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Ferroptosis is an iron-dependent form of cell death that results from excess lipid peroxidation in cellular membranes. Within the last decade, physiological and pathological roles for ferroptosis have been uncovered in autoimmune diseases, inflammatory conditions, infection, and cancer biology. Excitingly, cancer cell metabolism may be targeted to induce death by ferroptosis in cancers that are resistant to other forms of cell death. Ferroptosis sensitivity is regulated by oxidative stress, lipid metabolism, and iron metabolism, which are all influenced by the tumor microenvironment (TME). Whereas some cancer cell types have been shown to adapt to these stressors, it is not clear how immune cells regulate their sensitivities to ferroptosis. In this review, we discuss the mechanisms of ferroptosis sensitivity in different immune cell subsets, how ferroptosis influences which immune cells infiltrate the TME, and how these interactions can determine epithelial-to-mesenchymal transition (EMT) and metastasis. While much focus has been placed on inducing ferroptosis in cancer cells, these are important considerations for how ferroptosis-modulating strategies impact anti-tumor immunity. From this perspective, we also discuss some promising immunotherapies in the field of ferroptosis and the challenges associated with targeting ferroptosis in specific immune cell populations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Lupica-Tondo, Arner, Mogilenko and Voss.)

Extensive, large-scale single-cell profiling of healthy human blood at different ages is one of the critical pending tasks required to establish a framework for the systematic understanding of human aging. Here, using single-cell RNA/T cell receptor (TCR)/BCR-seq with protein feature barcoding, we profiled 317 samples from 166 healthy individuals aged 25-85 years old. From this, we generated a dataset from ∼2 million cells that described 55 subpopulations of blood immune cells. Twelve subpopulations changed with age, including the accumulation of GZMK + CD8 + T cells and HLA-DR + CD4 + T cells. In contrast to other T cell memory subsets, transcriptionally distinct NKG2C + GZMB - CD8 + T cells counterintuitively decreased with age. Furthermore, we found a concerted age-associated increase in type 2/interleukin (IL)4-expressing memory subpopulations across CD4 + and CD8 + T cell compartments (CCR4 + CD8 + Tcm and Th2 CD4 + Tmem), suggesting a systematic functional shift in immune homeostasis with age. Our work provides novel insights into healthy human aging and a comprehensive annotated resource., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background & Aims: Roux-en-Y gastric bypass (RYGB), the most effective surgical procedure for weight loss, decreases obesity and ameliorates comorbidities, such as non-alcoholic fatty liver (NAFLD) and cardiovascular (CVD) diseases. Cholesterol is a major CVD risk factor and modulator of NAFLD development, and the liver tightly controls its metabolism. How RYGB surgery modulates systemic and hepatic cholesterol metabolism is still unclear., Methods: We studied the hepatic transcriptome of 26 patients with obesity but not diabetes before and 1 year after undergoing RYGB. In parallel, we measured quantitative changes in plasma cholesterol metabolites and bile acids (BAs)., Results: RYGB surgery improved systemic cholesterol metabolism and increased plasma total and primary BA levels. Transcriptomic analysis revealed specific alterations in the liver after RYGB, with the downregulation of a module of genes implicated in inflammation and the upregulation of three modules, one associated with BA metabolism. A dedicated analysis of hepatic genes related to cholesterol homeostasis pointed towards increased biliary cholesterol elimination after RYGB, associated with enhancement of the alternate, but not the classical, BA synthesis pathway. In parallel, alterations in the expression of genes involved in cholesterol uptake and intracellular trafficking indicate improved hepatic free cholesterol handling. Finally, RYGB decreased plasma markers of cholesterol synthesis, which correlated with an improvement in liver disease status after surgery., Conclusions: Our results identify specific regulatory effects of RYGB on inflammation and cholesterol metabolism. RYGB alters the hepatic transcriptome signature, likely improving liver cholesterol homeostasis. These gene regulatory effects are reflected by systemic post-surgery changes of cholesterol-related metabolites, corroborating the beneficial effects of RYGB on both hepatic and systemic cholesterol homeostasis., Impact and Implications: Roux-en-Y gastric bypass (RYGB) is a widely used bariatric surgery procedure with proven efficacy in body weight management, combatting cardiovascular disease (CVD) and non-alcoholic fatty liver disease (NAFLD). RYGB exerts many beneficial metabolic effects, by lowering plasma cholesterol and improving atherogenic dyslipidemia. Using a cohort of patients undergoing RYGB, studied before and 1 year after surgery, we analyzed how RYGB modulates hepatic and systemic cholesterol and bile acid metabolism. The results of our study provide important insights on the regulation of cholesterol homeostasis after RYGB and open avenues that could guide future monitoring and treatment strategies targeting CVD and NAFLD in obesity., (Copyright © 2023 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Over the last decade, immunometabolism has emerged as a novel interdisciplinary field of research and yielded significant fundamental insights into the regulation of immune responses. Multiple classical approaches to interrogate immunometabolism, including bulk metabolic profiling and analysis of metabolic regulator expression, paved the way to appreciating the physiological complexity of immunometabolic regulation in vivo. Studying immunometabolism at the systems level raised the need to transition towards the next-generation technology for metabolic profiling and analysis. Spatially resolved metabolic imaging and computational algorithms for multi-modal data integration are new approaches to connecting metabolism and immunity. In this review, we discuss recent studies that highlight the complex physiological interplay between immune responses and metabolism and give an overview of technological developments that bear the promise of capturing this complexity most directly and comprehensively.

