Transcriptional and Epigenetic Regulation of Gasdermins.

Gasdermins (GSDM) are a family of six homologous proteins (GSDMA to E and Pejvakin) in humans. GSDMA-E are pore-forming proteins targeting the plasma membrane to trigger a rapid cell death termed pyroptosis or bacterial membranes to promote antibacterial immune defenses. Activation of GSDM relies on a proteolytic cleavage but is highly dependent on GSDM expression levels. The different GSDM genes have tissue-specific expression pattern although metabolic, environmental signals, cell stress and numerous cytokines modulate their expression levels in tissues. Furthermore, expression of GSDM genes can be modulated by polymorphisms and have been associated with susceptibility to asthma, inflammatory bowel diseases and rhinovirus wheezing illness. Finally, the expression level of GSDMs controls the balance between apoptosis and pyroptosis affecting both the response and the toxicity to chemotactic drugs and antitumoral treatments. Numerous cancer demonstrate positive or negative modulation of GSDM expression levels correlating with distinct tumor-specific prognosis. In this review, we present the transcriptional and epigenetic mechanisms controlling GSDM levels and their functional consequences in asthma, infection, cancers and inflammatory bowel disease to highlight how this first layer of regulations has key consequences on disease susceptibility and response to treatment.

Transcriptional Regulation of Inflammasomes.

Inflammasomes are multimolecular complexes with potent inflammatory activity. As such, their activity is tightly regulated at the transcriptional and post-transcriptional levels. In this review, we present the transcriptional regulation of inflammasome genes from sensors (e.g., NLRP3) to substrates (e.g., IL-1ß). Lineage-determining transcription factors shape inflammasome responses in different cell types with profound consequences on the responsiveness to inflammasome-activating stimuli. Pro-inflammatory signals (sterile or microbial) have a key transcriptional impact on inflammasome genes, which is largely mediated by NF-κB and that translates into higher antimicrobial immune responses. Furthermore, diverse intrinsic (e.g., circadian clock, metabolites) or extrinsic (e.g., xenobiotics) signals are integrated by signal-dependent transcription factors and chromatin structure changes to modulate transcriptionally inflammasome responses. Finally, anti-inflammatory signals (e.g., IL-10) counterbalance inflammasome genes induction to limit deleterious inflammation. Transcriptional regulations thus appear as the first line of inflammasome regulation to raise the defense level in front of stress and infections but also to limit excessive or chronic inflammation.

Publisher Correction: Human CD45 is an F-component-specific receptor for the staphylococcal toxin Panton-Valentine leukocidin.

In the version of this Article originally published, the name of author Robert Jan Lebbink was coded wrongly, resulting in it being incorrect when exported to citation databases. This has now been corrected, though no visible changes will be apparent.

A proximity-dependent biotinylation (BioID) approach flags the p62/sequestosome-1 protein as a caspase-1 substrate.

The inflammasome is a major component of the innate immune system, and its main function is to activate caspase-1, a cysteine protease that promotes inflammation by inducing interleukin-1ß (IL-1ß) maturation and release into the extracellular milieu. To prevent uncontrolled inflammation, this complex is highly regulated. When it is assembled, the inflammasome is insoluble, which has long precluded the analysis of its interactions with other proteins. Here we used the proximity-dependent biotinylation assay (BioID) to identify proteins associated with caspase-1 during inflammasome activation. Using the BioID in a cell-free system in which the inflammasome had been activated, we found that a caspase-1-biotin ligase fusion protein selectively labeled 111 candidates, including the p62/sequestosome-1 protein (p62). Using co-immunoprecipitation experiments, we demonstrated that p62 interacts with caspase-1. This interaction promoted caspase-1-mediated cleavage of p62 at Asp-329. Mechanistic and functional analyses revealed that caspase-1-mediated cleavage of p62 leads to loss of its interaction with the autophagosomal protein microtubule-associated protein 1 light chain 3 ß (LC3B). Strikingly, overexpression of a p62 N-terminal fragment generated upon caspase-1 cleavage decreased IL-1ß release, whereas overexpression of p62's C-terminal portion enhanced IL-1ß release, by regulating pro-IL1ß levels. Overall, the overexpression of both fragments together decreased IL-1ß release. Taken together, our results indicate that caspase-1-mediated p62 cleavage plays a complex role in balancing caspase-1-induced inflammation.

