Date of Award

January 2017

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Marie E. Egan

Abstract

We hypothesize that intrinsic defects in cystic fibrosis (CF) macrophages affect their ability to adequately respond to extracellular stimuli, affecting their phenotypic response and contributing to a hyper-inflammatory state.

Peripheral blood-derived monocytes from healthy donors (HD) (n=10) as well as CF subjects (n=10) were differentiated into macrophages using RPMI media with 50 ng/mL of MCSF for 10 days. Cells were treated with 100 ng/mL of LPS plus 20 ng/mL of INF-gamma to activate the M1 phenotype or 20 ng/mL of IL-4 to activate the M2 phenotype. Cells were treated for 24 hours in preparation for RNA isolation and 3 hours in preparation for protein isolation. RNA was isolated and reverse transcribed to cDNA in preparation for qualitative, reverse transcription polymerase chain reaction (qRT-PCR). qRT-PCR was performed using IL-6, IL-1B, and TNF-alpha primers as M1 markers and TGF-B1, MRC, PPAR-gamma as M2 markers. Expression levels were performed in seven experiments, each using a different set of HD and CF subjects, utilizing 8.0 x 10^5 to 1.0 x 10^6 cells per well, while three protein experiment were done using different sets of HD and CF subjects.

Stimulation of CF macrophages induces greater phosphorylation of proteins that regulate macrophage activation, as compared to HD macrophages. When CF macrophages are challenged with LPS/INF-gamma, they show greater phosphorylation of STAT1 protein as compared to HD macrophages. Similarly, when CF macrophages are challenged with IL-4, they exhibit greater phosphorylation of STAT6 protein, as compared to HD macrophages. Finally, CF macrophages show greater phosphorylation of AKT protein as compared to HD macrophages when stimulated with both M1 and M2 cytokines. As expected, HD macrophages (n=7, experiments) demonstrated appropriate plasticity and polarization with increased expression of M1 markers and decreased expression of M2 markers in response to LPS/INF-gamma. Furthermore, they demonstrated modestly increased expression of M2 markers and decreased expression of M1 markers in response to IL-4. In marked contrast, when stimulated with LPS/INF-gamma CF macrophages (n=7, experiments) demonstrated hyper-inflammation with dramatically increased expression levels of M1 markers as well as aberrant polarization as evidenced by increased expression levels of some M2 makers. Aberrant polarization was further characterized by increased expression of M1 markers in the presence of IL-4 in addition to increased expression of M2 markers.

These data suggest that lack of functioning CFTR in macrophages leads to the inability of macrophages to adequately respond to environmental cues and activate into appropriate phenotypes suggesting there is an intrinsic cellular defect. Moreover, the mechanism(s) that underlie these aberrant responses likely involve altered intracellular signal transduction which is currently under investigation.

Comments

This thesis is restricted to Yale network users only. It will be made publicly available on 06/12/2019

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