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Understanding the signaling cascades that govern adipocyte metabolism and differentiation is necessary for the development of therapies for obesity. Toll-like receptors (TLRs) are key mediators in adipogenesis, but their specific role is not completely understood. In this study, siRNA knockdown of Tlr2 in 3T3-L1 cells allowed them to differentiate more efficiently into adipocytes, whereas the opposite was observed for the knockdown of Tlr4 . At the same time, we show that TLR2 knock-out mice spontaneously developed mature-onset obesity and insulin resistance. Besides a higher incidence of hyperplasia and hypertrophy in white adipose tissue (WAT), we found a significantly increased number of adipocyte precursor cells in TLR2 -/- mice compared to TLR4 -/- mice. Interestingly, genetic inactivation of Tlr4 in TLR2 -/- mice reverted their increased adiposity, insulin resistance, and restored normal levels of adipocyte precursor cells. These findings provide evidence that TLR2 and TLR4 play opposing roles in WAT homeostasis and point to the existence of cross-regulation among TLR2 and TLR4 during adipocyte differentiation both in vitro and in vivo.

The innate immunity toll-like receptor 4 (TLR4) system is a receptor of paramount importance as a therapeutic target. Virtual screening following a "computer-aided drug repurposing" approach was applied to the discovery of novel TLR4 modulators with a non-lipopolysaccharide-like structure. We screened almost 29,000 approved drugs and drug-like molecules from commercial, public, and in-house academia chemical libraries and, after biological assays, identified several compounds with TLR4 antagonist activity. Our computational protocol showed to be a robust approach for the identification of hits with drug-like scaffolds as possible inhibitors of the TLR4 innate immune pathways. Our collaborative work broadens the chemical diversity for inspiration of new classes of TLR4 modulators.

The Toll-like receptor 4 (TLR4)/myeloid differentiation protein-2 (MD-2) complex is considered the major receptor of the innate immune system to recognize lipopolysaccharides (LPSs). However, some atypical LPSs with different lipid A and core saccharide moiety structures and compositions than the well-studied enterobacterial LPSs can induce a TLR2-dependent response in innate immune cells. Ochrobactrum intermedium , an opportunistic pathogen, presents an atypical LPS. In this study, we found that O. intermedium LPS exhibits a weak inflammatory activity compared to Escherichia coli LPS and, more importantly, is a specific TLR4/TLR2 agonist, able to signal through both receptors. Molecular docking analysis of O. intermedium LPS predicts a favorable formation of a TLR2/TLR4/MD-2 heterodimer complex, which was experimentally confirmed by fluorescence resonance energy transfer (FRET) in cells. Interestingly, the core saccharide plays an important role in this interaction. This study reveals for the first time TLR4/TLR2 heterodimerization that is induced by atypical LPS and may help to escape from recognition by the innate immune system., Competing Interests: Authors CP, SF and JM were employed by company Diomune SL. Author MF was an advisor of company Diomune SL. The remaining 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 © 2022 Francisco, Billod, Merino, Punzón, Gallego, Arranz, Martin-Santamaria and Fresno.)

Toll-like receptors (TLRs) play a crucial role in the recognition of pathogen-derived components as a first line of defense against infections. It has been suggested that depending on the nature of the pathogens, TLRs activation induce a distinct cytokine profile that may contribute to the polarization of the acquired immune response. Here, we investigated the early MAPK signaling activation via TLR4 and TLR2 receptors and its impact in differential cytokine profile by macrophages. We found that TLR2 ligands activated MAPKs p38 and ERK earlier compared to the TLR4 ligand LPS in macrophages. Higher IL-10/IL-12 and IL-10/TNF-α ratios were also observed at later time points in response to TLR2 ligands compared to LPS. The results also indicate an earlier activation of the phosphatase MKP-1 and that MKP-1 KO macrophages show a prolongation in p38 phosphorylation in response to TLR2 stimulation. Furthermore, p38 is critical for IL-10 expression in response to TLR2 ligands, which triggers the macrophage change to a M2 and regulatory phenotype in contrast to the M1 phenotype induced by TLR4 activation. Therefore, the early TLR2-mediated p38 induction contributes for the high IL-10 production, likely as a virulence strategy to suppress host Th1 response against certain types of pathogens., 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 © 2021 Francisco, Arranz, Merino, Punzón, Perona and Fresno.)

