Correction: Entamoeba lysyl-tRNA Synthetase Contains a Cytokine-Like Domain with Chemokine Activity towards Human Endothelial Cells.

[This corrects the article DOI: 10.1371/journal.pntd.0001398.].

Distinct Responses to IL4 in Macrophages Mediated by JNK.

IL(Interleukin)-4 is the main macrophage M2-type activator and induces an anti-inflammatory phenotype called alternative activation. The IL-4 signaling pathway involves the activation of STAT (Signal Transducer and Activator of Transcription)-6 and members of the MAPK (Mitogen-activated protein kinase) family. In primary-bone-marrow-derived macrophages, we observed a strong activation of JNK (Jun N-terminal kinase)-1 at early time points of IL-4 stimulation. Using selective inhibitors and a knockout model, we explored the contribution of JNK-1 activation to macrophages' response to IL-4. Our findings indicate that JNK-1 regulates the IL-4-mediated expression of genes typically involved in alternative activation, such as Arginase 1 or Mannose receptor, but not others, such as SOCS (suppressor of cytokine signaling) 1 or p21Waf-1 (cyclin dependent kinase inhibitor 1A). Interestingly, we have observed that after macrophages are stimulated with IL-4, JNK-1 has the capacity to phosphorylate STAT-6 on serine but not on tyrosine. Chromatin immunoprecipitation assays revealed that functional JNK-1 is required for the recruitment of co-activators such as CBP (CREB-binding protein)/p300 on the promoter of Arginase 1 but not on p21Waf-1. Taken together, these data demonstrate the critical role of STAT-6 serine phosphorylation by JNK-1 in distinct macrophage responses to IL-4.

IRF1 Is Required for MDA5 (IFIH1) Induction by IFN-α, LPS, and poly(I:C) in Murine Macrophages.

Melanoma differentiation-associated protein 5 (MDA5) induces type I interferons (IFNs) after the recognition of viral RNA. In addition, gain-of-function mutations in the interferon induced with helicase C domain 1 (IFIH1) gene, which encodes MDA5, lead to type I interferonopathies. Here, we show that Mda5 is highly expressed in murine macrophages and is regulated by pro-inflammatory stimuli such as the cytokines IFN-α and IFN-γ, the TLR ligand LPS, and a mimic of dsRNA, poly(I:C). Mda5 induction is mediated through the production of reactive oxygen species. The induction by IFN-α or LPS occurs at the transcriptional level since the Mda5 mRNA half-life before and after induction is very stable. Interestingly, STAT1 is required for Mda5 induction by IFN-α, LPS, or poly(I:C). The time course of induction of at least 3 h and the need for protein synthesis indicate that Mda5 requires an intermediate protein for transcription. In transient transfection experiments, we found that a 105-bp fragment of this gene, between -1153 and -1258 bp relative to the transcription start site, is required for transcription. In this specific region, we observed a sequence containing an IRF-binding motif, which, when mutated, abolishes the induction of Mda5. This sequence is strongly conserved in the IFIH1 promoters of eutherian mammals and in other distant species. Kinetic experiments, chromatin immunoprecipitation assays, and gene-silencing experiments revealed that IRF1 is required for induction of Mda5 expression.

GM-CSF Protects Macrophages from DNA Damage by Inducing Differentiation.

At inflammatory loci, pro-inflammatory activation of macrophages produces large amounts of reactive oxygen species (ROS) that induce DNA breaks and apoptosis. Given that M-CSF and GM-CSF induce two different pathways in macrophages, one for proliferation and the other for survival, in this study we wanted to determine if these growth factors are able to protect against the DNA damage produced during macrophage activation. In macrophages treated with DNA-damaging agents we found that GM-CSF protects better against DNA damage than M-CSF. Treatment with GM-CSF resulted in faster recovery of DNA damage than treatment with M-CSF. The number of apoptotic cells induced after DNA damage was higher in the presence of M-CSF. Protection against DNA damage by GM-CSF is not related to its higher capacity to induce proliferation. GM-CSF induces differentiation markers such as CD11c and MHCII, as well as the pro-survival Bcl-2A1 protein, which make macrophages more resistant to DNA damage.

Induction of CIITA by IFN-γ in macrophages involves STAT1 activation by JAK and JNK.

