The role of glycosylation in allergen recognition

This project is an attempt to have a better understanding of the role of carbohydrates in recognition and uptake of allergens by the innate immune system. Glycosylation analysis of different allergens like Der p 1, Fel d 1, Ara h 1, Ber e 1, Der p 2, Bla g 2, Can f 1, Bromelain and Papain was made using labelled lectins that can detect specific carbohydrate moieties on proteins to reveal their pattern of glycosylation. These experiments showed that all major allergens are glycosylated and this represented a major first step towards demonstrating a link between glycosylation and allergen recognition by the innate immune system. N- and O-glycosylation patterns were predicted in different allergens and a difference in mannosylation and fucosylation was detected between allergens and non-allergenic proteins. We found that the main dominant sugars on allergens are 1,2 1,3 and 1,6 mannose, as detected by GNA lectin. We have also showed that Der p 1 and Der p 2 possess 1,3 fucose in their natural forms, thus concluding that Der p1 and Der p 2 have part of the CCDs which are epitope structures for IgE. O-glycosylation in allergens was also studied giving a better understanding of the whole glycan structure in allergens. The role of mannosylation in allergen recognition by the immune system was investigated further. Different methods, including recombinant expression, enzymatic and chemical deglycosylation, were optimised to produce glycoforms of Der p 1. A recombinant preparation of Der p 1 produced in Pichia pastoris was used as a hypermannosylated form of the allergen. These glycoforms served as useful tools in addressing the nature of glycoallergen recognition by looking at the uptake of hyper- and hypo-glycosylated preparations by DCs, with confocal microscopy, ELISA and FACS as readouts. Results indicate that deglycosylated forms of Der p 1 exhibited minimal uptake by DCs compared to the natural and hyperglycosylated recombinant allergen. Comparative analysis of the hypermannosylated preparation of Der p 1 and its natural counterpart, possessing less mannan, showed that the recombinant form was taken up more readily by DCs at 37°C and at 4°C. We also showed that these glycoforms bind to the MR subfragment CTLD 4-7-FC, the C-type lectin carbohydrate recognition domain. This binding significantly decreased when the Der p 1 allergen was deglycosylated. These results were confirmed further using confocal microscopy imaging which also showed that recombinant Der p 1 uptake is immediate, starting at 5 mins of incubation and that a higher quantity accumulates inside the DC compared to natural allergen. Recombinant and natural Der p 1 both co-localised with MR, DC-SIGN and LAMP-2 lysosomal marker, suggesting a key role for these receptors in allergen uptake and a common fate for these preparations inside the DC. Further experiments were done to show the effect of Der p 1 and Der p 1 glycoforms on TSLP secretion by epithelial cells, which is known to induce Th2 driven immune responses. The results show that TSLP secretion decreases significantly when epithelial cells are challenged with deglycosylated preparations of the same allergen. This may indicate a change in the outcome of adaptive immune responses when a deglycosylated allergen challenges epithelial cells. In conclusion, this work has demonstrated a link between allergenicity and glycosylation patterns in allergens. It therefore appears that mannosylation is the dominant sugar moiety associated with allergen uptake and recognition by humans DCs, and this is in line with MR being the main receptor involved in allergen binding by these cells