Human CD45 is an F-component-specific receptor for the staphylococcal toxin Panton-Valentine leukocidin.

The staphylococcal bi-component leukocidins Panton-Valentine leukocidin (PVL) and γ-haemolysin CB (HlgCB) target human phagocytes. Binding of the toxins' S-components to human complement C5a receptor 1 (C5aR1) contributes to cellular tropism and human specificity of PVL and HlgCB. To investigate the role of both leukocidins during infection, we developed a human C5aR1 knock-in (hC5aR1KI) mouse model. HlgCB, but unexpectedly not PVL, contributed to increased bacterial loads in tissues of hC5aR1KI mice. Compared to humans, murine hC5aR1KI neutrophils showed a reduced sensitivity to PVL, which was mediated by the toxin's F-component LukF-PV. By performing a genome-wide CRISPR-Cas9 screen, we identified CD45 as a receptor for LukF-PV. The human-specific interaction between LukF-PV and CD45 provides a molecular explanation for resistance of hC5aR1KI mouse neutrophils to PVL and probably contributes to the lack of a PVL-mediated phenotype during infection in these mice. This study demonstrates an unsuspected role of the F-component in driving the sensitivity of human phagocytes to PVL.

Familial Mediterranean fever mutations are hypermorphic mutations that specifically decrease the activation threshold of the Pyrin inflammasome.

Objectives: FMF is the most frequent autoinflammatory disease and is associated in most patients with bi-allelic MEFV mutations. MEFV encodes Pyrin, an inflammasome sensor activated following RhoGTPase inhibition. The functional consequences of MEFV mutations on the ability of Pyrin variants to act as inflammasome sensors are largely unknown. The aim of this study was to assess whether MEFV mutations affect the ability of Pyrin to detect RhoGTPase inhibition and other inflammasome stimuli. Methods: IL-1ß and IL-18 released by monocytes from healthy donors (HDs) and FMF patients were measured upon specific engagement of the Pyrin, NLRP3 and NLRC4 inflammasomes. Cell death kinetics following Pyrin activation was monitored in real time. Results: Monocytes from FMF patients secreted significantly more IL-1ß and IL-18 and died significantly faster than HD monocytes in response to low concentrations of Clostridium difficile toxin B (TcdB), a Pyrin-activating stimulus. Monocytes from patients bearing two MEFV exon 10 pathogenic variants displayed an increased Pyrin inflammasome response compared with monocytes from patients with a single exon 10 pathogenic variant indicating a gene-dosage effect. Using a short priming step, the response of monocytes from FMF patients to NLRP3- and NLRC4-activating stimuli was normal indicating that MEFV mutations trigger a specific hypersensitivity of monocytes to low doses of a Pyrin-engaging stimulus. Conclusion: Contrary to the NLRP3 mutations described in cryopyrin-associated periodic syndrome, FMF-associated MEFV mutations do not lead to a constitutive activation of Pyrin. Rather, FMF-associated mutations are hypermorphic mutations that specifically decrease the activation threshold of the Pyrin inflammasome without affecting other canonical inflammasomes.

Geoepidemiology and Immunologic Features of Autoinflammatory Diseases: a Comprehensive Review.