The induction of major histocompatibility complex (MHC) class II proteins by interferon gamma (IFN-γ) in macrophages play an important role during immune responses. Here we explore the signaling pathways involved in the induction by IFN-γ of the MHC II transactivator (CIIta) required for MHC II transcriptional activation. Cyclophilin A (CypA) is required for IFN-γ-dependent induction of MHC II in macrophages, but not when it is mediated by GM-CSF. The effect of CypA appears to be specific because it does not affect the expression of other molecules or genes triggered by IFN-γ, such as FcγR, NOS2, Lmp2, and Tap1. We found that CypA inhibition blocked the IFN-γ-induced expression of CIIta at the transcriptional level in two phases. In an early phase, during the first 2 h of IFN-γ treatment, STAT1 is phosphorylated at Tyrosine 701 and Serine 727, residues required for the induction of the transcription factor IRF1. In a later phase, STAT1 phosphorylation and JNK activation are required to trigger CIIta expression. CypA is needed for STAT1 phosphorylation in this last phase and to bind the CIIta promoter. Our findings demonstrate that STAT1 is required in a two-step induction of CIIta, once again highlighting the significance of cross talk between signaling pathways in macrophages.

The Translational Relevance of Human Circulating Memory Cutaneous Lymphocyte-Associated Antigen Positive T Cells in Inflammatory Skin Disorders.

Circulating memory T cells are heterogeneous in their tissue tropism. The skin-seeking T cell subset expresses the cutaneous lymphocyte-associated antigen (CLA) on their surface. CLA+ memory T cells not only migrate from blood to skin but also recirculate between blood and skin. Studying CLA+ memory T cells in cutaneous diseases has allowed a better understanding of immune-inflammatory mechanisms that take place. The analysis of the phenotypical features of these cells, their antigen specificity, cytokine production profile, and changes in relationship to clinical status and therapies among other characteristics have led to the concept that they constitute peripheral cellular biomarkers in T cell-mediated cutaneous conditions. CLA+ memory T cells are of relevance in the pathogenesis of several cutaneous diseases, such as psoriasis (PSO), atopic dermatitis, vitiligo, and drug-induced allergic reactions, to name a few. The interaction of circulating CLA+ T cells with skin-resident cells has been investigated in different ex vivo coculture models made out of clinical samples. Interestingly, microbes that are present in the skin or related with human skin diseases are preferentially recognized by CLA+ T cells. Thus, the interaction of Streptococcus pyogenes with CLA+ T cells in PSO is providing novel concepts that help to understand disease immunopathogenesis. The goal of this review is to present latest results in the field of CLA+ T cells in T cell-mediated inflammatory skin diseases and their translational relevance for human immunodermatology.

Interplay between Humoral and CLA+ T Cell Response against Candida albicans in Psoriasis.

Candida albicans (CA) infections have been associated with psoriasis onset or disease flares. However, the integrated immune response against this fungus is still poorly characterized in psoriasis. We studied specific immunoglobulins in plasma and the CA response in cocultures of circulating memory CD45RA- cutaneous lymphocyte antigen (CLA)+/- T cell with autologous epidermal cells from plaque and guttate psoriasis patients (cohort 1, n = 52), and also healthy individuals (n = 17). A complete proteomic profile was also evaluated in plaque psoriasis patients (cohort 2, n = 114) regarding their anti-CA IgA levels. Increased anti-CA IgA and IgG levels are present in the plasma from plaque but not guttate psoriasis compared to healthy controls. CA cellular response is confined to CLA+ T cells and is primarily Th17. The levels of anti-CA IgA are directly associated with CLA+ Th17 response in plaque psoriasis. Proteomic analysis revealed distinct profiles in psoriasis patients with high anti-CA IgA. C-C motif chemokine ligand 18, chitinase-3-like protein 1 and azurocidin were significantly elevated in the plasma from plaque psoriasis patients with high anti-CA levels and severe disease. Our results indicate a mechanism by which Candida albicans exposure can trigger a clinically relevant IL-17 response in psoriasis. Assessing anti-CA IgA levels may be useful in order to evaluate chronic psoriasis patients.

