Peripheral immune traffic to the AD brain: role of soluble TNF signaling and peripheral inflammation Open Access

Abstract

Recent evidence has shifted our understanding of the origin and longevity of microglia and perivascular macrophages as well as the role the immune system plays in brain health and disease. However, it remains unclear when during the course of disease peripheral immune cells traffic to the brain. Subsets of immune cells from both the innate and adaptive immune system have proposed protective and harmful roles in mediation of neuroinflammatory disease, while chronic peripheral inflammation has been linked to increased risk of developing neuroinflammatory disease. We hypothesize that the inflammatory phenotype of peripheral immune cells changes as a function of age and in response to progressive neurodegeneration. However, it is not yet understood how chronically inflamed central and peripheral immune cell populations impact brain health and disease course outcome. At terminal stages of neuroinflammatory diseases such as Alzheimer's disease (AD), evidence suggests that populations of peripheral immune cells exist within the parenchyma; however, the kinetics of trafficking have yet to be established. Understanding when subsets of immune cells traffic to the brain will reveal novel opportunities for disease-modulating therapies. Here we used mouse models to investigate the extent to which markers of inflammation and immune cell population profiles centrally and peripherally change as a function of AD-like pathology progression, and whether these changes represent adaptive or maladaptive responses. Trafficking of peripheral immune cells to inflamed tissues is regulated by several mechanisms, including cytokine and chemokine signaling. The cytokine soluble Tumor Necrosis Factor (sTNF) has been shown in multiple studies to be elevated in patients with AD, and sTNF regulates the permeability of the blood-brain barrier (BBB). sTNF is produced by both brain-resident and peripheral immune cells. Therefore, we hypothesize that sTNF is a key mediator of peripheral immune cell contributions to AD-like pathology. Here we have investigated the extent to which inhibition of sTNF signaling in the periphery and within the brain modulates not only neuroinflammation and peripheral immune cell trafficking to the CNS, but also the extent of progression of AD-like pathology. In addition, we investigated the extent to which chronic peripheral inflammation induced by high-caloric diet or lipopolysaccharide, alters brain neuroinflammatory kinetics and AD-like pathology to support a role for therapeutic intervention of sTNF signaling in the clinic to delay onset or slow progression of AD.

Table of Contents

Chapter 1: Peripheral and brain immune mediators in brain health and AD

1.1 Introduction 2

1.2 Contributors to the CNS inflammatory environment 3

1.2.1 Microglia 3

1.2.2 Macrophages 6

1.2.3 T cells 9

1.2.4 Astrocytes 13

1.3 Regulation of Neuroinflammation 16

1.3.1 Lymphatics and Deep Cervical Lymph Nodes 17

1.3.2 Blood-brain Barrier 19

1.3.3 Cytokine signaling - focus on tumor necrosis factor 21

1.4 Role of peripheral inflammation in AD 23

1.4.1 Inflammatory risk factors for AD 24

1.4.2 Alteration in trafficking of peripheral immune cells to the brain 25

1.5 Dissertation overview 27

Chapter 2: Peripheral administration of the soluble TNF inhibitor XPro1595 modifies brain immune cell profiles, decreases beta-amyloid plaque load, and rescues impaired long-term potentiation in 5xFAD mice

2.1 Context, Author's Contribution, and Acknowledgement of Reproduction 31

2.2 Abstract 31

2.3 Significance statement 32

2.4 Introduction 33

2.5 Methods 35

2.5.1 Animals 35

2.5.2 Multiplexed Immunoassays 36

2.5.3 Brain Dissociation for Immune Cell Isolation 37

2.5.4 Spleen Dissociation for Immune Cell Isolation 38

2.5.5 Deep Cervical Lymph Node Dissociation (DCLN) for Immune Cell Isolation 38

2.5.6 Multi-color Flow Cytometry 38

2.5.7 XPro1595 Administration 40

2.5.8 Electrophysiology 41

2.5.9 Brain Dissection for RNA Extraction 43

2.5.10 Real-time PCR (QPCR) 44

2.5.11 Brain Dissection for Immunohistochemistry and Histological Analysis 45

2.5.12 Statistical Analysis 46

2.6 Results 46

2.6.1 Age-dependent pro-inflammatory cytokine expression in 5xFAD mice 46

2.6.2 Altered peripheral immune cell populations in the pro-inflammatory Tg CNS environment 47

2.6.3 Altered naïve and effector T cell populations in deep cervical lymph node of Tg mice 49

2.6.4 Inhibition of soluble TNF decreases brain populations of activated CD11b⁺ immune cells and CD4⁺ T cells 50

2.6.5 Inhibition of soluble TNF increases the naïve T cell populations in the DCLNs of Tg mice 53

2.6.6 Inhibition of soluble TNF decreases amyloid beta and cytokine expression in the hippocampus of Tg mice 54

2.6.7. XPro1595 blocked LTP deficits in 5xFAD mice 55

2.7 Discussion 56

2.8 Summary 64

Chapter 3: The role of high caloric diet on central and peripheral inflammation and immune cell profiles