Background: Psoriasis is a frequent inflammatory skin disease that is mainly mediated by IL-23, IL-1β, and IL-17 cytokines. Although psoriasis is a hyperproliferative skin disorder, the possible role of amino acid transporters has remained unexplored., Objective: We sought to investigate the role of the essential amino acid transporter L-type amino acid transporter (LAT) 1 (SLC7A5) in psoriasis., Methods: LAT1 floxed mice were crossed to Cre-expressing mouse strains under the control of keratin 5, CD4, and retinoic acid receptor-related orphan receptor γ. We produced models of skin inflammation induced by imiquimod (IMQ) and IL-23 and tested the effect of inhibiting LAT1 (JPH203) and mammalian target of rapamycin (mTOR [rapamycin])., Results: LAT1 expression is increased in keratinocytes and skin-infiltrating lymphocytes of psoriatic lesions in human subjects and mice. LAT1 deletion in keratinocytes does not dampen the inflammatory response or their proliferation, which could be maintained by increased expression of the alternative amino acid transporters LAT2 and LAT3. Specific deletion of LAT1 in γδ and CD4 T cells controls the inflammatory response induced by IMQ. LAT1 deletion or inhibition blocks expansion of IL-17-secreting γ4 + δ4 + and CD4 T cells and dampens the release of IL-1β, IL-17, and IL-22 in the IMQ-induced model. Moreover, inhibition of LAT1 blocks expansion of human γδ T cells and IL-17 secretion by human CD4 T cells. IL-23 and IL-1β stimulation upregulates LAT1 expression and induces mTOR activation in IL-17 + γδ and T H 17 cells. Deletion or inhibition of LAT1 efficiently controls IL-23- and IL-1β-induced phosphatidylinositol 3-kinase/AKT/mTOR activation independent of T-cell receptor signaling., Conclusion: Targeting LAT1-mediated amino acid uptake is a potentially useful immunosuppressive strategy to control skin inflammation mediated by the IL-23/IL-1β/IL-17 axis., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Visceral leishmaniasis is a neglected parasitic disease with no vaccine available and its pharmacological treatment is reduced to a limited number of unsafe drugs. The scarce readiness of new antileishmanial drugs is even more alarming when relapses appear or the occurrence of hard-to-treat resistant strains is detected. In addition, there is a gap between the initial and late stages of drug development, which greatly delays the selection of leads for subsequent studies., Methodology/principal Findings: In order to address these issues, we have generated a red-shifted luminescent Leishmania infantum strain that enables long-term monitoring of parasite burden in individual animals with an in vivo limit of detection of 106 intracellular amastigotes 48 h postinfection. For this purpose, we have injected intravenously different infective doses (104-5x108) of metacyclic parasites in susceptible mouse models and the disease was monitored from initial times to 21 weeks postinfection. The emission of light from the target organs demonstrated the sequential parasite colonization of liver, spleen and bone marrow. When miltefosine was used as proof-of-concept, spleen weight parasite burden and bioluminescence values decreased significantly., Conclusions: In vivo bioimaging using a red-shifted modified Leishmania infantum strain allows the appraisal of acute and chronic stage of infection, being a powerful tool for accelerating drug development against visceral leishmaniasis during both stages and helping to bridge the gap between early discovery process and subsequent drug development., Competing Interests: The authors have declared that no competing interests exist.

The activation marker CD69 is expressed by skin γδ T cells. Here we found that CD69 controlled the aryl hydrocarbon receptor (AhR)-dependent secretion of interleukin 22 (IL-22) by γδ T cells, which contributed to the development of psoriasis induced by IL-23. CD69 associated with the aromatic-amino-acid-transporter complex LAT1-CD98 and regulated its surface expression and uptake of L-tryptophan (L-Trp) and the intracellular quantity of L-Trp-derived activators of AhR. In vivo administration of L-Trp, an inhibitor of AhR or IL-22 abrogated the differences between CD69-deficient mice and wild-type mice in skin inflammation. We also observed LAT1-mediated regulation of AhR activation and IL-22 secretion in circulating Vγ9(+) γδ T cells of psoriatic patients. Thus, CD69 serves as a key mediator of the pathogenesis of psoriasis by controlling LAT1-CD98-mediated metabolic cues., Competing Interests: Authors declare no competing financial interest.

