Adenosine Regulates the Formation of Macrophage Extracellular Traps Through the A2a Signaling Pathway Open Access

Cicka, Markus (Spring 2022)

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Heart disease is one of the leading causes of death in developed countries across the world. During a myocardial infarction, immune cells, such as neutrophils and macrophages enter the site of ischemia and undergo various processes leading to increased inflammation in the heart and worse patient outcomes. Neutrophil extracellular traps or NETs, which are a mechanism where neutrophils eject their DNA into the extracellular space ensnaring pathogens, are currently recognized to play an important role in myocardial infarctions. More recently, macrophages have been discovered to undergo a process of DNA extrusion similar to NETosis. Here, I characterize murine bone marrow derived macrophages and their ability to produce extracellular traps (METs), stimulated by LPS treatment. I also investigate if the anti-inflammatory compound, adenosine, can inhibit the formation of these extracellular traps through the adenosine receptor signaling pathways. Determining how MET formation occurs will allow greater understanding of the pathogenesis of aberrant inflammation and assist in developing new therapies to increase the survivability of myocardial infarctions in patients.

Table of Contents

Table of Contents


1a. Immune Cell Recruitment in Cardiac Inflammation and Repair...1

1b. Neutrophil Extracellular Traps: A Newly Recognized Form of Neutrophil Activation...2

1c. Macrophages Also Produce Extracellular Traps...3

1d. The Advantages of Bone Marrow Derived Macrophages in Experimental Models...4

1e. Macrophages Exist in Different Polarized States...5

1f. Limitations of Macrophage M1 and M2 Classification...5

1g. NETs and METs are Stimulated by Similar Compounds...6

1h. Extracellular Trap Inhibitors...7

1i. Adenosine’s Role as an Anti-Inflammatory Nucleoside...7

2. Experimental Aims...9

3. Materials and Methods...11

3a. Animal Model...11

3b. Macrophage Media...11

3c. Bone Marrow Macrophage Culturing...11

3d. Macrophage Harvesting...12

3e. Immunofluorescent Staining...13

3f. Macrophage SYTOX assay...15

3g. Western Blotting...16

3h. Two Step Reverse Transcription Quantitative Polymerase Chain Reaction...17

3i. Cell Fluorescence Calculations...20

3j. Image Processing and Statistical Analysis...20

4. Results...22

4a. Characterization of Bone Marrow Derived Macrophages...22

4b. Adenosine Inhibits Macrophages Produce Extracellular Traps Induced by LPS...23

4c. Expression of CitH3 in Response to Various Treatments...24

4d. Increased M1-Like Gene Expression of LPS-Treated Macrophages...25

5. Discussion...26

6. Figures...29

6a. Figure 1: Extracellular Trap Formation Schematic...29

6b. Figure 2: Macrophage Differentiation and Culturing Schematic...30

6c. Figure 3: 100 ng/mL M-CSF Produces Greatest Macrophage Confluence on Day 7...31

6d. Figure 4: Murine Bone Marrow Derived Macrophages Express Macrophage Markers CD11b and F4/80 and Extracellular Trap Enzyme PAD4...32

6e. Figure 5: Murine Bone Marrow Derived Macrophages Express A1, A2a, A2b, and A3 Adenosine Receptors Through IF Staining..33

6f. Figure 6: Western Blots of Murine Macrophage A1, A2a, A2b, and A3 Adenosine Receptors...34

6g. Figure 7: Adenosine Inhibit LPS-Induced Murine Macrophage Extracellular Trap Formation Through the A2a Adenosine Receptor...35

6h. Figure 8: A1, A2b, and A3 Adenosine Receptors Do Not Play a Key Role of Modulating Macrophage Extracellular Trap Formation...36

6i. Figure 9: Citrullination of the H3 Histone Increases in Response to 60 Minutes LPS Treatment...38 

6j. Figure 10: Macrophages Treated With LPS Express M1-Like Genes...39

7. Non-Standard Abbreviations...40

8. References...42 

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