Novel adjuvants in allergen-specific immunotherapy: where do we stand?

Type I hypersensitivity, or so-called type I allergy, is caused by Th2-mediated immune responses directed against otherwise harmless environmental antigens. Currently, allergen-specific immunotherapy (AIT) is the only disease-modifying treatment with the potential to re-establish clinical tolerance towards the corresponding allergen(s). However, conventional AIT has certain drawbacks, including long treatment durations, the risk of inducing allergic side effects, and the fact that allergens by themselves have a rather low immunogenicity. To improve AIT, adjuvants can be a powerful tool not only to increase the immunogenicity of co-applied allergens but also to induce the desired immune activation, such as promoting allergen-specific Th1- or regulatory responses. This review summarizes the knowledge on adjuvants currently approved for use in human AIT: aluminum hydroxide, calcium phosphate, microcrystalline tyrosine, and MPLA, as well as novel adjuvants that have been studied in recent years: oil-in-water emulsions, virus-like particles, viral components, carbohydrate-based adjuvants (QS-21, glucans, and mannan) and TLR-ligands (flagellin and CpG-ODN). The investigated adjuvants show distinct properties, such as prolonging allergen release at the injection site, inducing allergen-specific IgG production while also reducing IgE levels, as well as promoting differentiation and activation of different immune cells. In the future, better understanding of the immunological mechanisms underlying the effects of these adjuvants in clinical settings may help us to improve AIT.

A flagellin-conjugate protein induces dual NLRC4- and NLRP3-inflammasome activation which modulates inflammatory cytokine secretion from macrophages.

Background: A recombinant fusion protein combining the adjuvant and TLR5-ligand flagellin with the major birch pollen allergen Bet v 1 (rFlaA:Betv1) has been suggested to prevent the manifestation of birch allergy. Noteworthy, rFlaA:Betv1 induced both pro- and anti-inflammatory responses which were differentially regulated. However, the mechanism by which flagellin fusion proteins modulate allergen-specific immune responses, especially the mechanisms underlying IL-1ß secretion and their contribution to the overall immune responses remains elusive. Objective: To investigate the mechanisms underlying the production of IL-1ß from rFlaA:Betv1 stimulated macrophages. Methods: Macrophages were derived from mouse peritoneal-, human buffy-coat-, and PMA-differentiated THP-1 (wild type or lacking either ASC, NLRP3, or NLRC4) cells. Macrophages were stimulated with non-modified rFlaA:Betv1, mutant variants lacking either the flagellin DC0 domain or a sequence motif formerly described to mediate TLR5-activation, and respective controls in the presence or absence of inhibitors interfering with MAPK- and NFκB-signaling. Cytokine secretion was analyzed by ELISA and intracellular signaling by Western Blot. To study the contribution of IL-1ß to the overall immune responses, IL1R-deficient mouse peritoneal macrophages were used. Results: rFlaA:Betv1 consistently activated all types of investigated macrophages, inducing higher IL-1ß secretion compared with the equimolar mixture of both proteins. rFlaA:Betv1-induced activation of THP-1 macrophages was shown to be independent of either the TLR5-activating sequence motif or the flagellin DC0 domain but depended on both NLRP3- and NLRC4-inflammasomes. In addition, NFκB and SAP/JNK MAP kinases regulated rFlaA:Betv1-induced inflammasome activation and cytokine secretion by modulating pro-Caspase-1- and pro-IL-1ß-expression in THP-1 macrophages. Finally, lack of IL-1ß positive feedback via the IL1R strongly diminished the rFlaA:Betv1-induced secretion of IL-1ß, IL-6, and TNF-α from peritoneal macrophages. Conclusion: The mechanisms contributing to rFlaA:Betv1-induced IL-1ß secretion from macrophages were shown to be complex, involving both NLRC4- and NLRP3-inflammsomes, as well as NFκB- and SAP/JNK MAP kinase-signaling. Better understanding the mechanisms regulating the activation of immune cells by novel therapeutic candidates like the rFlaA:Betv1 fusion protein will allow us to further improve and develop new treatment strategies when using flagellin as an adjuvant.

Role of Glycolysis and Fatty Acid Synthesis in the Activation and T Cell-Modulating Potential of Dendritic Cells Stimulated with a TLR5-Ligand Allergen Fusion Protein.