The knowledge on systemic autoinflammatory disorders (SAID) is expanding rapidly and new signalling pathways are being decrypted. The concept of autoinflammation has been proposed since 1999, to define a group of diseases with abnormal innate immunity activation. Since then, more than 30 monogenic SAID have been described. In this review, we first describe inflammasomopathies and SAID related to the interleukin-1 pathway. Recent insights into the pathogenesis of familial Mediterranean fever and the function of Pyrin are detailed. In addition, complex or polygenic SAID, such as Still's disease or PFAPA syndrome, are also discussed. Then, major players driving autoinflammation, such as type-1 interferonopathies (including the recently described haploinsuffiency in A20 and otulipenia), TNF-associated periodic syndromes, defects in ubiquitination, and SAID with overlapping features of autoimmunity or immunodeficiency. Discoveries of the pathogenic role of mosaicism, intronic defects coupled to the likelihood to identify digenic or polygenic diseases are providing new challenges for physicians and geneticists. This comprehensive review depicts the various SAID, presenting them according to their predominant pathophysiological mechanism, with a particular emphasis on recent findings. Epidemiologic data are also presented. Finally, we propose a practical diagnostic approach to the most common monogenic SAID, based on the most characteristic clinical presentation of these disorders.

Alveolar Epithelial Cell-Derived Prostaglandin E2 Serves as a Request Signal for Macrophage Secretion of Suppressor of Cytokine Signaling 3 during Innate Inflammation.

Preservation of gas exchange mandates that the pulmonary alveolar surface restrain unnecessarily harmful inflammatory responses to the many challenges to which it is exposed. These responses reflect the cross-talk between alveolar epithelial cells (AECs) and resident alveolar macrophages (AMs). We recently determined that AMs can secrete suppressor of cytokine signaling (SOCS) proteins within microparticles. Uptake of these SOCS-containing vesicles by epithelial cells inhibits cytokine-induced STAT activation. However, the ability of epithelial cells to direct AM release of SOCS-containing vesicles in response to inflammatory insults has not been studied. In this study, we report that SOCS3 protein was elevated in bronchoalveolar lavage fluid of both virus- and bacteria-infected mice, as well as in an in vivo LPS model of acute inflammation. In vitro studies revealed that AEC-conditioned medium (AEC-CM) enhanced AM SOCS3 secretion above basal levels. Increased amounts of PGE2 were present in AEC-CM after LPS challenge, and both pharmacologic inhibition of PGE2 synthesis in AECs and neutralization of PGE2 in AEC-CM implicated this prostanoid as the major AEC-derived factor mediating enhanced AM SOCS3 secretion. Moreover, pharmacologic blockade of PGE2 synthesis or genetic deletion of a PGE2 synthase similarly attenuated the increase in bronchoalveolar lavage fluid SOCS3 noted in lungs of mice challenged with LPS in vivo. These results demonstrate a novel tunable form of cross-talk in which AECs use PGE2 as a signal to request SOCS3 from AMs to dampen their endogenous inflammatory responses during infection.

Catch me if you can.

Direct contact between host cells allows some bacteria to spread within the body without being attacked by the immune system.

Transcellular delivery of vesicular SOCS proteins from macrophages to epithelial cells blunts inflammatory signaling.

JAK-STAT signaling mediates the actions of numerous cytokines and growth factors, and its endogenous brake is the family of SOCS proteins. Consistent with their intracellular roles, SOCS proteins have never been identified in the extracellular space. Here we report that alveolar macrophages can secrete SOCS1 and -3 in exosomes and microparticles, respectively, for uptake by alveolar epithelial cells and subsequent inhibition of STAT activation. Secretion is tunable and occurs both in vitro and in vivo. SOCS secretion into lung lining fluid was diminished by cigarette smoking in humans and mice. Secretion and transcellular delivery of vesicular SOCS proteins thus represent a new model for the control of inflammatory signaling, which is subject to dysregulation during states of inflammation.

Prostaglandin E2 reduces Toll-like receptor 4 expression in alveolar macrophages by inhibition of translation.

