Human Polymorphonuclear Cells Research Articles

The Response of Paracoccidioides lutzii to the Interaction with Human Neutrophils.

The fungal pathogen Paracoccidioides lutzii causes systemic mycosis Paracoccidioidomycosis (PCM), which presents a broad distribution in Latin America. Upon infection, the fungus undergoes a morphological transition to yeast cells and provokes an inflammatory granulomatous reaction with a high number of neutrophils in the lungs. In this work, we employed proteomic analysis to investigate the in vitro response of the fungus to the interaction with human neutrophils. Proteomic profiling of P. lutzii yeast cells harvested at 2 and 4 h post interaction with human polymorphonuclear cells allowed the identification of 505 proteins differentially accumulated. The data indicated that P. lutzii yeast cells underwent a shift in metabolism from glycolysis to Beta oxidation, increasing enzymes of the glyoxylate cycle and upregulating enzymes related to the detoxification of oxidative and heat shock stress. To our knowledge, this is the first study employing proteomic analysis in the investigation of the response of a member of the Paracoccidioides genus to the interaction with neutrophils.

Peptides Derived from Soybean β-Conglycinin Induce the Migration of Human Peripheral Polymorphonuclear Leukocytes.

Food-derived peptides have various biological activities. When food proteins are ingested orally, they are digested into peptides by endogenous digestive enzymes and absorbed by the immune cell-rich intestinal tract. However, little is known about the effects of food-derived peptides on the motility of human immune cells. In this study, we aimed to understand the effects of peptides derived from a soybean protein β-conglycinin on the motility of human peripheral polymorphonuclear leukocytes. We illustrated that MITL and MITLAIPVNKPGR, produced by digestion using in-vivo enzymes (trypsin and pancreatic elastase) of β-conglycinin, induces the migration of dibutyryl cAMP (Bt2 cAMP)-differentiated human promyelocytic leukemia 60 (HL-60) cells and human polymorphonuclear leukocytes in a dose- and time-dependent manner. This migration was more pronounced in Bt2 cAMP-differentiated HL-60 cells; mRNA expression of formyl peptide receptor (FPR) 1 increased significantly than in all-trans-retinoic acid (ATRA)-differentiated HL-60 cells. This migration was inhibited by tert-butoxycarbonyl (Boc)-MLP, an inhibitor of FPR, and by pretreatment with pertussis toxin (PTX). However, the effect was weak when treated with WRW4, a selective inhibitor of the FPR2. We then demonstrated that MITLAIPVNKPGR induced intracellular calcium responses in human polymorphonuclear leukocytes and Bt2 cAMP-HL60 cells. Furthermore, pre-treatment by fMLP desensitized the calcium response of MITLAIPVNKPGR in these cells. From the above, MITLAIPVNKPGR and MITL derived from soybean β-conglycinin induced polymorphonuclear leukocyte migration via the FPR1-dependent mechanism. We found chemotactic peptides to human polymorphonuclear leukocytes, which are the endogenous enzyme digests of soybean protein.

Complement dependent TNFα production in neutrophil-like HL60 cells

Neutrophils develop in the bone marrow (BM) from hematopoietic stem cells (HSCs) through a series of progenitor cells and mature neutrophils play a critical role in the human immune system. Previous studies revealed that tumor necrosis factor α (TNFα) produced by immature neutrophils contributes to HSCs development and vascular regeneration in the BM niche. However, the precise mechanism of TNFα production in immature neutrophils remains unclear. This study aims to assess the relationship between complement C3 activation and TNFα production from immature neutrophils. We investigated the regulatory mechanism of TNFα production by complement components in neutrophil-like HL60 cells. Flow cytometric analysis showed that C3a receptor (C3aR) and C3bi receptor (CR3, Mac-1, CD11b/CD18, integrin αMβ2) are expressed on the surface of neutrophil-like HL60 cells. We found that zymosan-treated human serum leads to TNFα production in neutrophil-like HL60 cells, but not in human polymorphonuclear cells (PMNs). A C3-convertase inhibitor, compstatin suppresses TNFα production. These data suggest that the TNFα production is mediated by complement C3 activation. Furthermore, the TNFα production is enhanced by Ca2+ elevating agents, thapsigargin (TG), but is suppressed by treatment with Ca2+ chelators, EGTA, or BAPTA-AM. In addition, the soluble TNFα production is suppressed by treatment with immobilized-fibrinogen or -fibronectin. Thus, the TNFα production is enhanced by intracellular Ca2+ elevation and is negatively regulated by the interaction between the neutrophil-like HL60 cells and fibrinogen or fibronectin.

