Central carbon metabolism exhibits unique characteristics during the handling of fungal patterns by monocyte-derived dendritic cells.

Monocyte-derived dendritic cells (MDDCs) are key players in the defense against fungal infection because of their outstanding capacity for non-opsonic phagocytosis and phenotypic plasticity. Accordingly, MDDCs rewire metabolism to meet the energetic demands for microbial killing and biomass synthesis required to restore homeostasis. It has been commonplace considering the metabolic reprogramming a mimicry of the Warburg effect observed in tumor cells. However, this may be an oversimplification since the offshoots of glycolysis and the tricarboxylic acid (TCA) cycle are connected in central carbon metabolism. Zymosan, the external wall of Saccharomyces cerevisiae, contains ß-glucan and α-mannan chains that engage the C-type lectin receptors dectin-1/2 and Toll-like receptors. This makes it an optimal fungal surrogate for experimental research. Using real-time bioenergetic assays and [U-13C]glucose labeling, central hubs connected to cytokine expression were identified. The pentose phosphate pathway (PPP) exhibited a more relevant capacity to yield ribose-5-phosphate than reducing equivalents of NADPH, as judged from the high levels of isotopologues showing 13C-labeling in the ribose moiety and the limited contribution of the oxidative arm of the PPP to the production of ROS by NADPH oxidases (NOX). The finding of 13C-label in the purine ring and in glutathione unveiled the contribution of serine-derived glycine to purine ring and glutathione synthesis. Serine synthesis also supported the TCA cycle. Zymosan exhausted NAD+ and ATP, consistent with intracellular consumption and/or extracellular export. Poly-ADP-ribosylated proteins detected in the nuclear fractions of MDDCs did not show major changes upon zymosan stimulation, which suggests its dependence on constitutive Fe(II)/2-oxoglutarate-dependent demethylation of 5-methylcytosine by TET translocases and/or demethylation of histone H3 lysine 27 by JMJD demethylases rather than on NOX activities. These results disclose a unique pattern of central carbon metabolism following fungal challenge, characterized by the leverage of glycolysis offshoots and an extensive recycling of NAD+ and poly(ADP-ribose).

The role of PAF in immunopathology: From immediate hypersensitivity reactions to fungal defense.

Platelet-activating factor (PAF, 1-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) was discovered when the mechanisms involved in the deposition of immune complex in tissues were being scrutinized in the experimental model of rabbit serum sickness. The initial adscription of PAF to IgE-dependent anaphylaxis was soon extended after disclosing its release from phagocytes stimulated by calcium mobilizing agents, formylated peptides, and phagocytosable particles. This explains why ongoing research in the field turned to the analysis of immune cell types and stimuli involved in PAF production with the purpose of establishing its role in pathology. This was spurred by the identification of the chemical structure of PAF and the enzymic mechanisms involved in its biosynthesis and degradation, which showed commonalities with those involved in eicosanoid production and the Lands' cycle of phospholipid fatty acid remodeling. The reassignment of PAF function in immunopathology is explained by the finding that the most robust mechanisms leading to PAF production are associated with opsonic and non-opsonic phagocytosis, depending on the cell type. While polymorphonuclear leukocytes exhibit opsonic phagocytosis, monocyte-derived dendritic cells show a marked preference for non-opsonic phagocytosis associated with C-type lectin receptors. This is particularly relevant to the defense against fungal invasion and explains why PAF exerts an autocrine feed-forwarding mechanism required for the selective expression of some cytokines.

PSTPIP1-LYP phosphatase interaction: structural basis and implications for autoinflammatory disorders.