Endothelial cells (EC) are able to actively control vascular permeability, coagulation, blood pressure and angiogenesis. Most recently, a role for endothelial cells in the immune response has been described. Therefore, the endothelium has a dual role controlling homeostasis but also being the first line for host defence and tissue damage repair thanks to its ability to mount an inflammatory response. Endothelial cells have been shown to express pattern-recognition receptors (PRR) including Toll-like receptors (TLR) that are activated in response to stimuli within the bloodstream including pathogens and damage signals. TLRs are strategic mediators of the immune response in endothelial cells but they also regulate the angiogenic process critical for tissue repair. Nevertheless, endothelial activation and angiogenesis can contribute to some pathologies. Thus, inappropriate endothelial activation, also known as endothelial dysfunction, through TLRs contributes to tissue damage during autoimmune and inflammatory diseases such as atherosclerosis, hypertension, ischemia and diabetes associated cardiovascular diseases. Also TLR induced angiogenesis is required for the growth of some tumors, atherosclerosis and rheumatoid arthritis, among others. In this review we discuss the importance of various TLRs in modulating the activation of endothelial cells and their importance in immunity to infection and vascular disease as well as their potential as therapeutic targets., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Macrophage migration to the focus of infection is a hallmark of the innate immune response. Macrophage spreading, adhesion, and migration through the extracellular matrix require dynamic remodeling of the actin cytoskeleton associated to integrin clustering in podosomes and focal adhesions. Here, we show that prostaglandin E2 (PGE2 ), the main prostaglandin produced by macrophages during inflammation, promote the distinctive dose-dependent formation of podosomes or focal adhesions in macrophages. Low concentrations of PGE2 increased p110γ PI3K expression, phosphorylation of actin-related protein 2, and formation of podosomes, which enhanced macrophage migration in response to chemokines. However, high doses of PGE2 increased phosphorylation of paxillin and focal adhesion kinase, the expression of serine/threonine protein kinase 1, and promoted focal adhesion formation and macrophage adhesion, reducing macrophage chemotaxis. In summary, we describe the dual role of PGE2 as a promoter of macrophage chemotaxis and adhesion, proposing a new model of macrophage migration to the inflammatory focus in the presence of a gradient of PGE2 ., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Understanding the regulation of T-cell responses during inflammation and auto-immunity is fundamental for designing efficient therapeutic strategies against immune diseases. In this regard, prostaglandin E2 (PGE2) is mostly considered a myeloid-derived immunosuppressive molecule. We describe for the first time that T cells secrete PGE2 during T-cell receptor stimulation. In addition, we show that autocrine PGE2 signaling through EP receptors is essential for optimal CD4(+) T-cell activation in vitro and in vivo, and for T helper 1 (Th1) and regulatory T cell differentiation. PGE2 was found to provide additive co-stimulatory signaling through AKT activation. Intravital multiphoton microscopy showed that triggering EP receptors in T cells is also essential for the stability of T cell-dendritic cell (DC) interactions and Th-cell accumulation in draining lymph nodes (LNs) during inflammation. We further demonstrated that blocking EP receptors in T cells during the initial phase of collagen-induced arthritis in mice resulted in a reduction of clinical arthritis. This could be attributable to defective T-cell activation, accompanied by a decline in activated and interferon-γ-producing CD4(+) Th1 cells in draining LNs. In conclusion, we prove that T lymphocytes secret picomolar concentrations of PGE2, which in turn provide additive co-stimulatory signaling, enabling T cells to attain a favorable activation threshold. PGE2 signaling in T cells is also required for maintaining long and stable interactions with DCs within LNs. Blockade of EP receptors in vivo impairs T-cell activation and development of T cell-mediated inflammatory responses. This may have implications in various pathophysiological settings.

Background: Visceral leishmaniasis (VL) is hypoendemic in the Mediterranean region, where it is caused by the protozoan Leishmania infantum. An effective vaccine for humans is not yet available and the severe side-effects of the drugs in clinical use, linked to the parenteral administration route of most of them, are significant concerns of the current leishmanicidal medicines. New drugs are desperately needed to treat VL and phenotype-based High Throughput Screenings (HTS) appear to be suitable to achieve this goal in the coming years., Methodology/principal Findings: We generated two infrared fluorescent L. infantum strains, which stably overexpress the IFP 1.4 and iRFP reporter genes and performed comparative studies of their biophotonic properties at both promastigote and amastigote stages. To improve the fluorescence emission of the selected reporter in intracellular amastigotes, we engineered distinct constructs by introducing regulatory sequences of differentially-expressed genes (A2, AMASTIN and HSP70 II). The final strain that carries the iRFP gene under the control of the L. infantum HSP70 II downstream region (DSR), was employed to perform a phenotypic screening of a collection of small molecules by using ex vivo splenocytes from infrared-infected BALB/c mice. In order to further investigate the usefulness of this infrared strain, we monitored an in vivo infection by imaging BALB/c mice in a time-course study of 20 weeks., Conclusions/significance: The near-infrared fluorescent L. infantum strain represents an important step forward in bioimaging research of VL, providing a robust model of phenotypic screening suitable for HTS of small molecule collections in the mammalian parasite stage. Additionally, HSP70 II+L. infantum strain permitted for the first time to monitor an in vivo infection of VL. This finding accelerates the possibility of testing new drugs in preclinical in vivo studies, thus supporting the urgent and challenging drug discovery program against this parasitic disease.

Sigma (σ) receptor ligands are essentially known for their effects on the nervous system although recent studies have shown their potential effects modulating some other pathophysiological processes as cell proliferation, cancer, and the immune response. Here, we have analyzed the actions of σ-1 and σ-2 receptors ligands on T cell activation. Our results show that treatment of Jurkat T cells with σ-2 agonists decreased the induction of the expression of Interleukin (IL)-2, Tumor necrosis factor (TNF)-α, and Cyclooxygenase (COX)-2 by activated T cells in a dose-dependent manner. These effects take place at the transcriptional level since σ-2 agonists BD-737 and CB-184 diminished the activity of the promoters of those genes. Those immunosuppressive effects could be attributable to interference with transcription factor activation. Induced transcription mediated by Nuclear factor (NF)-κB or Nuclear Factor of Activated T cells (NFAT) was inhibited by σ-2 agonists. These effects seem to be specific for σ-2 agonists as no significant effects on T cell activation by σ-1 ligands PRE-084 and BD-1063 were found. Our results provide new insights into the immunomodulatory actions of σ ligands and describe a new property of σ-2 agonists, through inhibition of activation of transcription factors as NFAT by which these compounds are regulating gene expression. This may have important consequences on the possible therapeutic use of those compounds.