A novel bioassay to detect Nociceptin/Orphanin FQ release from single human polymorphonuclear cells.

Nociceptin/Orphanin FQ (N/OFQ) is the endogenous opioid agonist for the N/OFQ receptor or NOP. This receptor system is involved in pain processing but also has a role in immune regulation. Indeed, polymorphonuclear cells (PMNs) express mRNA for N/OFQ precursor and are a potential source for circulating N/OFQ. Current measurements are based on ELISA and RIA techniques. In this study we have designed a bioassay to measure N/OFQ release from single PMNs. Chinese Hamster Ovary (CHO) cells transfected with the human (h) NOP receptor and Gαiq5 chimera force receptor coupling in biosensor cells to increase intracellular Ca2+; this can be measured with FLUO-4 dye. If isolated PMNs from healthy human volunteers are layered next to CHOhNOPGαiq5 biosensor cells then stimulated with the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) we hypothesise that released N/OFQ will activate the biosensor. PMNs also release ATP and CHO cells express purinergic receptors coupled to elevated Ca2+. In a system where these receptors (P2Y1, P2Y2 and P2X7) are blocked with high concentrations of PPADS and oATP, PMN stimulation with fMLP increases Ca2+ in PMNs then shortly afterwards the biosensor cells. Our data therfore reports detection of single cell N/OFQ release from immune cells. This was absent when cells were preincubated with the selective NOP antagonist; SB-612111. Collectively this is the first description of single cell N/OFQ release. We will deploy this assay with further purified individual cell types and use this to further study the role of the N/OFQ-NOP system in disease; in particular sepsis where there is strong evidence for increased levels of N/OFQ worsening outcome.

Effects of Modified Melatonin Release on Human Colostrum Neutrophils to Induce Death in the MCF-7 Cell Line.

Cancer is one of the diseases with the highest mortality rate today, with breast cancer being the second most common type among the Brazilian population. Due to its etiological complexity and inefficiency of treatments, studies have focused on new forms of treatment. Among these forms of treatment, hormonal therapy seems to be an excellent auxiliary mechanism in tumoricidal activity, and melatonin has great potential as a modulator of the immune system. Thus, the present study is aimed at evaluating the effect of the hormone melatonin on the coculture of colostrum polymorphonuclear cells and MCF-7 cancer cells and evaluates the effect of this hormone using a modified transport system. A feasibility analysis was performed by fluorescence microscopy at three cell incubation times, 2 hours, 24 hours, and 72 hours. The measurement of cytokines in the cell supernatant occurred in 24 hours, and the apoptosis assay was performed in 72 hours using flow cytometry. The results showed higher levels of cell viability in groups treated with melatonin and less viability in groups containing a coculture of polymorphonuclear cells and MCF-7 after 72 hours of incubation. Furthermore, the apoptosis and necrosis rates were higher in coculture polymorphonuclear and MCF-7 cells, especially in groups containing microemulsion as a modified release agent. These data suggest that melatonin, especially if associated with a modified release system, has immunomodulatory effects on human colostrum polymorphonuclear cells. These cells can play a crucial role in the resolution of the tumor through their mediation and inflammatory action.