Mutations in the adaptor protein PSTPIP1 cause a spectrum of autoinflammatory diseases, including PAPA and PAMI; however, the mechanism underlying these diseases remains unknown. Most of these mutations lie in PSTPIP1 F-BAR domain, which binds to LYP, a protein tyrosine phosphatase associated with arthritis and lupus. To shed light on the mechanism by which these mutations generate autoinflammatory disorders, we solved the structure of the F-BAR domain of PSTPIP1 alone and bound to the C-terminal homology segment of LYP, revealing a novel mechanism of recognition of Pro-rich motifs by proteins in which a single LYP molecule binds to the PSTPIP1 F-BAR dimer. The residues R228, D246, E250, and E257 of PSTPIP1 that are mutated in immunological diseases directly interact with LYP. These findings link the disruption of the PSTPIP1/LYP interaction to these diseases, and support a critical role for LYP phosphatase in their pathogenesis.

Interferons Are Pro-Inflammatory Cytokines in Sheared-Stressed Human Aortic Valve Endothelial Cells.

Calcific aortic valve disease (CAVD) is an athero-inflammatory process. Growing evidence supports the inflammation-driven calcification model, mediated by cytokines such as interferons (IFNs) and tumor necrosis factor (TNF)-α. Our goal was investigating IFNs' effects in human aortic valve endothelial cells (VEC) and the potential differences between aortic (aVEC) and ventricular (vVEC) side cells. The endothelial phenotype was analyzed by Western blot, qPCR, ELISA, monocyte adhesion, and migration assays. In mixed VEC populations, IFNs promoted the activation of signal transducers and activators of transcription-1 and nuclear factor-κB, and the subsequent up-regulation of pro-inflammatory molecules. Side-specific VEC were activated with IFN-γ and TNF-α in an orbital shaker flow system. TNF-α, but not IFN-γ, induced hypoxia-inducible factor (HIF)-1α stabilization or endothelial nitric oxide synthase downregulation. Additionally, IFN-γ inhibited TNF-α-induced migration of aVEC. Also, IFN-γ triggered cytokine secretion and adhesion molecule expression in aVEC and vVEC. Finally, aVEC were more prone to cytokine-mediated monocyte adhesion under multiaxial flow conditions as compared with uniaxial flow. In conclusion, IFNs promote inflammation and reduce TNF-α-mediated migration in human VEC. Moreover, monocyte adhesion was higher in inflamed aVEC sheared under multiaxial flow, which may be relevant to understanding the initial stages of CAVD.

Clinically used JAK inhibitor blunts dsRNA-induced inflammation and calcification in aortic valve interstitial cells.

Calcific aortic valve disease (CAVD) is the most prevalent valvulopathy worldwide. Growing evidence supports a role for viral and cell-derived double-stranded (ds)-RNA in cardiovascular pathophysiology. Poly(I:C), a dsRNA surrogate, has been shown to induce inflammation, type I interferon (IFN) responses, and osteogenesis through Toll-like receptor 3 in aortic valve interstitial cells (VIC). Here, we aimed to determine whether IFN signaling via Janus kinase (JAK)/Signal transducers and activators of transcription (STAT) mediates dsRNA-induced responses in primary human VIC. Western blot, ELISA, qPCR, calcification, flow cytometry, and enzymatic assays were performed to evaluate the mechanisms of dsRNA-induced inflammation and calcification. Poly(I:C) triggered a type I IFN response characterized by IFN-regulatory factors gene upregulation, IFN-ß secretion, and STAT1 activation. Additionally, Poly(I:C) promoted VIC inflammation via NF-κB and subsequent adhesion molecule expression, and cytokine secretion. Pretreatment with ruxolitinib, a clinically used JAK inhibitor, abrogated these responses. Moreover, Poly(I:C) promoted a pro-osteogenic phenotype and increased VIC calcification to a higher extent in cells from males. Inhibition of JAK with ruxolitinib or a type I IFN receptor blocking antibody blunted Poly(I:C)-induced calcification. Mechanistically, Poly(I:C) promoted VIC apoptosis in calcification medium, which was inhibited by ruxolitinib. Moreover, Poly(I:C) co-operated with IFN-γ to increase VIC calcification by synergistically activating extracellular signal-regulated kinases and hypoxia-inducible factor-1α pathways. In conclusion, JAK/STAT signaling mediates dsRNA-triggered inflammation, apoptosis, and calcification and may contribute to a positive autocrine loop in human VIC in the presence of IFN-γ. Blockade of dsRNA responses with JAK inhibitors may be a promising therapeutic avenue for CAVD.