Macrophage mitochondrial MFN2 (mitofusin 2) links immune stress and immune response through reactive oxygen species (ROS) production.

MFN2 (mitofusin 2) is required for mitochondrial fusion and for mitochondria-endoplasmic reticulum interaction. Using myeloid-conditional KO mice models, we found that MFN2 but not MFN1 is a prerequisite for the adaptation of mitochondrial respiration to stress conditions as well as for the production of reactive oxygen species (ROS). The deficient ROS production in the absence of MFN2 impairs the induction of cytokines and nitric oxide, and is associated with dysfunctional autophagy, apoptosis, phagocytosis, and antigen processing. The lack of MFN2 in macrophages causes an impaired response in a model of non-septic inflammation in mice, as well as a failure in protection from Listeria, Mycobacterium tuberculosis or LPS endotoxemia. These results reveal an unexpected role of MFN2 to ROS production in macrophages affecting natural and acquired immunity and the immune response.

Mitofusin 2 in Macrophages Links Mitochondrial ROS Production, Cytokine Release, Phagocytosis, Autophagy, and Bactericidal Activity.

Mitofusin 2 (Mfn2) plays a major role in mitochondrial fusion and in the maintenance of mitochondria-endoplasmic reticulum contact sites. Given that macrophages play a major role in inflammation, we studied the contribution of Mfn2 to the activity of these cells. Pro-inflammatory stimuli such as lipopolysaccharide (LPS) induced Mfn2 expression. The use of the Mfn2 and Mfn1 myeloid-conditional knockout (KO) mouse models reveals that Mfn2 but not Mfn1 is required for the adaptation of mitochondrial respiration to stress conditions and for the production of reactive oxygen species (ROS) upon pro-inflammatory activation. Mfn2 deficiency specifically impairs the production of pro-inflammatory cytokines and nitric oxide. In addition, the lack of Mfn2 but not Mfn1 is associated with dysfunctional autophagy, apoptosis, phagocytosis, and antigen processing. Mfn2floxed;CreLysM mice fail to be protected from Listeria, Mycobacterium tuberculosis, or LPS endotoxemia. These results reveal an unexpected contribution of Mfn2 to ROS production and inflammation in macrophages.

IL-15 and IL-23 synergize to trigger Th17 response by CLA+ T cells in psoriasis.

IL-15 has emerged as a potentially relevant target in the IL-17 response in psoriasis. However, its mechanism is poorly characterized in humans. IL-15 and IL-23 are constitutively expressed in the psoriatic lesion. Also, IL-15 is considered a susceptibility-associated gene in psoriasis, as are IL-23R, and HLACW6. Here, we studied the effect of IL-15 and IL-23 stimulation on the cytokine response of CLA+/CLA- T cells from 9 psoriasis patients and 3 healthy control subjects. To this end, CLA + and CLA- T cells from blood samples were cultured with epidermal cells from skin biopsies and treated with IL-15 and IL-23. After five days of culture, cytokines in supernatant were measured by ELISA or fluorescent bead-based immunoassay. There was a statistically significant increase in IL-17F and IL-17A production (P < .001) in cocultures of psoriasis skin-homing CLA + T cells with epidermal cells when stimulated with IL-15 and IL-23, but this effect was not observed in the cells of healthy controls. Interestingly, this response was reduced by around 50 to 80% by blocking HLA class I and II molecules. Our results point to the synergic action of IL-15 and IL-23 selectively for CLA + cells in psoriasis, leading to the induction of Th17 cell-related cytokines.

Induction of Samhd1 by interferon gamma and lipopolysaccharide in murine macrophages requires IRF1.

SAMHD1 is an enzyme with phosphohydrolase activity. Mutations in SAMHD1 have been linked to the development of Aicardi-Goutières syndrome in humans. This enzyme also has the capacity to restrict HIV virus replication in macrophages. Here, we report that Samhd1 is highly expressed in murine macrophages and is regulated by proinflammatory (IFN-γ and LPS) but not by anti-inflammatory (IL-4 or IL-10) activators. The induction of Samhd1 follows the pattern of an intermediate gene that requires protein synthesis. In transient transfection experiments using the Samhd1 promoter, we found that a fragment of 27 bps of this gene, falling between -937 and -910 bps relative to the transcription start site, is required for IFN-γ-dependent activation. Using EMSAs, we determined that IFN-γ treatment led to the elimination of a protein complex. Chromatin immunoprecipitation assays and siRNA experiments revealed that IRF1 is required for IFN-γ- or LPS-induced Samhd1 expression. Therefore, our results indicate that Samhd1 is stimulated by proinflammatory agents IFN-γ and LPS. Moreover, they reveal that these two agents, via IRF1, eliminate a protein complex that may be related to a repressor, thereby, triggering Samhd1 expression.