Background: Leishmania major cutaneous leishmaniasis is an infectious zoonotic disease. It is produced by a digenetic parasite, which resides in the phagolysosomal compartment of different mammalian macrophage populations. There is an urgent need to develop new therapies (drugs) against this neglected disease that hits developing countries. The main goal of this work is to establish an easier and cheaper tool of choice for real-time monitoring of the establishment and progression of this pathology either in BALB/c mice or in vitro assays. To validate this new technique we vaccinated mice with an attenuated Δhsp70-II strain of Leishmania to assess protection against this disease., Methodology: We engineered a transgenic L. major strain expressing the mCherry red-fluorescent protein for real-time monitoring of the parasitic load. This is achieved via measurement of fluorescence emission, allowing a weekly record of the footpads over eight weeks after the inoculation of BALB/c mice., Results: In vitro results show a linear correlation between the number of parasites and fluorescence emission over a range of four logs. The minimum number of parasites (amastigote isolated from lesion) detected by their fluorescent phenotype was 10,000. The effect of antileishmanial drugs against mCherry+L. major infecting peritoneal macrophages were evaluated by direct assay of fluorescence emission, with IC(50) values of 0.12, 0.56 and 9.20 µM for amphotericin B, miltefosine and paromomycin, respectively. An experimental vaccination trial based on the protection conferred by an attenuated Δhsp70-II mutant of Leishmania was used to validate the suitability of this technique in vivo., Conclusions: A Leishmania major strain expressing mCherry red-fluorescent protein enables the monitoring of parasitic load via measurement of fluorescence emission. This approach allows a simpler, faster, non-invasive and cost-effective technique to assess the clinical progression of the infection after drug or vaccine therapy.

Trypanosoma cruzi, the protozoan parasite responsible for Chagas' disease, causes severe myocarditis often resulting in death. Here, we report that Slamf1-/- mice, which lack the hematopoietic cell surface receptor Slamf1, are completely protected from an acute lethal parasite challenge. Cardiac damage was reduced in Slamf1-/- mice compared to wild type mice, infected with the same doses of parasites, as a result of a decrease of the number of parasites in the heart even the parasitemia was only marginally less. Both in vivo and in vitro experiments reveal that Slamf1-defIcient myeloid cells are impaired in their ability to replicate the parasite and show altered production of cytokines. Importantly, IFN-γ production in the heart of Slamf1 deficient mice was much lower than in the heart of wt mice even though the number of infiltrating dendritic cells, macrophages, CD4 and CD8 T lymphocytes were comparable. Administration of an anti-Slamf1 monoclonal antibody also reduced the number of parasites and IFN-γ in the heart. These observations not only explain the reduced susceptibility to in vivo infection by the parasite, but they also suggest human Slamf1 as a potential target for therapeutic target against T. cruzi infection.

Glomerular diseases are characterized by a sustained synthesis and accumulation of abnormal extracellular matrix proteins, such as collagen type I. The extracellular matrix transmits information to cells through interactions with membrane components, which directly activate many intracellular signaling events. Moreover, accumulating evidence suggests that eicosanoids derived from cyclooxygenase (COX)-2 participate in a number of pathological processes in immune-mediated renal diseases, and it is known that protein kinase B (AKT) may act through different transcription factors in the regulation of the COX-2 promoter. The present results show that progressive accumulation of collagen I in the extracellular medium induces a significant increase of COX-2 expression in human mesangial cells, resulting in an enhancement in PGE(2) production. COX-2 overexpression is due to increased COX-2 mRNA levels. The study of the mechanism implicated in COX-2 upregulation by collagen I showed focal adhesion kinase (FAK) activation. Furthermore, we observed that the activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway by collagen I and collagen I-induced COX-2 overexpression was abolished by PI3K and AKT inhibitors. Additionally, we showed that the cAMP response element (CRE) transcription factor is implicated. Finally, we studied COX-2 expression in an animal model, N(G)-nitro-l-arginine methyl ester hypertensive rats. In renal tissue and vascular walls, COX-2 and collagen type I content were upregulated. In summary, our results provide evidence that collagen type I increases COX-2 expression via the FAK/PI3K/AKT/cAMP response element binding protein signaling pathway.