Tricarboxylic Acid Cycle Activity and Remodeling of Glycerophosphocholine Lipids Support Cytokine Induction in Response to Fungal Patterns.

Increased glycolysis parallels immune cell activation, but the role of pyruvate remains largely unexplored. We found that stimulation of dendritic cells with the fungal surrogate zymosan causes decreases of pyruvate, citrate, itaconate, and α-ketoglutarate, while increasing oxaloacetate, succinate, lactate, oxygen consumption, and pyruvate dehydrogenase activity. Expression of IL10 and IL23A (the gene encoding the p19 chain of IL-23) depended on pyruvate dehydrogenase activity. Mechanistically, pyruvate reinforced histone H3 acetylation, and acetate rescued the effect of mitochondrial pyruvate carrier inhibition, most likely because it is a substrate of the acetyl-CoA producing enzyme ACSS2. Mice lacking the receptor of the lipid mediator platelet-activating factor (PAF; 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) showed reduced production of IL-10 and IL-23 that is explained by the requirement of acetyl-CoA for PAF biosynthesis and its ensuing autocrine function. Acetyl-CoA therefore intertwines fatty acid remodeling of glycerophospholipids and energetic metabolism during cytokine induction.

Lipopolysaccharide and interferon-γ team up to activate HIF-1α via STAT1 in normoxia and exhibit sex differences in human aortic valve interstitial cells.

In early stages of calcific aortic valve disease (CAVD), immune cells infiltrate into the valve leaflets and release cytokines such as interferon (IFN)-γ. IFN-γ has context-dependent direct effects, and also regulates other immune pathways. The purpose of this study was addressing the effects of IFN-γ on human aortic valve interstitial cells (AVICs), focusing on the pathogenic processes underlying CAVD. Strikingly, under normoxic conditions, IFN-γ induced hypoxia inducible factor (HIF)-1α expression, an effect strongly potentiated by the Toll-like receptor (TLR)-4 ligand lipopolysaccharide (LPS). Immunodetection studies confirmed the nuclear translocation of HIF-1α. Gene silencing showed that HIF-1α expression is dependent on signal transducer and activator of transcription (STAT)-1 expression. Consistent with HIF-1α induction, the secretion of the endothelial growth factor was detected by ELISA, and downregulation of the antiangiogenic factor chondromodulin-1 gene was observed by qPCR. Results also disclosed IFN-γ as a proinflammatory cytokine that cooperates with LPS to induce the expression of adhesion molecules, prostaglandin E2 and interleukins. Moreover, IFN-γ induced an osteogenic phenotype and promoted in vitro calcification that were markedly potentiated by LPS. Pharmacological experiments disclosed the involvement of Janus Kinases (JAK)/STATs as well as ERK/HIF-1α routes on the induction of calcification. Notably, IFN-γ receptor 1 expression, as well as ERK/HIF-1α activation, and the subsequent responses were more robust in male AVICs. This is the first report uncovering an immune and non-hypoxic activation of HIF-1α via STAT1 in AVIC. The aforementioned results and the sex-differential responses may be potentially relevant to better understand CAVD pathogenesis.

Polymorphisms in Receptors Involved in Opsonic and Nonopsonic Phagocytosis, and Correlation with Risk of Infection in Oncohematology Patients.

