DYNAMIC INTRAVITAL IMAGING of IMMUNE CELLS DURING the INITIATING EVENTS of EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS By

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DYNAMIC INTRAVITAL IMAGING OF IMMUNE CELLS DURING THE INITIATING EVENTS OF EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS by

DEBORAH SIM BARKAUSKAS

Submitted in partial fulfillment of the requirements

For the degree of Doctor of Philosophy

Dissertation Adviser: Dr. Alex Y. Huang

Department of Biomedical Engineering

CASE WESTERN RESERVE UNIVERSITY

May, 2014

CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

Deborah Sim Barkauskas

Candidate for the Doctor of Philosophy degree *

Signed James Basilion

(Chair of the committee)

Alex Y. Huang

John Letterio

Jeff Capadona

Date: March 17, 2014

We also certify that written approval has been obtained for any proprietary material contained therein.

DEDICATION

I dedicate this work to my father, Boon Kheng, who because of his own experience of

opportunities gained through education, ultimately made life sacrifices along with my mother, Lucy, to ensure that my siblings and I were able to receive a higher education.

I hope I have fulfilled his vision of empowerment through education. I also dedicate

this work to my siblings, for their love, support and who have influenced my life in so

many ways. Agnes for being the calming voice within me when I am stressed; Elizabeth

for being my rock and the best role model for women in engineering; David for being

consistently concerned for me and so supportive; Sandra, who in my mind was my first

teacher and who has continued to teach me throughout life; and Timothy who not

only showed me the rigor to excel but helped me make sense of life. Most of all, I

dedicated this work to Kestutis, the love of my life, who not only helped me analyze

hours of imaging data with Matlab, helped me construct microscope environment

chambers but provided me with delicious food, many laughs and most of all love and

support, I could not have done this without you.

Table of Contents

List of Figures ...... v

List of Supplemental Movies ...... vii

Acknowledgements ...... viii

Abstract ...... 1

1 Introduction ...... 2

Models for Studying Multiple Sclerosis in Rodents ...... 6

Anatomy of the Blood Brain Barrier ...... 8

Antigen Presenting Cells of the CNS ...... 11

Trafficking of Peripheral Immune Cells into the CNS ...... 17

Vessel Permeability is implicated in CNS Pathologies ...... 21

Role of Toxins in EAE Induction ...... 23

Role of Histamine in the CNS ...... 25

Summary ...... 29

2 Focal Intermittent CNS Vascular Leak Contributes to Sequential Immune Cell

Infiltration during the Asymptomatic Induction Phase of Experimental

Autoimmune Encephalomyelitis ...... 32

2.1 Abstract ...... 32

2.2 Introduction ...... 32

2.3 RESULTS ...... 36

2.3.1 Transient focal leaks in leptomeningeal vessels occur early during

EAE induction ...... 36

2.4 Discussion ...... 59

3 Extravascular CX3CR1+ Cells Extend Intravascular Dendritic Processes into

Intact CNS Vessel Lumen ...... 67

3.1 Abstract ...... 67

3.2 Introduction ...... 68

3.3 Results ...... 71

3.4 Discussion ...... 89

4 Image Analysis of Dynamic Intravital Multi-photon Imaging...... 96

4.1 Introduction ...... 96

4.2 Chemokine Influence on Motile Cells ...... 98

4.3 Point of Reference ...... 101

4.4 Angle of Approach ...... 102

4.5 Other Motility Parameters ...... 104

4.6 Conclusion ...... 110

5. Conclusion and Future Direction ...... 112

5.1. Conclusion ...... 112

5.2. Imaging to Detect Vessel Leaks in a Relapsing-Remitting Model ...... 113

5.3. Imaging of CX3CR1 Knock-out Mice ...... 114

iii

5.4. Signaling of Vessel Projections ...... 114

5.5. Determining the Role of Microglia in the Balance of T cell Regulation

versus Autoimmune Destruction of the CNS...... 118

APPENDIX I Material and Methods ...... 121

Mice ...... 121

EAE Induction ...... 122

Priming of Antigen-specific T cells...... 123

Spinal Trauma Model ...... 123

Tumor Cell Preparation and Injection ...... 124

Mouse Surgery and Preparation for Intravital Imaging ...... 125

Spinal Imaging ...... 126

Two-Photon Imaging Equipment, Data Acquisition ...... 127

Image Analysis ...... 127

Electron Microscopy ...... 130

Blood brain barrier leakiness analysis ...... 130

Immunofluorescence Histology ...... 131

Flow cytometry analysis ...... 131

Statistical consideration ...... 132

References ...... 134

List of Figures Figure 1: Proposed cellular infiltration events in EAE...... 5

Figure 2: Cellular components of the blood brain barrier. Adapted from Guilleman

60 and Ransohoff 6...... 10

Figure 3: Trafficking of immune cells from the periphery to the CNS. Adapted

from Goverman103 ...... 19

Figure 4: Sequence of events in the initiating phase of EAE ...... 31

Figure 5: PTx administration causes transient and focal CNS vessel leak...... 38

Figure 6: Insignificant number of leaks detected in control mouse...... 41

Figure 7: Focal vessel leaks induce localized activation of endogenous CNS

immune cells and increased DC accumulation...... 44

Figure 8: Peripherally activated T cells accumulate and migrate near sites of vessel leak and DC aggregate in an antigen-dependent manner...... 48

Figure 9: Characterization of OTII and 2D2 cells when co-activated during induction...... 50

Figure 10: Histamine receptor blockade inhibits early activation of CNS

phagocytes, diminishes DC and 2D2 accumulation, and blunts EAE severity. ... 55

Figure 11: Additional effects of HXYZ on immune cells during EAE induction. .. 57

Figure 12: Comparison of CX3CR1+ cellular morphology and baseline projection

frequency using different tissue preparations...... 73

Figure 13: Visualization, identification, and analysis of parenchymal CX3CR1+

cells with Intravital imaging methods...... 75

Figure 14: Intravital microscopy reveals persistent intravascular CX3CR1+

dendritic projections into intact CNS vessels...... 76

Figure 15: Immuno-histology and TEM confirm intravascular CX3CR1+ dendritic projections into intact CNS vessels...... 79

Figure 16: Vessel boundary outlined by different in vivo labeling techniques. .... 81

Figure 17: Increasing number of extravascular CX3CR1+ cells with intravascular dendritic projections in the brain during early EAE induction...... 82

Figure 18: Increasing number of extravascular CX3CR1+ cells with intravascular dendritic projections in the spine during early EAE induction...... 84

Figure 19: Intravascular projections decrease and then recover in aseptic traumatic spinal cord injury and do not increase in the CNS tumor microenvironment...... 86

