Date of Award

2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Microbiology, Immunology, and Biochemistry

Research Advisor

Ae-Kyung Yi

Committee

David Brand; Elizabeth Fitzpatrick; Marko Radic; Maureen McGargill

Keywords

Hypersensitivity, Myeloid, PKD1, Pneumonitis

Abstract

Introduction: Hypersensitivity pneumonitis (HP) is an inflammation driven interstitial lung disease characterized by inflammation of the interstitium, bronchioles, and alveoli of the lung that leads to granuloma formation. Myeloid lineage immune cells play a critical role in the early and late stages of the disease, and this activity is mediated through toll-like receptors (TLRs) expressed by these cells. Protein kinase D1 (PKD1) has previously been identified as a critical signaling molecule in the TLR signaling pathways of all TLR family members except for TLR3. Our previous research found that activation of protein kinase D1 (PKD1) in the lungs following exposures to Saccharopolyspora rectivirgula, a common antigen trigger for HP, contributes to acute pulmonary inflammation, IL-17A expression in the lungs, and development of HP. The purpose of our research is to elucidate the impact of PKD1 activation in myeloid cells on the onset, progression, and severity of S. rectivirgula induced HP. The method Methods: WT mice and mice in which PKD1 is knocked out in myeloid cells (the PKD1mKO group) were exposed intranasally to S. rectivirgula over short (2 hr, 6 hr, 24 hr) or extended periods of time (3-15 weeks). Disease onset and severity was analyzed by measuring mRNA expression and protein levels of inflammatory cytokines and chemokines in the lung using quantitative real-time PCR and enzyme-linked immunosorbent assays respectively. Flow cytometry was used to assess populations of different immune cells as well as surface marker expression. Masson’s Trichrome staining and hematoxylin and eosin staining was performed on tissue sections from the lung to assess alveolitis, granuloma formation, and fibrosis. Results: Compared to PKD1-sufficient WT controls, PKD1mKO animals displayed a more suppressed immune response, with lower levels of cytokines such as IL-6, IL-17A, TNFα, and IFNγ. Chemokine levels of CCL2, CCL3, CCL4, CXCL1, CXCL2, and CXCL10 were also lowered. Neutrophilic alveolitis was decreased, as was granuloma formation and fibrosis over longer exposure periods. Minor alterations in B cell populations and levels of immunoglobulin subtypes were altered, with PKD1mKO lungs containing more immature B cells and less plasma cells than the WT counterparts, and immunoglobulin M being more expressed in the PKD1mKO group. In the PKD1mKO group, MHC-II expression was reduced on myeloid cells, which corresponded T cell activation marker CD69 also being reduced in this group. Finally, CXCR3+CCR6+ nonconventional Th1 cells in the lungs were significantly reduced in PKD1mKO mice after repeated S. rectivirgula exposures. Discussion: Our results demonstrate that PKD1 in myeloid-lineage cells plays an essential role in the onset and progression of HP caused by repeated exposure to S. rectivirgula. The mechanisms by which this occurs include: contributing to Th1/Th17 polarizing proinflammatory responses, alveolitis, the accumulation of pathogenic nonconventional Th1 cells in the lungs, impacting B cell maturation and immunoglobulin class switching, and granuloma formation and fibrosis in the airways.

ORCID

0000-0003-3545-9420

DOI

10.21007/etd.cghs.2025.0700

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