Anti-inflammatory efficacy of non-steroidal anti-inflammatory drugs (NSAIDs) has been related to their properties as inhibitors of cyclooxygenase (COX)-mediated prostaglandin (PG) synthesis. However, recent studies have suggested that variations of the in vivo anti-inflammatory actions among different NSAIDs could not be solely explained by COX inhibition. Here, we have analyzed the effects on T cell activation of novel 4,5-dihydro-3 trifluoromethyl pyrazole anti-inflammatory drugs with different potencies as COX-2 inhibitors, namely E-6087, E-6232, E-6231, E-6036 and E-6259 as well as the chemically related COX-2 inhibitor Celecoxib. These drugs inhibited mitogen-mediated T cell proliferation as well as Interleukin (IL)-2, tumor necrosis factor (TNF)-α and Interferon (IFN)-γ synthesis by activated T cells, independently of their ability to inhibit COX-2 enzymatic activity. Immunosuppressive effects of these drugs seem to be due to their interference on transcription factor activation as induced transcription from Nuclear Factor (NF)-κB and Nuclear Factor of Activated T cells (NFAT)-dependent enhancers was inhibited in a dose-dependent manner, being the latter effect the most sensitive to the action of those compounds. Both NFAT dephosphorylation, required for its nuclear translocation, as well as transcriptional activity of a GAL4-NFAT chimera were diminished in the presence of these compounds. These findings provide new insights into the molecular mechanisms involved in the immunomodulatory and anti-inflammatory actions of NSAIDs, which may have important implications in anti-inflammatory therapy, through inhibition of NFAT., (Copyright © 2010 Elsevier B.V. All rights reserved.)

We have investigated the role of intracellular HIV-1 Tat on CXCR4 expression on T cells. We found that stable or doxycycline-regulated expression of HIV-1 Tat on Jurkat T cells results in lower cell surface expression of CXCR4, but not of other chemokine receptors. This effect was not due to an alteration in CXCR4 transcription, and total CXCR4 levels remained unaltered. Rather, when cells were treated with CXCL12/Stromal Cell-Derived Factor 1, a faster downmodulation of CXCR4 was observed although resurfacing was unaffected. Similar effect was seen in peripheral human T cells transiently transfected with Tat. At the molecular level Tat did not alter cellular levels of G-coupled receptor kinases 2 and 6 and beta-arrestin, proteins involved in CXCR4 downregulation. Neither Tat significantly affected phosphatidylinositol 3-kinase activation in response to CXCL12. Interestingly, in Jurkat cell clones stably expressing both Protein kinase (PK)-Czeta and HIV-1 Tat, CXCL12 induced a faster CXCR4 internalization than in cells only expressing HIV-1 Tat. In contrast in Jurkat cell stably expressing a dominant negative PKCzeta, Tat enhancement of CXCR4 internalization was abrogated. Thus, our results show a new function of HIV-1 Tat, its ability to regulate CXCR4 expression via PKCzeta. The significance of those results is discussed.

We have previously described that IFN-gamma induces cyclooxygenase 2 and inducible NO synthase expression by a mechanism that involved endogenously produced TNF-alpha. In this study, we report that TNF-alpha production is induced by IFN-gamma treatment in the murine macrophage cell line RAW 264.7. TNF-alpha mRNA levels are increased in cells treated with IFN-gamma in a time-dependent manner and IFN-gamma also increased human TNF-alpha promoter-dependent transcription. Two regions in the TNF-alpha promoter seem to be responsible for the IFN-gamma response: a distal region between -1311 and -615 bp of the human TNF-alpha promoter, and a proximal region located between -95 and -36 bp upstream of the transcriptional start. In contrast, IFN-gamma stimulation induces the expression of the transcription factors IRF-1 and IRF-8. Overexpression of these transcription factors produces an increase in the transcriptional activity of the human TNF-alpha promoter. There is a correlation between the regions of the TNF-alpha promoter responsible of the transcriptional activation elicited by IRF-1 and IRF-8 and those required for IFN-gamma response. In addition, IRF-1 and IRF-8 are recruited to the TNF-alpha promoter in IFN-gamma-treated RAW 264.7 cells, as demonstrated by chromatin immunoprecipitation assays. Moreover, overexpression of IRF-1 and IRF-8 induces TNF-alpha production in unstimulated RAW 264.7 macrophages, comparable to the production of TNF-alpha elicited by IFN-gamma stimulation, and silencing of IRF-1 and/or IRF-8 with specific small interfering RNAs, decreases IFN-gamma-elicited TNF-alpha production. In summary, IFN-gamma treatment induces TNF-alpha expression at transcriptional level requiring the coordinate action of IRF-1 and IRF-8.

Chronicity and Th2 immune responses are features of helminth infections in humans. The liver fluke promotes its own survival through several strategies to down-regulate the immune response of the host during the early phase of infection. However, there is no evidence that this modulation occurs much later. The immune response in advanced chronic fascioliasis was analyzed in an experimental rat model at 20 weeks after infection. Cytokine quantification in infected rat serum revealed basal levels. The predominant immunoglobulin (Ig) isotype was IgG1. Flow cytometry analysis of T cell (CD3(+), CD4(+), and CD8a(+)), B cell (CD45R(+)), and macrophage (CD11b(+)) populations in spleens showed no significant differences between infected and control rats. Mononuclear cell proliferation in the spleen in response to T and B mitogens was strongly inhibited in infected versus control rats. During early chronic infection, there is a predominance of a Th2 response, which decreases in advanced chronic infection characterized by a persistent immune suppression.