Figure 20: From Matheu. Two-photon multidimensional data analysis parameters

302...... 97

Figure 21: Types of T Cell tracks ...... 105

Figure 22: Three phases of naıve T-cell priming by dendritic cells in peripheral lymph nodes, as revealed by multiphoton intravital microscopy from Mempel 318

...... 109

List of Supplemental Movies Supplemental movie 1: Illustration of a transient vessel leak with vessel

content persistence in parenchyma and of multiple transient vessel leaks

from the same point of origin...... 65

Supplemental movie 2: Transient vessel leaks originate from outside the dynamic field of imaging...... 65

Supplemental movie 3: Only CX3CR1+/GFP cells phagocytose TRITC vessel

dye...... 65

Supplemental movie 4: Infiltrating myelin specific T cells traffic to locations of previous vessel leak and CD11c+ accumulations...... 65

Supplemental movie 5: HXYZ treatment of EAE induced mice does not prevent

vessel leak but does prevent phagocytosis...... 66

Supplemental movie 6: Microglia morphology and distribution in non-inflamed

CNS parenchyma of a Cx3cr1+/GFP mouse...... 95

Supplemental movie 7: Dynamic dendritic motility of stationary extravascular

CX3CR1+ cells...... 95

Supplemental movie 8: Extravascular CX3CR1+ cells project dendrites into CNS

vessels...... 95

Supplemental movie 9: Three-dimensional view of the intravascular dendritic extension by extravascular CX3CR1+ cells...... 95

vii

Acknowledgements I first need to acknowledge the influence of the De La Salle Brothers of Australia

in my education. All the Brothers but especially Brother Ambrose Payne who first

believed in my academic abilities and Brother Vincent Cotter who spent many,

many hours teaching me physics and introducing me to basic electronics.

I would not be here at this point of my life without Dr. Roly Biggs and the

Movement Disorder Foundation of Australia who generously took a chance on

me and provided me with the scholarship to attend Case Western Reserve

University, to learn Functional Electrical Stimulation, and a chance to make a difference in the lives of people with tetraplegia.

I would like to thank Dr. Hunter Peckham who provided many words of wisdom, and the FES Center, which is composed of many knowledgeable and kind hearted people. They guided me through projects and understanding the clinical rehabilitation and academic systems.

I would like to thank my friends Dr. Chris Flask, Dr. Mark Griswold, Dr. Gheorghe

Mattescu and Dr. David Wilson from the Case Comprehensive Imaging Center, for generously providing me with medical imaging research experience, knowledge and equipment to my side projects, and NIH training support.

I would like to thank my PhD thesis committee: Dr. Jim Basilion, Dr. Jeff

Capadona, Dr. John Letterio, Dr. Niloy Bhadra for pushing me to be more

scientifically rigorous. In addition, I would like to thank the administrative staff and

viii

faculty of the Biomedical Engineering department for all the help over the years,

and the Pediatric and Pathology departments for their support.

I would like to thank Jay Myers for his wealth of knowledge, for allowing me to bounce ideas and for being my sanity check. Together we learnt Intravital surgeries, imaging, and analysis, I truly could not have done this work without you.

I need to thank Teresa Evans for all her neuroscience knowledge and her considerable IHC and EM expertise, I am still amazed we captured that EM data and didn’t get the champagne. Thank you for being a good friend especially in cookies and crafts. By association, I would like to thank Ross Anderson for helping with computers when we were freaking out about losing precious intravital data.

Thank you to Rachel Liou for being like a sister in the lab and such a great friend,

thank you for your help on so many experiments, your ability to help me

understand the boss was immensely helpful.

I would like to thank the hematology oncology fellows Stephanos Intzes, Agne

Petrosiute, Youmna Othman, internal medicine resident and post-doc Caryn

Tong, and surgery resident Iuli Bobanga for not only being great friends but for

allowing me to learn more about clinical life, and helping me through my multiple

hospital stays.

I would like to thank fellow graduate students for being such great friends and

making my research experience so rich and enjoyable. Dixon Dorand was a

Godsend as a neuro enthusiast and editor of all my final documents before boss got to read it, Nile Chang was such a brilliant mind and great support, Fred Allen had such amazing surgery hands, Alex Tong gave me comfort that the lab will be in good hands. In addition, I would like to thank graduate students that rotated through the lab that I maintained friendships with Bradley Martin, Ling Wu and

Charles Su.

I would like to thank Francesca Scrimieri and David Askew, post-docs who in addition to being great friends giving me lots of academic and career advice showed me how real science should be conducted and gave me very high standards to strive for.

Finally, I would like to thank the following research staff and students for their wonderful help, Joseph Nthale, Saada Eid, Matthew Tsao, Susie Min, Emi Bays,

Jennifer Tsai, Katherine Davis, Corrina Wolf, Yoshi Mondal, Jason Ashe, Ryan

Stultz, Tim Welliver, Nathan Teich, Rob Purgert.

Most of all I would like to thank you for being my mentor, Dr. Alex Huang. Thank you for giving me the opportunity to learn so much knowledge from you, you have gone above and beyond for me. Thank you for believing in me, I am eternally grateful. You are truly a remarkable and gifted man.

For the experiments and manuscript presented in Chapter 2, the authors

acknowledge the following for critique of this work: R. Ransohoff, F. Scrimieri, T.

Welliver, A. Tong, A. Petrosiute, J. Tsai, E. Bays, and Y. Othman. This work is

supported by the following grants: T32 NIH EB007509 (DSB); T32 NIH

GM007250 and the Wolstein Research Scholarship (RDD); T32 NIH GM007250

(TA); NIH R00EB011527 (KB); Steven G. Giallourakis AYA Cancer Research

Foundation (DA and RP); and to AYH from the Dana Foundation, the Gabrielle’s

Angels Foundation, the St. Baldrick’s Foundation, the Hyundai “Hope-on-Wheels”

Program and the Alex’s Lemonade Stand Foundation.

For the experiments and manuscript presented in Chapter 3, the authors

acknowledge the technical help of M. Yin and J. Drazba (Imaging Core,

Cleveland Clinic Foundation), N. Avishai and A. Avishai (Swagelock Center for

Surface Analysis, Case Western Reserve University), S. Min and R.D. Dorand in

obtaining EM and IHC data. The authors are grateful to D. Askew, K. Cooke, J.

Letterio, R. Liou, F. Scrimieri, D. Stearns and S.C. Wang for thorough review and thoughtful critiques of this manuscript. The following agencies provided funding support for this study: Dana Foundation (AYH), St. Baldrick’s Foundation (AYH &

AP), Hyundai Hope-on-Wheel’s Program (AYH & AP), Gabrielle’s Angel

Foundation (AYH), Cancer Research Institute (AYH), FRAP (AP), Case

Comprehensive Cancer Center P30CA043703 (AYH), 5T32EB7509 (DSB);

5K12HD057581 (AP), Alex’s Lemonade Stand Foundation (AYH), NCI R01

CA154656 (AYH), NIAID R21 AI092299 (AYH), NINDS RO1 NS25713 (JS) and

MSTP T32 GM007250 (TAE).

