An allergen-fused dendritic cell-binding peptide enhances in vitro proliferation of equine T-cells and cytokine production.

Allergen-specific immunotherapy (AIT) constitutes the only curative approach for allergy treatment. There is need for improvement of AIT in veterinary medicine, such as in horses suffering from insect bite hypersensitivity, an IgE-mediated dermatitis to Culicoides. Dendritic cell (DC)-targeting represents an efficient method to increase antigen immunogenicity. It is studied primarily for its use in improvement of cancer therapy and vaccines, but may also be useful for improving AIT efficacy. Immunomodulators, like the Toll-like receptor 4 (TLR-4) agonist monophosphoryl lipid-A (MPLA) has been shown to enhance the IL-10 response in horses, while CpG-rich oligonucleotides (CpG-ODN), acting as TLR-9 agonists, have been shown to induce Th1 or regulatory responses in horses with equine asthma. Our aim was to evaluate in vitro effects of antigen-targeting to equine DC with an antigen-fused peptide known to target human and mouse DC and investigate whether addition of MPLA or CpG-ODN would further improve the induced immune response with regard to finding optimal conditions for equine AIT. For this purpose, DC-binding peptides were fused to the model antigen ovalbumin (OVA) and to the recombinant Culicoides allergen Cul o3. Effects of DC-binding peptides on cellular antigen uptake and induction of T cell proliferation were assessed. Polarity of the immune response was analysed by quantifying IFN-γ, IL-4, IL-10, IL-17 and IFN-α in supernatants of antigen-stimulated peripheral blood mononuclear cells (PBMC) in presence or absence of adjuvants. Fusion of DC-binding peptides to OVA significantly enhanced antigen-uptake by equine DC. DC primed with DC-binding peptides coupled to OVA or Cul o3 induced a significantly higher T-cell proliferation compared to the corresponding control antigens. PBMC stimulation with DC-binding peptides coupled to Cul o3 elicited a significant increase in the pro-inflammatory cytokines IFN-γ, IL-4, IL-17, as well as the anti-inflammatory IL-10, but not of IFN-α. Adjuvant addition further enhanced the effect of the DC-binding peptides by significantly increasing the production of IFN-γ, IL-4, IL-10 and IFN-α (CpG-ODN) and IL-10 (MPLA), while simultaneously suppressing IFN-γ, IL-4 and IL-17 production (MPLA). Targeting equine DC with allergens fused to DC-binding peptides enhances antigen-uptake and T-cell activation and may be useful in increasing the equine immune response against recombinant antigens. Combination of DC-binding peptide protein fusions with adjuvants is necessary to appropriately skew the resulting immune response, depending on intended use. Combination with MPLA is a promising option for improvement of AIT efficacy in horses, while combination with CpG-ODN increases the effector immune response to recombinant antigens.

Identification and characterization of equine blood plasmacytoid dendritic cells.

Dendritic cells (DC) are antigen-presenting cells that can be classified into three major cell subsets: conventional DC1 (cDC1), cDC2 and plasmacytoid DCs (pDC), none of which have been identified in horses. Therefore, the objective of this study was to identify and characterize DC subsets in equine peripheral blood, emphasizing on pDC. Surface marker analysis allowed distinction of putative DC subsets, according to their differential expression of CADM-1 and MHC class II. Equine pDC were found to be Flt3(+) CD4(low) CD13(-) CD14(-) CD172a(-) CADM-1(-) MHCII(low). The weak expression of CD4 on equine pDC contrasts with findings in several other mammals. Furthermore, pDC purified by fluorescence-activated cell sorting were found to be the only cell subset able to produce large amounts of IFN-α upon TLR9-agonist stimulation. The pDC identity was confirmed by demonstrating high-levels of PLAC8, RUNX2 and TCF4 expression, showing pDC-restricted expression in other mammals.

In vivo phosphoenolpyruvate carboxykinase promoter mapping identifies disrupted hormonal synergism as a target of inflammation during sepsis in mice.