Complement Factor H Immunoproteins Increase Complement-mediated Opsonophagocytic Killing of Methicillin-resistant Staphylococcus aureus

Abstract Background As a major cause of a myriad of superficial and invasive infections worldwide, Staphylococcus aureus is a master of immune evasion, with the complement system being a primary target. Previously, we have shown that S. aureus surface protein SdrE binds the regulator Factor H (FH) to inhibit complement. Taking advantage of this interaction, we tested the effect of a fusion protein comprised of FH(18–20) fused to an IgG Fc on complement-mediated opsonophagocytosis and killing of methicillin resistant S. aureus. Methods Fusions were produced via the ΔXT/FT Nicotiana benthamiana plant expression system. Binding dynamics were examined via immunoblotting, fluorescence and ELISA. The effect of fusions on complement activation was determined by examining C3-fragment opsonization of S. aureus (total C3, C3b, iC3b) and subsequent C5a generation. The effect of fusions on S. aureus survival was assessed using an opsonphagocytosis assay with human polymorphonuclear cells. Results S. aureus bound significantly more FH-Fc compared to Fc-controls, and competed with serum FH for S. aureus binding. FH-Fc outperformed a complement inactive fusion variant in C3-fragment deposition and C5a generation, signifying an active role for the Fc region of FH-Fc. For 75% (3/4) of clinical isolates tested, FH-Fc significantly reduced S. aureus survival. Clinical isolates were all sdrE positive, sequence type 8, SCCmec IVa and Panton-Valentine Leukocidin (pvl) positive). Conclusion FH-Fc competitively inhibited serum FH from S. aureus binding and increased complement-mediated opsonophagocytosis and killing of CA-MRSA. Future studies will focus on enhancing the efficacy of FH-Fc fusion molecules as potential anti-staphylococcal therapeutics. Supported by a grant from the US Department of Defense, USAMRDC (W81XWH-19-1-0119).

Inhibitory effects of Syzygium jambos extract on biomarkers of endothelial cell activation

BackgroundDisordered endothelial cell activation plays an important role in the pathophysiology of atherosclerosis, cancer, sepsis, viral infections, and inflammatory responses. There is interest in developing novel therapeutics to regulate endothelial cell function in atherothrombotic, metabolic, vascular, and hematological diseases. Extracts from leaves of the Syzygium jambos (L.) Alston (S. jambos) trees have been proposed to treat cardiovascular diseases and diabetes through unclear mechanisms. We investigated the effects of the S. jambos extract on biomarkers of endothelial dysfunction and immune responses in the human endothelial cell line, EA.hy926.MethodsLeaves of S. jambos were collected, concocted and lyophilized. To study the effects of S. jambos on endothelial cell activation, we used the human endothelial cell line. IL-6 levels were measured using qPCR and ELISA. PDI activity was measured using Insulin Turbidity and Di-E-GSSG assays. CM-H2DCFDA was used to study ROS levels. Migration assay was used to study S. jambos effect on ex vivo human polymorphonuclear and human mononuclear cells.ResultsOur results show that incubation of EA.hy926 cells with ET-1 led to a 6.5 ± 1.6 fold increase in IL-6 expression by qPCR, an event that was blocked by S. jambos. Also, we observed that ET-1 increased extracellular protein disulfide isomerase (PDI) activity that was likewise dose-dependently blocked by S. jambos (IC50 = 14 μg/mL). Consistent with these observations, ET-1 stimulated ex vivo human polymorphonuclear and mononuclear cell migration that also was dose-dependently blocked by S. jambos. In addition, ET-1 stimulation led to significant increases in ROS production that were sensitive to S. jambos.ConclusionOur results suggest that the S. jambos extract represents a novel cardiovascular protective pharmacological approach to regulate endothelial cell activation, IL-6 expression, and immune-cell responses.

Cold Atmospheric Plasma Promotes the Immunoreactivity of Granulocytes In Vitro.

Cold atmospheric plasma (CAP) reduces bacteria and interacts with tissues and cells, thus improving wound healing. The CAP-related induction of neutrophils was recently described in stained sections of wound tissue in mice. Consequently, this study aimed to examine the functionality of human polymorphonuclear cells (PMN)/granulocytes through either a plasma-treated solution (PTS) or the direct CAP treatment with different plasma modes and treatment durations. PTS analysis yielded mode-dependent differences in the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) after CAP treatment. Live-cell imaging did not show any chemo-attractive or NETosis-inducing effect on PMNs treated with PTS. The time to maximum ROS production (TmaxROS) in PMNs was reduced by PTS and direct CAP treatment. PMNs directly treated with CAP showed an altered cell migration dependent on the treatment duration as well as decreased TmaxROS without inducing apoptosis. Additionally, flow cytometry showed enhanced integrin and selectin expression, as a marker of activation, on PMN surfaces. In conclusion, the modification of PMN immunoreactivity may be a main supporting mechanism for CAP-induced improvement in wound healing.