Dynamic Intravital Imaging of Immune Cells During the Initiating

Events of Experimental Autoimmune Encephalomyelitis

ABSTRACT By

DEBORAH SIM BARKAUSKAS

Experimental autoimmune encephalomyelitis (EAE) is a murine model of central

nervous system (CNS) inflammatory disease resulting in immune cell

accumulation, neuronal tissue destruction and paralysis.

With this work, we endeavor to reconcile the literature on EAE, from the action of

pertussis toxin (PTx) in the initiation of inflammation through to infiltration of CD4+

T cells. We show for the first time that localized, transient vessel leak initiates an inflammatory state within the CNS that results in microglia activation, and recruitment of dendritic cells (DCs). We also show for the first time that microglia are able to project extensions into the blood vessel with an intact blood brain barrier under non-inflammatory conditions and that the number of projections increase during the first 12 days of EAE induction. These resident microglia and peripheral DCs accumulate near vessel leaks and recruit myelin-specific T cells to cause subsequent tissue damage and plaque formation. This inflammatory process, serial immune cell infiltration and tissue destruction can be dampened by the use of anti-histamines to inhibit the activation of microglia at the onset of disease initiation.

1 Introduction EAE is driven by a population of myelin-specific, auto-reactive T cell and aspects

resemble multiple sclerosis (MS). EAE presents as a disease of the subpial

spinal cord in the lumbar region in areas in contact with the CSF, whereas MS

lesions are found in the brain with demyelination of the cerebral and cerebellar

cortex1. Involvement of the cortex in EAE has been difficult to prove given

standard techniques and therefore it has not been well studied. Via bioluminescence imaging of these myelin-specific T cells, it was shown that in

EAE, T cells do enter the brain by day 3 followed by infiltration into the spine by day 7 and both of these events happen before clinical disease onset around day

2, 3 10 , Microglia are the dominant surveying immune cell population in the CNS

parenchyma4. DCs are bone marrow derived professional antigen presenting

cells (APCs) and are virtually non-existent in the CNS parenchyma under non-

inflammatory conditions3, 5. In EAE, peripherally activated or transferred effector

T cells cross from blood circulation through either the choroid plexus into the

cerebrospinal fluid (CSF) or at Virchow-Robin Spaces (VRS) to a perivascular

location. The re-activation of T cells by macrophages or DCs in the CSF or VRS

are postulated to play an important role during the induction of EAE6, 7, 8.

However, antigen surveillance and presentation in the CNS is unique from other

tissues of the body due to: (1) suppression of microglia responses by neurons9,

and; (2) the blood-brain barrier (BBB) which poses a structural impediment for

peripheral antigen presenting cells (APCs)10, 11, 12. It has been shown that

parenchyma infiltrating DC are required for induction and progression of disease

in EAE5, 13, 14 although the route of entry of these cells into the CNS have not

been identified. Thus taken together, current dogma suggests that the

presentation of CNS tissue-derived myelin peptides by APCs and subsequent re- activation of myelin-specific T cells can occur randomly in the CNS and are not restricted to specific locations during inflammation.

Histologic studies of static brain tissues provide evidence that perivascular

CD11c+ APCs help to orchestrate immune responses by extending dendritic extensions into CNS parenchyma, presumably to sample and present brain- derived antigens to other immune cells in the VRS15. The observation that APCs

can insert dendritic extensions for antigen sampling in a separate anatomic

compartment has been shown in many organs of the body in contact with the

external environment16, 17, 18, 19, 20, 21, 22, 23. This new phenomenon provides a

location-specific mechanism by which T cells could enter the CNS. We hypothesize that microglia can sample directly into the blood vessel lumen without breaching vessel integrity, where circulating antigen-specific lymphocytes could potentially directly recognize antigens on the intravascular dendrites to result in intraluminal activation and subsequent extravasation.

Post mitotic neurons in the CNS are protected from peripheral pathogens and immune reactions by physical barriers and a host of immunosuppressive

cytokines including TGF-β24, 25. Intravital imaging has shown that in areas of microglia-macrophage clusters, there are vessel leaks during EAE25, 26, 27, 28, 29, 30.

Factors found in blood such as platelets31, fibrinogen32, 33, and complement

factors34, 35, have been shown to activate microglia within the CNS and lead to

neuronal destruction whereas erythropoietin36, 37 and von Willebrand factor38 have been shown to be beneficial for remyelination. There is growing evidence of the role of vascular leakiness in the etiology of neurodegenerative diseases39, in

particular MS, vascular dementia, epilepsy, Alzheimers, Parkinson’s and most

obviously stroke27, 28, 29, 30, 40. We propose that vascular dysfunction induced by

paracrine factors and toxins within circulation allow solutes to enter the CNS

parenchyma, initiating an inflammatory environment that leads to microglia

activation, infiltration of APCs and T cells, resulting in glia and neuronal

destruction (Fig 1.)

Together, these two hypotheses study the communication of the immune system

from both sides of the solute and cellular gate keeper of the CNS, the BBB. First,

we explore during a peripheral immune response against myelin antigen that is

largely found on the other side of the BBB, how and when the circulating immune

cells gain access to the CNS. Second, we investigate how microglia behind a

tightly regulated wall are able to either survey peripheral systemic pathogens or

recruit peripheral immune cells into the CNS during disease. Are vascular leaks a

cause or response to microglia extensions, or is there no correlation?

Figure 1: Proposed cellular infiltration events in EAE.

We propose that vascular dysfunction induced by paracrine factors within circulation allow solutes and toxins to enter the CNS parenchyma, initiating an inflammatory environment that leads to microglia activation, infiltration of peripheral APCs and T cells, resulting in glia and neuronal destruction

Models for Studying Multiple Sclerosis in Rodents

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system

(CNS). MS is a progressive neurodegenerative disease initiated by CNS

inflammation that is characterized by extensive immune cell infiltration, which

results in myelin and in some cases axonal destruction41, 42. The exact cause for

the inflammation remains unclear and the heterogeneity of demyelination

patterns coupled with differences in surrounding oligodendrocytes and microglia suggests that the initiating pathology may also be heterogeneous43 but is thought

to be an inflammatory T cell initiated and mediated disease44, 45. Experimental

autoimmune encephalomyelitis (EAE) is a murine model of CNS inflammatory

disease resulting in peripheral immune cell accumulation, neuronal tissue

destruction and ascending paralysis which resembles clinical MS41, 42. Even though induction of the disease is reproducible, by combining myelin peptides with an adjuvant to activate T cells in peripheral lymph nodes (LNs) in conjunction with a systemic inflammatory stimulus of pertussis toxin (PTx), the exact initiation events remain unknown.

