Nicotinamide Antagonizes Lipopolysaccharide-Induced Hypoxic Cell Signals in Human Macrophages.

Mechanisms to control the immune response are important to pathogen evasion and host defense. Gram-negative bacteria are common pathogens that can activate host immune responses through their outer membrane component, LPS. Macrophage activation by LPS induces cell signals that promote hypoxic metabolism, phagocytosis, Ag presentation, and inflammation. Nicotinamide (NAM) is a vitamin B3 derivative and precursor in the formation of NAD, which is a required cofactor in cellular function. In this study, treatment of human monocyte-derived macrophages with NAM promoted posttranslational modifications that antagonized LPS-induced cell signals. Specifically, NAM inhibited AKT and FOXO1 phosphorylation, decreased p65/RelA acetylation, and promoted p65/RelA and hypoxia-inducible transcription factor-1α (HIF-1α) ubiquitination. NAM also increased prolyl hydroxylase domain 2 (PHD2) production, inhibited HIF-1α transcription, and promoted the formation of the proteasome, resulting in reduced HIF-1α stabilization, decreased glycolysis and phagocytosis, and reductions in NOX2 activity and the production of lactate dehydrogenase A. These NAM responses were associated with increased intracellular NAD levels formed through the salvage pathway. NAM and its metabolites may therefore decrease the inflammatory response of macrophages and protect the host against excessive inflammation but potentially increase injury through reduced pathogen clearance. Continued study of NAM cell signals in vitro and in vivo may provide insight into infection-associated host pathologies and interventions.

Inflammation and DKK1-induced AKT activation contribute to endothelial dysfunction following NR2F2 loss.

NR2F2 is expressed in endothelial cells (ECs) and Nr2f2 knockout produces lethal cardiovascular defects. In humans, reduced NR2F2 expression is associated with cardiovascular diseases including congenital heart disease and atherosclerosis. Here, NR2F2 silencing in human primary ECs led to inflammation, endothelial-to-mesenchymal transition (EndMT), proliferation, hypermigration, apoptosis-resistance, and increased production of reactive oxygen species. These changes were associated with STAT and AKT activation along with increased production of DKK1. Co-silencing DKK1 and NR2F2 prevented NR2F2-loss-induced STAT and AKT activation and reversed EndMT. Serum DKK1 concentrations were elevated in patients with pulmonary arterial hypertension (PAH) and DKK1 was secreted by ECs in response to in vitro loss of either BMPR2 or CAV1, which are genetic defects associated with the development of PAH. In human primary ECs, NR2F2 suppressed DKK1, whereas its loss conversely induced DKK1 and disrupted endothelial homeostasis, promoting phenotypic abnormalities associated with pathologic vascular remodeling. Activating NR2F2 or blocking DKK1 may be useful therapeutic targets for treating chronic vascular diseases associated with EC dysfunction.NEW & NOTEWORTHY NR2F2 loss in the endothelial lining of blood vessels is associated with cardiovascular disease. Here, NR2F2-silenced human endothelial cells were inflammatory, proliferative, hypermigratory, and apoptosis-resistant with increased oxidant stress and endothelial-to-mesenchymal transition. DKK1 was induced in NR2F2-silenced endothelial cells, while co-silencing NR2F2 and DKK1 prevented NR2F2-loss-associated abnormalities in endothelial signaling and phenotype. Activating NR2F2 or blocking DKK1 may be useful therapeutic targets for treating vascular diseases associated with endothelial dysfunction.

The complexity of nicotinamide adenine dinucleotide (NAD), hypoxic, and aryl hydrocarbon receptor cell signaling in chronic kidney disease.