Alveolar macrophages (AMs) represent the first line of innate immune defense in the lung. AMs use pattern recognition receptors (PRRs) to sense pathogens. The best studied PRR is Toll-like receptor (TLR)4, which detects LPS from gram-negative bacteria. The lipid mediator prostaglandin (PG)E2 dampens AM immune responses by inhibiting the signaling events downstream of PRRs. We examined the effect of PGE2 on TLR4 expression in rat AMs. Although PGE2 did not reduce the mRNA levels of TLR4, it decreased TLR4 protein levels. The translation inhibitor cycloheximide reduced TLR4 protein levels with similar kinetics as PGE2, and its effects were not additive with those of the prostanoid, suggesting that PGE2 inhibits TLR at the translational level. The action of PGE2 could be mimicked by the direct stimulator of cAMP formation, forskolin, and involved E prostanoid receptor 2 ligation and cAMP-dependent activation of unanchored type I protein kinase A. Cells pretreated with PGE2 for 24 hours exhibited decreased TNF-α mRNA and protein levels in response to LPS stimulation. Knockdown of TLR4 protein by small interfering RNA to the levels achieved by PGE2 treatment likewise decreased TNF-α mRNA and protein in response to LPS, establishing the functional significance of this PGE2 effect. We provide the first evidence of a lipid mediator acting through its cognate G protein-coupled receptor to affect PRR translation. Because PGE2 is produced in abundance at sites of infection, its inhibitory effects on AM TLR4 expression have important implications for host defense in the lung.

Prostaglandin E2 suppresses allergic sensitization and lung inflammation by targeting the E prostanoid 2 receptor on T cells.

BACKGROUND: Endogenous prostanoids have been suggested to modulate sensitization during experimental allergic asthma, but the specific role of prostaglandin (PG) E2 or of specific E prostanoid (EP) receptors is not known. OBJECTIVE: Here we tested the role of EP2 signaling in allergic asthma. METHODS: Wild-type (WT) and EP2(-/-) mice were subjected to ovalbumin sensitization and acute airway challenge. The PGE2 analog misoprostol was administered during sensitization in both genotypes. In vitro culture of splenocytes and flow-sorted dendritic cells and T cells defined the mechanism by which EP2 exerted its protective effect. Adoptive transfer of WT and EP2(-/-) CD4 T cells was used to validate the importance of EP2 expression on T cells. RESULTS: Compared with WT mice, EP2(-/-) mice had exaggerated airway inflammation in this model. Splenocytes and lung lymph node cells from sensitized EP2(-/-) mice produced more IL-13 than did WT cells, suggesting increased sensitization. In WT but not EP2(-/-) mice, subcutaneous administration of misoprostol during sensitization inhibited allergic inflammation. PGE2 decreased cytokine production and inhibited signal transducer and activator of transcription 6 phosphorylation by CD3/CD28-stimulated CD4(+) T cells. Coculture of flow cytometry-sorted splenic CD4(+) T cells and CD11c(+) dendritic cells from WT or EP2(-/-) mice suggested that the increased IL-13 production in EP2(-/-) mice was due to the lack of EP2 specifically on T cells. Adoptive transfer of CD4(+) EP2(-/-) T cells caused greater cytokine production in the lungs of WT mice than did transfer of WT CD4(+) T cells. CONCLUSION: We conclude that the PGE2-EP2 axis is an important endogenous brake on allergic airway inflammation and primarily targets T cells and that its agonism represents a potential novel therapeutic approach to asthma.

EP4 and EP2 receptor activation of protein kinase A by prostaglandin E2 impairs macrophage phagocytosis of Clostridium sordellii.

PROBLEM: Clostridium sordellii causes endometrial infections, but little is known regarding host defenses against this pathogen. METHOD OF STUDY: We tested the hypothesis that the immunoregulatory lipid prostaglandin (PG) E2 suppresses human macrophage clearance of C. sordellii through receptor-induced increases in intracellular cyclic adenosine monophosphate (cAMP). The THP-1 macrophage cell line was used to quantify C. sordellii phagocytosis. RESULTS: PGE2 increased cAMP levels, activated protein kinase A (PKA), and inhibited the class A scavenger receptor-dependent phagocytosis of C. sordellii. Activation of the EP2 and EP4 receptors increased intracellular cAMP and inhibited phagocytosis, with evidence favoring a more important role for EP4 over EP2. This was supported by EP receptor expression data and the use of pharmacological receptor antagonists. In addition, the PKA isoform RI appeared to be more important than RII in mediating the suppression of ingestion of C. sordellii. CONCLUSION: The endogenous lipid mediator PGE2 impairs human innate immune responses against C. sordellii.