Effective Photodynamic Therapy with Ir(III) for Virulent Clinical Isolates of Extended-Spectrum Beta-Lactamase Klebsiella pneumoniae.

Background: The extended-spectrum beta-lactamase (ESBL) Klebsiella pneumoniae is one of the leading causes of health-associated infections (HAIs), whose antibiotic treatments have been severely reduced. Moreover, HAI bacteria may harbor pathogenic factors such as siderophores, enzymes, or capsules, which increase the virulence of these strains. Thus, new therapies, such as antimicrobial photodynamic inactivation (aPDI), are needed. Method: A collection of 118 clinical isolates of K. pneumoniae was characterized by susceptibility and virulence through the determination of the minimum inhibitory concentration (MIC) of amikacin (Amk), cefotaxime (Cfx), ceftazidime (Cfz), imipenem (Imp), meropenem (Mer), and piperacillin–tazobactam (Pip–Taz); and, by PCR, the frequency of the virulence genes K2, magA, rmpA, entB, ybtS, and allS. Susceptibility to innate immunity, such as human serum, macrophages, and polymorphonuclear cells, was tested. All the strains were tested for sensitivity to the photosensitizer PSIR-3 (4 µg/mL) in a 17 µW/cm2 for 30 min aPDI. Results: A significantly higher frequency of virulence genes in ESBL than non-ESBL bacteria was observed. The isolates of the genotype K2+, ybtS+, and allS+ display enhanced virulence, since they showed higher resistance to human serum, as well as to phagocytosis. All strains are susceptible to the aPDI with PSIR-3 decreasing viability in 3log10. The combined treatment with Cfx improved the aPDI to 6log10 for the ESBL strains. The combined treatment is synergistic, as it showed a fractional inhibitory concentration (FIC) index value of 0.15. Conclusions: The aPDI effectively inhibits clinical isolates of K. pneumoniae, including the riskier strains of ESBL-producing bacteria and the K2+, ybtS+, and allS+ genotype. The aPDI with PSIR-3 is synergistic with Cfx.

ERBB1/2/3 Expression, Prognosis, and Immune Infiltration in Cutaneous Melanoma.

