SGK1 Governs the Reciprocal Development of Th17 and Regulatory T Cells.

A balance between Th17 and regulatory T (Treg) cells is critical for immune homeostasis and tolerance. Our previous work has shown Serum- and glucocorticoid-induced kinase 1 (SGK1) is critical for the development and function of Th17 cells. Here, we show that SGK1 restrains the function of Treg cells and reciprocally regulates development of Th17/Treg balance. SGK1 deficiency leads to protection against autoimmunity and enhances self-tolerance by promoting Treg cell development and disarming Th17 cells. Treg cell-specific deletion of SGK1 results in enhanced Treg cell-suppressive function through preventing Foxo1 out of the nucleus, thereby promoting Foxp3 expression by binding to Foxp3 CNS1 region. Furthermore, our data suggest that SGK1 also plays a critical role in IL-23R-mediated inhibition of Treg and development of Th17 cells. Therefore, we demonstrate that SGK1 functions as a pivotal node in regulating the reciprocal development of pro-inflammatory Th17 and Foxp3+ Treg cells during autoimmune tissue inflammation.

Arabidopsis MAP-Kinase 3 Phosphorylates UDP-Glucose Dehydrogenase: a Key Enzyme Providing UDP-Sugar for Cell Wall Biosynthesis.

The enzyme UDP-glucose dehydrogenase (UGD) competes with sucrose-phosphate synthase for the common photosynthesis product UDP-glucose. Sucrose-phosphate synthase is part of a pathway for the export of sucrose from source leaves to neighboring cells or the phloem. UGD is a central enzyme in a pathway for many nucleotide sugars used in local cell wall biosynthesis. Here, we identify a highly conserved phosphorylation site in UGD which is readily phosphorylated by MAP-kinase 3 in Arabidopsis. Phosphorylation occurs at a surface-exposed extra loop in all plant UGDs that is absent in UGDs from bacteria or animals. Phosphorylated sucrose-phosphate synthase is shifted to an inactive form which we did not measure for phosphorylated UGD. Plant UGDs have an extra loop which is phosphorylated by AtMPK3. Phosphorylation is not causing a reduction of UGD activity as found for the competitor enzymes and thus sets a preference for maintaining UDP-sugars at a constant level to prioritize cell wall biosynthesis.

The transcription factor musculin promotes the unidirectional development of peripheral Treg cells by suppressing the TH2 transcriptional program.

Although master transcription factors (TFs) are key to the development of specific T cell subsets, whether additional transcriptional regulators are induced by the same stimuli that dominantly repress the development of other, non-specific T cell lineages has not been fully elucidated. Through the use of regulatory T cells (Treg cells) induced by transforming growth factor-ß (TGF-ß), we identified the TF musculin (MSC) as being critical for the development of induced Treg cells (iTreg cells) by repression of the T helper type 2 (TH2) transcriptional program. Loss of MSC reduced expression of the Treg cell master TF Foxp3 and induced TH2 differentiation even under iTreg-cell-differentiation conditions. MSC interrupted binding of the TF GATA-3 to the locus encoding TH2-cell-related cytokines and diminished intrachromosomal interactions within that locus. MSC-deficient (Msc-/-) iTreg cells were unable to suppress TH2 responses, and Msc-/- mice spontaneously developed gut and lung inflammation with age. MSC therefore enforced Foxp3 expression and promoted the unidirectional induction of iTreg cells by repressing the TH2 developmental program.

Skin vaccination via fractional infrared laser ablation - Optimization of laser-parameters and adjuvantation.