Regulation of alveolar macrophage p40phox: hierarchy of activating kinases and their inhibition by PGE2.

PGE(2), produced in the lung during infection with microbes such as Klebsiella pneumoniae, inhibits alveolar macrophage (AM) antimicrobial functions by preventing H(2)O(2) production by NADPH oxidase (NADPHox). Activation of the NADPHox complex is poorly understood in AMs, although in neutrophils it is known to be mediated by kinases including PI3K/Akt, protein kinase C (PKC) δ, p21-activated protein kinase (PAK), casein kinase 2 (CK2), and MAPKs. The p40phox cytosolic subunit of NADPHox has been recently recognized to function as a carrier protein for other subunits and a positive regulator of oxidase activation, a role previously considered unique to another subunit, p47phox. The regulation of p40phox remains poorly understood, and the effect of PGE(2) on its activation is completely undefined. We addressed these issues in rat AMs activated with IgG-opsonized K. pneumoniae. The kinetics of kinase activation and the consequences of kinase inhibition and silencing revealed a critical role for a PKCδ-PAK-class I PI3K/Akt1 cascade in the regulation of p40phox activation upon bacterial challenge in AMs; PKCα, ERK, and CK2 were not involved. PGE(2) inhibited the activation of p40phox, and its effects were mediated by protein kinase A type II, were independent of interactions with anchoring proteins, and were directed at the distal class I PI3K/Akt1 activation step. Defining the kinases that control AM p40phox activation and that are the targets for inhibition by PGE(2) provides new insights into immunoregulation in the infected lung.

PTEN directly activates the actin depolymerization factor cofilin-1 during PGE2-mediated inhibition of phagocytosis of fungi.

Macrophage ingestion of the yeast Candida albicans requires its recognition by multiple receptors and the activation of diverse signaling programs. Synthesis of the lipid mediator prostaglandin E(2) (PGE(2)) and generation of cyclic adenosine monophosphate (cAMP) also accompany this process. Here, we characterized the mechanisms underlying PGE(2)-mediated inhibition of phagocytosis and filamentous actin (F-actin) polymerization in response to ingestion of C. albicans by alveolar macrophages. PGE(2) suppressed phagocytosis and F-actin formation through the PGE(2) receptors EP2 and EP4, cAMP, and activation of types I and II protein kinase A. Dephosphorylation and activation of the actin depolymerizing factor cofilin-1 were necessary for these inhibitory effects of PGE(2). PGE(2)-dependent activation of cofilin-1 was mediated by the protein phosphatase activity of PTEN (phosphatase and tensin homolog deleted on chromosome 10), with which it directly associated. Because enhanced production of PGE(2) accompanies many immunosuppressed states, the PTEN-dependent pathway described here may contribute to impaired antifungal defenses.

Arsenic increases lipopolysaccharide-dependent expression of interleukin-8 gene by stimulating a redox-sensitive pathway that strengthens p38-kinase activation.

