Pharmacological Regulation of Macrophage Polarization Restricted; Files Only

Abstract

The tumor microenvironment has multiple types of immune cells that have specific functions of eliminating tumor from our body. Macrophages play an important role in regulating tumor growth and terminating tumor cells. M1 macrophages are known as anti-tumor macrophages and M2 macrophages are known as pro-tumor macrophages. Due to the effects of increased lactic acid in the tumor microenvironment, macrophages are driven to a M2 polarization state that favors tumor growth. Hence, we used the enzyme LOXCAT to decrease cellular lactic acid levels to attempt to repolarize macrophages from M2 to M1. We found that with the treatment LOXCAT, we repolarized M2 macrophages to express M1-specific phenotypes, including iNOS expression, elevated levels of M1-specific metabolites, and M1-associated cytokine expression. We also found that treating M0 unpolarized macrophages with LOXCAT has the effect of increasing itaconic acid, an important immune metabolite that has shown beneficial effects in treating certain cancer types. This study can serve as a basis for developing a new pharmacological regulation of macrophage polarization as well as itaconic acid production.

Table of Contents

Table of Contents

1.    Introduction ……1

1.1. The tumor microenvironment and macrophage cells ……1

1.2. Macrophage polarizations ……2

1.3. Lactic acid induced macrophage polarization ……4

1.4. Regulating macrophage polarizations ……5

2.    Results and Discussion ……7

2.1. Polarization specific metabolite detection in RAW264.7 macrophages ……7

2.2. Polarized RAW264.7 macrophages treated with LOXCAT ……8

2.3. Polarized RAW264.7 macrophages treated with LOXCAT and LOXCATmut ……10

3.    Conclusion and Future Directions ……14

4.    Materials and Methods ……15

4.1. Assay development for the macrophage polarization ……15

4.1.1.    Experimental setup ……15

4.1.2.    Sample prep ……15

4.2. Polarizing macrophages and treating them with LOXCAT ……16

4.2.1.    Western blot ……16

4.2.2.    Liquid chromatography- mass spectrometry ……16

4.2.3.    Nitric Oxide assay ……17

4.2.4.    Cytokine array ……17

4.3. Liquid chromatography- mass spectrometry data analysis ……17

References  ……18

About this Honors Thesis

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School	Emory College
Department	Chemistry
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Chemistry, Biochemistry
Stichwort	Macrophage polarization
Committee Chair / Thesis Advisor	Anupam Patgiri, Emory University
Committee Members	Antonio Brathwaite, Emory University Richard Himes, Emory University

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