Susanne C. Diesner

Sphingosine kinase 1 and sphingosine-1-phosphate receptor 2 are vital to recovery from anaphylactic shock in mice

Sphingosine kinase 1 (SphK1) and SphK2 are ubiquitous enzymes that generate sphingosine-1-phosphate (S1P), a ligand for a family of G protein-coupled receptors (S1PR1-S1PR5) with important functions in the vascular and immune systems. Here we explore the role of these kinases and receptors in recovery from anaphylaxis in mice. We found that Sphk2-/- mice had a rapid recovery from anaphylaxis. In contrast, Sphk1-/- mice showed poor recovery from anaphylaxis and delayed histamine clearance. Injection of S1P into Sphk1-/- mice increased histamine clearance and promoted recovery from anaphylaxis. Adoptive cell transfer experiments demonstrated that SphK1 activity was required in both the hematopoietic and nonhematopoietic compartments for recovery from anaphylaxis. Mice lacking the S1P receptor S1PR2 also showed a delay in plasma histamine clearance and a poor recovery from anaphylaxis. However, S1P did not promote the recovery of S1pr2-/- mice from anaphylaxis, whereas S1pr2+/- mice showed partial recovery. Unlike Sphk2-/- mice, Sphk1-/- and S1pr2-/- mice had severe hypotension during anaphylaxis. Thus, SphK1-produced S1P regulates blood pressure, histamine clearance, and recovery from anaphylaxis in a manner that involves S1PR2. This suggests that specific S1PR2 agonists may serve to counteract the vasodilation associated with anaphylactic shock.

Dose-dependent food allergy induction against ovalbumin under acid-suppression: A murine food allergy model

BACKGROUND Animal models are essential for analyzing the allergenic potential of food proteins and for investigating mechanisms underlying food allergy. Based on previous studies revealing acid-suppression medication as risk factor for food allergy induction, we aimed to establish a mouse model mimicking the natural route of sensitization in patients. METHODS The effect of acid-suppressing medication on murine gastric pH was assessed by intragastric pH measurements after two injections of a proton pump inhibitor (PPI). To investigate dose-dependency, mice were fed different concentrations of ovalbumin (OVA; 0.2, 0.5, 1.0, 2.5 or 5.0mg) either with or without anti-ulcer medication. Additionally, different routes of exposure (i.p. vs. oral) were compared in a second immunization experiment. Sera were screened for OVA-specific antibody titers (IgG1, IgG2a and IgE) in ELISA and RBL assay. Clinical reactivity was evaluated by measuring rectal temperature after oral challenge and by type I skin tests. RESULTS Two intravenous injections of PPI significantly elevated the gastric pH from 2.97 to 5.3. Only oral immunization with 0.2mg OVA under anti-acid medication rendered elevated IgG1, IgG2a and IgE titers compared to all other concentrations. Protein feeding alone altered antibody titers only marginally. Even though also i.p. immunizations induced high levels of specific IgE, only oral immunizations under anti-acids induced anaphylactic reactions evidenced by a significant decrease of body temperature. CONCLUSION Only low-dosage ovalbumin feedings under anti-acid medication resulted in IgE mediated food allergy. Based on this knowledge we have established a suitable food allergy model for further investigations of food adverse reactions.

Antacids and dietary supplements with an influence on the gastric pH increase the risk for food sensitization

Background Elevation of the gastric pH increases the risk for sensitization against food allergens by hindering protein breakdown. This can be caused by acid‐suppressing medication like sucralphate, H2‐receptor blockers and proton pump inhibitors, as shown in recent murine experimental and human observational studies.

Protamine nanoparticles with CpG-oligodeoxynucleotide prevent an allergen-induced Th2-response in BALB/c mice

