a dose- and time-dependent manner. In contrast, the cell proliferation of mouse splenocytes (SPLs) and bone marrow cells (BMCs) were stimulated by the treatment with PCEPS. The treatment with PCEPS also increased specific subpopulations of the cells in BMCs: antigen presenting cells (CD19+ B cells, 33D1+ dendritic cells and CD68+ macrophage) and CD8+ cytotoxic T cells. In three-dimensional spheroid culture, spheroid growth of CT26 cells co-cultured with HL-60 human neutrophilic promyeloblasts and Jurkat cells (human T lymphoblasts), but not THP-1 human monocytes/macrophages, was significantly attenuated by PCEPS treatment. In transwell co-culture of CT26 and HL-60, THP-1 or Jurkat cells, mRNA level of NOX2 in HL-60 and THP-1 cells, and TNFα, IFNγ, IL-2 and granzyme B (GZMB) in Jurkat cells was significantly increased by PCEPS treatment. In a mouse CT26 colon carcinoma peritoneal dissemination model, intraperitoneal injection of PCEPS (10 mg/kg, started from 1 week after CT26 inoculation for 11 days, twice per week) significantly attenuated the growth of CT26 colon carcinoma in syngeneic mice by PCEPS treatment-associated apoptosis without any noticeable adverse effect. Analysis of subpopulation of intraperitoneally infiltrating immune cells collected from CT26 cell tumor-bearing mice found that PCEPS treatment increased CD4+ and CD8+ T lymphocytes, CD19+ B cell, and CD68+ macrophage populations. In contrast, 33D1+ dendritic cell and LY6G+ neutrophil populations were decreased as compared to PBS-treated group. The present study suggests that PCEPS inhibits colon carcinoma growth via direct cell growth inhibition and a stimulation of the host antitumor immune responses. Taken together, the current study suggests that exopolysaccharides derived from Parachlorella kessleri contain significant bioactive materials that inhibit colon carcinoma growth. This study was supported by Kansas State University Johnson Cancer Research Center 2011JCRC-EQ (MT), 2014CRA (ZG and MT), 2015CRA (JH, HH, AN, and MT), 2016CRA (HH and MT), College of Veterinary Medicine Dean's funds 2010CVM-EQ (MT), and NIH grant P20 RR017686 (MT). Citation Format: Susumu Ishiguro, Deepthi Uppalapati, Zachary Goldsmith, Dana Robertson, Jacob Hodge, Hayley Holt, Arashi Nakashima, Katie Turner, Masaaki Tamura. Extracellular polysaccharides extracted from Parachlorella kessleri enhance host antitumor immune responses and inhibit colon carcinoma growth in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4706.

Effects of 18β-Glycyrrhetinic Acid on Fungal Protease-Induced Airway Inflammatory Responses.

Airway epithelial cells secrete diverse inflammatory mediators in response to various stimuli. Thus, early regulation of immune responses in the airway epithelium is likely critical for the control of chronic inflammatory diseases. The purpose of the present study was to evaluate the effects of 18β-glycyrrhetinic acid (GA) on inflammatory responses generated in response to a fungal protease allergen that induces epithelial damage. To understand the underlying mechanisms, we also investigated the inhibitory effects of GA on the production of mitochondrial reactive oxygen species (ROS) in the human bronchial epithelial cell line BEAS2B. In this study, GA treatment reduced cytokine production and the human neutrophil cell line HL60 migration through decreased mitochondrial ROS production. In addition, GA significantly reduced inflammatory cell infiltration and cytokine levels in the bronchoalveolar lavage (BAL) fluid of fungal allergen-administered mice. Inhibitory effects of GA are dependent on the mitochondrial ROS/MAPK axis. Moreover, the effect of GA on the regulation of mitochondrial ROS depends on the expression of uncoupling protein-2 (UCP-2). Taken together, GA might represent a potential therapeutic agent for blocking inflammatory responses in airways.

Anti-Inflammatory Lobane and Prenyleudesmane Diterpenoids from the Soft Coral Lobophytum varium.

New lobane-based diterpenoids lobovarols A–D (1–4) and a prenyleudesmane-type diterpenoid lobovarol E (5) along with seven known related diterpenoids (6–12) were isolated from the ethyl acetate extract of a Taiwanese soft coral Lobophytum varium. Their structures were identified on the basis of multiple spectroscopic analyses and spectral comparison. The absolute configuration at C-16 of the known compound 11 is reported herein for the first time. The anti-inflammatory activities of compounds 1–12 were assessed by measuring their inhibitory effect on N-formyl-methionyl-leucyl-phenyl-alanine/cytochalasin B (fMLP/CB)-induced superoxide anion generation and elastase release in human neutrophils. Metabolites 2, 5, and 11 were found to show moderate inhibitory activity on the generation of superoxide anion, while compounds 5, 8, 11, and 12 could effectively suppress elastase release in fMLP/CB-stimulated human neutrophil cells at 10 μM. All of the isolated diterpenoids did not exhibit cytotoxic activity (IC50 > 50 μM) towards a limited panel of cancer cell lines.