Objective: Obesity is associated with metabolic dysfunction of white adipose tissue (WAT). Activated adipocytes secrete pro-inflammatory cytokines resulting in the recruitment of pro-inflammatory macrophages, which contribute to WAT insulin resistance. The bile acid (BA)-activated nuclear Farnesoid X Receptor (FXR) controls systemic glucose and lipid metabolism. Here, we studied the role of FXR in adipose tissue function., Methods: We first investigated the immune phenotype of epididymal WAT (eWAT) from high fat diet (HFD)-fed whole-body FXR-deficient (FXR -/- ) mice by flow cytometry and gene expression analysis. We then generated adipocyte-specific FXR-deficient (Ad-FXR -/- ) mice and analyzed systemic and eWAT metabolism and immune phenotype upon HFD feeding. Transcriptomic analysis was done on mature eWAT adipocytes from HFD-fed Ad-FXR -/- mice., Results: eWAT from HFD-fed whole-body FXR -/- and Ad-FXR -/- mice displayed decreased pro-inflammatory macrophage infiltration and inflammation. Ad-FXR -/- mice showed lower blood glucose concentrations, improved systemic glucose tolerance and WAT insulin sensitivity and oxidative stress. Transcriptomic analysis identified Gsta4, a modulator of oxidative stress in WAT, as the most upregulated gene in Ad-FXR -/- mouse adipocytes. Finally, chromatin immunoprecipitation analysis showed that FXR binds the Gsta4 gene promoter., Conclusions: These results indicate a role for the adipocyte FXR-GSTA4 axis in controlling HFD-induced inflammation and systemic glucose homeostasis., (Copyright © 2023 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

The diversity of mononuclear phagocyte (MNP) subpopulations across tissues is one of the key physiological characteristics of the immune system. Here, we focus on understanding the metabolic variability of MNPs through metabolic network analysis applied to three large-scale transcriptional datasets: we introduce (1) an ImmGen MNP open-source dataset of 337 samples across 26 tissues; (2) a myeloid subset of ImmGen Phase I dataset (202 MNP samples); and (3) a myeloid mouse single-cell RNA sequencing (scRNA-seq) dataset (51,364 cells) assembled based on Tabula Muris Senis. To analyze such large-scale datasets, we develop a network-based computational approach, genes and metabolites (GAM) clustering, for unbiased identification of the key metabolic subnetworks based on transcriptional profiles. We define 9 metabolic subnetworks that encapsulate the metabolic differences within MNP from 38 different tissues. Obtained modules reveal that cholesterol synthesis appears particularly active within the migratory dendritic cells, while glutathione synthesis is essential for cysteinyl leukotriene production by peritoneal and lung macrophages., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Adiponectin is an adipose tissue hormone, participating in energy metabolism and involved in atherogenesis. Previously, it was found that adiponectin increases expression of the APOA1 (apolipoprotein A-1) gene in hepatocytes, but the mechanisms of this effect remained unexplored. Our aim was to investigate the role of adiponectin receptors AdipoR1/R2, AMP-activated protein kinase (AMPK), nuclear peroxisome proliferator-activated receptor alpha (PPARα) and liver X receptors (LXRs) in mediating the action of adiponectin on hepatic APOA1 expression in human hepatoma HepG2 cells. The level of APOA1 expression was determined by RT-qPCR and ELISA. We showed that the siRNA-mediated knockdown of genes coding for AdipoR1, AdipoR2, AMPK, PPARα, and LXRα and β prevented adiponectin-induced APOA1 expression in HepG2 cells and demonstrated that interaction of PPARα and LXRs with the APOA1 gene hepatic enhancer is important for the adiponectin-dependent APOA1 transcription. The results of this study point out to the involvement of both types of adiponectin receptors, AMPK, PPARα, and LXRs in the adiponectin-dependent upregulation of the APOA1 expression.

Ageing leads to profound alterations in the immune system and increases susceptibility to some chronic, infectious and autoimmune diseases. In recent years, widespread application of single-cell techniques has enabled substantial progress in our understanding of the ageing immune system. These comprehensive approaches have expanded and detailed the current views of ageing and immunity. Here we review a body of recent studies that explored how the immune system ages using unbiased profiling techniques at single-cell resolution. Specifically, we discuss an emergent understanding of age-related alterations in innate and adaptive immune cell populations, antigen receptor repertoires and immune cell-supporting microenvironments of the peripheral tissues. Focusing on the results obtained in mice and humans, we describe the multidimensional data that align with established concepts of immune ageing as well as novel insights emerging from these studies. We further discuss outstanding questions in the field and highlight techniques that will advance our understanding of immune ageing in the future., (© 2021. Springer Nature Limited.)

Anaphylatoxin C3a is a small signaling polypeptide that is generated during complement activation. C3a is involved in the regulation of various innate and adaptive immune system processes; however, the role of C3a in macrophage differentiation and polarization is poorly elucidated. Here we showed that C3a impairs alternative M2 polarization of human macrophages and suppressed CD206, IL1Ra and CCL22 expression. C3a leads to a decrease of nuclear receptor PPARγ expression via the ERK1/2 signaling pathway, resulting in repressed PPARγ-dependent activation of CD36, FABP4 and LXRα genes and blunted response to an LXR ligand TO901317. Using small interfering RNA and agonist/antagonist approaches we showed that C3a decreases CD206, IL1Ra and CCL22 transcription at least partly in a PPARγ-dependent manner in M2 macrophages. Moreover, C3a impairs efferocytosis by M2 macrophages and inhibits their migratory activity. By contrast, macrophages treated with C3a during differentiation show blunted response to lipopolysaccharide stimulation owing to downregulation of TLR4 and lipid raft content. At the same time, differentiation of macrophages with C3a does not change M1 polarization in interferon gamma (IFNγ) and IFNγ + lipopolysaccharide-treated macrophages. These data provide a novel role of complement system and C3a in the regulation of M2 macrophage polarizations and suggest crosstalk between C3a, TLR4, PPARγ and LXR signaling pathways., (© 2022 Australian and New Zealand Society for Immunology, Inc.)

