Date of Defense
8-11-2024 9:00 AM
Location
F3-238
Document Type
Dissertation Defense
Degree Name
Doctor of Philosophy (PhD)
College
COS
Department
Biology
First Advisor
Dr. Khalid Muhammad
Keywords
Atopic Dermatitis (AD), Regulatory B cells (Bregs), IL-10, NFATc1, IgE
Abstract
Atopic dermatitis (AD) is the most prevalent non-infectious skin allergies with approximately 10-30% adults and 40% children being affected worldwide. It is clinically characterized by eczema tic lesions and elevated IgE levels and is a multifactorial disease that is heterogeneous in nature. AD results in a dominant Th2 polarized immune response. Until date, there is no cure for this disease. B cells are key players of humoral immune responses and contribute significantly to allergic responses through IgE secretion. However, recently a subset of B cells termed regulatory B cells (Bregs) were discovered that were shown to have a protective role by secretion of anti-inflammatory cytokine IL-10. Furthermore, NFATc1 is a transcription factor that functions through a calcium dependent calcineurin/calmodulin pathway to exert its effects on target genes. Nfatc1 has an important role in the development and function of the immune system. Previous studies suggested that NFATc1 can inhibit IL-10 by binding to its gene in association with HDAC1. Hence the aim of this research was to study the role of B cells during allergic responses using AD as a model for skin allergies. To achieve this, we used transgenic mouse lacking Nfatc1 in B cells (Nfatc1f/f x mb1cre) and wild-type (WT) mice to induce AD model and study the outcome of the lacking NFATc1 in B cells responses during allergies. AD was induced by calcipotriol, a vitamin D3 analogue, on mouse ears by a method that was previously established. The calcipotriol induced AD response was evaluated through ear thickness to measure inflammation followed by histological examination. IgE levels in serum were also determined by ELISA. The ear draining lymph nodes (dLNs) were then used to study the various immune cell populations through immunophenotyping by flow cytometry. Finally, B cells were isolated from dLNs, and RNA sequencing was performed to study in detail the mechanisms that were involved in B cells during such immune responses at a transcriptome level. It was seen that Nfatc1f/f x mb1cre AD mice exhibited a diminished AD phenotype compared to WT AD mice displaying weaker ear swelling and lower epidermal thickening and cellular infiltration in histological analysis and no change in IgE levels at the start and end of calcipotriol mediated AD induction. Further the Nfatc1f/f x mb1cre AD mice showcased a higher percentage of IL-10 producing B220+CD5+CD1d+ Breg cells supporting the fact that lack of NFATc1 promoted B cells to differentiate into Bregs producing more anti-inflammatory cytokine IL-10 and hence alleviated the AD symptoms. At the transcriptome level, compared to WT AD mice, Nfatc1f/f x mb1cre AD mice demonstrated genes that were expressed differently which promoted B cell development and enhanced its response to stress and stimulus. This research sheds light on the possibility of developing NFATc1 as a potential molecular target to alleviate AD responses without compromising B cell functions and by promoting higher endogenous production of anti-inflammatory IL-10. However further research is required on models of human cells and strategies to manipulate NFATc1 in-vivo to establish this putative molecular target.
Included in
ROLE OF B CELL MEDIATED IMMUNE RESPONSES DURING SKIN ‘ATOPIC DERMATITIS’ DISEASE
F3-238
Atopic dermatitis (AD) is the most prevalent non-infectious skin allergies with approximately 10-30% adults and 40% children being affected worldwide. It is clinically characterized by eczema tic lesions and elevated IgE levels and is a multifactorial disease that is heterogeneous in nature. AD results in a dominant Th2 polarized immune response. Until date, there is no cure for this disease. B cells are key players of humoral immune responses and contribute significantly to allergic responses through IgE secretion. However, recently a subset of B cells termed regulatory B cells (Bregs) were discovered that were shown to have a protective role by secretion of anti-inflammatory cytokine IL-10. Furthermore, NFATc1 is a transcription factor that functions through a calcium dependent calcineurin/calmodulin pathway to exert its effects on target genes. Nfatc1 has an important role in the development and function of the immune system. Previous studies suggested that NFATc1 can inhibit IL-10 by binding to its gene in association with HDAC1. Hence the aim of this research was to study the role of B cells during allergic responses using AD as a model for skin allergies. To achieve this, we used transgenic mouse lacking Nfatc1 in B cells (Nfatc1f/f x mb1cre) and wild-type (WT) mice to induce AD model and study the outcome of the lacking NFATc1 in B cells responses during allergies. AD was induced by calcipotriol, a vitamin D3 analogue, on mouse ears by a method that was previously established. The calcipotriol induced AD response was evaluated through ear thickness to measure inflammation followed by histological examination. IgE levels in serum were also determined by ELISA. The ear draining lymph nodes (dLNs) were then used to study the various immune cell populations through immunophenotyping by flow cytometry. Finally, B cells were isolated from dLNs, and RNA sequencing was performed to study in detail the mechanisms that were involved in B cells during such immune responses at a transcriptome level. It was seen that Nfatc1f/f x mb1cre AD mice exhibited a diminished AD phenotype compared to WT AD mice displaying weaker ear swelling and lower epidermal thickening and cellular infiltration in histological analysis and no change in IgE levels at the start and end of calcipotriol mediated AD induction. Further the Nfatc1f/f x mb1cre AD mice showcased a higher percentage of IL-10 producing B220+CD5+CD1d+ Breg cells supporting the fact that lack of NFATc1 promoted B cells to differentiate into Bregs producing more anti-inflammatory cytokine IL-10 and hence alleviated the AD symptoms. At the transcriptome level, compared to WT AD mice, Nfatc1f/f x mb1cre AD mice demonstrated genes that were expressed differently which promoted B cell development and enhanced its response to stress and stimulus. This research sheds light on the possibility of developing NFATc1 as a potential molecular target to alleviate AD responses without compromising B cell functions and by promoting higher endogenous production of anti-inflammatory IL-10. However further research is required on models of human cells and strategies to manipulate NFATc1 in-vivo to establish this putative molecular target.