BackgroundThe four ERBB tyrosine kinase family members [ERBB1 (epidermal growth factor receptor, EGFR), ERBB2 (HER2), ERBB3 (HER3), and ERBB4 (HER4)] (ERBB receptor family) have been shown, according to previous studies, to be related to the cutaneous melanoma. ERBB3 is the only member of the ERBBs that lacks tyrosine kinase activity and thus needs to dimer with other tyrosine kinases receptors to trigger the signaling pathway, while ERBB3 may dimer with all members of the ERBB family. Melanoma progression depends on activation of ERBB signaling, especially the ERBB3/ERBB2 cascade. There are lymphocytes and T cell infiltrates in melanoma. Numerous pieces of evidences indicate that local immune status plays an important role in the formation of anti-tumor immune responses. However, the relationship between the ERBBs and prognosis and immune infiltration in cutaneous melanoma is not completely clear.MethodsThe expression of the ERBBs was analyzed through the Oncomine database, Gene Expression Profiling Interactive Analysis (GEPIA), respectively. Immunohistochemistry of ERBBs was obtained from the Human Protein Atlas is increased before HPA database. ERBBs genes expression and mutation analysis in cutaneous melanoma from the cBioPortal. Functional annotation and Kyoto Encyclopedia of Genes and Genomes is increased before KEGG pathway enrichment analysis from the Metascape. Correlations between ERBBs and 31 genes that were close to each other and frequently altered were explored by GEPIA. Using the GEPIA database, we also investigated the relationship between ERBBs and myeloid-derived suppressor cells (MDSC) in cutaneous melanoma. The disease-free survival and different tumor stages of ERBBs were evaluated by GEPIA. The correlation of ERBBs and tumor-infiltrating immune cells and prognostic(5 years survival rates) was tested by the Tumor Immune Estimation Resource (TIMER).ResultsIn general, the expression levels of ERBB1/2 in cutaneous melanoma were lower than those in normal skin tissue. By contrast, the ERBB3 expression level was higher in cutaneous melanoma than in normal skin tissue. Low expression of ERBB1/2 and high expression of ERBB3 were detrimental to the 5 years survival of cutaneous melanoma patients (ERBB1: log-rank P: 0.03; ERBB2: log-rank P: 0.008; ERBB3: log-rank P: 0.039). ERBB4 expression may not affect the prognosis of patients with cutaneous melanoma. ERBBs may not play a role in the tumor stage and disease-free survival in cutaneous melanoma patients. The relationship between the ERBB family and 31 genes that were close to each other and frequently altered is demonstrated as the genes regulated by the ERBB family being mainly concentrated in the RAS/RAF/MEK/ERK signaling pathway. ERBB2 can induce infiltration of CD8+ T cells and B cells, while ERBB3 can induce infiltration of CD4+ T cells, CD8+ T cells, and Neutrophil cells. ERBBs are more significantly associated with M1 macrophages, dendritic cells, Th1, Th2, Th17, and Treg cellular immune markers (Cor > 0.2). ERBB2/3 were related to MDSC in cutaneous melanoma, including human mononuclear myeloid-derived suppressor cells (M-MDSC) and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC), and may influence the progression of cutaneous melanoma through MDSC, but the conclusion needs further probing.ConclusionThis study investigated the prognosis and immune infiltration of the ERBB family in cutaneous melanoma. Our results suggest that ERBB1/2/3 may serve as early prognostic markers and potential therapeutic targets in cutaneous melanoma.

Cytokine Regulation from Human Peripheral Blood Leukocytes Cultured In Vitro with Silver Doped Bioactive Glasses Microparticles.

Bioactive glasses (BG) applications include tissue engineering for bone regeneration, coating for implants, and scaffolds for wound healing. BG can be conjugated to ions like silver, which might add some antimicrobial properties to this biomaterial. The immunomodulatory activity of ion-doped bioactive glasses particles was not investigated before. The aim of this work was to evaluate the cytotoxic and immunomodulatory effect of BG and silver-doped bioactive glass (BGAg) in human peripheral blood cells. BG and BGAg samples belonging to the system 58SiO2•(36-x)CaO·6P2O5·xAg2O, where x = 0 and 1 mol%, respectively, were synthesized via sol–gel method and characterized. Cytotoxicity, modulation of cytokine production (TNF-α, IL-1β, IL-6, IL-4, and IL-10), and oxidative stress response were investigated in human polymorphonuclear cells (PMNs) and peripheral blood mononuclear cells (PBMCs) cultures. Cell viability in the presence of BG or BGAg was concentration-dependent. In addition, BGAg presented higher PBMCs toxicity (LC50 = 0.005%) when compared to BG (LC50 = 0.106%). Interestingly, interleukin4 was produced by PBMCs in response to BG and BGAg in absence of phytohemagglutinin (PHA) and did not modulate PHA-induced cytokine levels. Subtoxic concentrations (0.031% for BG and 0.0008% for BGAg) did not change other cytokines in PBMCs nor reactive oxygen species (ROS) production by PMN. However, BG and BGAg particles decreased zymosan-induced ROS levels in PMN. Although ion incorporation increased BG cytotoxicity, the bioactive glass particles demonstrated a in vitro anti-inflammatory potencial. Future studies are needed to clarify the scavenger potential of the BG/BGAg particles/scaffolds as well as elucidate the effect of the anti-inflammatory potential in modulating tissue growth in vivo.