In Vitro Oxidation of Collagen Promotes the Formation of Advanced Oxidation Protein Products and the Activation of Human Neutrophils.

The accumulation of advanced oxidation protein products (AOPPs) has been linked to several pathological conditions. Here, we investigated collagen as a potential source for AOPP formation and determined the effects of hypochlorous acid (HOCl)-treated collagen (collagen-AOPPs) on human neutrophil activity. We also assessed whether alpha-tocopherol could counteract these effects. Exposure to HOCl increased the levels of collagen-AOPPs. Collagen-AOPPs also stimulated the production of AOPPs, nitric oxide (NO), superoxide radicals (O2(-)), and HOCl by neutrophils. Collagen-AOPPs induced apoptosis and decreased the number of viable cells. Alpha-tocopherol prevented the formation of collagen-AOPPs, strongly inhibited the collagen-AOPP-induced production of O2(-) and HOCl, and increased the viability of neutrophils. Our results suggest that collagen is an important protein that interacts with HOCl to form AOPPs, and consequently, collagen-AOPP formation is related to human neutrophil activation and cell death.

Ptdglc-Enriched Lipid Rafts Are a Novel Platform for Apoptosis Signaling in Human Neutrophilic Lineage Cells

Phosphatidylglucoside (PtdGlc) is a recently discovered type of cell surface glycophospholipid that was originally detected in human cord red cells. PtdGlc was also detected in early erythroblastic leukemia cells, astroglial cells from fetal rat brains and the HL-60 acute promyelocytic leukemia (APL) cell line, and thought to be associated with their differentiation (Biochim Biophys Acta. 1851: 90, 2015). Indeed, the recombinant anti-PtdGlc Fab antibody rGL-7 induced HL-60 cells to differentiate into neutrophilic cells along with inducing the phosphorylation of Lyn and Hck (PNAS 100: 7454, 2003). These activities were suppressed by methyl-β-cyclodextrin, which reduced endogenous cholesterol, suggesting that PtdGlc forms lipid raft-like domains on HL-60 cells. Another anti-PtdGlc monoclonal antibody DIM21 induced the formation of FAS-colocalized clusters of PtdGlc on plasma membranes and the apoptosis of human neutrophils[RJ1]. However, it is still unclear about the molecular connection of PtdGlc, inserted into the external leaflet of the membrane bilayer, with signal transduction molecules located at the cytoplasmic side. In this study, we investigated the differentiation effect of DIM21 by comparing the ones of dimethyl sulfoxide (DMSO) and all-trans retinoic acid (ATRA), and identified the extracellular proteins associate with PtdGlc-enriched domains on plasma membranes during the activation of apoptosis signaling in neutrophilic lineage cells.HL60 cells or chronic neutrophilic leukemia (MOLM-20) cells were treated with DMSO or ATRA in the absence or presence of DIM21, and the expression of differentiation markers was determined. DIM21 induced the neutrophilic differentiation marker CD11b by itself, and synergistically enhanced DMSO-induced CD11b expression in HL-60 and MOLM-20 cells. DIM21 significantly enhanced annexin V positive DMSO-treated neutrophilic differentiated HL-60 (D-HL60) cells, but not MOLM-20 cells. FITC BrdU Flow analysis showed that the both ATRA and DIM21 induced the cell cycle accumulation in G0/G1 phase and reduced the percentage in S phase, suggesting that not only ATRA but also DIM21 is capable of reducing cell proliferation. Treatment with ATRA or DMSO increased the expression of PtdGlc and FAS in HL60, while DIM21 in itself is not capable of inducing the PtdGlc expression. To isolate the extracellular proteins associate with PtdGlc-enriched domains on plasma membranes, PtdGlc associated molecules on plasma membranes were labeled with FITC by the enzyme-mediated activation of radical sources (EMARS) method. Silver staining and western blotting analyses showed that the treatment with DIM21 enhanced the expression of several proteins in HL60, including FAS, integrin αM/β2 (CD11b/CD18). Furthermore, about five FITC-labeled proteins were immunoprecipitated with anti-FITC antibody and detected by sequenced SDS-PAGE. The shot-gun proteomics analysis detected an approximately equal number of FITC-labeled CD18 peptides in the resting and DIM21-stimulated HL60 cells. Other FITC-labeled proteins detected by proteomics analysis were included insulin receptor and Insulin like growth factor-1-receptor which is responsible in cell growth and survival control. These findings indicate that PtdGlc might have an intimate involvement in cell survival through the direct interaction with integrins and other transmembrane receptors. The novel strategies targeting PtdGlc by monoclonal antibody DIM21 warrant further exploration in patients with leukemia including APL.[RJ1] DisclosuresEkyalongo:ONO PHARMACEUTICAL CO., LTD: Research Funding. Iwabuchi:ONO PHARMACEUTICAL CO., LTD: Research Funding.