Early-stage detection of chronic kidney diseases (CKD) is important to treatment that may slow and occasionally halt CKD progression. CKD of diverse etiologies share similar histologic patterns of glomerulosclerosis, tubular atrophy, and interstitial fibrosis. Macro-vascular disease and micro-vascular disease promote tissue ischemia, contributing to injury. Tissue ischemia promotes hypoxia, and this in turn activates the hypoxia-inducible transcription factors (HIFs). HIF-1α and HIF-2α, share a dimer partner, HIF-1ß, with the aryl hydrocarbon receptor (AHR) and are each activated in CKD and associated with kidney cellular nicotinamide adenine dinucleotide (NAD) depletion. The Preiss-Handler, salvage, and de novo pathways regulate NAD biosynthesis and gap-junctions regulate NAD cellular retention. In the Preiss-Handler pathway, niacin forms NAD. Niacin also exhibits crosstalk with HIF and AHR cell signals in the regulation of insulin sensitivity, which is a complication in CKD. Dysregulated enzyme activity in the NAD de novo pathway increases the levels of circulating tryptophan metabolites that activate AHR, resulting in poly-ADP ribose polymerase activation, thrombosis, endothelial dysfunction, and immunosuppression. Therapeutically, metabolites from the NAD salvage pathway increase NAD production and subsequent sirtuin deacetylase activity, resulting in reduced activation of retinoic acid-inducible gene I, p53, NF-κB and SMAD2 but increased activation of FOXO1, PGC-1α, and DNA methyltransferase-1. These post-translational responses may also be initiated through non-coding RNAs (ncRNAs), which are additionally altered in CKD. Nanoparticles traverse biological systems and can penetrate almost all tissues as disease biomarkers and drug delivery carriers. Targeted delivery of non-coding RNAs or NAD metabolites with nanoparticles may enable the development of more effective diagnostics and therapies to treat CKD.

Anti-PD-L1 Therapy Does Not Improve Survival in a Murine Model of Lethal Staphylococcus aureus Pneumonia.

BACKGROUND: Staphylococcus aureus (SA) bacterial pneumonia is a common cause of sepsis in intensive care units. Immune checkpoint inhibitors (CPIs) that target programmed cell death protein 1 (PD-1) and its ligand (PD-L1) have been proposed for the treatment of sepsis. However, in our systematic review of sepsis preclinical models, none of the models examined CPIs in pneumonia. METHODS: Mice were inoculated intratracheally with vehicle control, low dose (LD)- or high dose (HD)-SA. Immune cell recruitment and checkpoint molecule expression were examined at 4, 24, and 48 hours after infection. Infected animals, treated with control or anti-PD-L1 antibodies, were assessed for survival, bacterial burden, lung immunophenotypes, and mediator production. RESULTS: LD-SA and HD-SA produced lethality of 15% and 70%, respectively, by 168 hours. At 24 hours, LD-infected animals exhibited increased lung monocyte PD-L1 expression (P = .0002) but lower bacterial counts (P = .0002) compared with HD animals. By 48 hours, either infection induced lung neutrophil and macrophage PD-L1 expression (P < .0001). Anti-PD-L1 treatment at the time of infection and at 24 hours following infection with low to high doses of SA reduced PD-L1 detection but did not affect survival or bacterial clearance. CONCLUSIONS: Anti-PD-L1 therapy did not alter survival in this pneumonia model. Preclinical studies of additional common pathogens and septic foci are needed.

Report on the 2018 Cancer, Autoimmunity, and Immunology Conference.

With the increased use of cancer immunotherapy, a number of immune-related adverse events (irAEs) are being identified. These irAEs can be compared with known autoimmune disorders in similar tissues, with important similarities and differences. Understanding the etiology of irAEs may bring to light concepts applicable to immune responses in cancer, autoimmunity, and infectious disease. This immunobiology is especially relevant to cancer patients with preexisting allogeneic transplants or autoimmune disease who are undergoing cancer immunotherapy. To address these facets of cancer immunotherapy, academic leaders from these various disciplines discussed current irAE basic and clinical research, irAE diagnosis and management, and the need for biomarkers and algorithms to identify individuals at risk for irAEs at a conference jointly sponsored by the National Cancer Institute, National Institute of Allergy and Infectious Diseases, and National Institute of Arthritis and Musculoskeletal and Skin Diseases in Bethesda, MD, on March 22-23, 2018. Mechanisms and models to characterize irAEs, standardize protocols, store biospecimens, and capture and analyze irAE data were also reviewed during the inaugural Cancer, Autoimmunity, and Immunology Conference. This summary highlights cancer immunotherapy-induced irAEs, the challenges ahead, and the opportunities for greater understanding of autoimmune conditions.

