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.

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.

The kinase p38 alpha serves cell type-specific inflammatory functions in skin injury and coordinates pro- and anti-inflammatory gene expression.

The mitogen-activated protein kinase p38 mediates cellular responses to injurious stress and immune signaling. Among the many p38 isoforms, p38 alpha is the most widely expressed in adult tissues and can be targeted by various pharmacological inhibitors. Here we investigated how p38 alpha activation is linked to cell type-specific outputs in mouse models of cutaneous inflammation. We found that both myeloid and epithelial p38 elicit inflammatory responses, yet p38 alpha signaling in each cell type served distinct inflammatory functions and varied depending on the mode of skin irritation. In addition, myeloid p38 alpha limited acute inflammation via activation of anti-inflammatory gene expression dependent on mitogen- and stress-activated kinases. Our results suggest a dual function for p38 alpha in the regulation of inflammation and show mixed potential for its inhibition as a therapeutic strategy.

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.

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.

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.

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.

Reciprocal negative cross-talk between liver X receptors (LXRs) and STAT1: effects on IFN-γ-induced inflammatory responses and LXR-dependent gene expression.

Liver X receptors (LXRs) exert key functions in lipid homeostasis and in control of inflammation. In this study we have explored the impact of LXR activation on the macrophage response to the endogenous inflammatory cytokine IFN-γ. Transcriptional profiling studies demonstrate that ∼38% of the IFN-γ-induced transcriptional response is repressed by LXR activation in macrophages. LXRs also mediated inhibitory effects on selected IFN-γ-induced genes in primary microglia and in a model of IFN-γ-induced neuroinflammation in vivo. LXR activation resulted in reduced STAT1 recruitment to the promoters tested in this study without affecting STAT1 phosphorylation. A closer look into the mechanism revealed that SUMOylation of LXRs, but not the presence of nuclear receptor corepressor 1, was required for repression of the NO synthase 2 promoter. We have also analyzed whether IFN-γ signaling exerts reciprocal effects on LXR targets. Treatment with IFN-γ inhibited, in a STAT1-dependent manner, the LXR-dependent upregulation of selective targets, including ATP-binding cassette A1 (ABCA1) and sterol response element binding protein 1c. Downregulation of ABCA1 expression correlated with decreased cholesterol efflux to apolipoprotein A1 in macrophages stimulated with IFN-γ. The inhibitory effects of IFN-γ on LXR signaling did not involve reduced binding of LXR/retinoid X receptor heterodimers to target gene promoters. However, overexpression of the coactivator CREB-binding protein/p300 reduced the inhibitory actions of IFN-γ on the Abca1 promoter, suggesting that competition for CREB-binding protein may contribute to STAT1-dependent downregulation of LXR targets. The results from this study suggest an important level of bidirectional negative cross-talk between IFN-γ/STAT1 and LXRs with implications both in the control of IFN-γ-mediated immune responses and in the regulation of lipid metabolism.

Arginine transport is impaired in C57Bl/6 mouse macrophages as a result of a deletion in the promoter of Slc7a2 (CAT2), and susceptibility to Leishmania infection is reduced.

Host genetic factors play a crucial role in immune response. To determine whether the differences between C57Bl/6 and BALB-C mice are due only to the production of cytokines by T-helper 1 cells or T-helper 2 cells, we obtained bone marrow-derived macrophages from both strains and incubated them with these cytokines. Although the induction of Nos2 and Arg1 was similar in the 2 strains, infectivity to Leishmania major differed, as did macrophage uptake of arginine, which was higher in BALB-C macrophages. The levels of interferon γ- and interleukin 4-dependent induction of the cationic amino acid transporter SLC7A2 (also known as "cationic amino acid transporter 2," or "CAT2") were decreased in macrophages from C57Bl/6 mice. This reduction was a result of a deletion in the promoter of one of the 4 AGGG repeats. These results demonstrate that the availability of arginine controls critical aspects of macrophage activation and reveal a factor for susceptibility to Leishmania infection.

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.].

MKP-1: a critical phosphatase in the biology of macrophages controlling the switch between proliferation and activation.

Macrophages play a central role in the immune response. These cells either proliferate in response to, for example, growth factors or become activated in response to, for example, LPS and develop functional activities. Experiments carried out in mice showed that macrophage proliferation requires a short period of ERK phosphorylation, while an extended period is required for macrophage activation. The length of phosphorylation is controlled by the MAPK phosphatase-1 (MKP-1), a nuclear-localized dual-specificity phosphatase that dephosphorylates the MAPKs ERK, p38, and c-Jun NH(2) -terminal kinase (JNK). MKP-1 is induced in macrophages by growth factors, as well as by activators such as LPS, but with different kinetics; to achieve the different functional outcomes (proliferation versus activation), the inhibition of MKP-1 by cytokines such as IFN-γ blocks macrophage proliferation and induces activation. The data presented in this review show that this phosphatase is the switch between macrophage proliferation and activation.

Deacetylation of C/EBPß is required for IL-4-induced arginase-1 expression in murine macrophages.