UNLABELLED: In mammals, proper maintenance of blood glucose levels within narrow limits is one of the most critical prerequisites for healthy energy homeostasis and body function. Consequently, hyper- and hypoglycemia represent hallmarks of severe metabolic pathologies, including type II diabetes and acute sepsis, respectively. Although the liver plays a crucial role in the control of systemic glucose homeostasis, the molecular mechanisms of aberrant hepatic glucose regulation under metabolic stress conditions remain largely unknown. Here we report the development of a liver-specific adenoviral in vivo system for monitoring promoter activity of the key gluconeogenic enzyme gene phosphoenolpyruvate carboxykinase (PEPCK) in mice. By employing in vivo promoter deletion technology, the glucocorticoid response unit (GRU) and the cyclic adenosine monophosphate (cAMP)-responsive element (CRE) were identified as critical cis-regulatory targets of proinflammatory signaling under septic conditions. In particular, both elements were found to be required for inhibition of PEPCK transcription during sepsis, thereby mediating endotoxic hypoglycemia. Indeed, expression of nuclear receptor cofactor peroxisome proliferator-activator receptor coactivator 1alpha (PGC-1alpha), the molecular mediator of GRU/CRE synergism on the PEPCK promoter, was found to be specifically repressed in septic liver, and restoration of PGC-1alpha in cytokine-exposed hepatocytes blunted the inhibitory effect of proinflammatory signaling on PEPCK gene expression. CONCLUSION: The dysregulation of hormonal synergism through the repression of PGC-1alpha as identified by in vivo promoter monitoring may provide a molecular rationale for hypoglycemia during sepsis, thereby highlighting the importance of hepatic glucose homeostasis for metabolic dysfunction in these patients.

Nuclear receptor cofactor receptor interacting protein 140 controls hepatic triglyceride metabolism during wasting in mice.

UNLABELLED: In mammals, triglycerides (TG) represent the most concentrated form of energy. Aberrant TG storage and availability are intimately linked to the negative energy balance under severe clinical conditions, such as starvation, sepsis, or cancer cachexia. Despite its crucial role for energy homeostasis, molecular key determinants of TG metabolism remain enigmatic. Here we show that the expression of nuclear receptor cofactor receptor interacting protein (RIP) 140 was induced in livers of starved, septic, and tumor-bearing mice. Liver-specific knockdown of RIP140 led to increased hepatic TG release and alleviated hepatic steatosis in tumor-bearing, cachectic animals. Indeed, hepatic RIP140 was found to control the expression of lipid-metabolizing genes in liver. CONCLUSION: By preventing the mobilization of hepatic TG stores, the induction of RIP140 in liver provides a molecular rationale for hepatic steatosis in starvation, sepsis, or cancer cachexia. Inhibition of hepatic RIP140 transcriptional activity might, thereby, provide an attractive adjunct scheme in the treatment of these conditions.

Signal-dependent control of gluconeogenic key enzyme genes through coactivator-associated arginine methyltransferase 1.

Together with impaired glucose uptake in skeletal muscle, elevated hepatic gluconeogenesis is largely responsible for the hyperglycemic phenotype in type II diabetic patients. Intracellular glucocorticoid and cyclic adenosine monophosphate (cAMP)/protein kinase A-dependent signaling pathways contribute to aberrant hepatic glucose production through the induction of gluconeogenic enzyme gene expression. Here we show that the coactivator-associated arginine methyltransferase 1 (CARM1) is required for cAMP-mediated activation of rate-limiting gluconeogenic phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) and glucose-6-phosphatase genes. Mutational analysis showed that CARM1 mediates its effect via the cAMP-responsive element within the PEPCK promoter, which is identified here as a CARM1 target in vivo. In hepatocytes, endogenous CARM1 physically interacts with cAMP-responsive element binding factor CREB and is recruited to the PEPCK and glucose-6-phosphatase promoters in a cAMP-dependent manner associated with increased promoter methylation. CARM1 might, therefore, represent a critical component of cAMP-dependent glucose metabolism in the liver.