PD-1 immunobiology in glomerulonephritis and renal cell carcinoma.

BACKGROUND: Programmed cell death protein (PD)-1 receptors and ligands on immune cells and kidney parenchymal cells help maintain immunological homeostasis in the kidney. Dysregulated PD-1:PD-L1 binding interactions occur during the pathogenesis of glomerulopathies and renal cell carcinoma (RCC). The regulation of these molecules in the kidney is important to PD-1/PD-L1 immunotherapies that treat RCC and may induce glomerulopathies as an adverse event. METHODS: The expression and function of PD-1 molecules on immune and kidney parenchymal cells were reviewed in the healthy kidney, PD-1 immunotherapy-induced nephrotoxicity, glomerulopathies and RCC. RESULTS: PD-1 and/or its ligands are expressed on kidney macrophages, dendritic cells, lymphocytes, and renal proximal tubule epithelial cells. Vitamin D3, glutathione and AMP-activated protein kinase (AMPK) regulate hypoxic cell signals involved in the expression and function of PD-1 molecules. These pathways are altered in kidney disease and are linked to the production of vascular endothelial growth factor, erythropoietin, adiponectin, interleukin (IL)-18, IL-23, and chemokines that bind CXCR3, CXCR4, and/or CXCR7. These factors are differentially produced in glomerulonephritis and RCC and may be important biomarkers in patients that receive PD-1 therapies and/or develop glomerulonephritis as an adverse event CONCLUSION: By comparing the functions of the PD-1 axis in glomerulopathies and RCC, we identified similar chemokines involved in the recruitment of immune cells and distinct mediators in T cell differentiation. The expression and function of PD-1 and PD-1 ligands in diseased tissue and particularly on double-negative T cells and parenchymal kidney cells needs continued exploration. The possible regulation of the PD-1 axis by vitamin D3, glutathione and/or AMPK cell signals may be important to kidney disease and the PD-1 immunotherapeutic response.

The Mammary Tumor Microenvironment.

A developing breast tumor is regulated by its tumor microenvironment (TME) which includes various immune cell subsets, fibroblasts, adipocytes, and endothelial and epithelial cells surrounded by an extracellular matrix (ECM). Breast tissue density is also a defining feature of breast cancer and plays an integral role in the exchange of biochemical cues between cells and the ECM. Cell signals from these interactions regulate tumor growth, metabolism, immunity, and invasion. The distinct organization of cells in the ECM generates structural patterns that are important in understanding disease development and progression. In this chapter, we discuss this complex interplay between the ECM and cells in the TME. Various models that mimic density are described to more fully understand the effect of ECM density on immunity, metabolism, tumorigenesis, and dormancy. Continued study of the interactions between cells and the ECM in the TME may provide needed biomarkers and therapeutic targets in breast cancer.

PD-1 immunobiology in systemic lupus erythematosus.

Programmed death (PD)-1 receptors and their ligands have been identified in the pathogenesis and development of systemic lupus erythematosus (SLE). Two key pathways, toll-like receptor and type I interferon, are significant to SLE pathogenesis and modulate the expression of PD-1 and the ligands (PD-L1, PD-L2) through activation of NF-κB and/or STAT1. These cell signals are regulated by tyrosine kinase (Tyro, Axl, Mer) receptors (TAMs) that are aberrantly activated in SLE. STAT1 and NF-κB also exhibit crosstalk with the aryl hydrocarbon receptor (AHR). Ligands to AHR are identified in SLE etiology and pathogenesis. These ligands also regulate the activity of the Epstein-Barr virus (EBV), which is an identified factor in SLE and PD-1 immunobiology. AHR is important in the maintenance of immune tolerance and the development of distinct immune subsets, highlighting a potential role of AHR in PD-1 immunobiology. Understanding the functions of AHR ligands as well as AHR crosstalk with STAT1, NF-κB, and EBV may provide insight into disease development, the PD-1 axis and immunotherapies that target PD-1 and its ligand, PD-L1.