From the first attempts at producing an animal model for MS, CNS homogenates were used in xenograft immunization protocols to induce paralysis41, 46. Iterations from these first animal models have been small and today there are four main myelin peptide based immunization protocols used to induce EAE:

(a) myelin basic protein (MBP) in Lewis rats induces a monophasic, self-

remitting form of paralysis47, 48

(b) myelin oligodendrocyte glycoprotein 35-55 (MOG) in C57BL/6J mice

induces a chronic-progressive form49

of disease50

(d) myelin-associated oligodendrocytic basic protein and 20,30-cyclic

nucleotide 30-phosphodiesterase for Balb/C and SJL/J mice51

Survey of methodology in the literature shows that there is a wide range of induction variables with differing amounts of peptide, complete Freuds adjuvant, amount and dosing schedule of pertussis toxin, and some include irradiation of the recipient animal. The interpretation of results becomes more problematic when it was discovered that primed, activated myelin specific T cells could be isolated from a donor rat, in vitro expanded and transferred into a recipient rat to cause disease52. Thus more variables were introduced including in vitro expansion conditions, the number of transferred T cells and the inflammatory conditions of the recipient mouse. Key features common to all models include:

(a) Ascending paralysis

(b) Peripherally activated or transferred effector myelin-specific, auto-reactive

T cells cross from blood circulation through the choroid plexus and Virchow-

Robin space into the parenchyma or CSF

peripherally activated or transferred effector T cells into the parenchyma or

CSF during the effector phase of disease induction

(d) Some toxic agent or irradiation is required to cause dysfunction of the BBB

There are viral models that induce CNS demyelinating lesions even though there is weak evidence of viral persistence as a cause of MS1. Theiler’s encephalomyelitis virus is the most common53 model used to study some aspects of CNS inflammation.

Anatomy of the Blood Brain Barrier

The pathway for antigen surveillance and presentation in the CNS is unique from other tissues of the body due to the BBB, which poses a structural impediment for APCs as well as antigens.

The BBB is a functional unit comprised of: endothelia; astrocyte end feet and

microglia that together, highly regulate the solutes and cells passing from the

blood vessel into the parenchyma54 (Fig. 2a). The endothelia of the CNS have

complex tight junctions, low endocytic activity and an absence of fenestrations, that prevent the passage of most polar and hydrophilic solutes from the blood into the brain55. Astrocytes have two main roles within the CNS: they regulate

ions and uptake excess neurotransmitters at the synapse56 they regulate blood

flow57 and the passage of cells and solutes into the parenchyma58. The basal

lamina surrounding the endothelial cells is contiguous with the plasma membrane

of astrocyte end-feet. Overlapping astrocytic end-feet have been shown by

ultrastructure 3D reconstruction to cover the entire length of endothelial basal lamina, with exception of areas with pericytes, and where microglia connect with the basal lamina59. The meninges surrounding the CNS consists of three distinct

layers: (1) the outermost dura mater; (2) the arachnoid mater; and (3) the pia

mater; collectively termed the leptomeninges. Astrocyte foot processes are found

up against the collagen rich pia mater, forming a barrier termed the glia limitans,

and the astrocytic end-feet ensheath blood vessels as they penetrate the

parenchyma from the sub arachnoid space (SAS) in the leptomeninges60 (Fig.

2b).

Whereas peripherally activated APCs and soluble antigens can drain directly to

the closest LN, in the CNS the cerebral spinal fluid (CSF) acts as the lymphatic

fluid. Clearance of soluble protein waste products occurs through the removal of

Figure 2: Cellular components of the blood brain barrier. Adapted from Guilleman61 and Ransohoff6.

(a) (b)

(a) The BBB is comprised of endothelial cells, astrocyte end feet and microglia. Pericytes and peripheral immune cells such as macrophages can be found distributed along the blood vessel between the endothelium and basal lamina61. (b) The blood-CSF (BCSF) barrier is different from the BBB in that astrocytes in the CNS parenchyma ensheath vessels with foot processes as they penetrate the CNS from the meninges, through the pial layer. The vessel on the subarachnoid side of the pial layer does not have the same barrier regulation6.

10 interstitial fluid termed the glymphatic system62. Para-arterial influx of CSF was

shown to enter the parenchyma, and wash through extracellular solutes from the

interstitial space to drain into the para-venous space where it is routed to cervical

lymph nodes and to the CSF at the glia limitans. Up to 50% of the CSF soluble

contents exit via the cribriform plate into deep cervical LNs, and the rest that

contains solutes and cells, drains at the spinal roots or the venous sinus directly

into venous circulation10, 11, 12. It was shown that activated T cells can be found in

the cervical lymph node in the early days of EAE induction and that removing the

cervical lymph nodes result in delayed onset and decrease in disease severity63 but how APCs in the parenchyma relate to the APCs in the cervical lymph node and their role in licensing and activation of T cells in the parenchyma is still unknown. Vessels that carry CSF along the cribriform plate are only 100-150 nm wide which is too small for dendritic cells, macrophage or microglia to migrate to the deep cervical lymph node11, 12.

Antigen Presenting Cells of the CNS

The antigen presenting cells (APCs) that have access to the brain parenchyma

are microglia, macrophages and dendritic cells. Microglia, defined as

Ly6Clo/CD45lo/Iba-1+/CX3CR1+, constitute ~10% of the cells in the CNS and

are the main immune cell population in the parenchyma under non-inflamed

conditions61, 64. Microglia are derived from the yolk sac before embryonic day 8,

once they have migrated to the CNS they are walled off from the blood vessel

11 and peripheral system by astrocytes65, 66. Microglia are self-renewing, replaced

by local proliferation throughout life67, 68. Microglia are evenly distributed throughout the CNS and have a ramified morphology to survey all of the CNS64.

There is heterogeneity in the protein expression and functional responses of

microglia based on location within the CNS4, 69. Microglia are also the first cells to respond to injury in the CNS and are thought to play a role in antigen presentation in the CNS70, 71, even though they have been shown to not be as

effective at presenting antigen as macrophages and dendritic cells. CX3CR1 is a

fractalkine receptor almost exclusively expressed in microglia in the CNS, while

NK cells, activated CD8 T cells, dendritic cells, and a subset of monocytes also

express the GFP marker in the peripheral tissues72.

