ALTERNATIVELY ACTIVATED MACROPHAGES IN PSEUDOMONAS AERUGINOSA PNEUMONIA: MODULATION OF THE NF-ΚB SIGNALING PATHWAY AND THE IMMUNOMODULATORY ROLE OF ARGINASE-1

Author

Dalia Haydar, University of KentuckyFollow

Author ORCID Identifier

https://orcid.org/0000-0002-7959-2699

Date Available

7-25-2018

Year of Publication

2018

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Pharmacy

Department/School/Program

Pharmaceutical Sciences

First Advisor

Dr. David Feola

Abstract

Background: Azithromycin polarizes macrophages into an alternative phenotype and promotes a regulated immunity. Arginase is an important effector of these macrophages believed to play an essential role in decreasing injury and promoting repair.

Hypothesis: Decreases in inflammation in response to Pseudomonas aeruginosa (PA) pneumonia achieved by polarizing macrophages to an alternative phenotype is dependent upon the production of arginase.

Methods: Requirement of arginase was examined by pharmacological inhibition using S-(2-boronoethyl)- l-cysteine (BEC) or l-norvaline and by infecting arginase-1 conditional knock-out mice (Arg1^flox/flox;Lyz2-cre (Arg1^Δm)) with PA intratracheally. Arg1^ΔM and control Arg1^flox/flox mice were then dosed with azithromycin daily via oral gavage beginning four days prior to infection. Analysis of weight loss in addition to characterization of inflammatory cells and cytokine production via flow cytometry was performed. Macrophages were then stimulated with LPS and polarized with IL4/13, IFNγ, or azithromycin plus IFNγ. Western blot for signaling mediators, p65 translocation assay, and immunofluorescence were performed.

Results: Myeloid arginase-1 deletion resulted in greater morbidity along with more severe inflammatory response compared to the Arg1^flox/flox mice. Arg1^Δm mice had greater numbers of neutrophils, macrophages, and lymphocytes in their airways and lymph nodes compared to the Arg1^flox/flox mice. Conversely, global arginase inhibition resulted in greater weight loss along with greater neutrophil and macrophage infiltration compared to Arg1^Δm mice. BEC and l-norvaline treated mice had higher numbers of lymphocytes in their lymph nodes with variable effects on airway lymphocyte counts. Azithromycin treatment comparably reduced the acute inflammatory responses in both Arg1^Δm and Arg1^flox/flox mice. To evaluate this mechanism, we show in vitro that azithromycin decreases NF-κB activation by preventing p65 nuclear translocation and by decreasing STAT1 activation in a concentration-dependent manner. These effects were reversed with IKKβ inhibition.

Conclusions: Myeloid arginase is essential for control of inflammatory responses in PA pneumonia with potentially different effects of other cellular sources demonstrated with global arginase inhibition. Azithromycin reduces excessive inflammation even in the absence of arginase, potentially through a cross-inhibitory mechanism involving STAT1 and NF-κB pathways through IKKβ.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2018.328

Recommended Citation

Haydar, Dalia, "ALTERNATIVELY ACTIVATED MACROPHAGES IN PSEUDOMONAS AERUGINOSA PNEUMONIA: MODULATION OF THE NF-ΚB SIGNALING PATHWAY AND THE IMMUNOMODULATORY ROLE OF ARGINASE-1" (2018). Theses and Dissertations--Pharmacy. 90.
https://uknowledge.uky.edu/pharmacy_etds/90