BACKGROUND: Methods to deliver an antigen into the skin in a painless, defined, and reproducible manner are essential for transcutaneous immunization (TCI). Here, we employed an ablative fractional infrared laser (P.L.E.A.S.E. Professional) to introduce clinically relevant vaccines into the skin. To elicit the highest possible antibody titers with this system, we optimized different laser parameters, such as fluence and pore number per area, and tested various adjuvants. METHODS: BALB/c mice were immunized with Hepatitis B surface antigen (HBsAg) by laser-microporation. Adjuvants used were alum, CRM197, monophosphoryl lipid A, heat-labile enterotoxin subunit B of E. coli (LT-B), and CpG ODN1826. The influence of different fluences (2.1 to 16.8J/cm2) and pore densities (5-15%) was investigated. Furthermore, immunogenicity of HBsAg and the commercially available conjugate vaccines ActHIB® and Menveo® applied via TCI was compared to standard i.m. injection. Antigen-specific antibody titers were assessed by luminometric ELISA. RESULTS: Antibody titers against HBsAg were dependent on pore depth and peaked at a fluence of 8.4J/cm2. Immunogenicity was independent of pore density. Adjuvantation with alum significantly reduced antibody titers after TCI, whereas other adjuvants only induced marginal changes in total IgG titers. LT-B and CpG shifted the polarization of the immune response as indicated by decreased IgG1/IgG2a ratios. HBsAg/LT-B applied via TCI induced similar antibody titers compared to i.m. injection of HBsAg/alum. In contrast to i.m. injection, we observed a dose response from 5 to 20µg after TCI. Both, ActHIB® and Menveo® induced high antibody titers after TCI, which were comparable to i.m. injection. CONCLUSIONS: Alum, the most commonly used adjuvant, is contraindicated for transcutaneous vaccination via laser-generated micropores. TCI with optimized laser parameters induces high antibody titers, which cannot be significantly increased by the tested adjuvants. Commercially available vaccines formulated without alum have the potential for successful TCI via laser-generated micropores, without the need for reformulation.

Fold stability during endolysosomal acidification is a key factor for allergenicity and immunogenicity of the major birch pollen allergen.

BACKGROUND: The search for intrinsic factors, which account for a protein's capability to act as an allergen, is ongoing. Fold stability has been identified as a molecular feature that affects processing and presentation, thereby influencing an antigen's immunologic properties. OBJECTIVE: We assessed how changes in fold stability modulate the immunogenicity and sensitization capacity of the major birch pollen allergen Bet v 1. METHODS: By exploiting an exhaustive virtual mutation screening, we generated mutants of the prototype allergen Bet v 1 with enhanced thermal and chemical stability and rigidity. Structural changes were analyzed by means of x-ray crystallography, nuclear magnetic resonance, and molecular dynamics simulations. Stability was monitored by using differential scanning calorimetry, circular dichroism, and Fourier transform infrared spectroscopy. Endolysosomal degradation was simulated in vitro by using the microsomal fraction of JAWS II cells, followed by liquid chromatography coupled to mass spectrometry. Immunologic properties were characterized in vitro by using a human T-cell line specific for the immunodominant epitope of Bet v 1 and in vivo in an adjuvant-free BALB/c mouse model. RESULTS: Fold stabilization of Bet v 1 was pH dependent and resulted in resistance to endosomal degradation at a pH of 5 or greater, affecting presentation of the immunodominant T-cell epitope in vitro. These properties translated in vivo into a strong allergy-promoting TH2-type immune response. Efficient TH2 cell activation required both an increased stability at the pH of the early endosome and efficient degradation at lower pH in the late endosomal/lysosomal compartment. CONCLUSIONS: Our data indicate that differential pH-dependent fold stability along endosomal maturation is an essential protein-inherent determinant of allergenicity.

Galectin-9-CD44 interaction enhances stability and function of adaptive regulatory T cells.

The ß-galactoside-binding protein galectin-9 is critical in regulating the immune response, but the mechanism by which it functions remains unclear. We have demonstrated that galectin-9 is highly expressed by induced regulatory T cells (iTreg) and was crucial for the generation and function of iTreg cells, but not natural regulatory T (nTreg) cells. Galectin-9 expression within iTreg cells was driven by the transcription factor Smad3, forming a feed-forward loop, which further promoted Foxp3 expression. Galectin-9 increased iTreg cell stability and function by directly binding to its receptor CD44, which formed a complex with transforming growth factor-ß (TGF-ß) receptor I (TGF-ßRI), and activated Smad3. Galectin-9 signaling was further found to regulate iTreg cell induction by dominantly acting through the CNS1 region of the Foxp3 locus. Our data suggest that exogenous galectin-9, in addition to being an effector molecule for Treg cells, acts synergistically with TGF-ß to enforce iTreg cell differentiation and maintenance.

Metallothioneins negatively regulate IL-27-induced type 1 regulatory T-cell differentiation.