Mechanisms and Targeted Therapies for Pseudomonas aeruginosa Lung Infection.

Pseudomonas aeruginosa is a complex gram-negative facultative anaerobe replete with a variety of arsenals to activate, modify, and destroy host defense mechanisms. The microbe is a common cause of nosocomial infections and an antibiotic-resistant priority pathogen. In the lung, P. aeruginosa disrupts upper and lower airway homeostasis by damaging the epithelium and evading innate and adaptive immune responses. The biology of these interactions is essential to understand P. aeruginosa pathogenesis. P. aeruginosa interacts directly with host cells via flagella, pili, lipoproteins, lipopolysaccharides, and the type III secretion system localized in the outer membrane. P. aeruginosa quorum-sensing molecules regulate the release of soluble factors that enhance the spread of infection. These characteristics of P. aeruginosa differentially affect lung epithelial, innate, and adaptive immune cells involved in the production of mediators and the recruitment of additional immune cell subsets. Pathogen interactions with individual host cells and in the context of host acute lung infection are discussed to reveal pathways that may be targeted therapeutically.

Designing a broad-spectrum integrative approach for cancer prevention and treatment.

Targeted therapies and the consequent adoption of "personalized" oncology have achieved notable successes in some cancers; however, significant problems remain with this approach. Many targeted therapies are highly toxic, costs are extremely high, and most patients experience relapse after a few disease-free months. Relapses arise from genetic heterogeneity in tumors, which harbor therapy-resistant immortalized cells that have adopted alternate and compensatory pathways (i.e., pathways that are not reliant upon the same mechanisms as those which have been targeted). To address these limitations, an international task force of 180 scientists was assembled to explore the concept of a low-toxicity "broad-spectrum" therapeutic approach that could simultaneously target many key pathways and mechanisms. Using cancer hallmark phenotypes and the tumor microenvironment to account for the various aspects of relevant cancer biology, interdisciplinary teams reviewed each hallmark area and nominated a wide range of high-priority targets (74 in total) that could be modified to improve patient outcomes. For these targets, corresponding low-toxicity therapeutic approaches were then suggested, many of which were phytochemicals. Proposed actions on each target and all of the approaches were further reviewed for known effects on other hallmark areas and the tumor microenvironment. Potential contrary or procarcinogenic effects were found for 3.9% of the relationships between targets and hallmarks, and mixed evidence of complementary and contrary relationships was found for 7.1%. Approximately 67% of the relationships revealed potentially complementary effects, and the remainder had no known relationship. Among the approaches, 1.1% had contrary, 2.8% had mixed and 62.1% had complementary relationships. These results suggest that a broad-spectrum approach should be feasible from a safety standpoint. This novel approach has potential to be relatively inexpensive, it should help us address stages and types of cancer that lack conventional treatment, and it may reduce relapse risks. A proposed agenda for future research is offered.

The effect of environmental chemicals on the tumor microenvironment.

Potentially carcinogenic compounds may cause cancer through direct DNA damage or through indirect cellular or physiological effects. To study possible carcinogens, the fields of endocrinology, genetics, epigenetics, medicine, environmental health, toxicology, pharmacology and oncology must be considered. Disruptive chemicals may also contribute to multiple stages of tumor development through effects on the tumor microenvironment. In turn, the tumor microenvironment consists of a complex interaction among blood vessels that feed the tumor, the extracellular matrix that provides structural and biochemical support, signaling molecules that send messages and soluble factors such as cytokines. The tumor microenvironment also consists of many host cellular effectors including multipotent stromal cells/mesenchymal stem cells, fibroblasts, endothelial cell precursors, antigen-presenting cells, lymphocytes and innate immune cells. Carcinogens can influence the tumor microenvironment through effects on epithelial cells, the most common origin of cancer, as well as on stromal cells, extracellular matrix components and immune cells. Here, we review how environmental exposures can perturb the tumor microenvironment. We suggest a role for disrupting chemicals such as nickel chloride, Bisphenol A, butyltins, methylmercury and paraquat as well as more traditional carcinogens, such as radiation, and pharmaceuticals, such as diabetes medications, in the disruption of the tumor microenvironment. Further studies interrogating the role of chemicals and their mixtures in dose-dependent effects on the tumor microenvironment could have important general mechanistic implications for the etiology and prevention of tumorigenesis.

