Streamlined Approach to Identify Novel Immunomodulatory Agents to Mitigate Ischemic Stroke in a Primary Microglia Model Público

Abstract

Background

Microglia activation is associated with neuroinflammation, pathogenesis of secondary brain damage, and cerebral ischemia reperfusion injury post ischemic stroke. Baricitinib, a selective Janus kinase 1/2 inhibitor with potent anti-inflammation effects in treating rheumatoid arthritis, alopecia areata, and hospitalized COVID-19, may mitigate inflammation induced by microglia polarization, especially to classic pro-inflammatory M1 phenotype. This study aimed to investigate if baricitinib can prevent or reverse neuroinflammation in a primary cell model of ischemic stroke.

Methods

A primary cell model of ischemic stroke was established using differentiated monocyte-derived microglia-like cells (MDMG) from human blood buffy coat. The microglia activation marker expression profile was assessed by flow cytometry analysis. Further sub-gating with Flow Jo was performed to determine the microglia activation changes morphologically. The cytotoxicity of baricitinib and microglia viability inhibition were analyzed by the MTS assay.

Results

The data revealed that baricitinib did not confer apparent toxicity in microglia at physiologically relevant concentrations in humans. Baricitinib significantly suppressed LPS-induced upregulation of microglia M1 phenotype-related markers CD40, CD32, CD86, CD16, HLA-DR, and activated state marker CD163 when administered as a post-treatment. Pre-treatment with baricitinib attenuated the expression of microglia activation markers CD40, CD16, CD163, and HLA-DR. Furthermore, the morphological activation was ameliorated by the baricitinib treatment at clinically relevant dosages.

Conclusion

Baricitinib could significantly reduce key markers of neuroinflammation and suppress polarization morphologically in a primary microglia model. Baricitinib can reduce ischemic stroke-like, LPS-induced inflammation in microglia, a proof of principle that baricitinib has therapeutic potential in mitigating ischemia-triggered neuronal inflammation.

Table of Contents

Table of Contents

Introduction 1

Methods 5

Blood Collection and PBMC isolation 5

Monocyte Isolation and Monocyte-Derived Microglia-like cells differentiation 6

In vitro treatment of MDMG with baricitinib and LPS for activation markers 7

Assessment of cell viability 8

Flow cytometry 9

Immunofluorescence Staining 12

Statistical analysis 12

Results 12

Modified Gavegnano differentiation method yields MDMG with microglia morphological phenotypes 12

Effects of baricitinib on the viability of microglia 14

Baricitinib attenuates the activation of LPS stimulated microglia 15

Discussion 22

Reference 29

About this Honors Thesis

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School	Emory College
Department	Neuroscience and Behavioral Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Neuroscience Health Sciences, Immunology Health Sciences, Pharmacology
Palabra Clave	Ischemic Stroke Neuroinflammation
Committee Chair / Thesis Advisor	Gavegnano, Christina, Emory University
Committee Members	Thompson, Rick , Emory University Grizzell, Alex , Emory University

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