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Supervisors:
Dr Claire Smith and Dr Tereza Masonou
Project Description:
Background
CD44 is a marker of airway inflammation and a crucial neutrophil ligand. Recent research in our laboratory using a neutrophil-epithelial migration model highlights the role of CD44 in influencing neutrophil adherence and promoting an activated neutrophil phenotype. Our work showed that older adults have higher CD44 levels in nasal airway cells, resulting in fewer viable neutrophils adhering to the airway epithelium after SARS-CoV-2 infection. These neutrophils exhibit higher expression of CD11b, MPO, and NE, releasing elevated levels of myeloperoxidase and LDH, a cell death marker.
We have developed a CRISPR-Cas9 CD44 knockout respiratory epithelial cell line (Calu3) to understand the mechanisms driving CD44-mediated inflammation during infection and in chronic inflammatory airway diseases, such as asthma and cystic fibrosis.
Why do this?
Identifying CD44 as a key player in airway inflammation opens the door to developing new therapeutic strategies targeting CD44. Current treatments, such as corticosteroids, often have significant side effects, especially with long-term use. Targeting CD44 specifically may lead to treatments with fewer side effects, providing safer options for patients.
Aims/Objectives
• Investigate CD44's role in promoting an inflammatory phenotype in neutrophils in the airway.
• Examine the functional properties of neutrophils migrating in a CD44-deficient airway.
• Provide insights into potential therapeutics targeting CD44-related airway pathologies by evaluating the inflammatory response and mediator release in a CD44 knockout model.
Methods
• Use a neutrophil-epithelial migration model to study the interaction between neutrophils and CRISPR-Cas9 CD44 knockout epithelial cells.
• Investigate the impact of CD44 deficiency on neutrophil adherence and function, including measuring IL8, IL6, and myeloperoxidase activity in response to LPS stimulation, and assessing phagocytic ability and NET production of neutrophils.
• Examine CD44 expression in primary respiratory epithelial cells from children with chronic airway inflammation, asthma, and cystic fibrosis. Use siRNA to generate CD44 knockouts to assess neutrophil functionality differences.
• Integrate existing scRNAseq datasets from children with inflammatory airway disease using bioinformatics approaches.
• Test the efficacy of hyaluronan inhibitors to block CD44 interactions, reducing inflammatory responses and improving neutrophil function in healthy and asthmatic airway cells.
Timeline
Year 1:
• Months 1-3: Develop and validate CRISPR-Cas9 CD44 knockout Calu3 cell lines.
• Months 3-6: Optimize the neutrophil-epithelial migration model.
• Months 6-12: Conduct preliminary experiments on neutrophil adherence, phenotype, and inflammatory mediator release in response to LPS.
Year 2:
• Months 12-18: Quantify and characterize inflammatory responses in CD44 knockout models.
• Months 18-24: Test the efficacy of hyaluronan inhibitors in blocking CD44 interactions and reducing inflammation.
Year 3:
• Months 24-30: Confirm findings and validate therapeutic approaches.
• Months 30-36: Prepare and submit manuscripts, disseminate findings through conferences, and finalize the PhD thesis.
References
1. Borregaard, N. (2010) Immunity, 33(5), pp. 657–670. doi: 10.1016/j.immuni.2010.11.011.
2. Leir, S. H. et al. (2000) ‘American Journal of Physiology - Lung Cellular and Molecular Physiology, 278(6), pp. 1129–1137. doi: 10.1152/ajplung.2000.278.6.l1129.
3. Reeves, S. R. et al. (2018) Respiratory Research, 19(1), pp. 1–11. doi: 10.1186/s12931-018-0849-1.
4. Robinson, E., et al(2023) Thorax. doi: 10.1136/thorax-2023-220081
Contact Information:
Dr Claire Smith AND/OR Dr Tereza Msonou