IL-27-induced type 1 regulatory T (Tr1) cells suppress autoimmunity by producing IL-10. Signal transducer and activator of transcription (STAT) 1 and STAT3 have been described as key transcription factors that promote IL-10 secretion from Tr1 cells induced by IL-27. However, the molecular pathways for negatively regulating Tr1 cell differentiation remain elusive. Here, we show that IL-27 induces metallothioneins (MTs) that in turn prevent Tr1 cell development. MT expression leads to the reduction of STAT1 and STAT3 phosphorylation under Tr1 differentiation condition, resulting in impaired IL-10 production. Accordingly, Tr1 cells derived from MT-deficient mice showed an increased ability to produce IL-10 and potently suppress experimental autoimmune encephalomyelitis upon adoptive transfer. Moreover, activation of STAT1 and/or STAT3 can overcome the suppression of IL-10 by MTs, indicating a dynamic balance between STATs and MTs in regulating IL-10 during Tr1 cell differentiation.

Induction of pathogenic TH17 cells by inducible salt-sensing kinase SGK1.

TH17 cells (interleukin-17 (IL-17)-producing helper T cells) are highly proinflammatory cells that are critical for clearing extracellular pathogens and for inducing multiple autoimmune diseases. IL-23 has a critical role in stabilizing and reinforcing the TH17 phenotype by increasing expression of IL-23 receptor (IL-23R) and endowing TH17 cells with pathogenic effector functions. However, the precise molecular mechanism by which IL-23 sustains the TH17 response and induces pathogenic effector functions has not been elucidated. Here we used transcriptional profiling of developing TH17 cells to construct a model of their signalling network and nominate major nodes that regulate TH17 development. We identified serum glucocorticoid kinase 1 (SGK1), a serine/threonine kinase, as an essential node downstream of IL-23 signalling. SGK1 is critical for regulating IL-23R expression and stabilizing the TH17 cell phenotype by deactivation of mouse Foxo1, a direct repressor of IL-23R expression. SGK1 has been shown to govern Na(+) transport and salt (NaCl) homeostasis in other cells. We show here that a modest increase in salt concentration induces SGK1 expression, promotes IL-23R expression and enhances TH17 cell differentiation in vitro and in vivo, accelerating the development of autoimmunity. Loss of SGK1 abrogated Na(+)-mediated TH17 differentiation in an IL-23-dependent manner. These data demonstrate that SGK1 has a critical role in the induction of pathogenic TH17 cells and provide a molecular insight into a mechanism by which an environmental factor such as a high salt diet triggers TH17 development and promotes tissue inflammation.

Immune complex-mediated co-ligation of the BCR with FcγRIIB results in homeostatic apoptosis of B cells involving Fas signalling that is defective in the MRL/Lpr model of systemic lupus erythematosus.

Negative regulation of B cell activation by cognate immune complexes plays an important homeostatic role in suppressing B cell hyperactivity and preventing consequent autoimmunity. Immune complexes co-ligate the BCR and FcγRIIB resulting in both growth arrest and apoptosis. We now show that such apoptotic signalling involves induction and activation of p53 and its target genes, the pro-apoptotic Bcl-2 family members, Bad and Bid, as well as nuclear export of p53. Collectively, these events result in destabilisation of the mitochondrial and lysosomal compartments with consequent activation and interplay of executioner caspases and endosomal-derived proteases. In addition, the upregulation of Fas and FasL with consequent activation of caspase 8-dependent death receptor signalling is required to facilitate efficient apoptosis of B cells. Consistent with this role for Fas death receptor signalling, apoptosis resulting from co-ligation of the BCR and FcγRIIB is defective in B cells from Fas-deficient MRL/MpJ-Fas(lpr) mice. As these mice develop spontaneous, immune complex-driven lupus-like glomerulonephritis, targeting this FcγRIIB-mediated apoptotic pathway may therefore have novel therapeutic implications for systemic autoimmune disease.

Designing hypoallergenic derivatives for allergy treatment by means of in silico mutation and screening.

