Role of HLA-DR in dysregulation of B cell homeostasis in Dermatophagoides pteronyssinus-induced allergic rhinitis among Malays

Allergic rhinitis (AR) is an IgE-mediated nasal inflammatory disorder, commonly triggered by house dust mite, Dermatophagoides pteronyssinus. Der p 1 is a dominant allergenic protein derived from this species. The highly polymorphic human leukocyte antigen (HLA) genes were linked to AR predisposition/protection in different ethnicities. Variation in this gene alters epitope-binding affinities for HLA molecule, causing polarization of immune responses due to differential cytokines and antibodies secretions. The relationship between HLA and Der p 1 epitopes in the induction of specific cytokines and antibodies is unclear. During allergies, B cells perform various effector and regulatory functions. Dysregulation of B cells with Der p 1 stimulation under HLA regulation, remains unknown. This study aimed to use in silico approaches to predict relationship between Der p 1 epitopes and HLA-DR serotypes in cytokines/antibodies induction and evaluate in vitro changes in B and T cells of Der p 1-stimulated peripheral blood mononuclear cells (PBMCs) from AR patients and controls with selected HLADRB1 alleles. Full amino acid sequence of Der p 1 (NCBI protein database) was submitted into TepiTool, NetMHCIIpan 4.0, PREDIVAC and ABCpred, BCPREDS, BepiPred 2.0 to predict T and B cell epitopes, respectively. AllerTOP 2.0, AllergenFP 1.0 and AlgPred determined allergenicities of predicted peptides. IL4Pred, IL-10Pred and IFNepitope assessed T cell epitopes’ cytokine-inducing properties, while Bcipep and IgPred evaluated B cell epitopes’ antibody isotype secretions. Needleman-Wunsch alignment tool (NCBI BLAST) identified overlapping sequences between T and B cell epitopes. For in vitro study, Malay adults were recruited and screened for HLA-DRB1 alleles via polymerase chain reaction with sequence-specific primers using buccal DNA samples. Three subjects per HLA group for patients (DR7 and DR13) and controls (DR15) were selected for skin prick testing and blood sample collection. Der p 1- stimulated PBMCs were analyzed for B and T cells via flow cytometry. In silico analysis identified 32 CD4+ T cell epitopes, mostly IL-4 inducers (n = 11) and associated with allergenicity (p = 0.049). DR13 preferentially bound IL-4-inducing epitopes (75.00%) whereas DR7 mostly bound IL-10/IFN-γ-inducing epitopes (70.59%). DR15 bound almost equally IL-4 (42.86%) and IL-10/IFN-γ-inducing peptides (57.14%). From 13 predicted B cell epitopes, majority induced either IgE only (n = 4) or both IgE and IgA (n = 4). DR13-associated epitopes, cross-matched between T and B cell peptides for at least 60% similarities, were mostly IgE-inducing. Day 3 Der p 1-stimulated PBMCs showed lower percentages of IL-10-secreting T cells in AR patients (DR7+ and DR13+) compared to controls (DR15+) (27.08% vs 43.97%; p = 0.025). Memory B cell percentages were higher in DR13+ than DR7+ group (63.00% vs 10.77%; p = 0.022) whereas regulatory B cells were greater in DR7+ than DR13+ group (88.63% vs 32.53%; p = 0.022). In conclusion, in silico and in vitro studies supported HLA-DR13 as AR predisposing due to increased bindings of IL-4 and IgE-inducing epitopes, causing proliferation of memory B cells. Conversely, HLA-DR7 protected against AR via selective presentation of IL-10/IFN-γ-inducers, triggering differentiation of regulatory B cells. The DR15 controls maintained a healthy immune profile, suggesting a neutral outcome.

Text 116689 - edited.pdf Download (1MB) Official URL or Download Paper: http://ethesis.upm.edu.my/id/eprint/18292

Actions (login required)

View Item