The meninges contain adaptive immune cells that provide immunosurveillance of the central nervous system (CNS). These cells are thought to derive from the systemic circulation. Through single-cell analyses, confocal imaging, bone marrow chimeras, and parabiosis experiments, we show that meningeal B cells derive locally from the calvaria, which harbors a bone marrow niche for hematopoiesis. B cells reach the meninges from the calvaria through specialized vascular connections. This calvarial-meningeal path of B cell development may provide the CNS with a constant supply of B cells educated by CNS antigens. Conversely, we show that a subset of antigen-experienced B cells that populate the meninges in aging mice are blood-borne. These results identify a private source for meningeal B cells, which may help maintain immune privilege within the CNS., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background/objectives: Innate lymphoid cells (ILCs) play an important role in the maintenance of immune and metabolic homeostasis in adipose tissue (AT). The crosstalk between AT ILCs and adipocytes and other immune cells coordinates adipocyte differentiation, beiging, glucose metabolism and inflammation. Although the metabolic and homeostatic functions of mouse ILCs have been extensively investigated, little is known about human adipose ILCs and their roles in obesity and insulin resistance (IR)., Subjects/methods: Here we characterized T and NK cell populations in omental AT (OAT) from women (n = 18) with morbid obesity and varying levels of IR and performed an integrated analysis of metabolic parameters and adipose tissue transcriptomics., Results: In OAT, we found a distinct population of CD56 - NKp46 + EOMES + NK cells characterized by expression of cytotoxic molecules, pro-inflammatory cytokines, and markers of cell activation. AT IFNγ + NK cells, but not CD4, CD8 or γδ T cells, were positively associated with glucose levels, glycated hemoglobin (HbA1c) and IR. AT NK cells were linked to a pro-inflammatory gene expression profile in AT and developed an effector phenotype in response to IL-12 and IL-15. Moreover, integrated transcriptomic analysis revealed a potential implication of AT IFNγ + NK cells in controlling adipose tissue inflammation, remodeling, and lipid metabolism., Conclusions: Our results suggest that a distinct IFNγ-producing NK cell subset is involved in metabolic homeostasis in visceral AT in humans with obesity and may be a potential target for therapy of IR.

We examine the cellular and soluble determinants of coronavirus disease 2019 (COVID-19) relative to aging by performing mass cytometry in parallel with clinical blood testing and plasma proteomic profiling of ~4,700 proteins from 71 individuals with pulmonary disease and 148 healthy donors (25-80 years old). Distinct cell populations were associated with age (GZMK + CD8 + T cells and CD25 low CD4 + T cells) and with COVID-19 (TBET - EOMES - CD4 + T cells, HLA-DR + CD38 + CD8 + T cells and CD27 + CD38 + B cells). A unique population of TBET + EOMES + CD4 + T cells was associated with individuals with COVID-19 who experienced moderate, rather than severe or lethal, disease. Disease severity correlated with blood creatinine and urea nitrogen levels. Proteomics revealed a major impact of age on the disease-associated plasma signatures and highlighted the divergent contribution of hepatocyte and muscle secretomes to COVID-19 plasma proteins. Aging plasma was enriched in matrisome proteins and heart/aorta smooth muscle cell-specific proteins. These findings reveal age-specific and disease-specific changes associated with COVID-19, and potential soluble mediators of the physiological impact of COVID-19., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Itaconate is a unique regulatory metabolite that is induced upon Toll-like receptor (TLR) stimulation in myeloid cells. Here, we demonstrate major inflammatory tolerance and cell death phenotypes associated with itaconate production in activated macrophages. We show that endogenous itaconate is a key regulator of the signal 2 of NLR family pyrin domain containing 3 (NLRP3) inflammasome activation after long lipopolysaccharide (LPS) priming, which establishes tolerance to late NLRP3 inflammasome activation. We show that itaconate acts synergistically with inducible nitric oxide synthase (iNOS) and that the ability of various TLR ligands to establish NLRP3 inflammasome tolerance depends on the pattern of co-expression of IRG1 and iNOS. Mechanistically, itaconate accumulation upon prolonged inflammatory stimulation prevents full caspase-1 activation and processing of gasdermin D, which we demonstrate to be post-translationally modified by endogenous itaconate. Altogether, our data demonstrate that metabolic rewiring in inflammatory macrophages establishes tolerance to NLRP3 inflammasome activation that, if uncontrolled, can result in pyroptotic cell death and tissue damage., Competing Interests: Declaration of interests K.L., S.H., H.K., U.K., V.C., T.P.R., and D.M.S. are employees of Agios Pharmaceuticals. M.S.D. is a consultant for Inbios, Eli Lilly, Vir Biotechnology, and NGM Biopharmaceuticals; is on the Scientific Advisory Board of Moderna; and has received laboratory funding under unrelated sponsored research agreements from Moderna, Vir Biotechnology, and Emergent BioSolutions. All other authors declare no competing financial interest., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Systematic understanding of immune aging on a whole-body scale is currently lacking. We characterized age-associated alterations in immune cells across multiple mouse organs using single-cell RNA and antigen receptor sequencing and flow cytometry-based validation. We defined organ-specific and common immune alterations and identified a subpopulation of age-associated granzyme K (GZMK)-expressing CD8 + T (Taa) cells that are distinct from T effector memory (Tem) cells. Taa cells were highly clonal, had specific epigenetic and transcriptional signatures, developed in response to an aged host environment, and expressed markers of exhaustion and tissue homing. Activated Taa cells were the primary source of GZMK, which enhanced inflammatory functions of non-immune cells. In humans, proportions of the circulating GZMK + CD8 + T cell population that shares transcriptional and epigenetic signatures with mouse Taa cells increased during healthy aging. These results identify GZMK + Taa cells as a potential target to address age-associated dysfunctions of the immune system., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The impact of healthy aging on molecular programming of immune cells is poorly understood. Here, we report comprehensive characterization of healthy aging in human classical monocytes, with a focus on epigenomic, transcriptomic, and proteomic alterations, as well as the corresponding proteomic and metabolomic data for plasma, using healthy cohorts of 20 young and 20 older males (~27 and ~64 years old on average). For each individual, we performed eRRBS-based DNA methylation profiling, which allowed us to identify a set of age-associated differentially methylated regions (DMRs) - a novel, cell-type specific signature of aging in DNA methylome. Hypermethylation events were associated with H3K27me3 in the CpG islands near promoters of lowly-expressed genes, while hypomethylated DMRs were enriched in H3K4me1 marked regions and associated with age-related increase of expression of the corresponding genes, providing a link between DNA methylation and age-associated transcriptional changes in primary human cells., Competing Interests: COMPETING INTERESTS The authors declare no competing interests