Specific IgA and CLA+ T-Cell IL-17 Response to Streptococcus pyogenes in Psoriasis.

Streptococcus pyogenes tonsillar infection is well known to trigger and exacerbate psoriasis lesions in both guttate and plaque forms of the disease. Although mucosal and cutaneous tissues are closely involved in psoriasis pathology, the interaction between their specific immune responses has not been deeply explored. This work aims to address and characterize the presence of humoral responses against S. pyogenes in patients with psoriasis and its putative association with cytokine responses detected in vitro in our psoriasis ex vivo model, based on the coculture of cutaneous lymphocyte-associated antigen+/- T cells with autologous epidermal cells. Patients with psoriasis presented increased IgA response to S. pyogenes when compared with control subjects. In patients with plaque psoriasis, despite being negative for anti-streptolysin O antibody titer, IgA plasma levels against S. pyogenes correlated with cutaneous lymphocyte-associated antigen+ T-cell-dependent IL-17F response in vitro. No association is observed for IgG levels in plaque psoriasis. Similar association is observed for IgA anti-S. pyogenes extract and IL-17A in patients with guttate psoriasis. We propose S. pyogenes-specific IgA as a potential new perspective for better understanding the role of S. pyogenes in psoriasis development.

CLA+ T Cell Response to Microbes in Psoriasis.

Streptococcus pyogenes throat infection is a clinically relevant trigger of both guttate and chronic plaque psoriasis, and it provides an ideal context in which to study the pathogenesis of these diseases using an antigen-dependent approach. Circulating cutaneous lymphocyte-associated antigen (CLA) positive (+) memory T cells are a subset of peripheral lymphocytes whose phenotype and function are related to immunological mechanisms in the skin. These cells are considered peripheral biomarkers of T-cell-mediated skin diseases. The coculture of autologous epidermal cells with CLA+ T cells from psoriasis patients activated by S. pyogenes allows the reproduction of the ex vivo initial molecular events that occur during psoriatic lesion formation. With cooperation of autologous epidermal cells, S. pyogenes selectively activates CLA+ T cells both in guttate and plaque psoriasis, inducing key mediators, including an IL-17 response. Here, we explore potential new mechanisms of psoriasis development including the influence of HLA-Cw6 on S. pyogenes CLA+ T cell activation in guttate psoriasis, the relevance of IL-9 on microbe induced IL-17 response in guttate and plaque psoriasis, and novel effector functions of Candida albicans. This review will summarize recent knowledge of psoriatic mechanisms elicited by microbes that have been studied through an innovative translational perspective based on CLA+ T cell-mediated cutaneous immune response.

Microbe-Dependent Induction of IL-9 by CLA+ T Cells in Psoriasis and Relationship with IL-17A.

IL-9 is present in psoriatic lesions and is produced by lymphocytes. However, it is not known whether this cytokine is induced by relevant pathogenic triggers of psoriasis, such as Streptococcus pyogenes. Here we addressed the production of IL-9 in response to various pathogens in a psoriatic ex vivo model. Extracts of S. pyogenes and Candida albicans triggered the production of IL-9 and also IL-17A and IFN-γ. This induction was dependent on the interaction between CLA+ T cells and epidermal cells. Neutralization of IL-9 reduced S. pyogenes-induced IL-17A production by CLA+ T cells but had no effect on IFN-γ production. Also, IL-9 increased the survival of circulating psoriatic CLA+ T cells. Co-cultures from patients with guttate or plaque psoriasis with S. pyogenes produced similar amounts of IL-9. High cytokine responses in streptococcal-driven guttate patients paralleled peaks in Psoriasis Area Severity Index and anti-streptolysin O levels. Our results confirm that IL-9 promotes inflammation in psoriasis by up-regulating IL-17A production and support the clinical association of the immune response by streptococcal-sensitized CLA+ T cells with this cytokine, especially in guttate psoriasis.