Trained immune responses, based on metabolic and epigenetic changes in innate immune cells, are de facto innate immune memory and, therefore, are of great interest in vaccine development. In previous studies, the recombinant fusion protein rFlaA:Betv1, combining the adjuvant and toll-like receptor (TLR)5-ligand flagellin (FlaA) and the major birch pollen allergen Bet v 1 into a single molecule, significantly suppressed allergic sensitization in vivo while also changing the metabolism of myeloid dendritic cells (mDCs). Within this study, the immune-metabolic effects of rFlaA:Betv1 during mDC activation were elucidated. In line with results for other well-characterized TLR-ligands, rFlaA:Betv1 increased glycolysis while suppressing oxidative phosphorylation to different extents, making rFlaA:Betv1 a suitable model to study the immune-metabolic effects of TLR-adjuvanted vaccines. In vitro pretreatment of mDCs with cerulenin (inhibitor of fatty acid biosynthesis) led to a decrease in both rFlaA:Betv1-induced anti-inflammatory cytokine Interleukin (IL) 10 and T helper cell type (TH) 1-related cytokine IL-12p70, while the pro-inflammatory cytokine IL 1ß was unaffected. Interestingly, pretreatment with the glutaminase inhibitor BPTES resulted in an increase in IL-1ß, but decreased IL-12p70 secretion while leaving IL-10 unchanged. Inhibition of the glycolytic enzyme hexokinase-2 by 2-deoxyglucose led to a decrease in all investigated cytokines (IL-10, IL-12p70, and IL-1ß). Inhibitors of mitochondrial respiration had no effect on rFlaA:Betv1-induced IL-10 level, but either enhanced the secretion of IL-1ß (oligomycin) or decreased IL-12p70 (antimycin A). In extracellular flux measurements, mDCs showed a strongly enhanced glycolysis after rFlaA:Betv1 stimulation, which was slightly increased after respiratory shutdown using antimycin A. rFlaA:Betv1-stimulated mDCs secreted directly antimicrobial substances in a mTOR- and fatty acid metabolism-dependent manner. In co-cultures of rFlaA:Betv1-stimulated mDCs with CD4+ T cells, the suppression of Bet v 1-specific TH2 responses was shown to depend on fatty acid synthesis. The effector function of rFlaA:Betv1-activated mDCs mainly relies on glycolysis, with fatty acid synthesis also significantly contributing to rFlaA:Betv1-mediated cytokine secretion, the production of antimicrobial molecules, and the modulation of T cell responses.

The Flagellin:Allergen Fusion Protein rFlaA:Betv1 Induces a MyD88- and MAPK-Dependent Activation of Glucose Metabolism in Macrophages.

TLR5 ligand flagellin-containing fusion proteins are potential vaccine candidates for many diseases. A recombinant fusion protein of flagellin A and the major birch pollen allergen Bet v 1 (rFlaA:Betv1) modulates immune responses in vitro and in vivo. We studied the effects of rFlaA:Betv1 on bone marrow-derived macrophages (BMDMs). BMDMs differentiated from BALB/c, C57BL/6, TLR5-/-, or MyD88-/- mice were pre-treated with inhibitors, stimulated with rFlaA:Betv1 or respective controls, and analyzed for activation, cytokine secretion, metabolic state, RNA transcriptome, and modulation of allergen-specific Th2 responses. Stimulation of BMDMs with rFlaA:Betv1 resulted in MyD88-dependent production of IL-1ß, IL-6, TNF-α, IL-10, CD69 upregulation, and a pronounced shift towards glycolysis paralleled by activation of MAPK, NFκB, and mTOR signaling. Inhibition of either mTOR (rapamycin) or SAP/JNK-MAPK signaling (SP600125) resulted in dose-dependent metabolic suppression. In BMDM and T cell co-cultures, rFlaA:Betv1 stimulation suppressed rBet v 1-induced IL-5 and IL-13 secretion while inducing IFN-γ production. mRNA-Seq analyses showed HIF-1a, JAK, STAT, phagosome, NLR, NFκB, TNF, TLR, and chemokine signaling to participate in the interplay of cell activation, glycolysis, and immune response. rFlaA:Betv1 strongly activated BMDMs, resulting in MyD88-, MAPK-, and mTOR-dependent enhancement of glucose metabolism. Our results suggest macrophages are important target cells to consider during restauration of allergen tolerance during AIT.

Music therapy-induced changes in salivary cortisol level are predictive of cardiovascular mortality in patients under maintenance hemodialysis.

BACKGROUND: Music therapy has been applied in hemodialysis (HD) patients for relieving mental stress. Whether the stress-relieving effect by music therapy is predictive of clinical outcome in HD patients is still unclear. METHODS: We recruited a convenience sample of 99 patients on maintenance HD and randomly assigned them to the experimental (n=49) or control (n=50) group. The experimental group received relaxing music therapy for 1 week, whereas the control group received no music therapy. In the experimental group, we compared cardiovascular mortality in the patients with and without cortisol changes. RESULTS: The salivary cortisol level was lowered after 1 week of music therapy in the experimental group (-2.41±3.08 vs 1.66±2.11 pg/mL, P<0.05), as well as the frequency of the adverse reaction score (-3.35±5.76 vs -0.81±4.59, P<0.05), the severity of adverse reactions score (-1.93±2.73 vs 0.33±2.71, P<0.05), and hemodialysis stressor scale (HSS) score (-6.00±4.68 vs -0.877±7.08, P<0.05). The difference in salivary cortisol correlated positively with HD stress score scales (r=0.231, P<0.05), systolic blood pressure (r=0.264, P<0.05), and respiratory rates (r=0.369, P<0.05) and negatively with finger temperature (r=-0.235, P<0.05) in the total study population. The 5-year cardiovascular survival in the experimental group was higher in patients whose salivary cortisol lowered by <0.6 pg/mL than that in patients whose salivary cortisol lowered by >0.6 pg/mL (83.8% vs 63.6%, P<0.05). CONCLUSION: Providing music during HD is an effective complementary therapy to relieve the frequency and severity of adverse reactions, as well as to lower salivary cortisol levels. Differences in salivary cortisol after music therapy may predict cardiovascular mortality in patients under maintenance HD.