High-risk hematological malignancies are a privileged setting for infection by opportunistic microbes, with invasive mycosis being one of the most serious complications. Recently, genetic background has emerged as an unanticipated risk factor. For this reason, polymorphisms for genes encoding archetypal receptors involved in the opsonic and nonopsonic clearance of microbes, pentraxin-3 (PTX3) and Dectin-1, respectively, were studied and correlated with the risk of infection. Fungal, bacterial, and viral infections were registered for a group of 198 patients with high-risk hematological malignancies. Polymorphisms for the pentraxin-3 gene (PTX3) showed a significant association with the risk of fungal infection by Candida spp. and, especially, by Aspergillus spp. This link remained even for patients undergoing antifungal prophylaxis, thus demonstrating the clinical relevance of PTX3 in the defense against fungi. CLEC7A polymorphisms did not show any definite correlation with the risk of invasive mycosis, nor did they influence the expression of Dectin-1 isoforms generated by alternative splicing. The PTX3 mRNA expression level was significantly lower in samples from healthy volunteers who showed these polymorphisms, although no differences were observed in the extents of induction elicited by bacterial lipopolysaccharide and heat-killed Candidaalbicans, thus suggesting that the expression of PTX3 at the start of infection may influence the clinical outcome. PTX3 mRNA expression can be a good biomarker to establish proper antifungal prophylaxis in immunodepressed patients.

Calcification Induced by Type I Interferon in Human Aortic Valve Interstitial Cells Is Larger in Males and Blunted by a Janus Kinase Inhibitor.

Objective- Calcific aortic valve disease is the most prevalent valvulopathy in Western countries. An unanticipated pathogenetic clue involving IFN (interferon) was disclosed by the finding of constitutive type I IFN activity associated with aortic valve calcification in children with the atypical Singleton-Merten syndrome. On this basis, the role of type I IFN on inflammation and calcification in human aortic valve interstitial cells (AVIC) was examined. Approach and Results- IFN-α was weakly proinflammatory but potentiated lipopolysaccharide-mediated activation of NF (nuclear factor)-κB and the ensuing induction of proinflammatory molecules in human AVIC. Stimulation with IFN-α and in combination with lipopolysaccharide promoted osteoblast-like differentiation characterized by increased osteoblastic gene expression, BMP (bone morphogenetic protein)-2 secretion, and ectopic phosphatase activity. Sex differences were observed. Likewise, IFN-α treatment of human AVICs in osteogenic medium resulted in increased formation of calcific nodules. Strikingly, IFN-α-mediated calcification was significantly higher in AVICs from males, and was blocked by tofacitinib, a JAK (Janus kinase) inhibitor, and by a BMP antagonist. A female-specific protective mechanism involving the activation of PI3K-Akt (protein kinase B) pathways and cell survival was disclosed. Females exhibited higher levels of BCL2 in valve cells and tissues and lower annexin V staining on cell stimulation. Conclusions- IFN-α acts as a proinflammatory and pro-osteogenic cytokine in AVICs, its effects being potentiated by lipopolysaccharide. Results also uncovered sex differences with lower responses in female AVICs and sex-specific mechanisms involving apoptosis. Data point to JAK/STAT (signal transducer and activator of transcription) system as a potential therapeutic target for calcific aortic valve disease.

Toll-Like Receptors, Inflammation, and Calcific Aortic Valve Disease.

Inflammation, the primary response of innate immunity, is essential to initiate the calcification process underlying calcific aortic valve disease (CAVD), the most prevalent valvulopathy in Western countries. The pathogenesis of CAVD is multifactorial and includes inflammation, hemodynamic factors, fibrosis, and active calcification. In the development of CAVD, both innate and adaptive immune responses are activated, and accumulating evidences show the central role of inflammation in the initiation and propagation phases of the disease, being the function of Toll-like receptors (TLR) particularly relevant. These receptors act as sentinels of the innate immune system by recognizing pattern molecules from both pathogens and host-derived molecules released after tissue damage. TLR mediate inflammation via NF-κB routes within and beyond the immune system, and play a crucial role in the control of infection and the maintenance of tissue homeostasis. This review outlines the current notions about the association between TLR signaling and the ensuing development of inflammation and fibrocalcific remodeling in the pathogenesis of CAVD. Recent data provide new insights into the inflammatory and osteogenic responses underlying the disease and further support the hypothesis that inflammation plays a mechanistic role in the initiation and progression of CAVD. These findings make TLR signaling a potential target for therapeutic intervention in CAVD.