Progression of fatty liver to non-alcoholic steatohepatitis (NASH) is a rapidly growing health problem. Presence of inflammatory infiltrates in the liver and hepatocyte damage distinguish NASH from simple steatosis. However, the underlying molecular mechanisms involved in the development of NASH remain to be fully understood. Here we perform transcriptional and immune profiling of NASH patients before and after lifestyle intervention (LSI). Analysis of liver microarray data from a cohort of patients with histologically assessed NAFLD reveals a hepatic gene signature, which is associated with NASH and is sensitive to regression of NASH activity upon LSI independently of body weight loss. Enrichment analysis reveals the presence of immune-associated genes linked to inflammatory responses, antigen presentation and cytotoxic cells in the NASH-linked gene signature. In an independent cohort, NASH is also associated with alterations in blood immune cell populations, including conventional dendritic cells (cDC) type 1 and 2, and cytotoxic CD8 T cells. Lobular inflammation and ballooning are associated with the accumulation of CD8 T cells in the liver. Progression from simple steatosis to NASH in a mouse model of diet-driven NASH results in a comparable immune-related hepatic expression signature and the accumulation of intra-hepatic cDC and CD8 T cells. These results show that NASH, compared to normal liver or simple steatosis, is associated with a distinct hepatic immune-related gene signature, elevated hepatic CD8 T cells, and altered antigen-presenting and cytotoxic cells in blood. These findings expand our understanding of NASH and may identify potential targets for NASH therapy., Competing Interests: Competing interests: BS and SF are consultants for Genfit S.A. SF and LV are consultants for Inventiva. All other authors have nothing to declare.

Innate immune responses are intricately linked with intracellular metabolism of myeloid cells. Toll-like receptor (TLR) stimulation shifts intracellular metabolism toward glycolysis, while anti-inflammatory signals depend on enhanced mitochondrial respiration. How exogenous metabolic signals affect the immune response is unknown. We demonstrate that TLR-dependent responses of dendritic cells (DCs) are exacerbated by a high-fatty-acid (FA) metabolic environment. FAs suppress the TLR-induced hexokinase activity and perturb tricarboxylic acid cycle metabolism. These metabolic changes enhance mitochondrial reactive oxygen species (mtROS) production and, in turn, the unfolded protein response (UPR), leading to a distinct transcriptomic signature with IL-23 as hallmark. Interestingly, chemical or genetic suppression of glycolysis was sufficient to induce this specific immune response. Conversely, reducing mtROS production or DC-specific deficiency in XBP1 attenuated IL-23 expression and skin inflammation in an IL-23-dependent model of psoriasis. Thus, fine-tuning of innate immunity depends on optimization of metabolic demands and minimization of mtROS-induced UPR., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Human apolipoprotein A-I (ApoA-I) is a major structural and functional protein component of high-density lipoprotein (HDL). ApoA-I constitutes ~75% of the protein content of HDL. The main sites of ApoA-I synthesis in humans are the liver and the small intestine. The mechanisms that govern tissue-specific apoA-I transcription in tissues and organs other than the liver and the small intestine are poorly understood. It is known that the human apoA-I has two additional promoters, the proximal and the distal one. In this work these two alternative apoA-I promoters are characterized, their transcription start sites are mapped and their competition for apoA-Itranscription is demonstrated; the role of the alternative promoters in apoA-I expression in human cells and tissues other than hepatocytes and enterocytes is discussed.