Perivascular and meningeal macrophages are derived from circulating

Ly6Chi/CD45hi/CCR2+ monocytes and can sample CSF contents in the

arachnoid and Virchow-Robin space73. Monocyte development occurs initially in

the yolk sac like microglia but later in development, monocyte production moves

to the fetal liver, before finally residing in the bone marrow before birth74, 75. In the adult, monocytes arise from hematopoetic stem cells in the bone marrow76.

Interestingly, resident macrophages in peripheral tissue are primarily maintained

by local proliferation, similar to microglia61. Tissue resident macrophages are

exquisitely tuned immune cells that provide balanced homeostatic and regulatory

function (dead cell phagocytosis, antigen presentation, and soluble factor

regulation) within the organ systems they survey. However, subpopulations of

12 macrophages display a discrepancy in their antigen presentation capabilities

based on the tissue microenvironments in which they reside77. For example, lung

resident macrophages are exposed to the external environment and express high

amounts of esterase, TNF-α and MIP-1α; however, microglia do not express these cytokines in high amounts nor present antigen as efficiently because the majority of the available antigen under homeostatic conditions are self-derived, neurons regulate microglia function, and by-stander damage to neurons could result in severe deficits for the whole body.

Until recently, there were 2 known markers that differentiated microglia from peripheral macrophages because these cells express many of the same surface

receptors, including CD11b, CX3CR1, CD68 and Iba-14, 78. Distinction via

morphology has been generally accepted however could be problematic as

activated microglia have been reported to retract processes and either take on a

spindle shape or balled morphology61 similar to the morphology of peripheral

infiltrating macrophages. CD45 commonly assayed by FACS, is a membrane-

bound protein tyrosine phosphatase highly expressed on the surface of

monocytes and macrophages and in lower levels on microglia79, 80. However, it has been shown that levels of CD45 can increase on microglia during activation and inflammation81. The second marker is not widely used due to human toxicity

issues, mistletoe lectin is used in IHC and stains microglia but not

macrophages82. An extensive gene and microRNA array analysis of microglia

isolated from brain versus resident macrophages from various other organs,

13 identified P2ry12, Fcrls, Tmem119,Olfml3, Hexb and Tgfbr1 as unique microglial genes and 74 proteins that were uniquely expressed in microglia83. P2ry12 and

FCRLS were stringently analyzed for protein expression and FACS analysis revealed that antibodies to FCRLS and P2ry12 stained isolated adult microglia but did not stain CD11b-gated myeloid cells from murine spleen, bone marrow and peripheral blood. Via IHC, the P2ry12 signal co-localized with

CX3CR1GFP/+ microglia.

CD11c+ dendritic cells (DCs) are the most potent and effective antigen presenting cell84, defined as Ly6Chi /CD45hi/Iba-1-. DCs are bone marrow derived and are virtually non-existent in the CNS parenchyma under non-

inflamed conditions3, 85. It has also been shown that MHCII restricted to infiltrating

dendritic cells is required for induction and progression of disease in EAE13, 86.

There are 2 critical times within the induction of EAE where DCs play a pivotal

role: during the activation of T cells in the periphery, and; during re-activation of T

cells right before entry into the parenchyma. To date, the trafficking to and

specific interactions of these APCs and T cells within the CNS have not been

elucidated. In other tissues during inflammation, T cells interact with selectins,

adhesion molecules and chemokines expressed on endothelial cells to home to

the site of inflammation. In the CNS, it is thought that T cells require interaction

with an APC before entering the parenchyma. The idea of an APC needing to

license the T cell is born from the observation that in a chimera model where

peripheral cells lack MHCII and microglia have functional MHCII, transferred cells

14 are unable to infiltrate the CNS, whereas in a chimera where microglia lack

MHCII and peripheral cells have functional MHCII, these mice develop EAE normally. In addition, transferred activated T cells still require 3 or more days to gain access to the parenchyma even when the recipient is irradiated to cause openings in the BBB.

The role of APCs in EAE is still controversial. In contrast to the role of DCs described above, other studies have shown that infiltrating macrophages using the CCL2-CCR2 axis are required for disease41, 75. Once in the CSF the re-

activation of T cells occurs in locations in contact with CSF during inflammation,

where the presentation of CNS myelin peptides by meningeal macrophages is

postulated to play an important role during the induction of EAE87. Additionally, it

has been shown that increase in circulating inflammatory monocytes correlates

with relapses in EAE88, 89. Finally, activation of resident microglia have been shown to play a critical role in the destruction of axons in EAE25. Historically,

chimeric mouse models have been used to differentiate the APC contribution of microglia, which are radio resistant, from peripheral bone marrow derived cells that are more readily susceptible to irradiation or drug treatments such as macrophages and dendritic cells. The flaw in this methodology is that irradiation causes alterations in the BBB endothelium90, and in astrocyte function thus

allowing previously inhibited substances and cells to move from the blood stream into the CNS parenchyma91. This method can result in small numbers of donor

cells that take on microglia-like morphology in the parenchyma of the brain92.

Two methods that avoid irradiation are in utero chimeras and parabiosis. In utero

chimerism is achieved by transferring liver cells of a donor fetus into the recipient

fetus on E13 while hematopoiesis is transferring from the liver to the bone

marrow after the formation of microglia on E893, 94 but this method often results in

low efficiency. In parabiosis, two animals are surgically joined by the skin at the

side so they fuse circulation, eliminating the injection of donor hematopoetic stem

cells and allowing the naturally circulating hematopoetic stems cells to populate

both animals. In this method lower levels of chimerism are achieved unless one

of the animals is irradiated68, 91.

Exploiting the differential expression of CCR2 and CX3CR1 on macrophages and

microglia lead to the development of a double transgenic mice in which CCR2 is replaced by RFP and CX3CR1 is replaced by GFP72, 95. In these animals,

microglia express GFP and are RFP negative and newly infiltrating macrophages express RFP but emerging evidence shows CX3CR1 GFP expression increases over time while RFP expression decreases the longer the cell stays in the tissue96. The limitation of this mouse model is there may be a differential disease

dependency on heterozygote versus homozygote expression of the receptor. For

example, in some inflammatory models all infiltrating cells have been found to the

CCR2 positive, but others have shown a combination of CCR2+ and CCR2-

monocytes88.

To circumvent artifacts created by ex-vivo manipulation and irradiation we will

use APC fluorescent reporter mice, CX3CR1 GFP/+ for microglia and

macrophage or CD11c-GFP for dendritic cell. We will use the transgenic T cell

2D2 mouse where T cells have a TCR specific for myelin oligodendrocyte

glycoprotein (MOG35-55) and cross it to a mouse that express cyan fluorescent

protein under the beta actin promoter.