Mitogen-Activated Protein Kinases and Mitogen Kinase Phosphatase 1: A Critical Interplay in Macrophage Biology.

Macrophages are necessary in multiple processes during the immune response or inflammation. This review emphasizes the critical role of the mitogen-activated protein kinases (MAPKs) and mitogen kinase phosphatase-1 (MKP-1) in the functional activities of macrophages. While the phosphorylation of MAPKs is required for macrophage activation or proliferation, MKP-1 dephosphorylates these kinases, thus playing a balancing role in the control of macrophage behavior. MKP-1 is a nuclear-localized dual-specificity phosphatase whose expression is regulated at multiple levels, including at the transcriptional and post-transcriptional level. The regulatory role of MKP-1 in the interplay between MAPK phosphorylation/dephosphorylation makes this molecule a critical regulator of macrophage biology and inflammation.

MCPIP1 RNase Is Aberrantly Distributed in Psoriatic Epidermis and Rapidly Induced by IL-17A.

ZC3H12A, which encodes the RNase monocyte chemotactic protein-induced protein 1 (MCPIP1), is up-regulated in psoriatic skin and reduced to normal levels after clinical treatments with anti-IL-17A/IL-17R neutralizing antibodies. In IL-17A-stimulated keratinocytes, MCPIP1 is rapidly increased at the transcript and protein levels. Also, IL-17A was found to be the main inducer of ZC3H12A expression in keratinocytes treated with supernatants derived from a Streptococcus pyogenes-activated psoriatic ex vivo model based on the co-culture of psoriatic cutaneous lymphocyte-associated antigen (CLA(+)) T cells and lesional epidermal cells. Moreover, MCPIP1 was aberrantly distributed in the suprabasal layers of psoriatic epidermis. In psoriatic samples, IL-17A-stimulated epidermal cell suspensions showed an increased MCPIP1 expression, especially in the mid-differentiated cellular compartment. The knockdown of ZC3H12A showed that this RNase participates in the regulation of the mRNAs present in suprabasal differentiated keratinocytes. Furthermore, JAK/STAT3 inhibition prevented the IL-17A-dependent induction of MCPIP1. In the mouse model of imiquimod-induced psoriasis, Zc3h12a expression was abrogated in Il17ra(-/-) mice. These results support the notion that IL-17A-mediated induction of MCPIP1 is involved in the regulation of local altered gene expression in suprabasal epidermal layers in psoriasis.

Streptococcus pyogenes-induced cutaneous lymphocyte antigen-positive T cell-dependent epidermal cell activation triggers TH17 responses in patients with guttate psoriasis.

BACKGROUND: Guttate psoriasis (GP) is characterized by acute onset of small, rounded psoriatic lesions. Although this particular phenotype of psoriasis is usually associated with streptococcal throat infections and mainly occurs in HLA-Cw6(+) patients, the specific immunologic response to this innate stimulus that causes these skin lesions is poorly understood. OBJECTIVE: This study aims to elucidate how key cellular elements of patients with GP respond to Streptococcus pyogenes and whether this initial immune response is favored by the genetic and environmental background of these patients. METHODS: Circulating memory T cells and autologous epidermal cells from samples from either patients with GP (n = 14) or healthy control subjects (n = 6) were cocultured ex vivo in the presence of an S pyogenes extract. Levels of the psoriasis-associated cytokines IL-17A, IL-17F, IFN-γ, TNF-α, IL-6, and IL-8 were determined. The expression of several genes with increased (DEFB4, S100A7, LCN2, IL36G, IL8, CXCL9, CXCL10, and CXCL11) or decreased (FLG and LOR) transcripts in psoriatic lesions was examined in keratinocytes treated with coculture supernatants. RESULTS: When skin-homing effector memory cutaneous lymphocyte antigen-positive T cells were used in cocultures, a TH17-dominant response was observed, as reflected by the higher amounts of IL-17A and IL-17F than IFN-γ. Moreover, a higher TH17 response was observed in cells isolated from patients with flares associated with a streptococcal tonsillitis and with the HLA-Cw6 allele (cohort 1). In addition, in normal keratinocytes the supernatants from these cocultures induced an increase in IL-17-associated genes, such as DEFB4, S100A7, LCN2, IL36G, and IL8 but a decrease in FLG and LOR, thereby confirming the role of activated TH17 cells. CONCLUSION: This study reveals a dominant TH17 response of cutaneous lymphocyte antigen-positive T cells activated by epidermal cells and S pyogenes in patients with GP.