The role of glycosylation in allergen recognition

This project is an attempt to have a better understanding of the role of carbohydrates in recognition and uptake of allergens by the innate immune system. Glycosylation analysis of different allergens like Der p 1, Fel d 1, Ara h 1, Ber e 1, Der p 2, Bla g 2, Can f 1, Bromelain and Papain was made using labelled lectins that can detect specific carbohydrate moieties on proteins to reveal their pattern of glycosylation. These experiments showed that all major allergens are glycosylated and this represented a major first step towards demonstrating a link between glycosylation and allergen recognition by the innate immune system. N- and O-glycosylation patterns were predicted in different allergens and a difference in mannosylation and fucosylation was detected between allergens and non-allergenic proteins. We found that the main dominant sugars on allergens are 1,2 1,3 and 1,6 mannose, as detected by GNA lectin. We have also showed that Der p 1 and Der p 2 possess 1,3 fucose in their natural forms, thus concluding that Der p1 and Der p 2 have part of the CCDs which are epitope structures for IgE. O-glycosylation in allergens was also studied giving a better understanding of the whole glycan structure in allergens. The role of mannosylation in allergen recognition by the immune system was investigated further. Different methods, including recombinant expression, enzymatic and chemical deglycosylation, were optimised to produce glycoforms of Der p 1. A recombinant preparation of Der p 1 produced in Pichia pastoris was used as a hypermannosylated form of the allergen. These glycoforms served as useful tools in addressing the nature of glycoallergen recognition by looking at the uptake of hyper- and hypo-glycosylated preparations by DCs, with confocal microscopy, ELISA and FACS as readouts. Results indicate that deglycosylated forms of Der p 1 exhibited minimal uptake by DCs compared to the natural and hyperglycosylated recombinant allergen. Comparative analysis of the hypermannosylated preparation of Der p 1 and its natural counterpart, possessing less mannan, showed that the recombinant form was taken up more readily by DCs at 37°C and at 4°C. We also showed that these glycoforms bind to the MR subfragment CTLD 4-7-FC, the C-type lectin carbohydrate recognition domain. This binding significantly decreased when the Der p 1 allergen was deglycosylated. These results were confirmed further using confocal microscopy imaging which also showed that recombinant Der p 1 uptake is immediate, starting at 5 mins of incubation and that a higher quantity accumulates inside the DC compared to natural allergen. Recombinant and natural Der p 1 both co-localised with MR, DC-SIGN and LAMP-2 lysosomal marker, suggesting a key role for these receptors in allergen uptake and a common fate for these preparations inside the DC. Further experiments were done to show the effect of Der p 1 and Der p 1 glycoforms on TSLP secretion by epithelial cells, which is known to induce Th2 driven immune responses. The results show that TSLP secretion decreases significantly when epithelial cells are challenged with deglycosylated preparations of the same allergen. This may indicate a change in the outcome of adaptive immune responses when a deglycosylated allergen challenges epithelial cells. In conclusion, this work has demonstrated a link between allergenicity and glycosylation patterns in allergens. It therefore appears that mannosylation is the dominant sugar moiety associated with allergen uptake and recognition by humans DCs, and this is in line with MR being the main receptor involved in allergen binding by these cells

The matricellular protein TSP1 promotes human and mouse endothelial cell senescence through CD47 and Nox1.

Senescent cells withdraw from the cell cycle and do not proliferate. The prevalence of senescent compared to normally functioning parenchymal cells increases with age, impairing tissue and organ homeostasis. A contentious principle governing this process has been the redox theory of aging. We linked matricellular protein thrombospondin 1 (TSP1) and its receptor CD47 to the activation of NADPH oxidase 1 (Nox1), but not of the other closely related Nox isoforms, and associated oxidative stress, and to senescence in human cells and aged tissue. In human endothelial cells, TSP1 promoted senescence and attenuated cell cycle progression and proliferation. At the molecular level, TSP1 increased Nox1-dependent generation of reactive oxygen species (ROS), leading to the increased abundance of the transcription factor p53. p53 mediated a DNA damage response that led to senescence through Rb and p21cip, both of which inhibit cell cycle progression. Nox1 inhibition blocked the ability of TSP1 to increase p53 nuclear localization and p21cip abundance and its ability to promote senescence. Mice lacking TSP1 showed decreases in ROS production, p21cip expression, p53 activity, and aging-induced senescence. Conversely, lung tissue from aging humans displayed increases in the abundance of vascular TSP1, Nox1, p53, and p21cip Finally, genetic ablation or pharmacological blockade of Nox1 in human endothelial cells attenuated TSP1-mediated ROS generation, restored cell cycle progression, and protected against senescence. Together, our results provide insights into the functional interplay between TSP1 and Nox1 in the regulation of endothelial senescence and suggest potential targets for controlling the aging process at the molecular level

Current (Food) allergenic risk assessment: is it fit for novel foods? status quo and identification of gaps

Food allergies are recognized as a global health concern. In order to protect allergic consumers from severe symptoms, allergenic risk assessment for well-known foods and foods containing genetically modified ingredients is installed. However, population is steadily growing and there is a rising need to provide adequate protein-based foods, including novel sources, not yet used for human consumption. In this context safety issues such as a potential increased allergenic risk need to be assessed before marketing novel food sources. Therefore, the established allergenic risk assessment for genetically modified organisms needs to be re-evaluated for its applicability for risk assessment of novel food proteins. Two different scenarios of allergic sensitization have to be assessed. The first scenario is the presence of already known allergenic structures in novel foods. For this, a comparative assessment can be performed and the range of cross-reactivity can be explored, while in the second scenario allergic reactions are observed toward so far novel allergenic structures and no reference material is available. This review summarizes the current analytical methods for allergenic risk assessment, highlighting the strengths and limitations of each method and discussing the gaps in this assessment that need to be addressed in the near future.Austrian Science Fund [FWF SFB F4603]; Ministry of Education, Science and Technological Development of the Republic of Serbia [OI172024]; MINECO, Spain [AGL2014-59771-R]; PROMAR: Projetos Pilotos e a Transformacao de Embarcacoes de Pesca [31-03-05-FEP-0060]info:eu-repo/semantics/publishedVersio