The currently applied immunotherapy of type I allergy with aluminum hydroxide (alum) as adjuvant elicits - among other side effects - an initial IgE-boost. In contrast, CpG-oligodeoxynucleotides (ODNs) drive the immune response toward Th1. The biodegradable material protamine can spontaneously form nanoparticles together with such ODNs. Our aim was to investigate the immune response induced by protamine-based nanoparticles (proticles) with CpG-ODN as an allergen delivery system. Proticles complexed with Ara h 2 extracted from raw peanuts as model allergen were injected subcutaneously into naïve BALB/c mice. Ara h 2-specific antibodies were analyzed by ELISA and rat basophilic leukemia (RBL) cell assay. Cytokine levels were investigated in supernatants of stimulated splenocytes. The in vivo distribution after subcutaneous injection was examined via fluorescence imaging. BMDCs were stimulated with proticles, and expression of stimulation and maturation markers as well as cytokines in supernatants was investigated. A favorable increase in Ara h 2-specific IgG2a antibodies was found after immunization with proticles-Ara h 2, whereas Ara h 2-specific IgE was not detectable. Accordingly, the ratio of IL-5/IFN-gamma was low in this group. Granuloma formation was completely absent at injection sites of proticles. The distribution of Ara h 2 after subcutaneous injection was markedly decelerated when complexed to proticles. Stimulation of BMDCs with proticles-Ara h 2 caused upregulation of CD11c and CD80 as well as an increased IL-6 production. Our data suggest that biodegradable protamine-based nanoparticles with CpG-ODN counteract the Th2-dominated immune response induced by an allergen and therefore are suitable as novel carrier system for immunotherapy of allergy.

Nitration of the Egg-Allergen Ovalbumin Enhances Protein Allergenicity but Reduces the Risk for Oral Sensitization in a Murine Model of Food Allergy

Background Nitration of proteins on tyrosine residues, which can occur due to polluted air under “summer smog” conditions, has been shown to increase the allergic potential of allergens. Since nitration of tyrosine residues is also observed during inflammatory responses, this modification could directly influence protein immunogenicity and might therefore contribute to food allergy induction. In the current study we have analyzed the impact of protein nitration on sensitization via the oral route. Methodology/Principal Findings BALB/c mice were immunized intragastrically by feeding untreated ovalbumin (OVA), sham-nitrated ovalbumin (snOVA) or nitrated ovalbumin (nOVA) with or without concomitant acid-suppression. To analyze the impact of the sensitization route, the allergens were also injected intraperitoneally. Animals being fed OVA or snOVA under acid-suppressive medication developed significantly elevated levels of IgE, and increased titers of specific IgG1 and IgG2a antibodies. Interestingly, oral immunizations of nOVA under anti-acid treatment did not result in IgG and IgE formation. In contrast, intraperitoneal immunization induced high levels of OVA specific IgE, which were significantly increased in the group that received nOVA by injection. Furthermore, nOVA triggered significantly enhanced mediator release from RBL cells passively sensitized with sera from allergic mice. Gastric digestion experiments demonstrated protein nitration to interfere with protein stability as nOVA was easily degraded, whereas OVA and snOVA remained stable up to 120 min. Additionally, HPLC-chip-MS/MS analysis showed that one tyrosine residue (Y107) being very efficiently nitrated is part of an ovalbumin epitope recognized exclusively after oral sensitization. Conclusions/Significance These data indicated that despite the enhanced triggering capacity in existing allergy, nitration of OVA may be associated with a reduced de novo sensitizing capability via the oral route due to enhanced protein digestibility and/or changes in antibody epitopes.

Interleukin-10: An Anti-Inflammatory Marker To Target Atherosclerotic Lesions via PEGylated Liposomes

Atherosclerosis (AS) causes cardiovascular disease, which leads to fatal clinical end points like myocardial infarction or stroke, the most prevalent causes of death in developed countries. An early, noninvasive method of detection and diagnosis of atherosclerotic lesions is necessary to prevent and treat these clinical end points. Working toward this goal, we examined recombinant interleukin-10 (IL-10), stealth liposomes with nanocargo potency for NMRI relevant contrast agents, and IL-10 coupled to stealth liposomes in an ApoE-deficient mouse model using confocal laser-scanning microscopy (CLSM). Through ex vivo incubation and imaging with CLSM, we showed that fluorescently labeled IL-10 is internalized by AS plaques, and a low signal is detected in both the less injured aortic surfaces and the arteries of wild-type mice. In vivo experiments included intravenous injections of (i) fluorescent IL-10, (ii) IL-10 targeted carboxyfluorescin (CF−) labeled stealth liposomes, and (iii) untargeted CF-labeled stealth liposomes. Twenty-four hours after injection the arteries were dissected and imaged ex vivo. Compared to free IL-10, we observed a markedly stronger fluorescence intensity with IL-10 targeted liposomes at AS plaque regions. Moreover, untargeted CF-labeled liposomes showed only weak, unspecific binding. Neither free IL-10 nor IL-10 targeted liposomes showed significant immune reaction when injected into wild-type mice. Thus, the combined use of specific anti-inflammatory proteins, high payloads of contrast agents, and liposome particles should enable current imaging techniques to better recognize and visualize AS plaques for research and prospective therapeutic strategies.