NBS1 is required for macrophage homeostasis and functional activity in mice.

Nijmegen breakage syndrome 1 (NBS1) is a component of the MRE11 complex, which is a sensor of DNA double-strand breaks and plays a crucial role in the DNA damage response. Because activated macrophages produce large amounts of reactive oxygen species (ROS) that can cause DNA lesions, we examined the role of NBS1 in macrophage functional activity. Proliferative and proinflammatory (interferon gamma [IFN-γ] and lipopolysaccharide [LPS]) stimuli led to increased NBS1 levels in macrophages. In mice expressing a hypomorphic allele of Nbs1, Nbs1(∆B/∆B), macrophage activation-induced ROS caused increased levels of DNA damage that were associated with defects in proliferation, delayed differentiation, and increased senescence. Furthermore, upon stimulation, Nbs1(∆B/∆B) macrophages exhibited increased expression of proinflammatory cytokines. In the in vivo 2,4-dinitrofluorobenzene model of inflammation, Nbs1(∆B/∆B) animals showed increased weight and ear thickness. By using the sterile inflammation by zymosan injection, we found that macrophage proliferation was drastically decreased in the peritoneal cavity of Nbs1(∆B/∆B) mice. Our findings show that NBS1 is crucial for macrophage function during normal aging. These results have implications for understanding the immune defects observed in patients with NBS and related disorders.

The response of secondary genes to lipopolysaccharides in macrophages depends on histone deacetylase and phosphorylation of C/EBPß.

LPS induces the expression of NO synthase 2 (nos2) in macrophages. The expression of this molecule is one of the hallmarks of classical activation. In this paper, we describe that trichostatin A (TSA), which inhibits deacetylase activity, blocks LPS-dependent nos2 expression. TSA specifically inhibits LPS-dependent genes of secondary response, which require new protein synthesis for their induction but not those belonging to the primary response, which do not depend on this process. Deacetylase activity acts at the transcriptional level because RNA polymerase II was not bound after LPS stimulus when we added TSA. A link between the global acetylation caused by HDAC inhibitor and gene promoter recruitment of CDK8 was found. This Mediator complex subunit associates with Med 12, Med13, and cyclin C to form a submodule that is a transcriptional negative regulator. We also found that TSA reduces C/EBPß phosphorylation without affecting its binding to DNA. Taken together, these results shed light on the molecular mechanisms involved in the transcriptional regulation of LPS-treated macrophages and on how TSA targets critical LPS-induced genes, such as nos2 and tnf-α, in inflammatory macrophage response.

The exonuclease Trex1 restrains macrophage proinflammatory activation.

The three-prime repair exonuclease 1 (TREX1) is the most abundant exonuclease in mammalian cells. Mutations in Trex1 gene are being linked to the development of Aicardi-Goutières syndrome, an inflammatory disease of the brain, and systemic lupus erythematosus. In clinical cases and in a Trex1-deficient murine model, chronic production of type I IFN plays a pathogenic role. In this study, we demonstrate that Trex1(-/-) mice present inflammatory signatures in many different organs, including the brain. Trex1 is highly induced in macrophages in response to proinflammatory stimuli, including TLR7 and TLR9 ligands. Our findings show that, in the absence of Trex1, macrophages displayed an exacerbated proinflammatory response. More specifically, following proinflammatory stimulation, Trex1(-/-) macrophages exhibited an increased TNF-α and IFN-α production, higher levels of CD86, and increased Ag presentation to CD4(+) T cells, as well as an impaired apoptotic T cell clearance. These results evidence an unrevealed function of the Trex1 as a negative regulator of macrophage inflammatory activation and demonstrate that macrophages play a major role in diseases associated with Trex1 mutations, which contributes to the understanding of inflammatory signature in these diseases.