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead.

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology.

LPS modulates rhinovirus-induced chemokine secretion in monocytes and macrophages.

Recent studies suggest that both bacteria and rhinoviruses (RVs) contribute to asthma exacerbations. We hypothesized that bacteria might alter antiviral responses early in the course of infection by modifying monocyte-lineage chemokine responses to RV infection. To test this hypothesis, human blood monocytes or bronchoalveolar lavage (BAL) macrophages were treated with RV types A016, B014, A001, and/or A002 in the presence or absence of LPS, and secretion of chemokines (CXCL10, CXCL11, CCL2, and CCL8) and IFN-α was measured by ELISA. Treatment with RV alone induced blood monocytes and BAL macrophages to secrete CXCL10, CXCL11, CCL2, and CCL8. Pretreatment with LPS significantly attenuated RV-induced CXCL10, CXCL11, and CCL8 secretion by 68-99.9% on average (P < 0.0001, P < 0.004, and P < 0.002, respectively), but did not inhibit RV-induced CCL2 from blood monocytes. Similarly, LPS inhibited RV-induced CXCL10 and CXCL11 secretion by over 88% on average from BAL macrophages (P < 0.002 and P < 0.0001, respectively). Furthermore, LPS inhibited RV-induced signal transducer and activator of transcription 1 phosphorylation (P < 0.05), as determined by immunoblotting, yet augmented RV-induced IFN-α secretion (P < 0.05), and did not diminish expression of RV target receptors, as measured by flow cytometry. In summary, major and minor group RVs strongly induce chemokine expression and IFN-α from monocytic cells. The bacterial product, LPS, specifically inhibits monocyte and macrophage secretion of RV-induced CXCL10 and CXCL11, but not other highly induced chemokines or IFN-α. These effects suggest that airway bacteria could modulate the pattern of virus-induced cell recruitment and inflammation in the airways.

GM-CSF production by glioblastoma cells has a functional role in eosinophil survival, activation, and growth factor production for enhanced tumor cell proliferation.

Medicinal interventions of limited efficacy are currently available for the treatment of glioblastoma multiforme (GBM), the most common and lethal primary brain tumor in adults. The eosinophil is a pivotal immune cell in the pathobiology of atopic disease that is also found to accumulate in certain tumor tissues. Inverse associations between atopy and GBM risk suggest that the eosinophil may play a functional role in certain tumor immune responses. To assess the potential interactions between eosinophils and GBM, we cultured human primary blood eosinophils with two separate human GBM-derived cell lines (A172, U87-MG) or conditioned media generated in the presence or absence of TNF-α. Results demonstrated differential eosinophil adhesion and increased survival in response to coculture with GBM cell lines. Eosinophil responses to GBM cell line-conditioned media included increased survival, activation, CD11b expression, and S100A9 release. Addition of GM-CSF neutralizing Abs to GBM cell cultures or conditioned media reduced eosinophil adhesion, survival, and activation, linking tumor cell-derived GM-CSF to the functions of eosinophils in the tumor microenvironment. Dexamethasone, which has been reported to inhibit eosinophil recruitment and shrink GBM lesions on contrast-enhanced scans, reduced the production of tumor cell-derived GM-CSF. Furthermore, culture of GBM cells in eosinophil-conditioned media increased tumor cell viability, and generation of eosinophil-conditioned media in the presence of GM-CSF enhanced the effect. These data support the idea of a paracrine loop between GM-CSF-producing tumors and eosinophil-derived growth factors in tumor promotion/progression.

Anti-PD-L1 therapy altered inflammation but not survival in a lethal murine hepatitis virus-1 pneumonia model.