Sphingosine-kinase 1 and 2 contribute to oral sensitization and effector phase in a mouse model of food allergy

BACKGROUND Sphingosine-1-phosphate (S1P) influences activation, migration and death of immune cells. Further, S1P was proposed to play a major role in the induction and promotion of allergic diseases. However, to date only limited information is available on the role of S1P in food allergy. OBJECTIVE We aimed to investigate the role of sphingosine-kinase (SphK) 1 and 2, the enzymes responsible for endogenous S1P production, on the induction of food allergy. METHODS AND RESULTS Human epithelial colorectal CaCo2 cells stimulated in vitro with S1P revealed a decrease of transepithelial resistance and enhanced transport of FITC labeled OVA. We studied the effect of genetic deletion of the enzymes involved in S1P production on food allergy induction using a mouse model of food allergy based on intragastrically (i.g.) administered ovalbumin (OVA) with concomitant acid-suppression. Wild-type (WT), SphK1(-/-) and SphK2(-/-) mice immunized with OVA alone i.g. or intraperitoneally (i.p.) were used as negative or positive controls, respectively. SphK1- and SphK2-deficient mice fed with OVA under acid-suppression showed reduced induction of OVA specific IgE and IgG compared to WT mice, but had normal responses when immunized by the intraperitoneal route. Flow cytometric analysis of spleen cells revealed a significantly reduced proportion of CD4(+) effector T-cells in both SphK deficient animals after oral sensitization. This was accompanied by a reduced accumulation of mast cells in the gastric mucosa in SphK-deficient animals compared to WT mice. Furthermore, mouse mast cell protease-1 (mMCP-1) levels, an IgE-mediated anaphylaxis marker, were reliably elevated in allergic WT animals. CONCLUSION Modulation of the S1P homeostasis by deletion of either SphK1 or SphK2 alters the sensitization and effector phase of food allergy.

Preterm neonates display altered plasmacytoid dendritic cell function and morphology

Bacterial and viral infections cause high rates of morbidity and mortality in premature newborns. In the setting of viral infection, pDCs play a key role as strong producers of IFN‐α upon TLR9 activation. We analyzed pDC frequency, phenotype, morphology, and function in CB of preterm and term newborns in comparison with adults. Whereas all age groups show similar pDC numbers, BDCA‐2, CD123, and TLR9 levels, the expression of BDCA‐4 and capacity to produce IFN‐α upon TLR9 challenge were decreased significantly in preterm neonates. Furthermore, we show by means of electron microscopy that pDCs from preterm newborns exhibit a distinct, “immature” morphology. Taken together, these findings suggest decreased functionality of pDCs in the premature newborn. The reduced capacity to produce IFN‐α is likely to render such infants more susceptible to viral infections.

Characterization of intrinsic and extrinsic risk factors for celery allergy in immunosenescence

Recent studies indicated an underestimation of allergies in elderly. In our experimental food allergy model of protein feeding under acid-suppression we aimed to assess whether food allergy can be induced in immunosenescent mice. Furthermore, the impact of gastric digestion on celery allergenicity was evaluated in aged patients. Measurements of serum zinc and iron levels in senescent and adult BALB/c mice for definition of the nutritional status indicated a possible alteration of the immune response in the aged animals due to reduced zinc and iron levels. Feedings of mice with digestion-sensitive celery proteins under physiological gastric conditions induced IgG1 and IgG2a in the aged and preferentially IgG1 in the adult animals. In contrast, incomplete digestion due to acid-suppression rendered celery-specific IgE, positive skin tests and elevated IL-5 levels in both age groups. Also in aged celery allergic patients (mean age 72 years) properly digested celery showed decreased capacity to bind and crosslink IgE as evaluated by skin tests and IgE immunoblot. Thus, in the geriatric murine model, celery allergy was induced only if gastric digestion was hindered. Accordingly, gastric proteolysis decreased in vitro and in vivo IgE-reactivity against celery proteins in aged allergic patients.

Perspectives on immunomodulation early in life

To cite this article: Diesner SC, Förster‐Waldl E, Olivera A, Pollak A, Jensen‐Jarolim E, Untersmayr E. Perspectives on immunomodulation early in life. Pediatr Allergy Immunol 2012: 23: 210–223.