Trafficking of Peripheral Immune Cells into the CNS

Neuroscientists have spent hundreds of years dissecting and defining neuronal

regions and pathways within the CNS, while assuming that the accessory cells

function uniformly throughout the tissue. It is emerging that there is a diverse

response from microglia and astrocytes in different regions of the CNS and it

follows that during inflammatory conditions, varied outcomes can be observed97,

4, 24. Many immune cell subsets have been studied for relative contribution to

EAE and MS during the late phases of active CNS tissue destruction in an

attempt to identify the culprit and develop a therapeutic target. T cells have been

most intensely researched because: (1) a new category of autoimmune T helper

cells that express IL-17 were discovered using the EAE system98, 99 and (2) these

Th17 cells can be transferred to other animals to drive disease but it is still

uncertain exactly how these cells traffic from the peripheral location of activation

into the CNS8, 100.

Research has focused on the acute rather than induction phase of EAE, thus there are still many unanswered questions about the immune cells that play a role in initiating the disease. One current theory of immune cell trafficking during inflammation induced by EAE depicted in Fig. 3, is that T cells are activated in lymph nodes as described above and travel to the blood stream where they traffic to the CNS, through the choroid plexus into the cerebrospinal fluid, once within the CSF, they can encounter meningeal macrophages. Alternatively, T cells can move from the blood stream into perivascular spaces between the endothelial cell and the basal lamina, where they can encounter antigen presenting cells such as macrophages, dendritic cells and even microglia. Once the T cell has made contact with the APC, the T cell is allowed or licensed to move into the CNS parenchyma6.

The chemokines produced during inflammation in the CNS and chemokine

receptors expressed on circulating immune cells is essential to fully understand

trafficking into the parenchyma. With the availability of many chemokine receptor

knockout mice in the genetic background in which EAE is induced, many enlightening studies have been performed. It is important to know that inducing

EAE in CXCR3101, and CX3CR1102 knockout mice results in exacerbated

disease. However, when CXCR3 antibody therapy was explored, there was

exacerbation of disease when applied before onset of disease but conversely,

there was a decrease in disease when antibodies were applied during the acute

phase, thus the timing of use of this therapy would be critically important103.

Figure 3: Trafficking of immune cells from the periphery to the CNS. Adapted from Goverman104

Peripheral and CNS activation of myelin-specific CD4+ T cells. CD4+ T cells are primed in the periphery by

DCs presenting myelin antigen. (1). CD4+ T cells enter the subarachnoid space by crossing the blood– cerebrospinal fluid (CSF) barrier either in the choroid plexus or the meningeal venules. (2) the T cells are re‑ activated within the subarachnoid space by MHC class II‑expressing macrophages and DCs expressing myelin epitopes. (3) Reactivated T cells activate microglial cells in the subpial region, triggering activation of distal microglial cells and blood vessels. (4) Activated T cells adhere to and cross the activated blood–brain barrier, enter the perivascular space and are reactivated by perivascular macrophages and DCs. (5) T cells enter the parenchyma and, together with activated macrophages and microglial cells, secrete soluble mediators that trigger demyelination.

CCL21 has been shown to be constitutively expressed in the mouse CNS regardless of inflammation105, and infiltrating T cells express CCR7, but CCR7-

deficient mice have comparable disease to WT control animals106 thus it is not an important chemokine receptor in trafficking.

CCR6 deficient107 and CCR2 deficient mice108 are resistant to EAE but each

chemokine receptor are expressed by different cell types. Activated WT MOG

specific T cells transferred into a CCR6-/- mouse can enter the CNS and recruit

large numbers of effector CCR6-/- T cells into the parenchyma, therefore it is the

expression of CCR6 on T cells that mediate disease. Activated macrophages and

dendritic cells cross into the parenchyma using CCR266, 74, 75, 76. In EAE, it has

been shown that CCR2 expression is during the initiation and during the

spontaneous relapse of chronic relapsing increased EAE109 and CCL2 is

expressed by injured cells including neurons, astrocytes and endothelial cells76,

110, 111, 112 and correlates with disease severity in active EAE109. Taken together,

the infiltration of DCs and macrophages are critical to the induction of EAE and

they use CCR2 to traffic to the CNS at the critical times to activate T cells. In

addition, EAE disease is attenuated in CXCL13 deficient mice with fewer

infiltrating mononuclear cells into the CNS105. Finally, CCL3 and CCL5 were shown to be highly expressed in the CNS during inflammation109, disease is

attenuated in CCR1-deficient mice113 and a specific CCR1 antagonist was able to

ameliorate EAE114.

Vessel Permeability is implicated in CNS Pathologies

The cellular components of the BBB are redundant mechanisms to control the

passage of solutes and cells into the parenchyma. This neurovascular unit is

formed early in development such that the cells trapped behind this wall are

never exposed to most of the components comprising the blood, including

microglia. The outcome is a hyper response to blood vessel leaks that does not

occur in other tissues. It is becoming clear there is a role of blood vessel leaks in

the pathogenesis of neurodegenerative diseases39 including multiple sclerosis

(MS), vascular dementia, epilepsy, Alzheimer’s, and most obviously stroke26, 27,

28, 29, 30, 40.

Neuron depolarization occurs in patients following stroke, subarachnoid

hemorrhage and traumatic brain injury115, 116, 117. Indeed it has been shown that

breakdown of the BBB and astrocytic based dysregulation of extracellular

electrolytes causes neuron depolarizations116.

Inability of astrocytes to maintain BBB ion homeostasis and problems with

edema has been linked with epilepsy118. The same mechanism of breakdown of

the extracellular ionic gradients responsible for stroke and hemorrhage are also

responsible for epileptic seizures. MMP9 and albumin was increased in the CSF

of patients after epileptic seizures demonstrating the link between BBB leak and

21 seizures119, 120. It is assumed but not clear whether the BBB leak caused the seizure or vice versa.

In patients with Alzheimer’s, asymptomatic BBB leakage and diffusion

abnormality was detected via MRI121, 122. Transgenic mice that express human

amyloid precursor protein had increased permeability of brain microvessels

compared to WT controls, along with reduced tight junction proteins and

increased MMP expression121, 123, 124.

MRI of MS patients have shown abnormal BBB permeability in normal appearing

white matter125, 126. Gene chip analysis derived from the MOG EAE model in

NOD mice show that on day 12, non-specific neutrophil-related BBB impairment

was secondary to immunization with CFA127. It is possible to reverse the effect of

BBB breakdown on disease progression. Tetracycline induced expression of

claudin-1 in BBB tight junctions resulted in amelioration of chronic but not acute

EAE128. Others have extracted small molecules from Tuscan black kale and

shown it stops permeability of BBB in EAE129.

It is evident that MMPs BBB leaks and dysregulation can be identified and

correlated to multiple neurodegenerative diseases. It is encouraging that

22 therapies that target the endothelial cells rather than immune cells can have an

effect on disease outcome.