Keratinocytes are key players in chronic inflammatory skin diseases. A20 regulates NF-κB-dependent expression of proinflammatory genes and cell death, but the impact of its expression in keratinocytes on systemic inflammation and skin disorders has not been determined. Comparative transcriptomic analysis of microdissected epidermis showed that A20 is down-regulated in involved epidermis, but not in dermis, of psoriasis and atopic dermatitis patients, suggesting that loss of A20 expression in keratinocytes increases the vulnerability for psoriasis/atopic dermatitis induction. We have previously shown that epidermis-specific A20 knockout mice (A20 EKO ) develop mild epidermal hyperplasia but no macroscopic skin inflammation. We now show that various cytokines and chemokines are up-regulated in A20 EKO mouse skin. A20 EKO mice also display systemic proinflammatory changes, even in the absence of skin immune cell infiltration, and an exacerbated disease severity upon induction of experimental psoriasis, atopic dermatitis, or skin barrier disruption. Keratinocytes showed increased proinflammatory gene expression in the absence of A20 in unstimulated and IL-17A-stimulated conditions, in part resulting from uncontrolled MyD88-dependent signaling. Our findings indicate that absence of A20 in keratinocytes leads to systemic inflammation at homeostatic conditions and is sufficient to exacerbate inflammatory skin disorders associated with different immune profiles by increasing cytokine and chemokine expression., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Apolipoprotein A-I (ApoA-I) is the main structural and functional protein component of high-density lipoprotein. ApoA-I has been shown to regulate lipid metabolism and inflammation in macrophages. Recently, we found the moderate expression of endogenous apoA-I in human monocytes and macrophages and showed that pro-inflammatory cytokine tumor necrosis factor α (TNFα) increases apoA-I mRNA and stimulates ApoA-I protein secretion by human monocytes and macrophages. Here, we present data about molecular mechanisms responsible for the TNFα-mediated activation of apoA-I gene in human monocytes and macrophages. This activation depends on JNK and MEK1/2 signaling pathways in human monocytes, whereas inhibition of NFκB, JNK, or p38 blocks an increase of apoA-I gene expression in the macrophages treated with TNFα. Nuclear receptor PPARα is a ligand-dependent regulator of apoA-I gene, whereas LXRs stimulate apoA-I mRNA transcription and ApoA-I protein synthesis and secretion by macrophages. Treatment of human macrophages with PPARα or LXR synthetic ligands as well as knock-down of LXRα, and LXRβ by siRNAs interfered with the TNFα-mediated activation of apoA-I gene in human monocytes and macrophages. At the same time, TNFα differently regulated the levels of PPARα, LXRα, and LXRβ binding to the apoA-I gene promoter in THP-1 cells. Obtained results suggest a novel tissue-specific mechanism of the TNFα-mediated regulation of apoA-I gene in monocytes and macrophages and show that endogenous ApoA-I might be positively regulated in macrophage during inflammation.

Apolipoprotein A-I (ApoA-I) is the main anti-atherogenic component of human high-density lipoproteins (HDL). ApoA-I gene expression is regulated by several nuclear receptors, which are the sensors for metabolic changes during development of cardiovascular diseases. Activation of nuclear receptor PPARγ has been shown to impact lipid metabolism as well as inflammation. Here, we have shown that synthetic PPARγ agonist GW1929 decreases both ApoA-I mRNA and protein levels in HepG2 cells and the effect of GW1929 on apoA-I gene transcription depends on PPARγ. PPARγ binds to the sites A and C within the hepatic enhancer of apoA-I gene and the negative regulation of apoA-I gene transcription by PPARγ appears to be realized via the site C (-134 to -119). Ligand activation of PPARγ leads to an increase of LXRβ and a decrease of PPARα binding to the apoA-I gene hepatic enhancer in HepG2 cells. GW1929 abolishes the TNFα-mediated decrease of ApoA-I mRNA expression in both HepG2 and Caco-2 cells but does not block TNFα-mediated inhibition of ApoA-I protein secretion by HepG2 cells. These data demonstrate that complex of PPARγ with GW1929 is a negative regulator involved in the control of ApoA-I expression and secretion in human hepatocyte- and enterocyte-like cells. J. Cell. Biochem. 117: 2010-2022, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Interleukin-17A (IL-17A) is a pro-inflammatory cytokine that has an important role at mucosal sites in a wide range of immune responses including infection, allergy and auto-immunity. γδ T cells are recognized as IL-17 producers, but based on the level of CD3 expression, we now define the remarkable ability of a CD3(bright) γδ T-cell subset with an effector memory phenotype to rapidly produce IL-17A, but not interferon-γ. CD3(bright) γδ T cells uniformly express the canonical germline encoded Vγ6/Vδ1(+) T-cell receptor. They are widely distributed with a preferential representation in the lungs and skin are negatively impacted in the absence of retinoic acid receptor-related orphan receptor gammat expression or endogenous flora. This population responded rapidly to various stimuli in a mechanism involving IL-23 and NOD-like receptor family, pyrin domain containing 3 (NLRP3)-inflammasome-dependent IL-1β. Finally, we demonstrated that IL-17-producing CD3(bright) γδ T cells responded promptly and strongly to pneumococcal infection and during skin inflammation. Here, we propose a new way to specifically analyze IL-17-producing Vγ6/Vδ1(+) T cells based on the level of CD3 signals. Using this gating strategy, our data reinforce the crucial role of this γδ T-cell subset in respiratory and skin disorders.