Inorganic arsenic is an immunotoxic metalloid that causes or exacerbates deleterious inflammatory states. Notably, arsenic can increase inflammation-related gene expression induced by lipopolysaccharide (LPS) in monocytes/macrophages. Molecular mechanisms mediating such effects remain however poorly understood. In the present study, we determined molecular basis of arsenic trioxide (ATO) effects on LPS-dependent expression of interleukin-8 (IL-8) gene in human monocytic cells. Pre-treatment with non cytotoxic concentrations of ATO for 48h increase IL-8 gene expression induced by LPS in monocytic U937 cells and in some, but not all, primary cultures of blood monocytes. Actinomycin D blocks induction of IL-8 mRNA levels in LPS-stimulated U937 cells pre-treated or not with ATO, which suggests that the metalloid strengthens LPS-dependent transcriptional regulation of IL-8 expression. ATO increases LPS-dependent expression of IL-8 by enhancing p38-kinase activity induced by LPS in U937 cells. This increment of LPS-dependent p38-kinase activity is caused by the ATO-related production of reactive oxygen species (ROS) and the subsequent activation of the tyrosine kinase Src. The antioxidant N-acetylcysteine almost totally inhibits ROS production and Src kinase activation in ATO-pre-treated cells. In addition, it markedly prevents the increase of both p38-kinase phosphorylation and IL-8 gene expression in LPS-stimulated cells pre-treated with ATO. Finally, as observed with the metalloid, pre-treatment of U937 cells with hydrogen peroxide markedly increases LPS-dependent expression of IL-8 gene. In conclusion, our study demonstrates that ATO increases LPS-dependent expression of the inflammatory IL-8 gene by strengthening the p38 kinase activity induced by LPS through stimulation of a ROS/Src kinase signalling pathway.

Redox-sensitive regulation of gene expression in human primary macrophages exposed to inorganic arsenic.

Inorganic arsenic is an environmental contaminant toxic for key immune cells. We recently reported that low micromolar concentrations of arsenic trioxide (As(2)O(3)) alter functions and differentiation gene program of human macrophages. Particularly, prolonged treatment with As(2)O(3) concomitantly reverses expression of a macrophage-specific gene subset and triggers reactive oxygen species (ROS) production, suggesting a possible role of cell stress in As(2)O(3) gene effects. This study was thus designed to determine whether redox-sensitive signaling pathways could mediate gene expression in metalloid-exposed macrophages. Our results show that As(2)O(3)-dependent alterations of stress (HMOX1 and GCLM) and macrophage-specific (MMP9, CCL22, and CXCL2) gene expression are not mediated by ROS or related signaling pathways. Notably, As(2)O(3) alters neither activity of the redox-sensitive transcription factor Sp1 nor that of AP-1 or NF-kappaB. In contrast, N-acetylcysteine, a potent cysteine reductive compound, significantly prevents up-regulation of HMOX1, GCLM, and CXCL2 genes, and repression of MMP9 and CCL22 genes induced by As(2)O(3). In addition, we demonstrate that As(2)O(3) markedly alters nuclear levels of Nrf2 and Bach1, two redox-sensitive regulators of stress genes, and represses expression of the transcription factor EGR2 which is involved in mouse macrophage differentiation; such effects are reduced by N-acetylcysteine. Finally, we report that genetic invalidation of EGR2 gene partially mimics metalloid effects; it significantly represses CCL22 gene expression and weakly induces that of CXCL2. In conclusion, our results demonstrate that As(2)O(3) alters macrophage gene expression through redox-sensitive signaling pathways unrelated to ROS production and reveal the transcription factor EGR2 as a new molecular target of arsenic.

Global effects of inorganic arsenic on gene expression profile in human macrophages.

Inorganic arsenic, a major environmental contaminant, exerts immunosuppressive effects towards human cells. We previously demonstrated that relevant environmental concentrations of inorganic arsenic altered morphology and functions of human primary macrophages, suggesting interference with macrophage differentiation program. The goal of this study was to determine global effect of low concentrations of arsenic trioxide (As(2)O(3)) on gene expression profile in human primary macrophages, in order to identify molecular targets of inorganic arsenic, especially those relevant of macrophage differentiation process. Using a pan-genomic microarray, we demonstrate that exposure of human blood monocyte-derived macrophages to 1microM As(2)O(3) for 72h, a non-cytototoxic concentration, results in up-regulation of 32 genes and repression of 91 genes. Among these genes, 26 are specifically related to differentiation program of human macrophages. Particularly, we validated that As(2)O(3) strongly alters expression of MMP9, MMP12, CCL22, SPON2 and CXCL2 genes, which contribute to major macrophagic functions. Most of these metalloid effects were reversed when As(2)O(3)-treated macrophages were next cultured in arsenic-free medium. We also show that As(2)O(3) similarly regulates expression of this macrophagic gene subset in human alveolar macrophages, the phenotype of which closely resembles that of blood monocyte-derived macrophage. In conclusion, our study demonstrates that environmentally relevant concentrations of As(2)O(3) impair expression of macrophage-specific genes, which fully supports interference of metalloid with differentiation program of human macrophages.