The amount of arginine available at inflammatory loci is a limiting factor for the growth of several cells of the immune system. IL-4-induced activation of macrophages produced arginase-1, which converts arginine into ornithine, a precursor of polyamines and proline. Trichostatin A (TSA), a pan-inhibitor of histone deacetylases (HDACs), inhibited IL-4-induced arginase-1 expression. TSA showed promoter-specific effects on the IL-4-responsive genes. While TSA inhibited the expression of arginase-1, fizz1, and mrc1, other genes, such as ym,1 mgl1, and mgl2, were not affected. The inhibition of arginase-1 occurred at the transcriptional level with the inhibition of polymerase II binding to the promoter. IL-4 induced STAT6 phosphorylation and binding to DNA. These activities were not affected by TSA treatment. However, TSA inhibited C/EBPß DNA binding. This inhibitor induced acetylation on lysine residues 215-216, which are critical for DNA binding. Finally, using macrophages from STAT6 KO mice we showed that STAT6 is required for the DNA binding of C/EBPß. These results demonstrate that the acetylation/deacetylation balance strongly influences the expression of arginase-1, a gene of alternative activation of macrophages. These findings also provide a molecular mechanism to explain the control of gene expression through deacetylase activity.

Circulating CLA+ T lymphocytes as peripheral cell biomarkers in T-cell-mediated skin diseases.

T lymphocytes expressing the CLA antigen constitute a subset of effector memory lymphocytes that are functionally involved in T-cell-mediated cutaneous diseases. Skin-seeking lymphocytes recirculate between inflamed skin and blood during cutaneous inflammation. Many studies in different T-cell-mediated inflammatory cutaneous diseases have clearly related their pathologic mechanisms to CLA+ T cells. Based on common features of these cells in different cutaneous disorders mediated by T cells, we propose that circulating CLA+T cells could constitute very useful peripheral cellular biomarkers for T-cell-mediated skin diseases.

Characterization of Trex1 induction by IFN-γ in murine macrophages.

3' Repair exonuclease (Trex1) is the most abundant mammalian 3' → 5' DNA exonuclease with specificity for ssDNA. Trex1 deficiency has been linked to the development of autoimmune disease in mice and humans, causing Aicardi-Goutières syndrome in the latter. In addition, polymorphisms in Trex1 are associated with systemic lupus erythematosus. On the basis of all these observations, it has been hypothesized that Trex1 acts by digesting an endogenous DNA substrate. In this study, we report that Trex1 is regulated by IFN-γ during the activation of primary macrophages. IFN-γ upregulates Trex1 with the time course of an early gene, and this induction occurs at the transcription level. The half-life of mRNA is relatively short (half-life of 70 min). The coding sequence of Trex1 has only one exon and an intron of 260 bp in the promoter in the nontranslated mRNA. Three transcription start sites were detected, the one at -580 bp being the most important. In transient transfection experiments using the Trex1 promoter, we have found that two IFN-γ activation site boxes, as well as an adaptor protein complex 1 box, were required for the IFN-γ-dependent induction. By using EMSA assays and chromatin immune precipitation assays, we determined that STAT1 binds to the IFN-γ activation site boxes. The requirement of STAT1 for Trex1 induction was confirmed using macrophages from Stat1 knockout mice. We also establish that c-Jun protein, but not c-Fos, jun-B, or CREB, bound to the adaptor protein complex 1 box. Therefore, our results indicate that IFN-γ induces the expression of the Trex1 exonuclease through STAT1 and c-Jun.

Liver X receptors inhibit macrophage proliferation through downregulation of cyclins D1 and B1 and cyclin-dependent kinases 2 and 4.

Macrophages serve essential functions as regulators of immunity and homeostasis, and their proliferation contributes to pathogenesis of certain disorders. In this report, we show that induction of macrophage proliferation by the growth factor M-CSF is negatively modulated by agonists that activate the nuclear receptor liver X receptor (LXR), both in vitro and in vivo. Both isoforms LXR α and ß are involved in the antiproliferative actions of LXR ligands in macrophages. In contrast, M-CSF does not exert negative effects on LXR-mediated gene expression. Treatment with LXR agonists results in the accumulation of macrophages in the G(0)/G(1) phase of the cell cycle without affecting ERK-1/2 phosphorylation. The use of small interfering RNA or genetically modified mice revealed that, in contrast to other cellular models, functional expression of either the cyclin-dependent kinase inhibitor p27KIP1 or the cholesterol transporters ATP-binding cassette A1 or ATP-binding cassette G1 was not required for the antiproliferative effects of LXR agonists in macrophages. Western blot analysis revealed that protein expression of key molecules that regulate progression through the cell cycle, such as cyclins D1 and B1 and cyclin-dependent kinases 2 and 4, was downregulated upon LXR activation. These observations suggest a role for LXR agonists in limiting macrophage proliferative responses associated to pathogenic disorders.

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.

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.

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.

Telomere shortening and oxidative stress in aged macrophages results in impaired STAT5a phosphorylation.

Macrophages are an essential component of both innate and adaptive immunity, and altered function of these cells with aging may play a key role in immunosenescence. To determine the effect of aging on macrophages, we produced bone marrow-derived macrophages in vitro. In these conditions, we analyzed the effect of aging on macrophages without the influence of other cell types that may be affected by aging. We showed that telomeres shorten with age in macrophages leading to a decreased GM-CSF but not M-CSF-dependent proliferation of these cells as a result of decreased phosphorylation of STAT5a. Macrophages from aged mice showed increased susceptibility to oxidants and an accumulation of intracellular reactive oxygen species. In these macrophages STAT5a oxidation was reduced, which led to the decreased phosphorylation observed. Interestingly, the same cellular defects were found in macrophages from telomerase knockout (Terc(-/-)) mice suggesting that telomere loss is the cause for the enhanced oxidative stress, the reduced Stat5a oxidation and phosphorylation and, ultimately, for the impaired GM-CSF-dependent macrophage proliferation.