It has been shown that phosphorylation of p65/RelA and c-Rel plays a role in the regulation of transcriptional activity of NF-kappaB independent on IkappaB degradation. In this study, we show that anti CD3/CD28 activation induces the transactivation activity of both p65/RelA and c-Rel in T cells using Gal4 dependent assays. Moreover, protein kinase C (PKC)zeta, Cot kinase and NF-kappaB-inducing kinase (NIK) seem to be involved in those processes in a different manner. Thus, transfection of dominant negative forms of Cot and PKCzeta inhibits CD3/CD28 induction of Gal4-p65 transactivation, whereas the kinase inactive versions of the 3 kinases inhibit induction of Gal4-c-Rel. Cot induction of Gal4-c-Rel transactivating activity seems to be mediated sequentially through PKCzeta and NIK activation, since dominant negative form of NIK blocks Cot and PKCzeta induction, whereas kinase inactive PKCzeta only blocks Cot activity. In contrast, the contribution of NIK to the transactivation function of p65/RelA seems to be negligible and more importantly NIK-KD did not inhibit induction by Cot and PKCzeta. Besides, the enhancing effect of Cot on Gal4-p65 was not decreased in mouse embryo fibroblasts from NIK deficient aly/aly mice in contrast with a greatest reduction on Gal4-c-Rel. By using Ser to Ala mutants in p65 and c-Rel transactivation domains, PKCzeta and NIK activities seem to be dependent of a restricted set of Ser in both proteins. In contrast, the enhancing effect of Cot seems to be less dependent of a particular set of Ser residues being partially abrogated by mutation of several Ser residues.

Human immunodeficiency virus type 1 (HIV-1) Tat affects cellular gene expression through modulation of the activity of different transcription factors. Here, the role of Tat in the cooperation between nuclear factor of activated T cells (NFAT) and activator protein 1 (AP-1) transcription factors was investigated. Constitutive or transient Tat expression in Jurkat T cells enhanced cooperative NFAT/AP-1- but not AP-1-dependent transcription independent of its ability to transactivate the HIV-1 LTR. The enhancing effect of Tat took place after nuclear translocation of NFAT. Furthermore, transactivation of an NFAT/AP-1 reporter by transfection of NFAT and c-Jun was strongly enhanced by simultaneous Tat transfection. Moreover, intracellular Tat expression increased the binding of NFAT/AP-1 complexes to the interleukin 2 promoter without significantly altering NFAT- and AP-1-independent binding. HIV-1 Tat interacted with NFAT but not c-Jun. These results indicate that Tat interacts with NFAT, affecting its cooperation with AP-1, without altering independent binding of these transcription factors to DNA.

Recent evidence indicates that PPAR (peroxisome-proliferator-activated receptor) alpha ligands possess anti-inflammatory and antitumoural properties owing to their inhibitory effects on the expression of genes that are involved in the inflammatory response. However, the precise molecular mechanisms underlying these effects are poorly understood. In the present study, we show that tumour promoter PMA-mediated induction of genes that are significantly associated with inflammation, tumour growth and metastasis, such as COX-2 (cyclo-oxygenase 2) and VEGF (vascular endothelial growth factor), is inhibited by PPARalpha ligands in the human colorectal carcinoma cell line SW620. PPARalpha activators LY-171883 and WY-14,643 were able to diminish transcriptional induction of COX-2 and VEGF by inhibiting AP-1 (activator protein-1)-mediated transcriptional activation induced by PMA or by c-Jun overexpression. The actions of these ligands on AP-1 activation and COX-2 and VEGF transcriptional induction were found to be dependent on PPARalpha expression. Our studies demonstrate the existence of a negative cross-talk between the PPARalpha- and AP-1-dependent signalling pathways in these cells. PPARalpha interfered with at least two steps within the pathway leading to AP-1 activation. First, PPARalpha activation impaired AP-1 binding to a consensus DNA sequence. Secondly, PPARalpha ligands inhibited c-Jun transactivating activity. Taken together, these findings provide new insight into the anti-inflammatory and anti-tumoural properties of PPARalpha activation, through the inhibition of the induction of AP-1-dependent genes that are involved in inflammation and tumour progression.