Inorganic arsenic activates reduced NADPH oxidase in human primary macrophages through a Rho kinase/p38 kinase pathway.

Inorganic arsenic is an immunotoxic environmental contaminant to which millions of humans are chronically exposed. We recently demonstrated that human primary macrophages constituted a critical target for arsenic trioxide (As(2)O(3)), an inorganic trivalent form. To specify the effects of arsenic on macrophage phenotype, we investigated in the present study whether As(2)O(3) could regulate the activity of NADPH oxidase, a major superoxide-generating enzymatic system in human phagocytes. Our results show that superoxide levels were significantly increased in a time-dependent manner in blood monocyte-derived macrophages treated with 1 muM As(2)O(3) for 72 h. Concomitantly, As(2)O(3) induced phosphorylation and membrane translocation of the NADPH oxidase subunit p47(phox) and it also increased translocation of Rac1 and p67(phox). Apocynin, a selective inhibitor of NADPH oxidases, prevented both p47(phox) translocation and superoxide production. NADPH oxidase activation was preceded by phosphorylation of p38-kinase in As(2)O(3)-treated macrophages. The p38-kinase inhibitor SB-203580 prevented phosphorylation and translocation of p47(phox) and subsequent superoxide production. Pretreatment of macrophages with the Rho-kinase inhibitor Y-27632 was found to mimic inhibitory effects of SB-203580 and to prevent As(2)O(3)-induced phosphorylation of p38 kinase. Treatment with As(2)O(3) also resulted in an increased secretion of the proinflammatory chemokine CCL18 that was fully inhibited by both apocynin and SB-203580. Taken together, our results demonstrate that As(2)O(3) induced a marked activation of NADPH oxidase in human macrophages, likely through stimulation of a Rho-kinase/p38-kinase pathway, and which may contribute to some of the deleterious effects of inorganic arsenic on macrophage phenotype.

Human macrophages constitute targets for immunotoxic inorganic arsenic.

Chronic exposure to inorganic arsenic, a widely distributed environmental contaminant, can lead to toxic effects, including immunosuppression. Owing to the established roles of human macrophages in immune defense, we determined, in the present study, whether inorganic arsenic can affect these major immune cells. Our results demonstrate that noncytotoxic concentrations of arsenic trioxide (As2O3), an inorganic trivalent form, markedly impair differentiated features of human blood monocyte-derived macrophages. First, treatment of macrophages with 1 microM As2O3 induced a rapid cell rounding and a subsequent loss of adhesion. These morphologic alterations were associated with a marked reorganization of actin cytoskeleton, which includes retraction of peripheral actin extensions and formation of a cortical actin ring. In addition, As2O3 reduced expression of various macrophagic surface markers, enhanced that of the monocytic marker CD14, and altered both endocytosis and phagocytosis; unexpectedly, exposure of macrophages to the metalloid also strongly potentiated expression of TNFalpha and IL-8 induced by LPS. Finally, like monocytes, As2O3-treated macrophages can be differentiated into dendritic-like cells. Impairment of macrophage function by As2O3 mainly resulted from activation of a RhoA/Rho-associated kinase pathway; indeed, pretreatment of macrophages with the Rho-associated kinase inhibitor Y-27632 prevented metalloid effects on cytoskeleton and phagocytosis. Moreover, As2O3 was found to increase level of the active GTP-bound form of RhoA and that of phosphorylated-Moesin, a major cytoskeleton adaptor protein involved in RhoA regulation. Taken together, our results demonstrated that human macrophages constitute sensitive targets of inorganic arsenic, which may contribute to immunotoxicity of this environmental contaminant.