Complement C3 is involved in various protective and regulatory mechanisms of immune system. Recently it was established that C3 expression is regulated by nuclear receptors. Hepatic nuclear factor 4α (HNF4α) is a nuclear receptor critical for hepatic development and metabolism. We have shown that HNF4α is a positive regulator of C3 gene expression, realizing its effects through binding to two HNF4-response elements within the C3 promoter in HepG2 cells. TNFα is a well established positive regulator of C3 expression in hepatocytes during acute phase of inflammation. TNFα decreases the amount of HNF4α protein in HepG2 cells through NF-κB and MEK1/2 pathways thereby leading to a decrease in HNF4α bound to the C3 promoter. TNFα and HNF4α act in a synergetic way resulting in the potent activation of C3 transcription. These results suggest a novel mechanism of C3 regulation during acute phase response in HepG2 cells and display the mechanism of interaction of TNFα-induced pathways and HNF4α in transcriptional regulation of C3 gene., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Complement C3 is a pivotal component of three cascades of complement activation. The liver is the main source of C3 in circulation and expression and secretion of C3 by hepatocytes is increased during acute inflammation. However, the mechanism of the regulation of the C3 gene in hepatocytes is not well elucidated. We showed that the C3 gene is the direct target for peroxisome proliferator-activated receptor α (PPARα) in human hepatoma HepG2 cells and mouse liver. Using PPARα siRNA and synthetic PPARα agonist WY-14643 and antagonist MK886 we showed that activation of PPARα results in up-regulation of C3 gene expression and protein secretion by HepG2 cells. The PPAR response element (PPRE), which is able to bind PPARα in vitro and in vivo, was found in the human C3 promoter. PPRE is conserved between human and mouse, and WY-14643 stimulates mouse C3 expression in the liver. TNFα increases C3 gene via NF-κB and, to a lesser extent, MEK1/2 signaling pathways, whereas TNFα-mediated stimulation of C3 protein secretion depends on activation of MEK1/2, p38, and JNK in HepG2 cells. Activation of PPARα abolishes TNFα-mediated up-regulation of C3 gene expression and protein secretion due to interference with NF-κB via PPRE-dependent mechanism in HepG2 cells. TNFα decreases PPARα protein content via NF-κB and MEK1/2 signaling pathways and inhibits PPARα binding with the human C3 promoter in HepG2 cells. These results suggest novel mechanism controlling C3 expression in hepatocytes during acute phase inflammation and demonstrate a crosstalk between PPARα and TNFα in the regulation of complement system.

Apolipoprotein A-I (ApoA-I) is the main functional protein component of human high-density lipoproteins. ApoA-I shows various anti-inflammatory and atheroprotective properties toward macrophages; however, endogenous apoA-I expression has not been investigated in macrophages. We have shown that endogenous apoA-I gene is expressed in human macrophages at both mRNA and protein levels. Endogenous ApoA-I is localized in intracellular vesicles and at the external side of the plasma membrane in association with ATP-binding cassette transporter A1 (ABCA1) and lipid rafts in macrophages. We have shown that endogenous ApoA-I stabilizes ABCA1, moreover, down-regulation of ApoA-I by siRNA results in an increase of Toll-like receptor 4 (TLR4) mRNA and membrane surface protein expression, as well as an enhancement of bacterial lipopolysaccharide (LPS)-induced expression of tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), and inducible nitric oxide synthase (NOS2) genes in human macrophages. TNF-α stimulates ApoA-I expression and secretion (1.2±0.2 vs. 4.3±0.9 ng/mg total protein) in macrophages. Obtained results suggest that endogenous ApoA-I has anti-inflammatory properties, presumably due to ABCA1 stabilization in macrophages; these results elucidate the cell type-specific mechanism of the TNF-α-mediated regulation of apoA-I gene expression in monocytes and macrophages.

Complement C3 is a pivotal component of three cascades of complement activation. C3 is expressed in human atherosclerotic lesions and is involved in atherogenesis. However, the mechanism of C3 accumulation in atherosclerotic lesions is not well elucidated. We show that acetylated low density lipoprotein and oxidized low density lipoprotein (oxLDL) increase C3 gene expression and protein secretion by human macrophages. Modified LDL (mLDL)-mediated activation of C3 expression mainly depends on liver X receptor (LXR) and partly on Toll-like receptor 4 (TLR4), whereas C3 secretion is increased due to TLR4 activation by mLDL. LXR agonist TO901317 stimulates C3 gene expression in human monocyte-macrophage cells but not in human hepatoma (HepG2) cells. We find LXR-responsive element inside of the promoter region of the human C3 gene, which binds to LXRβ in macrophages but not in HepG2 cells. We show that C3 expression and secretion is decreased in IL-4-treated (M2) and increased in IFNγ/LPS-stimulated (M1) human macrophages as compared with resting macrophages. LXR agonist TO901317 potentiates LPS-induced C3 gene expression and protein secretion in macrophages, whereas oxLDL differently modulates LPS-mediated regulation of C3 in M1 or M2 macrophages. Treatment of human macrophages with anaphylatoxin C3a results in stimulation of C3 transcription and secretion as well as increased oxLDL accumulation and augmented oxLDL-mediated up-regulation of the C3 gene. These data provide a novel mechanism of C3 gene regulation in macrophages and suggest new aspects of cross-talk between mLDL, C3, C3a, and TLR4 during development of atherosclerotic lesions.