Role of Toxins in EAE Induction

During education of T cells in the thymus, generally self-reactive T cells are

eliminated. It is possible that in a normal mouse repertoire, MOG-reactive T cells

escape negative selection in the thymus and egress to the periphery, where they

then patrol both the secondary lymphoid tissues and the CNS. However, T cells

that are self-reactive generally have weak affinity to the peptide to have escaped this selection process130, 131, 132. As discussed previously in the “Animal Models”

section, early attempts at inducing a MS like paralysis in animal models started with multiple injections of whole brain homogenates. Upon injection of

homogenates with complete Freund’s adjuvant (CFA), disease induction was

achieved in several animal species133. This addition was borne from decades of

research that showed immunization with unrelated antigen could boosted the

immune response the pathogen134. When mycobacteria was resuspended in a

butter or paraffin oil emulsion, production of antigen-specific antibodies was

significantly increased. The actions of CFA are: 1) prolonged presence of

antigens at the site of injection; 2) enhanced antigen uptake of antigens and

promoting DC maturation; 3) increased antibody production; 4) PAMP receptors

recognize the mycobacteria and skew to a TH1 type response resulting in

increased IL-2 and IFN cytokine production135.

Pertussis toxin (PTX) is a major virulence factor of Bordetella pertussis, the causative agent of whooping cough. The use of PTx in immunology was traditionally to inhibit G protein signaling via the ability of PTx to ADP-ribosylate

G proteins. It is interesting that this toxin is used to induce EAE because PTx would affect all chemokine receptors on immune cells, thus inhibiting the migration of cells within the lymph node and trafficking of immune cells into the

CNS. PTx does affect lymphocyte accumulation in the lymph nodes but not the spleen however, in the spleen T cells and B cells are unable to migrate into the white pulp possibly contributing to activation of auto-reactive T cells136. However,

PTx has pleiotropic effects that last days after administration. First, ADP-

ribosylation results in a loss of inhibition of adenylate cyclase activity and causes

an increase of the intracellular concentration of cAMP. This accumulation of

cAMP disturbs cellular metabolic processes, including moderate endothelial

hypersensitivity to histamine, pancreatic islet activation and lymphocytosis137. It is generally accepted that mice injected with PTx have changes in brain endothelial permeability, but upon inspection of published results, the brain permeability measured by radioactively labelled BSA is subtle, approximately 1.3 fold increase compared to PBS control injection138. Second, PTX is able to activate both

MyD88-dependent and independent signalling pathways in DCs139 leading to

activation promotes the maturation of this antigen presenting cell, with upregulation of co-stimulatory molecules and the production of pro-inflammatory cytokines such as IL-6140.Third, apart from the enzymatic action, the B moiety of the PTx is preferentially mitogenic for T cells and activates TCR signaling similar

24 to that of anti-CD3, confirmed by signaling through Lck and ZAP-70141. The B moiety of the PTx causes internalization of CXCR4 and down-regulates signaling through the TCR signaling path142.

Role of Histamine in the CNS

Histamine is used by both the nervous system and the immune system.

Histamine has pleiotropic effects including neurotransmission, secretion of

pituitary hormones, and other brain functions such as sleep cycles, it regulates

gastrointestinal and circulatory functions143. Histamine is a potent mediator of

inflammation and a regulator of innate and adaptive immune responses144. Mast

cells and basophils are the major sources of stored histamine and it is rapidly released from these cells on demand145, 146.

All other immune cells, including lymphocytes, which lack histamine vesicles are

able to synthesize “inducible” histamine147, 148, 149, 150, 151, 152, 153.

Histamine has four receptors that are named H1, H2, H3, and H4154. H1R and

H2R are expressed on: mammalian brain; gastrointestinal tract; genitourinary

system; cardiovascular system; adrenal medulla; hepatocytes; nerve cells; airway and vascular smooth muscle cells; endothelial cells; eosinophils;

25 monocytes; neutrophils; dendritic cells; and lymphocytes143, 154. H3R expression

is restricted to neuronal cells in the brain and some peripheral tissues while H4R

is expressed exclusively on hematopoietic cells143.

H1R couples to Gαq/11 proteins143 and generally, activation of H1R leads to

stimulation of phospholipase C, resulting in calcium mobilization from intracellular stores and activation of protein kinase C (PKC)155. However, H1R ligation causes

signaling in other pathways: the production of phospholipase A2 and arachidonic

acid156; cGMP and nitric oxide155, 157, 158; the activation of NF-κB159, and

STAT1160. H1R-mediated PKCα stimulation activates MAP kinase pathways161,

162, 163. H2R couples to Gαs proteins that leads to increased cAMP production

and calcium mobilization156, 164, 165, 166. H3R and H4R couples Gαi/o proteins that leads to inhibition of cAMP and mobilization of calcium167, 168, 169, 170.

In endothelial cells, H1R-mediated signals lead to disassembly of VE-cadherin

complexes that regulate endothelial barrier function171, calcium mobilization and

PKC activation results in cytoskeletal and shape change172. These effects result

in increased vascular permeability. H1R signaling increases expression of

adhesion molecules ICAM-1, VCAM-1 and P-selectin on endothelial cells173, 174,

175, 176.

In dendritic cells, H1R ligation upregulates co- stimulatory molecules by increasing the production of proinflammatory cytokines such as IL-1, IL-6, IL-8,

MCP-1 and MIP-1α177, 178. It causes chemotaxis of immature dendritic cells by

inducing intracellular calcium flux and actin polymerization179. In B cells, H1R

signaling enhances anti-IgM mediated proliferation and antibody production180. In

CD4+ T cells, H1R signaling regulates IFN-γ and IL-4 production.

Mast cells accumulate at the site of inflammatory demyelination in the brain and

spinal cord both in animal models and in MS patients126, 181, 182, 183, 184, 185.

Histamine is significantly higher in CSF of MS patients186, 187. However, histidine

decarboxylase deficient mice which lack histamine develop a severe disease

than WT mice188. Thus histamine plays and immune regulatory role in the

development of EAE and thus histamine receptors are a better therapeutic target.

H1RKO mice develop an attenuated disease than WT mice189. Additionally,

H1H2RKO mice develop an attenuated disease while H3H4RKO mice develop

an exaggerated form of EAE when compared to WT mice190. Correspondingly,

the BBB permeability index of H1H2RKO mice is significantly lower than

H3H4RKO or WT mice. With the many locations H1R is expressed, it was

interesting to find that mice with endothelial cell expressing H1R under control of

the von Willebrand factor promoter exhibited decreased BBB permeability and

enhanced protection from EAE compared with H1RKO mice suggesting that

endothelial H1R signaling may be important in the maintenance of

cerebrovascular integrity191.