BACKGROUND: The incidence of allergic diseases in developed countries has been increasing constantly in the last 2 decades. The only curative treatment available thus far is specific immunotherapy (SIT). The problematic side effects that can occur during SIT with allergen extracts led to the search for safer alternatives, such as recombinant hypoallergenic proteins with reduced allergenic potential and preserved T-cell immunogenicity. OBJECTIVE: We created hypoallergenic variants of the allergens Bet v 1a and Phl p 5b by using in silico mutation and screening and characterized the biochemical and immunologic properties of selected mutant proteins. METHODS: Knowledge-based potentials were used to estimate structural changes of the protein structures under sequence variation. IgE antibodies and their cross-linking capacity were determined by using a basophil release assay, binding of human birch pollen-specific IgE was investigated by means of ELISA, and ELISPOT assays were performed to examine the T-cell immunogenicity. RESULTS: Selected mutated proteins showed significantly reduced IgE-binding and cross-linking ability but retained their T cell-stimulating properties. Immunization with the hypoallergenic mutants induced blocking antibodies against murine and human IgE epitopes. CONCLUSION: In silico calculation and selection of mutations that render a protein hypoallergenic represents a novel and rapid tool to create candidate molecules for SIT with recombinant allergens.

Allergy multivaccines created by DNA shuffling of tree pollen allergens.

BACKGROUND: The major allergens of trees belonging to the Fagales order are collectively known as the Bet v 1 family. Members of the Fagales order have distinct geographic distribution, and it is expected that depending on the exposure pattern of the individual, inclusion of other Bet v 1 family members might increase the efficacy of the treatment. OBJECTIVE: We aimed to generate molecules that are suitable for specific immunotherapy not only against birch pollen allergy but also against allergies caused by other cross-reactive tree pollens. METHODS: Fourteen genes of the Bet v 1 family were randomly recombined in vitro by means of DNA shuffling. This library of chimeric proteins was screened for molecules displaying low capacity to induce release of inflammatory mediators but with T-cell immunogenicity higher than that of the parental allergens. RESULTS: Two chimeric proteins were selected from the library of shuffled clones displaying low allergenicity and high immunogenicity, as determined in in vitro assays using human and animal cells and antibodies, as well as in vivo in animal models of allergy. CONCLUSION: Our results show that it is possible to randomly recombine in vitro T- and B-cell epitopes of a family of related allergens and to select chimeric proteins that perfectly match the criteria presently thought to be relevant for improving allergen-specific immunotherapy. CLINICAL IMPLICATIONS: The hypoallergenic chimeras described here recombine epitopes of the major Fagales pollen allergens and thus can efficiently substitute a mixture of extracts used for treating patients with tree pollen-induced spring pollinosis worldwide.

Generation of hypoallergenic DNA vaccines by forced ubiquitination: preventive and therapeutic effects in a mouse model of allergy.

BACKGROUND: Hypoallergenic immunotherapy of type I allergies aims at inducing T-cell immunity while avoiding cross-linking of pre-existing IgE. DNA-based immunotherapy depends on the recruitment of antigen-specific T(H)1 cells and therefore has to provide the whole repertoire of T-cell epitopes. Ubiquitination offers a general approach for the production of hypoallergenic DNA vaccines. OBJECTIVE: A DNA-based vaccine encoding the major birch pollen allergen Bet v 1 stably linked to ubiquitin was evaluated for its antiallergic potential in a BALB/c mouse model of allergy. METHODS: Plasmid DNA was applied to mice before (preventive) or after (therapeutic) sensitization with recombinant Bet v 1. In the preventive setting, mice were exposed to aerosolized allergen in addition. Cytokine production was monitored via ELISPOT and Luminex. IgG(1), IgG(2a), and IgE subclass antibody titers were determined by ELISA. In vitro antigen-specific cross-linking of IgE was measured in a degranulation assay. Bronchoalveolar lavages were analyzed for leukocyte subsets as well as for IFN-gamma and IL-5, and paraffin sections of lungs were examined for mucus production and endothelial damage. RESULTS: Prevaccination with ubiquitinated Bet v 1-stimulated T(H)1-biased immune responses with concomitant suppression of functional IgE, reduction of eosinophil counts in bronchoalveolar lavages, and alleviation of lung pathology, and could also suppress an ongoing IgE response in a therapeutic setting. CONCLUSION: The data clearly demonstrate that hypoallergenic DNA vaccines encoding ubiquitin fusion constructs induce effective antiallergic immune responses. CLINICAL IMPLICATIONS: Ubiquitination of allergen gene vaccines eliminates the risk of IgE cross-linking, thereby meeting the safety requirements for clinical applications.