The immunomodulatory activity of a standardized water soluble fraction of the fern Phlebodium decumanum (EXPLY-37) previously shown to have "in vivo" anti-inflammatory activity was analyzed "in vitro". This extract inhibited tumor necrosis factor (TNF) production by macrophages activated with lipopolysaccharide (LPS) or LPS plus interferon (IFN)-gamma. In contrast, nitric oxide (NO) and interleukin (IL)-1beta production were not affected in the same cultures, whereas IL-6 production was partially inhibited. More interestingly, EXPLY-37 increased the release of soluble TNF-receptor 2 (sTNFR2) and of IL-1R antagonist (IL-1Ra) but not of sTNFR1, by activated macrophages. EXPLY-37 had no effect on T lymphocyte activation, measured as proliferation as well as expression of early and late cell surface antigens CD69, CD25 (IL-2R-alpha) and CD71 (transferrin receptor) at the cell membrane. At the molecular level, EXPLY-37 did not inhibit the activation of the nuclear factor kappa B (NF-kappaB) transcription factor by TNF. In summary, EXPLY-37 has two anti-inflammatory activities "in vitro": it decreases TNF production and increases IL-1Ra and sTNFR2, which may be able to neutralize IL-1 and TNF activity, respectively.

Infection by HIV-1 is a major risk factor predisposing for fungal infection. However, few studies have addressed the immunological status of HIV-1 patients suffering fungal infections. This study examines the status of polymorphonuclear phagocytes (PMN) and T cells in HIV-1-infected patients suffering from mucosal Candida infections. These patients had a more immature population of blood PMN, as detected by lower CD18 expression, than HIV asymptomatics or healthy controls. They also had a selective defect in T cell activation in response to phytohemagglutinin (PHA), but not to stimulation through the T cell receptor by anti-CD3 crosslinking, when compared to HIV-1 asymptomatic patients. This was shown by a decrease in cellular proliferation and cell surface expression of CD69, CD25 and CD71 activation antigens. There was also a severe impairment of IL-2 production upon activation by PHA. IL-10, and TNF secretion was also reduced, whereas IFN-gamma and IL-5 production was not affected. No correlation with viral load, CD4 or CD8 T cell number or clinical stage was found. In conclusion, our results indicate that Candida-infected HIV patients have a selective defect, independent of viral load, CD4 or clinical status, involving some aspects of T cell activation, IL-2 production being severely impaired.

Blockade of phosphodiesterase 4 with rolipram reduced the production of tumor necrosis factor (TNF)-alpha, interleukin (IL)-5, IL-10, and IL-2 but poorly inhibited cell proliferation and interferon-gamma (IFN-gamma) production by activated human T cells. Addition of dibutyryl cAMP mimicked rolipram inhibitions on proliferation, IL-2, TNF-alpha, and IFN-gamma but not on IL-10 or IL-5 production. Moreover, the inhibitory effects of rolipram on proliferation, IFN-gamma, and TNF-alpha but not of IL-10 production can be prevented by a specific protein kinase A inhibitor. Rolipram and pentoxifylline, a nonspecific phosphodiesterase inhibitor, decreased transcription of IL-2 and TNF-alpha promoters in transiently transfected normal T cells. Moreover, they inhibited the activation of nuclear factor-kappaB (NF-kappaB) and nuclear factor of activated T cells (NFAT) and stimulated activator protein-1 (AP-1) and cAMP response element-binding proteins (CREBs). In contrast, dibutyryl cAMP inhibited NF-kappaB but not NFAT activation. Thus, our data indicate that blockade of phosphodiesterase 4 regulates transcription of a particular cytokine through inhibition of NF-kappaB and NFAT, and stimulation of AP-1 and CREB.

Dysregulation of cytokine secretion plays an important role in AIDS pathogenesis. Here, we demonstrate that expression of HIV-1 Tat protein in Jurkat cells induces a severe impairment of IL-2 but not TNF gene transcription. Interestingly, this inhibition correlates with the effect of the viral protein on the transactivation of the CD28RE/AP1 composite element (-164/-154), but not with that observed on the NFAT/AP1 site of the IL-2 gene promoter, neither with the effect on NF-kappa B- nor AP1-independent binding sites. Endogenous expression of Tat induced a decrease in the amount of the specific protein complex bound to the CD28RE/AP1 probe after PMA plus calcium ionophore stimulation. This effect was accompanied by qualitative alterations of the AP1 complex. Thus, in wild-type Jurkat cells, c-jun was absent from the complex, whereas in Tat-expressing cells, c-jun was increasingly recruited overtime. By contrast, similar amounts of c-rel and a small amount of NFAT1 were detected both in wild type and in Jurkat Tat(+) cells. Furthermore, Tat not only induced the participation of c-jun in the cooperative complex but also a decrease in its transactivation activity alone or in combination with c-rel. Thus, the interaction of Tat with the components of this rel/AP1 cooperative complex seems to induce quantitative and qualitative alterations of this complex as activation progresses, resulting in a decrease of IL-2 gene transcription. Altogether our results suggest the existence of tuned mechanisms that allow the viral protein to specifically affect cooperative interactions between transcription factors.