UVA and UVB radiation induce the formation of neutrophil extracellular traps by human polymorphonuclear cells

Prolonged exposure of the skin to ultraviolet radiation (UV) leads to its damage and loss of protective properties. This condition called photoaging of the skin is caused by a number of destructive factors, such as reactive oxygen species (ROS) and proteolytic enzymes that cause damage to the extracellular matrix, e.g. collagen fibers. Many cells of the immune system, including neutrophils, are involved in the photoaging process. The presence of neutrophils in the skin exposed to UV irradiation is known; however, the mechanism of neutrophil activity at these conditions remains unclear.In our study, we focused on the ability of neutrophils to release neutrophil extracellular traps (NETs) and the role of these structures in the photoaging process. NET release occurs in response to various stimuli; however, we hereby showed that the UVA and UVB radiation that reaches the Earth's surface could activate the mechanism of netosis. UV-induced netosis was much faster than that activated by chemical or biological factors; however, it also occurred due to the production of ROS, known signal mediators in netosis. In this work, we also identified the probable netosis signaling pathway involved in the neutrophil response to UV.The participation of NET components may explain the ongoing process of skin photoaging, but it is also important to indicate netosis as a potential target for skin protection therapy. Antioxidants tested in this work, such as N-acetylcysteine, ethamsylate, as well as vitamin B1 (thiamine), can successfully inhibit UV-induced netosis, and thus be used as protective components against the negative effects of solar radiation.

New Cadinane Sesquiterpenes from the Stems of Kadsura heteroclita.

As part of our continual efforts to exploit ‘Tujia Ethnomedicine’ for their pharmacophoric functionalities, we herein investigated Kadsura heteroclita collected from a deep Wulin mountain area in northern Hunan province. The current study resulted in the isolation of three new sesquiterpenes: 6α,9α,15-trihydroxycadinan-4-en-3-one (1), (+)-3,11,12-trihydroxycalamenene (2), (–)-3,10,11,12-tetrahydroxy-calamenene (3), along with four known sesquiterpenes (4–7), and a cytochalasin H (8). Their chemical structures were elucidated by 1D-, and 2D-NMR spectroscopy, and HRESI-MS, CD spectrometry. The antioxidant, and cytotoxic activities of the compounds were evaluated. Compound 8 exhibited a strong antioxidant effect with an IC50 value of 3.67 µM on isolated human polymorphonuclear cells or neutrophils.

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 Response of nor and nos Contributes to Staphylococcus aureus Virulence and Metabolism.

Staphylococcus aureus causes a wide spectrum of disease, with the site and severity of infection dependent on virulence traits encoded within genetically distinct clonal complexes (CCs) and bacterial responses to host innate immunity. The production of nitric oxide (NO) by activated phagocytes is a major host response to which S. aureus metabolically adapts through multiple strategies that are conserved in all CCs, including an S. aureus nitric oxide synthase (Nos). Previous genome analysis of CC30, a lineage associated with chronic endocardial and osteoarticular infections, revealed a putative NO reductase (Nor) not found in other CCs that potentially contributes to NO resistance and clinical outcome. Here, we demonstrate that Nor has true nitric oxide reductase activity, with nor expression enhanced by NO stress and anaerobic growth. Furthermore, we demonstrate that nor is regulated by MgrA and SrrAB, which modulate S. aureus virulence and hypoxic response. Transcriptome analysis of the S. aureus UAMS-1, UAMS-1 Δnor, and UAMS-1 Δnos strains under NO stress and anaerobic growth demonstrates that Nor contributes to nucleotide metabolism and Nos to glycolysis. We demonstrate that Nor and Nos contribute to enhanced survival in the presence of human human polymorphonuclear cells and have organ-specific seeding in a tail vein infection model. Nor contributes to abscess formation in an osteological implant model. We also demonstrate that Nor has a role in S. aureus metabolism and virulence. The regulation overlap between Nor and Nos points to an intriguing link between regulation of intracellular NO, metabolic adaptation, and persistence in the CC30 lineage.IMPORTANCEStaphylococcus aureus can cause disease at most body sites, and illness spans asymptomatic infection to death. The variety of clinical presentations is due to the diversity of strains, which are grouped into distinct clonal complexes (CCs) based on genetic differences. The ability of S. aureus CC30 to cause chronic infections relies on its ability to evade the oxidative/nitrosative defenses of the immune system and survive under different environmental conditions, including differences in oxygen and nitric oxide concentrations. The significance of this work is the exploration of unique genes involved in resisting NO stress and anoxia. A better understanding of the functions that control the response of S. aureus CC30 to NO and oxygen will guide the treatment of severe disease presentations.

