th2 Modulation CRSwNP

Overview

Chronic rhinosinusitis (CRS) is a condition of persistent sinonasal mucosal inflammation which affects 11.9% of the US population. Mepolizumab is newly approved to treat chronic rhinosinusitis with nasal polyps (CRSwNP, the spaces inside nose and head are swollen and inflamed) and acts booking interleukin-5 (IL-5) a protein implicated in the inflammatory process. We aim to use Single-cell RNA sequencing (RNA-Seq, a method of genetically 'barcoding' cells to allow gene expression to be profiled at the level of individual cells) to study the effects of IL-5 blockade on the generation and maintenance of nasal adaptive immune responses, in CRS subjects.

Purpose: CRS is a poorly understood disease with suboptimal therapeutic strategies and a high disease burden. Adaptive immunity in the form of Th2 polarized T cell responses and B cell IgE class switching, and plasma cell infiltration, are a core component of eosinophilic CRS and asthma, yet our understanding of adaptive immunity in CRS remains limited. Modulating the Th2 response, through IL-5 blockade, leads to mixed results for CRSwNP patients. There are a number of patients who achieve improved asthma control with IL-5 blockade therapies that fail to see the same improvements in their nasal disease, outlining the currently limited understanding of factors involved in disease activity, disease endotypes, and the effects of modulating the Th2 response in this tissue environment. Improved understanding of nasal tissue immunity modulation by IL-5 blockade at a cellular level is an essential step towards appropriate patient selection and development of new targeted therapeutics. Single-cell transcriptomics is powerful tool for characterizing tissue immune landscapes and functional variations, has as yet been poorly utilized beyond studies of nasal epithelium in CRS Hypothesis * IL-5 blockade stimulates increased activity of nasal tissue T cells \> peripheral blood T cells, and promotes increased tissue T cell Th2 polarization * IL-5 blockade stimulates increased B cell germinal center activity in nasal secondary and tertiary lymphoid structures, through increased Tfh-B cell interactions and increased maturation to tissue antibody-secreting cells through BLIMP1 upregulation in Germinal Centre B cells. Objectives 1. Characterise the local mucosal B cell response in tertiary (nasal) and secondary (postnasal space) lymphoid organs in CRSwNP, compared with circulating immunity, through visualization and modeling of germinal center activity, cellular support for germinal center activity and assessment of B cell clonal selection, class-switching, fate, and somatic hypermutation using single-cell transcriptomics, single-cell BCR (B- cell receptor) sequencing and spatial resolution through confocal microscopy. 2. Use single-cell transcriptomics and VDJ (variable, diversity, and joining) repertoire analysis to characterize the effects of Th2 modulation, through IL-5 blockade, on the formation and maintenance of nasal T and B cell adaptive immune memory. 3. Localize nasal sources of pro-inflammatory signaling in CRSwNP in the presence of IL-5 blockade, using single-cell transcriptomics, confocal microscopy, and nasal cytokine analysis. 4. Compare nasal pro-inflammatory cell subsets with paired peripheral blood samples by single cell transcriptomics, to correlate activity with biomarker candidates. Research design The proposed study is a prospective experimental medicine study using IL-5 blockade as a way of investigating the effects of Th2 modulation on the generation of local tissue adaptive immunity in CRSwNP. Participants will be classified in: * Treatment group:20 subjects with CRSwNP and asthma that will start Mepolizumab treatment * Disease control group: 10 subjects with CRSsNP without asthma that will not start Mepolizumab and will continue their standard of care treatment. * Control group: 10 healthy subjects without any sinuses disease This study involves three study visits for the Treatment arm (Baseline, weeks 6, and 30), two visits for disease control subjects, and one (Baseline) visit for Healthy control subjects. Participants on study medication will receive 100mg of mepolizumab every 4 weeks subcutaneously. Treatment Arm: CRSwNP (NPS 1-8) and asthma undergoing IL-5 blockade (Mepolizumab) • Week 0: Recruitment, screening, and initiating standard-of-care therapy o Nasal and blood samples Week 6 - Pre-Mepolizumab commencement (Pre-treatment) Week 30 - After Mepolizumab commencement (On treatment) Control Arm: 1. Diseased Control: CRSsNP without asthma • Week 0: Recruitment, screening, and initiating standard-of care therapy o Nasal and blood samples Week 6 - After 6 weeks standard of care therapy 2. Healthy control • Week 0: Recruitment and screening, Nasal and blood samples Statistical analysis Single cell RNA sequencing data Sequenced data will be aligned with CellRanger v7.0. The resulting output files will be processed using a standard filtering and QC pipeline in Scanpy. The batch correction will be performed using scVI, and Leiden clustering will be performed. Cluster labels will be assigned using canonical marker genes and published gene signatures, with validation using a published dataset of sorted immune cells via the SingleR package. The downstream analysis will include, but not be limited to, gene set expression analysis (GSEA) of Hallmark and Gene Ontology genesets, differential abundance testing, weighted gene co-expression network analysis, and imputation of cell-cell interaction using CellphoneDB. TCR analysis will be performed using Scirpy, and BCR analysis with the Dandelion, and Immcantation suites

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Outcomes