Fungal pattern receptors down-regulate the inflammatory response by a cross-inhibitory mechanism independent of interleukin-10 production.

Cyclic AMP regulatory element binding protein and signal transducer and activator of transcription 3 (STAT3) may control inflammation by several mechanisms, one of the best characterized is the induction of the expression of the anti-inflammatory cytokine interleukin-10 (IL-10). STAT3 also down-regulates the production of pro-inflammatory cytokines induced by immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors, a mechanism termed cross-inhibition. Because signalling via ITAM-dependent mechanisms is a hallmark of fungal pattern receptors, STAT3 activation might be involved in the cross-inhibition associated with invasive fungal infections. The fungal surrogate zymosan produced the phosphorylation of Y705-STAT3 and the expression of Ifnb1 and Socs3, but did not induce the interferon (IFN)-signature cytokines Cxcl9 and Cxcl10 in bone marrow-derived dendritic cells. Unlike lipopolysaccharide (LPS), zymosan induced IL-10 and phosphorylated Y705-STAT3 to a similar extent in Irf3 and Ifnar1 knockout and wild-type mice. Human dendritic cells showed similar results, although the induction of IFNB1 was less prominent. These results indicate that LPS and zymosan activate STAT3 through different routes. Whereas type I IFN is the main effector of LPS effect, the mechanism involved in Y705-STAT3 phosphorylation by zymosan is more complex, cannot be associated with type I IFN, IL-6 or granulocyte-macrophage colony-stimulating factor, and seems dependent on several factors given that it was partially inhibited by the platelet-activating factor antagonist WEB2086 and high concentrations of COX inhibitors, p38 mitogen-activate protein kinase inhibitors, and blockade of tumour necrosis factor-α function. Altogether, these results indicate that fungal pattern receptors share with other ITAM-coupled receptors the capacity to produce cross-inhibition through a mechanism involving STAT3 and induction of SOCS3 and IL-10, but that cannot be explained through type I IFN signalling.

Proline-serine-threonine phosphatase interacting protein 1 inhibition of T-cell receptor signaling depends on its SH3 domain.

Proline-serine-threonine phosphatase interacting protein 1 (PSTPIP1) is an adaptor protein associated with the cytoskeleton that is mainly expressed in hematopoietic cells. Mutations in PSTPIP1 cause the rare autoinflammatory disease called pyogenic arthritis, pyoderma gangrenosum, and acne. We carried out this study to further our knowledge on PSTPIP1 function in T cells, particularly in relation to the phosphatase lymphoid phosphatase (LYP), which is involved in several autoimmune diseases. LYP-PSTPIP1 binding occurs through the C-terminal homology domain of LYP and the F-BAR domain of PSTPIP1. PSTPIP1 inhibits T-cell activation upon T-cell receptor (TCR) and CD28 engagement, regardless of CD2 costimulation. This function of PSTPIP1 depends on the presence of an intact SH3 domain rather than on the F-BAR domain, indicating that ligands of the F-BAR domain, such as the PEST phosphatases LYP and PTP-PEST, are not critical for its negative regulatory role in TCR signaling. Additionally, PSTPIP1 mutations that cause the pyogenic arthritis, pyoderma gangrenosum and acne syndrome do not affect PSTPIP1 function in T-cell activation through the TCR.

Polarization of the innate immune response by prostaglandin E2: a puzzle of receptors and signals.