Role of Advanced Glycation End Products in Neutrophil Extracellular Trap Formation

During inflammatory responses, innate immune cells such as neutrophils are recruited to the site of infection to fight and kill foreign microbes. Neutrophils eliminate these potential pathogens in three distinct ways: phagocytosis, the generation of toxic reactive oxygen species (ROS), and the production of neutrophil extracellular traps (NETs). NETs, consisting of expelled DNA and associated proteins, bind and capture bacteria, making them easier for neutrophils to kill via ROS and antimicrobial histone functions. In cases involving microbial contamination, inflammation is beneficial and indeed necessary to prevent infection. However, in many disease contexts, inflammation persists despite an absence of pathogens, and the ROS and NETs that otherwise damage and kill pathogens instead damage and kill host tissues. This type of chronic inflammation is often present in Type II diabetics who have persistent high blood glucose levels or hyperglycemia. Hyperglycemia is correlated with neutrophil priming during NET formation and delayed wound healing, suggesting one mechanism linking hyperglycemia with chronic inflammation. However, the mechanisms underpinning hyperglycemia‐induced NET formation remain unclear. Hyperglycemic patients have increased concentrations of advanced glycation end products (AGEs), generated by sugars randomly and irreversibly attaching to the ends of proteins and lipids. AGEs then activate the receptor for advanced glycation end products (RAGE), resulting in pro‐inflammatory signaling that culminates in additional neutrophil recruitment and activation under hyperglycemic conditions. We are investigating whether the increases in NET production when cells are exposed to AGEs before treatment with NET‐inducing stimuli are mediated by RAGE, and found that inhibition of RAGE blocks the AGE‐enhanced, PMA‐induced NET formation in neutrophil‐like HL‐60 cells. Our results supports the therapeutic potential of RAGE inhibitors to reduce NET formation and thereby NET‐associated tissue inflammation and tissue damage in Type II diabetes. We are currently performing experiments to determine whether in normal human polymorphonuclear cells (PMNs), AGE‐enhanced NET production in response to lipopolysaccharide (LPS) is also mediated by RAGE to determine whether AGE‐RAGE interactions prime NET formation in a more physiological system.Support or Funding InformationDePauw University and the Arthur Vining Davis FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Gambogic Acid on the Production of Reactive Oxygen Species and Neutrophil Extracellular Traps

The innate immune system responds rapidly to the early signs of an infection or tissue damage by recruiting neutrophils to the affected area, where these inflammatory cells kill microbes through the production of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs). Although these normal inflammatory responses are necessary for the elimination of potential pathogens before they proliferate and establish an infection, ROS and NETs are non‐specific mechanisms that can also damage host tissues. In chronic inflammation associated with various diseases, such as rheumatoid arthritis or Crohn's disease, prolonged or non‐resolving inflammation can cause severe pain and permanent tissue damage. We are currently investigating the potential of gambogic acid, a compound derived from the Garcinia hanburyi plant used in Ayurvedic and traditional Thai medicines, to reduce inflammation or promote its resolution. Previous studies have suggested that gambogic acid promotes apoptosis in multiple cell types, suggesting that this molecule could promote neutrophil apoptosis and thereby inhibit their inflammatory functions. In support of this hypothesis, we found that gambogic acid decreases PMA‐induced ROS and NETs by neutrophil‐like HL‐60 cells. Gambogic acid also decreases survival of neutrophil‐like HL‐60 cells in a WST‐8 survival assay, suggesting that it may reduce pro‐inflammatory neutrophil functions by inducing their apoptosis. We are currently investigating whether gambogic acid exerts similar effects on NET and ROS production in normal human polymorphonuclear cells (PMNs), and whether the gambogic acid impacts on neutrophil function are dependent upon its induction of neutrophil apoptosis. If so, this would suggest a therapeutic, inflammation‐resolving potential of gambogic acid in diseases characterized by excessive inflammation.Support or Funding InformationDePauw University, Science Research FellowsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Light on In Vitro Production of Melatonin by Human Peripheral Blood Mononuclear, Polymorphonuclear, and Whole Blood Cells

