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.

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.

Endoplasmic Reticulum Stress Sensor IRE1α Enhances IL-23 Expression by Human Dendritic Cells.

Human monocyte-derived dendritic cells (DCs) exposed to pathogen-associated molecular patterns (PAMPs) undergo bioenergetic changes that influence the immune response. We found that stimulation with PAMPs enhanced glycolysis in DCs, whereas oxidative phosphorylation remained unaltered. Glucose starvation and the hexokinase inhibitor 2-deoxy-d-glucose (2-DG) modulated cytokine expression in stimulated DCs. Strikingly, IL23A was markedly induced upon 2-DG treatment, but not during glucose deprivation. Since 2-DG can also rapidly inhibit protein N-glycosylation, we postulated that this compound could induce IL-23 in DCs via activation of the endoplasmic reticulum (ER) stress response. Indeed, stimulation of DCs with PAMPs in the presence of 2-DG robustly activated inositol-requiring protein 1α (IRE1α) signaling and to a lesser extent the PERK arm of the unfolded protein response. Additional ER stressors such as tunicamycin and thapsigargin also promoted IL-23 expression by PAMP-stimulated DCs. Pharmacological, biochemical, and genetic analyses using conditional knockout mice revealed that IL-23 induction in ER stressed DCs stimulated with PAMPs was IRE1α/X-box binding protein 1-dependent upon zymosan stimulation. Interestingly, we further evidenced PERK-mediated and CAAT/enhancer-binding protein ß-dependent trans-activation of IL23A upon lipopolysaccharide treatment. Our findings uncover that the ER stress response can potently modulate cytokine expression in PAMP-stimulated human DCs.

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.

Pharmacological inhibition of eicosanoids and platelet-activating factor signaling impairs zymosan-induced release of IL-23 by dendritic cells.

The engagement of the receptors for fungal patterns induces the expression of cytokines, the release of arachidonic acid, and the production of PGE2 in human dendritic cells (DC), but few data are available about other lipid mediators that may modulate DC function. The combined antagonism of leukotriene (LT) B4, cysteinyl-LT, and platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) inhibited IL23A mRNA expression in response to the fungal surrogate zymosan and to a lower extent TNFA (tumor necrosis factor-α) and CSF2 (granulocyte macrophage colony-stimulating factor) mRNA. The combination of lipid mediators and the lipid extract of zymosan-conditioned medium increased the induction of IL23A by LPS (bacterial lipopolysaccharide), thus suggesting that unlike LPS, zymosan elicits the production of mediators at a concentration enough for optimal response. Zymosan induced the release of LTB4, LTE4, 12-hydroxyeicosatetraenoic acid (12-HETE), and PAF C16:0. DC showed a high expression and detectable Ser663 phosphorylation of 5-lipoxygenase in response to zymosan, and a high expression and activity of LPCAT1/2 (lysophosphatidylcholine acyltransferase 1 and 2), the enzymes that incorporate acetate from acetyl-CoA into choline-containing lysophospholipids to produce PAF. Pharmacological modulation of the arachidonic acid cascade and the PAF receptor inhibited the binding of P-71Thr-ATF2 (activating transcription factor 2) to the IL23A promoter, thus mirroring their effects on the expression of IL23A mRNA and IL-23 protein. These results indicate that LTB4, cysteinyl-LT, and PAF, acting through their cognate G protein-coupled receptors, contribute to the phosphorylation of ATF2 and play a central role in IL23A promoter trans-activation and the cytokine signature induced by fungal patterns.