Eicosanoids tailor the innate immune response by supporting local inflammation and exhibiting immunomodulatory properties. Prostaglandin (PG) E2 is the most abundant eicosanoid in the inflammatory milieu due to the robust production elicited by pathogen-associated molecular patterns on cells of the innate immune system. The different functions and cell distribution of E prostanoid receptors explain the difficulty encountered thus far to delineate the actual role of PGE2 in the immune response. The biosynthesis of eicosanoids includes as the first step the Ca(2+)- and kinase-dependent activation of the cytosolic phospholipase A2, which releases arachidonic acid from membrane phospholipids, and later events depending on the transcriptional regulation of the enzymes of the cyclooxygenase routes, where PGE2 is the most relevant product. Acting in an autocrine/paracrine manner in macrophages, PGE2 induces a regulatory phenotype including the expression of interleukin (IL)-10, sphingosine kinase 1, and the tumor necrosis factor family molecule LIGHT. PGE2 also stabilizes the suppressive function of myeloid-derived suppressor cells, inhibits the release of IL-12 p70 by macrophages and dendritic cells, and may enhance the production of IL-23. PGE2 is a central component of the inflammasome-dependent induction of the eicosanoid storm that leads to massive loss of intravascular fluid, increases the mortality rate associated with coinfection by Candida ssp. and bacteria, and inhibits fungal phagocytosis. These effects have important consequences for the outcome of infections and the polarization of the immune response into the T helper cell types 2 and 17 and can be a clue to develop pharmacological tools to address infectious, autoimmune, and autoinflammatory diseases.

The autoimmunity risk variant LYP-W620 cooperates with CSK in the regulation of TCR signaling.

The protein tyrosine phosphatase LYP, a key regulator of TCR signaling, presents a single nucleotide polymorphism, C1858T, associated with several autoimmune diseases such as type I diabetes, rheumatoid arthritis, and lupus. This polymorphism changes an R by a W in the P1 Pro rich motif of LYP, which binds to CSK SH3 domain, another negative regulator of TCR signaling. Based on the analysis of the mouse homologue, Pep, it was proposed that LYP and CSK bind constitutively to inhibit LCK and subsequently TCR signaling. The detailed study of LYP/CSK interaction, here presented, showed that LYP/CSK interaction was inducible upon TCR stimulation, and involved LYP P1 and P2 motifs, and CSK SH3 and SH2 domains. Abrogating LYP/CSK interaction did not preclude the regulation of TCR signaling by these proteins.

Apoptotic cells enhance IL-10 and reduce IL-23 production in human dendritic cells treated with zymosan.

Contact of apoptotic cells (AC) with phagocytes tilts the balance of pro-inflammatory and anti-inflammatory cytokines. To address the cell- and stimulus-dependency of this mechanism, human monocyte-derived dendritic cells were treated with Jurkat AC in the presence and absence of different stimuli. AC reduced the production of IL-23 and enhanced the production of IL-10 elicited by zymosan, but they did not influence IL-12 p70 production nor did they modify the effect of LPS. Since formation of lipid bodies (LB) and PGE(2) production have been associated with IL-10 induction, the effect of PGE(2), the formation of LB, and the role of PPAR-γ were assessed. Exogenous PGE(2) enhanced IL-10 expression, but no evidence of PGE(2) production elicited by AC was obtained. Inhibition of PPAR-γ activity reduced the production of IL-10 both in the presence and in the absence of AC, but formation of LB in response to zymosan and AC was not observed. Notably, AC induced a transient nuclear translocation of both the CREB coactivator CRTC2/TORC2 and the homeodomain protein PBX1, which are involved in the CREB/HOX/PBX/MEIS transcription complex. These data show a selective effect of AC on the production of cytokines elicited by the fungal surrogate zymosan through the enhancement of CREB-dependent transcription.

Notch- and transducin-like enhancer of split (TLE)-dependent histone deacetylation explain interleukin 12 (IL-12) p70 inhibition by zymosan.

