The Impact of Ptpn11(Shp2)E76K Mutation on Macrophage Phagocytosis Open Access

Abstract

Juvenile myelomonocytic leukemia (JMML) is an aggressive pediatric myeloproliferative disorder characterized by the overproduction of myelomonocytic cells, with approximately 34% of non-syndromic JMML cases arising from somatic mutations in Ptpn11, the gene encoding SHP-2. (Tartaglia et al. 2003; Loh et al. 2004) Macrophage-mediated phagocytosis is essential for the immune system's response to infections and the clearance of cellular debris. This study investigates the phagocytic activity of bone marrow-derived macrophages (BMDMs) from wild type and Shp2 E76K mice.

Macrophage-mediated phagocytosis is essential for the immune system's response to infections and the clearance of cellular debris. This study evaluated the phagocytic activities of BMDMs from wild type and Shp2 E76K mice against lineage-negative (Lin-) cells, HL60 cells, and E. coli particles utilizing a flow cytometry-based assay. BMDMs were cultivated in DMEM enriched with L-cell conditioned medium to promote differentiation. The phagocytic response was analyzed by exposing macrophages to varying ratios of Lin- and HL60 cells, as well as E. coli BioParticles. 

Our findings revealed that WT macrophages exhibited a significantly greater phagocytic rate towards HL60 cells and E. coli, highlighting their pivotal role in immune defense and the facilitation of antigen presentation, essential for triggering adaptive anti-tumor immunity. In contrast, the Ptpn11E76K mutation in macrophages did not significantly affect their phagocytic capacity against Lin- progenitor cells. Notably, it was not the mutations in the macrophages, but rather the Lin- target cells harboring the Ptpn11E76K mutation, that led to increased phagocytosis, suggesting an enhanced phagocytic attraction or identification due to the mutation. We hope to deepen our understanding of leukemia pathogenesis through the thorough exploration of these findings.

Table of Contents

Table of Contents

Introduction 1-4

Methods 5-13

Results 14-23

Discussion 24-25

References 26-29

Figures and Tables

1.    Figure 1. Flow Cytometry Gating Strategy for Cell Size, Singlet Discrimination, and Viability 10

2.    Figure 2. Gating of Phagocytic Macrophage Populations Using CD11b-PCy-7 and F4/80-APC Markers 11

3.    Figure 3. Gating of Phagocytic Macrophages 12

4.    Figure 4. Phagocytic Uptake of pHrodo™ Green E. coli BioParticles™ by Wild Type and Ptpn11E76K Mutated Macrophages at Different Particle Concentrations 14

5.    Figure 5. Phagocytotic Comparison between Wild Type and Ptpn11E76K Mutated Macrophages Engulfing HL60 Cells 16

6.    Table 1. Experimental Setup for the Assessment of Phagocytic Activity of Wild Type and Ptpn11E76K Mutated Macrophages against Wild Type and Ptpn11E76K Mutated Lineage-Negative Target Cells 18

7.    Table 2. Example Calculated Phagocytic Activity Percentage Across Experimental Conditions in Macrophages 20

8.    Figure 6. Comparison of Normalized Phagocytosis Ratios Across Different Experimental Conditions for Lineage-Negative Cells 21

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Cell
Keyword	Macrophage Phagocytosis
Committee Chair / Thesis Advisor	Cheng-Kui Qu, Emory University
Committee Members	LaTonia Taliaferro-Smith, Emory University David Civitello, Emory University

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