The mannose receptor negatively modulates the Toll-like receptor 4–aryl hydrocarbon receptor–indoleamine 2,3-dioxygenase axis in dendritic cells affecting T helper cell polarization

Background: Dendritic cells (DCs) are key players in the induction and re-elicitation of TH2 responses to allergens. We have previously shown that different C-type lectin receptors on DCs play a major role in allergen recognition and uptake. In particular, mannose receptor (MR), through modulation of Toll-like receptor (TLR) 4 signaling, can regulate indoleamine 2,3-dioxygenase (IDO) activity, favoring TH2 responses. Interestingly, the aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor with an emerging role in immune modulation, has been implicated in IDO activation in response to TLR stimulation. Objective: Here we investigated how allergens and lectins modulate the TLR4-AhR-IDO axis in human monocyte-derived DCs. Methods: Using a combination of genomics, proteomics, and immunologic studies, we investigated the role of MR and AhR in IDO regulation and its effect on T helper cell differentiation. Results: We have demonstrated that LPS induces both IDO isoforms (IDO1 and IDO2) in DCs, with partial involvement of AhR. Additionally, we found that, like mannan, different airborne allergens can effectively downregulate TLR4-induced IDO1 and IDO2 expression, most likely through binding to the MR. Mannose-based ligands were also able to downregulate IL-12p70 production by DCs, affecting T helper cell polarization. Interestingly, AhR and some components of the noncanonical nuclear factor κB pathway were shown to be downregulated after MR engagement, which could explain the regulatory effects of MR on IDO expression. Conclusion: Our work demonstrates a key role for MR in the modulation of the TLR4-AhR-IDO axis, which has a significant effect on DC behavior and the development of immune responses against allergens

MEF2C-MYOCD and Leiomodin1 Suppression by miRNA-214 Promotes Smooth Muscle Cell Phenotype Switching in Pulmonary Arterial Hypertension.

BACKGROUND: Vascular hyperproliferative disorders are characterized by excessive smooth muscle cell (SMC) proliferation leading to vessel remodeling and occlusion. In pulmonary arterial hypertension (PAH), SMC phenotype switching from a terminally differentiated contractile to synthetic state is gaining traction as our understanding of the disease progression improves. While maintenance of SMC contractile phenotype is reportedly orchestrated by a MEF2C-myocardin (MYOCD) interplay, little is known regarding molecular control at this nexus. Moreover, the burgeoning interest in microRNAs (miRs) provides the basis for exploring their modulation of MEF2C-MYOCD signaling, and in turn, a pro-proliferative, synthetic SMC phenotype. We hypothesized that suppression of SMC contractile phenotype in pulmonary hypertension is mediated by miR-214 via repression of the MEF2C-MYOCD-leiomodin1 (LMOD1) signaling axis. METHODS AND RESULTS: In SMCs isolated from a PAH patient cohort and commercially obtained hPASMCs exposed to hypoxia, miR-214 expression was monitored by qRT-PCR. miR-214 was upregulated in PAH- vs. control subject hPASMCs as well as in commercially obtained hPASMCs exposed to hypoxia. These increases in miR-214 were paralleled by MEF2C, MYOCD and SMC contractile protein downregulation. Of these, LMOD1 and MEF2C were directly targeted by the miR. Mir-214 overexpression mimicked the PAH profile, downregulating MEF2C and LMOD1. AntagomiR-214 abrogated hypoxia-induced suppression of the contractile phenotype and its attendant proliferation. Anti-miR-214 also restored PAH-PASMCs to a contractile phenotype seen during vascular homeostasis. CONCLUSIONS: Our findings illustrate a key role for miR-214 in modulation of MEF2C-MYOCD-LMOD1 signaling and suggest that an antagonist of miR-214 could mitigate SMC phenotype changes and proliferation in vascular hyperproliferative disorders including PAH

NADPH oxidases: key modulators in aging and age-related cardiovascular diseases?

Reactive oxygen species (ROS) and oxidative stress have long been linked to aging and diseases prominent in the elderly such as hypertension, atherosclerosis, diabetes and atrial fibrillation (AF). NADPH oxidases (Nox) are a major source of ROS in the vasculature and are key players in mediating redox signalling under physiological and pathophysiological conditions. In this review, we focus on the Nox-mediated ROS signalling pathways involved in the regulation of 'longevity genes' and recapitulate their role in age-associated vascular changes and in the development of age-related cardiovascular diseases (CVDs). This review is predicated on burgeoning knowledge that Nox-derived ROS propagate tightly regulated yet varied signalling pathways, which, at the cellular level, may lead to diminished repair, the aging process and predisposition to CVDs. In addition, we briefly describe emerging Nox therapies and their potential in improving the health of the elderly population

