Temporal regulation of human reactive astrocytes reveals their capacity for antigen presentation Restricted; Files Only
Hill, Emily (Spring 2025)
Abstract
Astrocytes respond to external or inflammatory stimuli, often produced by traumatic injury, ischemia, or neurological disease. These responses include transcriptomic, morphologic, and functional changes that together comprise a reactive phenotype. While reactive astrocytes can be induced via multiple intrinsic and extrinsic triggers, several important questions remain, including which genomic processes initiate and maintain the reactive state, how the temporal duration of stimuli affects astrocytes, and whether the process is reversible. To explore each of these questions, we investigated the plasticity of human astrocyte reactivity in both human cortical organoids (hCOs) and primary fetal tissues. We observed robust inflammatory transcriptomic and chromatin accessibility signatures when exposed to inflammatory cytokines. Importantly, however, these signatures were highly dependent on the duration of inflammatory exposure, ranging from acute (one day) to chronic (three months) durations. Both acute and chronic stages of human astrocyte reactivity possess uniquely accessible transcription factor binding motifs, coupled with distinct differential gene expression profiles. Next, to investigate the reversibility of human inflammatory astrocytes, we allowed hCOs exposed to either acute or chronic periods of inflammatory cytokines to undergo a period of withdrawal. Following removal of exogenous cytokines, both acute and chronically reactive human astrocytes display remarkable plasticity and return to a quiescent genomic state within days. One of our most surprising findings was that human astrocytes exposed to chronic durations of inflammatory cytokines greatly induce expression of major histocompatibility complex (MHC) class II genes, which encode receptors that typically only present on professional antigen-presenting cells. We confirmed human astrocyte MHC class II protein expression in both primary fetal tissue and hCOs via immunostaining and fluorescence-activated cell sorting (FACS). Finally, to understand the functionality of MHCII, we performed coimmunoprecipitation for MHCII and its invariant chain, CD74, as well as mass spectrometry to identify potential antigens MHCII can present. Altogether, this study offers new insight into the immune roles of human reactive astrocyte phenotypes after long-term inflammation.
Table of Contents
Chapter 1: Motivation and significance 1
1.1 Thesis motivation and significance of this work 1
1.2 Thesis roadmap 2
Chapter 2: Background on astrocyte biology and reactivity 4
2.1 Astrogenesis 4
2.2 Neuron-glia interactions 5
2.2.1 Formation of the synapse 5
2.2.2 Synaptic elimination 6
2.2.3 Synaptic function and regulation 7
2.2.4 Metabolic coupling 7
2.3 Astrocyte contributions to neurodevelopmental diseases 10
2.3.1 Dysfunction in timing of astrogenesis 10
2.3.2 Contributions of astrocytes to dysfunction in synapse formation/morphology 11
2.3.3 Dysfunction in cooperative metabolism between neurons and astrocytes 13
2.4 Astrocyte reactivity 14
2.4.1 MHC Class I and II presentation in the nervous system 16
2.5 In vitro models of human brain development and biology 17
2.6 Modeling human astrocyte reactivity 21
2.6.1 Astrocyte reactivity in hCOs 21
2.6.2 Astrocyte reactivity in human fetal cultures 21
Chapter 3: Plasticity of reactive astrocytes exposed to long-term inflammatory conditions 23
3.1 Introduction 23
3.2 Results 25
3.2.1 Exposure to inflammatory cytokines induces reactive astrocytes in hCOs 25
3.2.2 Temporal dynamics of inflammatory reactive astrocytes at genomic scales 29
3.2.3 Reactive astrocytes can return to quiescence after both acute and chronic inflammatory conditions. 33
Chapter 4: MHC class II expression and function in chronic inflammatory reactive astrocytes 37
4.1 MHCII expression in astrocytes 37
4.2 Astrocytes have detectable MHCII protein after chronic inflammation 37
4.3 Astrocytes can express both early inflammatory genes and MHCII 38
4.4 Inflammatory conditions lead to an increase in cell death, but not a rise in proliferation. 38
4.5 Reactive astrocytes present MHCII on cell surface. 43
4.6 MHCII complexes with functional molecules in reactive astrocytes, such as CD74, the invariant chain. 43
4.7: Reactive astrocytes may present endogenous peptide 47
Chapter 5: Materials and Methods 49
5.1 Human cortical organoid cultures 49
5.2 Tissue Dissociation 50
5.3 Immunopanning 50
5.4 RNA extraction 50
5.4.1 cDNA Library preparation 51
5.4.2 RNA-seq Analysis 51
5.5 ATAC-seq Library Preparation 51
5.5.1 ATAC Analysis 52
5.6 Single Cell Prep with CMOs 52
5.6.1 10X Analysis 53
5.7 Immunofluorescence 53
5.7.1 Imaging 54
5.7.2 Antibodies List: 54
5.8 EdU Staining for FACS 54
5.9 Fluorescence-activated cell sorting 54
5.9.1 FACS antibodies: 55
5.10 CoIP and Western Blot 55
5.11 Mass Spectrometry 56
Chapter 6: Discussion 57
6.1: Immature astrocytes can become reactive 57
6.2: Reactive astrocytes possess an extremely plastic genomic and transcriptomic landscape 58
6.3: Why do chronic reactive astrocytes upregulate MHCII? 58
6.4: Conclusions 60
References 62
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File download under embargo until 22 May 2026 | 2025-04-10 20:02:13 -0400 | File download under embargo until 22 May 2026 |
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