Recent studies have shown that melatonin can be secreted, in addition to other sources, by peripheral blood mononuclear cells (PBMCs). We investigated whether melatonin could also be secreted by cultured polymorphonuclear (PMNs) and whole blood (WB) cells under resting and stimulated conditions. In addition, influences of the intensity and color of light on the production of melatonin by these cells were estimated. Blood samples were collected from a healthy volunteer, and PBMCs and PMNs were isolated. The PBMC, PMN, and WB cultures were exposed for 8 h to light intensities of 0, 20, 50, 100, 300, and 500 lux and to white, red, blue, and green lights in the presence/absence of mitogens. Levels of melatonin in supernatants were detected by enzyme-linked immunosorbent assay (ELISA). In addition to PBMCs, PMNs and WB cells were found to also produce melatonin, and this production was increased due to mitogen activations. The most effective intensity of light, which suppresses melatonin secretion, was 100 lux. There were no significant differences between the influences of different monochromatic lights on the melatonin production by the examined blood cell cultures. Thus, PBMCs, PMNs, and WB cultures can produce melatonin, and mitogen activation causes a higher production of this hormone. Moreover, although white light suppressed melatonin secretion at 100 lux, there was no difference among various types of lights. © 2019, Springer Science+Business Media, LLC, part of Springer Nature.

Adaptation of the Staphylococcus aureus leukocidin LukGH for the rabbit host by protein engineering.

Host defense against Staphylococcus aureus greatly depends on bacterial clearance by phagocytic cells. LukGH (or LukAB) is the most potent staphylococcal leukocidin towards human phagocytes in vitro, but its role in pathogenesis is obscured by the lack of suitable small animal models because LukGH has limited or no cytotoxicity towards rodent and rabbit compared with human polymorphonuclear cells (PMNs) likely due to an impaired interaction with its cellular receptor, CD11b. We aimed at adapting LukGH for the rabbit host by improving binding to the rabbit homolog of CD11b, specifically its I-domain (CD11b-I). Targeted amino acid substitutions were introduced into the LukH polypeptide to map its receptor interaction site(s). We found that the binding affinity of LukGH variants to the human and rabbit CD11b-I correlated well with their PMN cytotoxicity. Importantly, we identified LukGH variants with significantly improved cytotoxicity towards rabbit PMNs, when expressed recombinantly (10-15-fold) or by engineered S. aureus strains. These findings support the development of small animal models of S. aureus infection with the potential for demonstrating the importance of LukGH in pathogenesis.

Human granulocytes undergo cell death via autophagy

Mature neutrophils must be quickly removed from inflammatory sites to prevent tissue damage. Neutrophil removal is thought to be accomplished primarily through caspase-dependent apoptosis, which involves several genes of mitochondrial origin. However, mature neutrophils show reduced gene transcription and mitochondrial numbers. We predicted that neutrophils utilize other cell death mechanisms and investigated programmed cell death in human peripheral blood mononuclear cells (MNCs) and polymorphonuclear cells (PMNCs or neutrophil fractions). Unlike MNCs, PMNCs did not undergo DNA fragmentation and were not TUNEL positive, but expressed LC3-II, an autophagy marker. We also found that during differentiation, autophagy inhibitor 3-MA, and not caspase inhibitor zVAD-fmk, prevented segmentation of the nucleus, indicating that these cells undergo autophagy during maturation. Therefore, human neutrophils may undergo spontaneous autophagic cell death rather than apoptosis, during which autophagy may be essential for both maturation and death.