Synthesis of ABCA1 protein in liver is necessary for high-density lipoproteins (HDL) formation in mammals. Nuclear receptor PPARγ is known as activator of ABCA1 expression, but details of PPARγ-mediated regulation of ABCA1 at both transcriptional and post-transcriptional levels in hepatocytes have not still been well elucidated. In this study we have shown, that PPARγ activates ABCA1 gene transcription in human hepatoma cells HepG2 through increasing of LXRβ binding with promoter region of ABCA1 gene. Treatment of HepG2 cells with PPARγ agonist GW1929 leads to dissociation of LXRβ from ABCA1/LXRβ complex and to nuclear translocation of this nuclear receptor resulting in reduction of ABCA1 protein level 24h after treatment. Inhibition of protein kinases MEK1/2 abolishes PPARγ-mediated dissociation of LXRβ from ABCA1/LXRβ complex, but does not block PPARγ-dependent down-regulation of ABCA1 protein in HepG2 cells. These data suggest that PPARγ may be important for regulation of the level of hepatic ABCA1 protein and indicate the new interplays between PPARγ, LXRβ and MEK1/2 in regulation of ABCA1 mRNA and protein expression., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Human apolipoprotein A-I (ApoA-I) is a major structural and functional protein component of high-density lipoproteins. The expression of the apolipoprotein A-I gene (apoA-I) in hepatocytes is repressed by pro-inflammatory cytokines such as IL-1beta and TNFalpha. Recently, two novel additional (alternative) promoters for human apoA-I gene have been identified. Nothing is known about the role of alternative promoters in TNFalpha-mediated downregulation of apoA-I gene. In this article we report for the first time about the different effects of TNFalpha on two alternative promoters of human apoA-I gene. Stimulation of HepG2 cells by TNFalpha leads to activation of the distal alternative apoA-I promoter and downregulation of the proximal alternative and the canonical apoA-I promoters. This effect is mediated by weakening of the promoter competition within human apoA-I 5'-regulatory region (apoA-I promoter switching) in the cells treated by TNFalpha. The MEK1/2-ERK1/2 cascade and nuclear receptors PPARalpha and LXRs are important for TNFalpha-mediated apoA-I promoter switching., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

The fragment of a homologue of complement component C3 gene has been cloned and sequenced from the starfish, Asterias rubens. Phylogenetic analysis of ArC3-like gene demonstrates that ArC3-like gene has close similarity to C3 gene homologues of Deuterostomia invertebrate animals. High level of ArC3-like gene expression was identified in circulating cells (coelomocytes), in a gut's derivate (hepatopancreas) and in male gonada but not in stomach, female gonad and rectal gland of A. rubens starfish. ArC3-like gene expression was shown in all types of starfish coelomocytes: in lymphocyte-like cells, granular and nongranular amebocytes. Injection of bacterial lipopolysaccharide (LPS) solution into the coelomic cavity of starfish leads to the increase of ArC3-like gene expression in coelomocytes and hepatopancreas over the control level of sterile sea water injection. The level of ArC3-like gene expression increased in response to LPS reaching the maximum 6 h after the stimulation, and decreased to basal level 24 h after the stimulation. Injection of LPS solution stimulated the increase of ArC3-like gene expression level in hepatopancreas reaching the maximum 6-12 h after the stimulation, and the level of mRNA of ArC3-like gene had still been increased 48 h after LPS injection. The data demonstrates sustained positive regulation of ArC3-like gene expression under the influence of LPS.

The rate and character of skin tissue regeneration after wounds, burns and other traumas depend on the cell proliferation within damaged area. Acceleration of healing by stimulation of cell proliferation and extracellular matrix synthesis is one of the most important tasks of modern medicine. There are gene therapy approaches to wound treatment consisting in the transfer of genes encoding mitogenic growth factors to wound area. The most important step in the development of gene therapy approaches is the design of gene delivery tools. In spite of high efficacy of viral vectors, the non-viral means have some preferences (low toxicity, low immunogenity, safety and the absence of backside effects). Among non-viral gene delivery tools, molecular conjugates are the most popular because of their efficacy, simplicity, and the capacity to the targeted gene transfer. In the present work we have developed two molecular conjugates--NLS-TSF7 and NLS-TSF12 consisting of the modified signal of nuclear localization of T-antigen of SV40 virus (cationic part) and the peptide ligands of mammalian transferrin receptor (ligand part). These conjugates bind to plasmid DNA with formation of polyelectrolytic complexes and are capable to deliver plasmid DNA into cells expressing transferrin receptors by receptor-mediated endocytosis. Transfer of the expression vector of luciferase gene in the complex with molecular conjugate NLS-TSF7 to murine surface tissues led to about 100 fold increasing of luciferase activity in comparison with the transfer of free expression vector. Treatment of slash wounds in mice with the complexes of expression vector of synthetic human gene encoding insulin-like growth factor 1 with molecular conjugates NLS-TSF7 led to acceleration of healing in comparison with mice treated with free expression vector. The results obtained confirm the high efficiency of the developed regenerative gene therapy approach for the treatment of damaged skin tissues in mammals.