The effects of music as therapy on the overall well-being of elderly patients on maintenance hemodialysis.

In this study, the authors explored the use of music during hemodialysis (HD) as a complementary therapy to improve overall well-being in elderly patients. The authors recruited a convenience sample of 88 patients on maintenance HD from a teaching hospital in northern Taiwan and randomly assigned them to either an experimental group (n = 44) or a control group (n = 44). In the first week, participants in the experimental group created their own music playlists. During the second week, these participants listened to music from their own playlists during each HD session (three times/week). The authors evaluated the effects of music as therapy by assessing its impact on perceived stressors and adverse reactions during HD (HD Adverse Reactions Self-Assessment Scale and HD Stressor Scale [HSS]) and measuring changes in physiological indices during the course of the music listening. After 1 week of the use of music as therapy during HD, the authors noted significant reductions in the frequency and severity of adverse reactions during dialysis and in scores on the HSS, p < .001. The authors also observed significantly decreased respiratory rate and significantly increased finger temperature and oxygen saturation, p < .001, during the same period. In conclusion, listening to music during HD may promote overall patient well-being. It may thus serve as a complementary form of therapy that facilitates care and delivery of adequate dialysis and thus improves overall patient well-being in the long run.

Mechanisms of Panax ginseng in preventing rat pheochromocytoma cells from apoptosis.

AIMS OF THE STUDY: Although ginseng root possesses dominant central therapeutic effects and has recently undergone investigations for treating different neuronal diseases, most of its mechanisms are still unknown. Therefore, the neuroprotective mechanisms of ginseng were studied. MATERIALS AND METHODS: The protection afforded by different methanol extracts of Panax ginseng (PG) was tested in a serum deprivation-induced apoptotic model using neuronal-like pheochromocytoma (PC12) cells. An MTT assay, annexin V-FITC staining, and Western blots were, respectively, applied to identify the viability of cells, the apoptotic form of cell death, and the activity of antiapoptotic signaling. RESULTS: The known antiapoptotic PI3-K/Akt and MEK/ERK pathways in this system were ruled out due to failure of LY 294002 and PD 98059 to block the protection by PG. A protein kinase A (PKA) inhibitor was found to block the protection by PG and PG-induced CREB phosphorylation, suggesting that the PKA/CREB pathway mediates the protective effect of PG. Downregulation of classical and novel PKCs failed to block the protection by PG, while an atypical PKC inhibitor blocked protection by PG. CONCLUSIONS: PKA and atypical PKC are important for the protection afforded by PG in preventing serum deprivation-induced PC12 cell apoptosis.

Ligusticum chuanxiong as a potential neuroprotectant for preventing serum deprivation-induced apoptosis in rat pheochromocytoma cells: functional roles of mitogen-activated protein kinases.

ETHNOPHARMACOLOGICAL RELEVANCE: Ligusticum chuanxiong (LC) as a common component in many traditional Chinese medicinal formulas and decoctions has been used to treat different central nervous diseases, suggesting a neuroprotective function. AIM OF THE STUDY: To investigate the functional roles of mitogen-activated protein kinases (MAPKs) in mediating the neuroprotection of LC. MATERIALS AND METHODS: Different extractions of LC were applied with or without MAPK inhibitor to test their protection against serum deprivation-induced apoptosis in rat neuronal-like pheochromocytoma (PC12) cells as revealed by an MTT assay or Hoechst staining. Western blot was used to identify the activations of MAPKs. RESULTS: The most effective butanol extraction (LC-BuOH) was used in the following experiments. LC-BuOH reversed serum deprivation-induced decreased phosphorylation of extracellular signal-regulated kinase (ERK) and increased phosphorylation of c-Jun NH(2)-terminal kinase (JNK) and p38, the family of MAPKs. A PKA inhibitor, blocked the protection of LC-BuOH and partially blocked LC-BuOH-induced alterations in MAPKs, suggesting that the LC-BuOH regulates MAPKs through both PKA-dependent and -independent pathways. Although PD 98059, an inhibitor of MEK which activates ERK, blocked LC-BuOH-induced ERK phosphorylation, it did not block the protection of LC-BuOH. CONCLUSIONS: LC-BuOH mediates protection by suppressing JNK/p38 instead of activating ERK activity.