The fungal analog zymosan induces IL-23 and low amounts of IL-12 p70. This study addresses the molecular mechanisms underlying this cytokine pattern in human monocyte-derived dendritic cells. The transcriptional regulation of il23a, one of the chains of IL-23, depended on the activation of c-Rel and histone H3 phosphorylation, as judged from the association of c-Rel with the il23a promoter and the correlation between IL-23 production and Ser-10-histone H3 phosphorylation. Consistent with its reduced ability to produce IL-12 p70, zymosan induced a transient occupancy of the il12a promoter by c-Rel, blocked the production of IL-12 p70 and the transcription of il12a induced by other stimuli, and triggered the expression and nuclear translocation of the transcriptional repressors of the Notch family hairy and enhancer of split (Hes)-1, Hes5, hairy/enhancer-of-split related with YRPW motif protein (Hey)-1, and transducin-like enhancer of split (TLE). Zymosan also induced the interaction of Hes1 and TLE with histone H3 phosphorylated on Ser-10 and deacetylated on Lys-14. Inhibition of class III histone deacetylases increased the production of IL-12 p70 and partially blunted the inhibitory effect of zymosan on the production of IL-12 p70 elicited by LPS and IFN-γ. These results indicate that the selective induction of IL-23 by ß-glucans is explained by the activation of c-Rel associated with Ser-10-histone H3 phosphorylation in the il23a promoter mediated by mitogen- and stress-activated kinase and/or protein kinase A and inhibition of il12a transcription by a mechanism involving activation of several corepressors with the ability to bind TLE and to promote histone deacetylation.

The induction of IL-10 by zymosan in dendritic cells depends on CREB activation by the coactivators CREB-binding protein and TORC2 and autocrine PGE2.

Stimulation of human monocyte-derived dendritic cells with the yeast extract zymosan is characterized by a predominant production of IL-10 and a strong induction of cyclooxygenase-2, but the molecular mechanisms underlying this response are only partially understood. To address this issue, the activation of transcription factors that may bind to the il10 proximal promoter was studied. Binding activity to Sp1, Sp3, NF-Y, and cAMP response element (CRE) sites was detected in the nuclear extracts of dendritic cells; however these binding activities were not influenced by zymosan. No binding activity to Stat1, Stat3, and c/EBP sites was detected. Notably, zymosan activated kappaB-binding activity, but inhibition of NF-kappaB was associated with enhanced IL-10 production. In sharp contrast, treatments acting on CREB (CRE binding protein), including 8-Br-cAMP, PGE(2), and inhibitors of PKA, COX, and glycogen-synthase kinase-3beta showed a direct correlation between CREB activation and IL-10 production. Zymosan induced binding of both P-CREB and CREB-binding protein (CBP) to the il10 promoter as judged from chromatin immunoprecipitation assays, whereas negative results were obtained with Ab reactive to Sp1, Sp3, c-Maf, and NF-Y. Zymosan also induced nuclear translocation of the CREB coactivator transducer of regulated CREB activity 2 (TORC2) and interaction of TORC2 with P-CREB coincidental with the association of CREB to the il10 promoter. Altogether, our data show that zymosan induces il10 transcription by a CRE-dependent mechanism that involves autocrine secretion of PGE(2) and a network of interactions of PKA, MAP/ERK, glycogen-synthase kinase-3beta, and calcineurin, which regulate CREB transcriptional activity by binding the coactivators CBP and TORC2 and inhibiting CBP interaction with other transcription factors.

Cyclooxygenase-2 induced by zymosan in human monocyte-derived dendritic cells shows high stability, and its expression is enhanced by atorvastatin.