Introduction: Because prior immune checkpoint inhibitor (ICI) therapy in cancer patients presenting with COVID-19 may affect outcomes, we investigated the beta-coronavirus, murine hepatitis virus (MHV)-1, in a lethal pneumonia model in the absence (Study 1) or presence of prior programmed cell death ligand-1 (PD-L1) antibody (PD-L1mAb) treatment (Study 2). Methods: In Study 1, animals were inoculated intratracheally with MHV-1 or vehicle and evaluated at day 2, 5, and 10 after infection. In Study 2, uninfected or MHV-1-infected animals were pretreated intraperitoneally with control or PD-L1-blocking antibodies (PD-L1mAb) and evaluated at day 2 and 5 after infection. Each study examined survival, physiologic and histologic parameters, viral titers, lung immunophenotypes, and mediator production. Results: Study 1 results recapitulated the pathogenesis of COVID-19 and revealed increased cell surface expression of checkpoint molecules (PD-L1, PD-1), higher expression of the immune activation marker angiotensin converting enzyme (ACE), but reduced detection of the MHV-1 receptor CD66a on immune cells in the lung, liver, and spleen. In addition to reduced detection of PD-L1 on all immune cells assayed, PD-L1 blockade was associated with increased cell surface expression of PD-1 and ACE, decreased cell surface detection of CD66a, and improved oxygen saturation despite reduced blood glucose levels and increased signs of tissue hypoxia. In the lung, PD-L1mAb promoted S100A9 but inhibited ACE2 production concomitantly with pAKT activation and reduced FOXO1 levels. PD-L1mAb promoted interferon-γ but inhibited IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) production, contributing to reduced bronchoalveolar lavage levels of eosinophils and neutrophils. In the liver, PD-L1mAb increased viral clearance in association with increased macrophage and lymphocyte recruitment and liver injury. PD-L1mAb increased the production of virally induced mediators of injury, angiogenesis, and neuronal activity that may play role in COVID-19 and ICI-related neurotoxicity. PD-L1mAb did not affect survival in this murine model. Discussion: In Study 1 and Study 2, ACE was upregulated and CD66a and ACE2 were downregulated by either MHV-1 or PD-L1mAb. CD66a is not only the MHV-1 receptor but also an identified immune checkpoint and a negative regulator of ACE. Crosstalk between CD66a and PD-L1 or ACE/ACE2 may provide insight into ICI therapies. These networks may also play role in the increased production of S100A9 and neurological mediators in response to MHV-1 and/or PD-L1mAb, which warrant further study. Overall, these findings support observational data suggesting that prior ICI treatment does not alter survival in patients presenting with COVID-19.

Eosinophils in glioblastoma biology.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. The development of this malignant glial lesion involves a multi-faceted process that results in a loss of genetic or epigenetic gene control, un-regulated cell growth, and immune tolerance. Of interest, atopic diseases are characterized by a lack of immune tolerance and are inversely associated with glioma risk. One cell type that is an established effector cell in the pathobiology of atopic disease is the eosinophil. In response to various stimuli, the eosinophil is able to produce cytotoxic granules, neuromediators, and pro-inflammatory cytokines as well as pro-fibrotic and angiogenic factors involved in pathogen clearance and tissue remodeling and repair. These various biological properties reveal that the eosinophil is a key immunoregulatory cell capable of influencing the activity of both innate and adaptive immune responses. Of central importance to this report is the observation that eosinophil migration to the brain occurs in response to traumatic brain injury and following certain immunotherapeutic treatments for GBM. Although eosinophils have been identified in various central nervous system pathologies, and are known to operate in wound/repair and tumorstatic models, the potential roles of eosinophils in GBM development and the tumor immunological response are only beginning to be recognized and are therefore the subject of the present review.

Human eosinophils express RAGE, produce RAGE ligands, exhibit PKC-delta phosphorylation and enhanced viability in response to the RAGE ligand, S100B.