The Glycosylation Pattern of Common Allergens: The Recognition and Uptake of Der p 1 by Epithelial and Dendritic Cells Is Carbohydrate Dependent

Allergens are initiators of both innate and adaptive immune responses. They are recognised at the site of entry by epithelial and dendritic cells (DCs), both of which activate innate inflammatory circuits that can collectively induce Th2 immune responses. In an attempt to have a better understanding of the role of carbohydrates in the recognition and uptake of allergens by the innate immune system, we defined common glycosylation patterns in major allergens. This was done using labelled lectins and showed that allergens like Der p 1 (Dermatophagoides pteronyssinus group 1), Fel d 1 (Felis domisticus), Ara h 1 (Arachis hypogaea), Der p 2 (Dermatophagoides pteronyssinus group 2), Bla g 2 (Blattella germanica) and Can f 1 (Canis familiaris) are glycosylated and that the main dominant sugars on these allergens are 1–2, 1–3 and 1–6 mannose. These observations are in line with recent reports implicating the mannose receptor (MR) in allergen recognition and uptake by DCs and suggesting a major link between glycosylation and allergen recognition. We then looked at TSLP (Thymic Stromal Lymphopoietin) cytokine secretion by lung epithelia upon encountering natural Der p 1 allergen. TSLP is suggested to drive DC maturation in support of allergic hypersensitivity reactions. Our data showed an increase in TSLP secretion by lung epithelia upon stimulation with natural Der p 1 which was carbohydrate dependent. The deglycosylated preparation of Der p 1 exhibited minimal uptake by DCs compared to the natural and hyperglycosylated recombinant counterparts, with the latter being taken up more readily than the other preparations. Collectively, our data indicate that carbohydrate moieties on allergens play a vital role in their recognition by innate immune cells, implicating them in downstream deleterious Th2 cell activation and IgE production

Photocrosslinkable gelatin hydrogels modulate the production of the major pro-inflammatory cytokine, TNF-α, by human mononuclear cells

Hydrogels are an attractive class of biomaterials in tissue engineering due to their inherently compatible properties for cell culture. Gelatin methacryloyl (GelMA) has shown significant promise in the fields of tissue engineering and drug delivery, as its physical properties can be precisely tuned depending on the specific application. There is a growing appreciation for the interaction between biomaterials and cells of the immune system with the increasing usage of biomaterials for in vivo applications. Here, we addressed the current lack of information regarding the immunogenicity of photocrosslinked GelMA. We investigated the ability of human mononuclear cells to mount inflammatory responses in the context of a GelMA hydrogel platform. Using lipopolysaccharide to stimulate a pro-inflammatory immune response, we found tumour necrosis factor-α (TNF-α) expression was suppressed in GelMA culture conditions. Our findings have important implications on the future use of GelMA and highlight the significance of investigating the potential immune-modulatory properties of biomaterials

Pulmonary vascular research institute GoDeep: a meta-registry merging deep phenotyping datafrom international PH reference centers

The Pulmonary Vascular Research Institute GoDeep meta-registry is a collaboration of pulmonary hypertension (PH) reference centers across the globe. Merging worldwide PH data in a central meta-registry to allow advanced analysis of the heterogeneity of PH and its groups/subgroups on a worldwide geographical, ethnical, and etiological landscape (ClinTrial. gov NCT05329714). Retrospective and prospective PH patient data (diagnosis based on catheterization; individuals with exclusion of PH are included as a comparator group) are mapped to a common clinical parameter set of more than 350 items, anonymized and electronically exported to a central server. Use and access is decided by the GoDeep steering board, where each center has one vote. As of April 2022, GoDeep comprised 15,742 individuals with 1.9 million data points from eight PH centers. Geographic distribution comprises 3990 enrollees (25%) from America and 11,752 (75%) from Europe. Eighty-nine perecent were diagnosed with PH and 11% were classified as not PH and provided a comparator group. The retrospective observation period is an average of 3.5 years (standard error of the mean 0.04), with 1159 PH patients followed for over 10 years. Pulmonary arterial hypertension represents the largest PH group (42.6%), followed by Group 2 (21.7%), Group 3 (17.3%), Group 4 (15.2%), and Group 5 (3.3%). The age distribution spans several decades, with patients 60 years or older comprising 60%. The majority of patients met an intermediate risk profile upon diagnosis. Data entry from a further six centers is ongoing, and negotiations with >10 centers worldwide have commenced. Using electronic interface-based automated retrospective and prospective data transfer, GoDeep aims to provide in-depth epidemiological and etiological understanding of PH and its various groups/subgroups on a global scale, offering insights for improved management