Cyclooxygenase (COX)-2 is a central enzyme of arachidonic acid metabolism, and its modulation by statins may explain some of the myocardial protective effects of these drugs. Dendritic cells (DCs) play a central role in microbial defense and in atherogenesis, and COX-2 expression in DCs is important for their migration to lymph nodes and antibody response, thus explaining why prostaglandin E(2) is a main component of the cocktails used to prepare DCs for clinical applications. On this basis, we addressed the effect of atorvastatin (ATV) on the release of arachidonic acid and on the expression of COX-2 in human monocyte-derived DCs. Although ATV on its own lacked any effect on COX-2 protein induction expression, it enhanced the release of arachidonic acid, the expression of COX-2 protein, and the production of prostaglandin E(2) induced by the fungal wall extract zymosan, and to a lower extent the effect of peptidoglycan. The effect on COX-2 protein was observed mainly 24 h after stimulation by zymosan and was not reverted by mevalonate, thus pointing to an effect unrelated to cholesterol metabolism. It is noteworthy that COX-2 protein showed a great stability, with a t((1/2)) of approximately 12 h, which was enhanced in the presence of ATV. In view of the important role played by COX-2 on DC function, these data indicate that ATV, by enhancing COX-2 stability, may increase DC function after infectious bouts and also counteract some of the risks associated with sustained inhibition of COX-2.

Characterization of new substrates targeted by Yersinia tyrosine phosphatase YopH.

YopH is an exceptionally active tyrosine phosphatase that is essential for virulence of Yersinia pestis, the bacterium causing plague. YopH breaks down signal transduction mechanisms in immune cells and inhibits the immune response. Only a few substrates for YopH have been characterized so far, for instance p130Cas and Fyb, but in view of YopH potency and the great number of proteins involved in signalling pathways it is quite likely that more proteins are substrates of this phosphatase. In this respect, we show here YopH interaction with several proteins not shown before, such as Gab1, Gab2, p85, and Vav and analyse the domains of YopH involved in these interactions. Furthermore, we show that Gab1, Gab2 and Vav are not dephosphorylated by YopH, in contrast to Fyb, Lck, or p85, which are readily dephosphorylated by the phosphatase. These data suggests that YopH might exert its actions by interacting with adaptors involved in signal transduction pathways, what allows the phosphatase to reach and dephosphorylate its susbstrates.

[Interleukin-8 expression in lung tissue during ischemia-reperfusion]. / Estudio de la expresión de interleucina-8 en el tejido pulmonar durante la isquemia-reperfusión.

OBJECTIVE: Local cytokine production is a pathogenic factor in ischemia-reperfusion injury in early graft dysfunction. This study analyzed interleukin 8 (IL-8) messenger RNA (mRNA) expression in lung tissue and the association between IL-8 mRNA levels and interstitial lung changes in an experimental model of warm lung ischemia-reperfusion. MATERIAL AND METHODS: We studied 16 New Zealand rabbits divided into 3 groups: control, ischemia (tissue taken from right lower lobe after 1, 2, or 3 hours of ischemia), and reperfusion (tissue taken from right upper and middle lobes after 1 hour of ischemia and 1, 2, or 3 hours of reperfusion). Expression of IL-8 mRNA was determined by reverse transcription and polymerase chain reaction. Interstitial infiltration by polymorphonuclear neutrophils was determined. The Mann-Whitney U-test was used for statistical comparisons, with P< .05 considered to indicate a significant result. RESULTS: During ischemia, IL-8 mRNA levels were elevated at the end of hour 1 (P=.009) with respect to the control group, but not thereafter. Interstitial changes were minimal. IL-8 mRNA levels during reperfusion were similar to those observed during ischemia, with a slight increase at the end of hour 2. There were no significant differences between hours 1, 2, and 3. Polymorphonuclear neutrophil recruitment occurred at the beginning of reperfusion (P=.014), but no significant differences were observed at hours 2 or 3. Progressive thickening of alveolar septa and edema was documented. CONCLUSIONS: Changes in IL-8 mRNA expression during ischemia precede interstitial infiltration by polymorphonuclear neutrophils during reperfusion, suggesting that the 2 processes are related. Quantification of IL-8 mRNA expression could facilitate early diagnosis of graft dysfunction.