This study tested the hypothesis that human eosinophils produce ligands for the receptor for advanced glycation end-products (RAGE), express RAGE and exhibit RAGE-mediated responses. In examining our microarray data, we identified the presence of RAGE and RAGE ligand (S100A4, S100A6, S100A8, S100A9, S100A11, S100P, HMGB1) transcripts. Expression of eosinophil RAGE mRNA was also compared with a known positive control and further assessed via bioinformatics and sequence analysis of RAGE cDNA. Positive and negative controls were used to identify RAGE, S100A8 and S100A9 protein in human primary eosinophils. Immunoblot assessment of eosinophils treated with cytokines (IL-5 or granulocyte macrophage colony-stimulating factor) indicated an up-regulation of S100A8 and S100A9 production, whereas co-treatment of eosinophils with a RAGE ligand and cytokines displayed a down-regulation in the levels of RAGE. Analysis of eosinophil-conditioned media revealed that eosinophils are capable of releasing RAGE, S100A8 and S100A9. To test the eosinophil response to RAGE activation, the most well-characterized RAGE ligand, S100B, was examined. Treatment of eosinophils with S100B resulted in RAGE-mediated PKC-delta phosphorylation, a 3-fold dose-dependent increase in cell survival and an increase in the level of cellular RAGE. Combined, these studies reveal eosinophil expression of RAGE, RAGE ligands and RAGE-mediated responses. The expression of eosinophil RAGE, soluble RAGE and RAGE ligands may be pivotal to the functions of eosinophils in various human diseases involving RAGE and S100 ligands.

Aryl Hydrocarbon Receptor Mechanisms Affecting Chronic Kidney Disease.

The aryl hydrocarbon receptor (AHR) is a basic helix-loop-helix transcription factor that binds diverse endogenous and xenobiotic ligands, which regulate AHR stability, transcriptional activity, and cell signaling. AHR activity is strongly implicated throughout the course of chronic kidney disease (CKD). Many diverse organic molecules bind and activate AHR and these ligands are reported to either promote glomerular and tubular damage or protect against kidney injury. AHR crosstalk with estrogen, peroxisome proliferator-activated receptor-γ, and NF-κB pathways may contribute to the diversity of AHR responses during the various forms and stages of CKD. The roles of AHR in kidney fibrosis, metabolism and the renin angiotensin system are described to offer insight into CKD pathogenesis and therapies.

RAGE pathway activation and function in chronic kidney disease and COVID-19.

The multi-ligand receptor for advanced glycation end-products (RAGE) and its ligands are contributing factors in autoimmunity, cancers, and infectious disease. RAGE activation is increased in chronic kidney disease (CKD) and coronavirus disease 2019 (COVID-19). CKD may increase the risk of COVID-19 severity and may also develop in the form of long COVID. RAGE is expressed in essentially all kidney cell types. Increased production of RAGE isoforms and RAGE ligands during CKD and COVID-19 promotes RAGE activity. The downstream effects include cellular dysfunction, tissue injury, fibrosis, and inflammation, which in turn contribute to a decline in kidney function, hypertension, thrombotic disorders, and cognitive impairment. In this review, we discuss the forms and mechanisms of RAGE and RAGE ligands in the kidney and COVID-19. Because various small molecules antagonize RAGE activity in animal models, targeting RAGE, its co-receptors, or its ligands may offer novel therapeutic approaches to slowing or halting progressive kidney disease, for which current therapies are often inadequate.

Observation and Quantification of Eosinophil Motility.

Eosinophils are important for tissue homeostasis and host responses to pathogens and allergens. The impact of eosinophils within tissues depends in part on whether cytotoxic proteins in crystalloid granules are released. Determinants of eosinophil motility and loss of granule contents are incompletely understood. The goal of this chapter is to present methods to study the effects of potential mediators on purified human blood eosinophils interacting with adhesive proteins found in extracellular matrix. We show that differential interference contrast video-enhanced microscopy and a bead-clearing assay provide complementary information about how different mediator-adhesive protein combinations direct eosinophil motility and granule fate. The former method is rich in information about cell shape, pattern of movement, and state of granules whereas the latter method lends itself to quantification and interrogation of multiple conditions in replicate.