The expression of the apolipoprotein A-I gene (apoA-I) in hepatocytes is repressed by pro-inflammatory cytokines such as IL-1beta and TNFalpha. In this work, we have demonstrated that treatment of HepG2 human hepatoma cells with chemical inhibitors for JNK, p38 protein kinases, and NFkappaB transcription factor abolishes the TNFalpha-mediated inhibition of human apoA-I gene expression in HepG2 cells. In addition, we have shown that TNFalpha decreases also the rate of secretion of apoA-I protein by HepG2 cells, and this effect depends on JNK and p38, but not on NFkappaB and MEK1/2 signaling pathways. The inhibitory effect of TNFalpha has been found to be mediated by the hepatic enhancer of the apoA-I gene. The decrease in the level of human apoA-I gene expression under the impact of TNFalpha appears to be partly mediated by the inhibition of HNF4alpha and PPARalpha gene expression. Treatment of HepG2 cells with PPARalpha antagonist (MK886) or LXR agonist (TO901317) abolishes the TNFalpha-mediated decrease in the level of apoA-I gene expression. PPARalpha agonist (WY-14643) abolishes the negative effect of TNFalpha on apoA-I gene expression in the case of simultaneous inhibition of MEK1/2, although neither inhibition of MEK1/2 nor addition of WY-14643 leads to the blocking of the TNFalpha-mediated decrease in the level of apoA-I gene expression individually. The ligand-dependent regulation of apoA-I gene expression by PPARalpha appears to be affected by the TNFalpha-mediated activation of MEK1/2 kinases, probably through PPARalpha phosphorylation. Treatment of HepG2 cells with PPARalpha and LXR synthetic agonists also blocks the inhibition of apoA-I protein secretion in HepG2 cells under the impact of TNFalpha. A chromatin immunoprecipitation assay demonstrates that TNFalpha leads to a 2-fold decrease in the level of PPARalpha binding with the apoA-I gene hepatic enhancer. At the same time, the level of LXRbeta binding with the apoA-I gene hepatic enhancer is increased 3-fold under the impact of TNFalpha. These results suggest that nuclear receptors HNF4alpha, PPARalpha, and LXRs are involved in the TNFalpha-mediated downregulation of human apoA-I gene expression and apoA-I protein secretion in HepG2 cells.

Several Ap1-like cis-acting elements were found within 5'-regulatory region (-2497...+173 versus transcription start point) of human apolipoprotein A-I gene (5'-apoA-I). Those elements are capable to interact with transcription factors belonging to Ap1 and CREB/ATF families. Those elements are localized outside of the hepatic enhancer (-220...-110) and the sequence responsible for apoA-I gene transcription in Caco2 cells (-595...-192). One of Ap1-like sites (5'-TGAGGTCT-3, Cre/jun2/apo) is present within 5'-apoA-I in two copies - distal (-1798 ...-1791) and proximal (+99...+106) ones. This and other Ap1-like sites - 5'-TGACTCT-3' (-1798...-1791, PF1) and 5'-TGACATCA-3' (-1171...-1163, Cre/jun1) were characterized by EMSA. It was shown by using the specific antibodies to c-Jun and ATF2 transcription factors in EMSA supershift experiments, that the DNA-protein complexes formed by Cre/jun2/apo, Cre/jun1 elements with nuclear proteins of human hepatoma HepG2 cells contain ATF2. The functional role of 5'-apoA-I regions containing Ap1-like sites was studied in cotransfection experiments of HepG2 cells (synthesize endogenous ApoA-I), human duodenum adenocarcinoma Hutu80 cells (do not synthesize endogenous ApoA-I), human neuroblastoma SK-N-SH cells (do not synthesize endogenous A-I) with expression vectors of c-jun and mekk1 genes. It was shown, that those Ap1-like sites appears to be responsible (the proximal Cre/jun2/apo is more efficient) for tissue-specific regulation of human apoA-I gene expression.

Human apolipoprotein A-I gene (apoA-I) plasmid expression vectors were transferred into mice by hydrodynamic injections into tail vein. Two types of expression vectors were used. First one -pCMVcapoAI contains cDNA of apo A-I driven by human cytomegalovirus early gene promoter (CMV). Second one--pAlg contains genomic locus of intron-containing apo A-I under control of own extended 5'-regulatory region (APOAI). Hydrodynamic intravenous injections of both expression vectors led to appearance of human apo A-I mRNA in the liver and human Apo A-I protein in the serum of injected mice. Dynamics of human Apo A-I content in the serum of mice injected by pCMVcapoAI and pAlg were different. When pCMVcapoAI was used, maximal concentration of human Apo A-I protein in the mouse serum was detected one day after injection with following decline to zero level during next two weeks. Under the same conditions injections of pAlg led to maximal level of human Apo A-I concentration in the mouse serum (up to 20 mkg/ml in some animals) on the 5th-7th day of experiment with following graduate decline during several months (human Apo A-I concentration in the serum of oldest analyzed mouse (6 months after injection) was about 25% of its maximal level in the same animal). Levels of human Apo A-I concentration in the mouse serum were compatible after injections of both expression vectors, in spite of much more strong activity of CMV promoter in comparison with APOAI in cultured human hepatoma cells HepG2. We ascribe the revealed difference in dynamics of human Apo A-I expression to delay of apo A-I transcription from pAlg vector, that was confirmed by nested RT-PCR. Significant level and long-term persistence of human Apo A-I in the serum of mice injected by pAlg could be explained by properties of APOAI or (and) exon-intron structure of genomic apo A-I gene. To test the role of APOAI in long-term expression of human Apo A-I in the mice we performed hydrodynamic injections of plasmid vectors containing cDNA of reporter gene encoding luciferase driven by variants of APOAI. No long-term expression of luciferase was found in the livers of injected mice. Therefore, our data suggest the role of exon-intron structure in maintaining of efficient and long-term expression of transferred human apo A-I.