We have previously reported that transcriptional induction of cyclooxygenase-2 (COX-2) isoenzyme occurs early after T cell receptor triggering, suggesting functional implications of cyclooxygenase activity in this process. Here, we identify the cis-acting elements responsible for the transcriptional activation of this gene in human T lymphocytes. COX-2 promoter activity was induced upon T cell activation both in primary resting T lymphocytes and in Jurkat cells. This induction was abrogated by inhibition of calcineurin phosphatase with the immunosuppressive drug cyclosporin A, whereas expression of an active calcineurin catalytic subunit enhanced COX-2 transcriptional activation. Moreover, cotransfection of nuclear factor of activated T cells (NFAT) wild type protein transactivated COX-2 promoter activity. Conversely, dominant negative mutants of NFATc or c-Jun proteins inhibited COX-2 induction. Electrophoretic mobility shift assays and site-directed mutagenesis allowed the identification of two regions of DNA located in the positions -117 and -58 relative to the transcriptional start site that serves as NFAT recognition sequences. These results emphasize the central role that the Ca(2+)/calcineurin pathway plays in COX-2 transcriptional regulation in T lymphocytes pointing to NFAT/activator protein-1 transcription factors as essential for COX-2 promoter regulation in these cells.

Immature neural cell lines could be productively infected by HIV-1. Interestingly, this infection was associated with a differentiation to a mature neuronal phenotype, characterized by the expression of mature neurofilaments and cell adhesion molecules, intercellular cell adhesion molecule-1, and vascular cell adhesion molecule-1. Infection also induced TNF-alpha and IL-1beta mRNA expression, as well as the synthesis of inducible nitric oxide synthase by neuroblastoma cells. Exogenous addition of TNF-alpha, but not of IL-1beta or many other cytokines, including nerve growth factor, mimicked those effects induced by infection. Moreover, blocking endogenous TNF-alpha or NO production in cultures of infected cells with a neutralizing anti-TNF-alpha antibody or inducible nitric oxide synthase inhibitors prevented the expression of the mature cell phenotype as well as expression of intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1. Addition of NO generators and TNF-alpha activated NF-kappaB- and intercellular cell adhesion molecule-1-dependent promoter transcription, whereas inducible nitric oxide synthase inhibitors prevented the transcriptional activation of intercellular cell adhesion molecule-1 promoter that was induced by TNF-alpha. Those results suggest that HIV can infect immature neural cells and this infection induces their neural development via a TNF-alpha- and NO-mediated mechanism., (Copyright 1999 Academic Press.)

Rolipram, a phosphosdiesterase type IV-specific inhibitor, prevented p24 antigen release from anti-CD3-activated human immunodeficiency virus (HIV)-infected T cells and CD4(+)-cell depletion associated with viral replication in a dose-responsive manner but minimally inhibited T-cell proliferation. Moreover, rolipram reduced the production of tumor necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10) by HIV-infected T cells. The transcriptional ability of a luciferase reporter gene under control of the HIV long terminal repeat, induced by phorbol myristic acetate plus ionomycin or by TNF-alpha, in primary T and Jurkat cells was also inhibited by rolipram. Rolipram inhibited NF-kappaB and NFAT activation induced by T-cell activation in Jurkat and primary T cells, as measured by transient transfection of reporter genes and electrophoretic mobility shift assays. Exogenous addition of TNF-alpha in the presence of rolipram restored NF-kappaB but not NFAT activation or p24 release. Addition of dibutyryl-cyclic AMP (dBcAMP) mimicked the effects of rolipram on p24 antigen release, NF-kappaB activation, and TNF-alpha secretion, but it did not affect NFAT activation or IL-10 production. The protein kinase A inhibitor KT5720 prevented the inhibition of TNF-alpha secretion but not that of HIV type 1 (HIV-1) replication caused by rolipram. Our data indicate that blockade of phosphodiesterase type IV could be of benefit against HIV-1 disease by modulating cytokine secretion and transcriptional regulation of HIV replication, and they suggest an important role of NFAT in HIV replication in primary T cells. Some of those activities cannot be ascribed solely to its ability to increase cAMP.

Tumor necrosis factor (TNF)-alpha controls T-cell activation and is a major inducer of human immunodeficiency virus (HIV)-1 replication in chronically infected cells. Therefore, we have investigated its role in primary cultures of HIV-infected human T lymphocytes by using neutralizing anti-TNF-alpha antibodies or TNF-alpha. Primary resting T lymphocytes produced TNF-alpha and supported HIV replication after T-cell receptor activation. Addition of neutralizing anti-TNF-alpha antibodies drastically reduced p24 antigen release and prevented CD4+ cell depletion associated with infection. Anti-TNF-alpha also prevented nuclear factor-kappa B (NF-kappa B) activation, and a good correlation between this inhibition and inhibition of HIV replication was observed. Moreover, supplementing the cultures with high doses of IL-2 reverted anti-TNF-alpha inhibition of cell proliferation but did not affect the inhibition of HIV p24 antigen release or NF-kappa B activation in the same cultures. Moreover, anti-TNF-alpha inhibited HIV-1 long terminal repeat (LTR)-driven transcription of a reporter gene in primary T cells in response to activation, either in the presence or the absence of HIV-1 Tat. Our results support an important role for autocrine TNF-alpha secretion in controlling HIV replication in primary T cells because of its ability to maintain NF-kappa B elevated in the nucleus of T cells.