3.1 Context, Author's Contribution, and Acknowledgement of Reproduction 88

3.2 Abstract 88

3.3 Introduction 89

3.4 Methods 92

3.4.1 Animals 92

3.4.2 High-fat high-fructose (HFHF) diet 93

3.4.3 Low-dose (LD) LPS administration 94

3.4.4 Brain Dissociation for Immune Cell Isolation 94

3.4.5 Plasma and Peripheral blood mononuclear cell (PBMC) isolation 95

3.4.6 Multi-color Flow Cytometry 95

3.4.7 Bioluminescence imaging (BLI) Measurements 97

3.4.8 Statistical Analysis 98

3.5 Results 99

3.5.1 PBMC populations in male mice are altered by low-dose LPS-induced peripheral inflammation 99

3.5.2 HFHF diet induced dynamic changes within CD11b⁺ myeloid PBMC populations in both male and female C57Bl/6Jmice 101

3.5.3 T cell populations show resistance to changes induced by HFHF-dependent peripheral inflammation 103

3.5.4 PBMC populations in female 5xSBE mice are resilient to changes induced by diet-dependent chronic peripheral inflammation 105

3.5.6 HFHF diet reduces frequency of CD3⁺ T cell populations trafficking to the brain in CCR2 RFP/+ mice 106

3.5.7 HFHF diet alters CD11b⁺ microglia/macrophage population within and trafficking to the brain 107

3.5.8 Peripheral inflammation from both LD LPS and HFHF diet alters the neuroinflammatory response in 5xTg_SBE mice 109

3.6 Discussion 110

3.7 Summary 116

Chapter 4: Conclusions

4.1 Summary of Results 142

4.1.1 Peripheral immune cell trafficking patterns in a mouse model of AD-like pathology 142

4.1.2 Regulation of peripheral immune cell trafficking patterns via soluble TNF (sTNF)-dependent signaling 143

4.1.3 Effects of chronic inflammation on peripheral immune cell trafficking and brain neuroinflammatory response 145

4.2 Implications and Future Directions 146

4.2.1 Modulation of peripheral immune cell activation and/or their trafficking may be a potential therapeutic target 146

4.2.2 Second-hit model of progression of AD-like pathology 148

4.2.3 Assessing changes in peripheral immune cell populations in at-risk human populations 149

4.2.4 Revitalization of an aging immune system 150

4.3 Conclusions 152

Appendix: Complete list of publications to which the author has contributed during her graduate training 153

References 154

Figure Index

Figure 1.1: Modeling the effects of peripheral inflammation and soluble TNF signaling on peripheral immune cell traffic to the brain and AD-like pathology 28

Supplementary figure S2.1. XPro1595 penetrates into the brain parenchyma when dosed peripherally 66

Table 2.1: Increased expression of inflammatory cytokines in the brain of Tg mice without evidence of inflammation in the CSF 68

Table 2.2: Inhibition of sTNF partially mitigates inflammatory profile in Tg mice 70

Figure 2.1. Altered peripheral immune cell populations in the pro-inflammatory AD-like brain environment 72

Figure 2.2. Altered naïve and effector T cell populations in deep cervical lymph nodes of 5xFAD mice 75

Figure 2.3. Inhibition of soluble TNF with XPro1595 decreases populations of activated CD11b⁺ immune cells and CD4⁺ T cells in the brain of 5xFAD mice 77

Figure 2.4. Inhibition of soluble TNF with XPro1595 increases the naïve T cell population in the deep cervical lymph nodes (DCLNs) of 5xFAD mice 79

Figure 2.5. Inhibition of soluble TNF with XPro1595 decreases amyloid beta in the subiculum and decreases pro-inflammatory mRNA in the hippocampus of 5xFAD mice 81

Figure 2.6. Inhibition of soluble TNF with peripheral XPro1595 administration in vivo rescued LTP impairment in 5xFAD mice 83

Figure 2.7. Specific targeting of sTNF/TNFR1 signaling rescues AD-like synaptic deficits and modulates AD-like associated immune cell population alterations 85

Table 3.1 - Special diet ingredient composition 118

Table 3.2 - Calculated nutrient composition and fatty acid profile 120

Supplementary figure S3.1 - PBMC gating 122

Supplementary figure S3.2 - CCR2 gating 124

Figure 3.1 PBMC populations in male mice are altered by low-dose LPS-induced peripheral inflammation 126

Figure 3.2 HFHF diet induced dynamic changes within CD11b⁺ myeloid PBMC populations in both male C57Bl/6Jmice 128

Figure 3.3 T cell populations show resistance to changes induced by HFHF-dependent peripheral inflammation 130

Figure 3.4 PBMC populations in female 5xSBE mice are resilient to changes induced by diet-dependent chronic peripheral inflammation 132

Figure 3.5 HFHF diet reduces frequency of CD3⁺ T cell populations trafficking to the brain in CCR2 RFP/+ mice 134

Figure 3.6 HFHF diet alters CD11b⁺ microglia/macrophage population within and trafficking to the brain 136

Figure 3.7 Peripheral inflammation from both LD LPS and HFHF diet alters the neuroinflammatory response in 5xTg_SBE mice 139

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience
Keyword	immune cell trafficking soluble TNF neuroinflammation T cells peripheral inflammation Alzheimer's disease
Committee Chair / Thesis Advisor	Tansey, Malu, Emory University
Committee Members	Walker, Lary C, Emory University Levey, Allan I, Emory University Ford, Mandy L, Emory University Caudle, William Michael, Emory University

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