Anti-histamine studies in EAE generally use H1R blockers, and show a reduction of EAE disease138, 184, 192, 193, 194, 195. In humans, administration of anti-H1R agents

either reduced the risk of MS196 or improved the neurological symptoms197. Mast cell-stabilizing drugs have been shown to improve disease symptoms in EAE184,

198, 199. C-kit knockout were thought to be mast cell-deficient mice and exhibited

delayed onset and reduced disease severity. However, characterization showed

that c-kit was expressed by many immune cells and although the results are

impressive, it cannot be attributed solely to mast cells.

Anti-histamine has proven to be a simple and effective way to treat EAE and MS,

however, it does not ameliorate disease and the multiple tissues that express

H1R makes it hard to determine the exact mechanism by which anti-histamine

reduces disease burden. We show that the anti-histamine, hydroxyzine, reduces

microglia phagocytosis and activation resulting in the reduction of peripherally

infiltrating APCs and T cells.

Summary

Studies using EAE have clarified our understanding of trafficking of immune cells

from the periphery into the CNS in response to inflammation, the important

cellular populations and their spatiotemporal role in the late stages of the

disease200. However, the earliest initiating cellular and tissue events that lead to massive immune cell recruitment prior to disease manifestation have not been described. For these reasons, we focus the current research on defining the earliest events of EAE with the intent to identify potential new therapeutic targets that can be applied to MS patients at diagnosis and recurrence in relapsing remitting MS. The goal is to reduce long term recurring inflammation and thus reduce neurologic deficits.

Through careful surgery and experimental design, we have confirmed a series of

events that lead to demyelination (Fig. 4):

1) Vascular dysfunction induced by PTx leads to localized, transient vessel

leaks

2) Leak of vessel contents causes microglia phagocytosis and activation

3) The phagocytosis overload possibly causes microglia to recruit peripheral

DCs and macrophages that are more proficient at phagocytosis

4) The inflammatory environment encountered by APCs causes recruitment

of T cells

5) Peripherally activated, myelin specific T cells and activated APCs in the

CNS are required for T cell licensing and motility within the parenchyma.

6) Microglia are able to extend processes across an intact BBB and that the

number of these projections found along the blood vessel doubles by days

9-12 of EAE induction.

Figure 4: Sequence of events in the initiating phase of EAE

(a) Vascular dysfunction induced by PTx leads to histamine release and localized, transient vessel leaks. (b)

Leak of vessel contents causes microglia phagocytosis and activation (c) Recruitment of peripheral DCs and macrophages. (d) The inflammatory environment encountered by infiltrating APCs results in recruitment of T cells (e) Microglia are able to extend processes across an intact BBB and the number of these projections doubles by days 9-12 of EAE induction.

2 Focal Intermittent CNS Vascular Leak Contributes to Sequential Immune Cell Infiltration during the Asymptomatic Induction Phase of Experimental Autoimmune Encephalomyelitis

2.1 Abstract Immune cell accumulation, neuronal destruction and paralysis are hallmarks of

experimental autoimmune encephalomyelitis, a central nervous system

inflammatory disease. Due to the spatial-temporal constrains of traditional

methods used to study this disease, debate continues as to whether vessel disruption represents a cause or a byproduct in the neuroinflammatory pathophysiology. Here, we provide dynamic, intravital, time-resolved single-cell information on the sequential structural and cellular events within the mouse cortex during the asymptomatic first 12 days of disease induction. Transient focal

vessel disruptions preceded microglia activation, followed by infiltration of and

interaction between circulating APC and T cells. Histamine H1 receptor blockade

prevents microglia activation, resulting in reduced immune cell accumulation,

disease incidence and clinical severity.

2.2 Introduction Mounting evidence suggests that peripheral immune cells are central to the

pathogenesis of multiple sclerosis (MS) and other neurodegenerative and

neuroinflammatory diseases80, 201, 202, 203, 204. Past studies have elucidated many

32 of the key features of experimental autoimmune encephalomyelitis (EAE), a widely studied animal model of neuroinflammation with features that mimic certain aspects of MS41, 205. However, the precise sequence of cellular events in

EAE pathogenesis during the early asymptomatic induction phase of the disease

remains poorly defined.

Current theory posits that disease induction is a two-step paradigm consisting of

an initiation stage and an infiltration stage for pathogenic T cells73, 206, 207. In the initiation stage, naïve myelin-specific CD4+ T cells that have escaped thymic

negative selection are induced to undergo proliferation and differentiation in lymph nodes (LNs) by activated myelin peptide-containing antigen presenting cells (APCs) are subsequently released into the systemic circulation. The blood-

CNS barrier, which normally protects the CNS from peripheral inflammatory cell infiltration, is rendered “leaky” by irradiation or pertussis toxin administration that

involves local histamine-mediated regulation of vessel integrity138. This breach in

the blood-CNS barrier then allows myelin-reactive T cells to traverse through the

blood-brain barrier (BBB) and/or the blood cerebrospinal fluid barrier (B-CSFB)

via chemokine guidance, including the action of CCR6208. These T cells

encounter another population of APCs presenting myelin peptides in the perivascular Virchow-Robin Space (VRS) and/or the subarachnoid space (SAS) and are then licensed for access to the CNS parenchyma6, 104. This model

supports an essential role for infiltrating DCs in the re-activation of myelin-

specific T cells within the CNS, although it is unclear what perivascular niche

33 factors drive circulating monocytes to become DCs and macrophages in the

CNS. Using chimeric models where the BBB has been compromised, it has been

shown that microglia are activated during the asymptomatic induction phase209,

but MHCII expression by microglia is not required for infiltration of cells210. This raises the question of whether other functions of microglia resulting in activation could play a role in disease progression, in particular the phagocytosis of myelin211 or neuron212 debris and potentially blood vessel contents25. Upon

exerting their effector function, the pathogenic CD4+ T cells recruit other effector

immune cells including additional CD4+ T cells, CD8+ T cells, neutrophils and B

cells that gain access through compromised CNS vessels, with ensuing

inflammation, focal neuronal demyelination and cell destruction resulting in

paralysis41, 213, 214.

The identity of initiating T cells in EAE has been widely studied63, 215. Current

evidence strongly suggests that IL-17-producing, myelin-specific CD4+ Th17 cells

play a critical role both in EAE and in MS132, 216 and that adoptive transfer of

these T cells into irradiated recipient mice induces neuronal destruction41, 132. In this passive transfer model that mimic the later infiltration stage of EAE, disease occurs either in the context of toll-like receptor (TLR) signaling in the CNS217, 218 and in recipients with vessel-CNS barrier dysfunction induced by pertussis toxin

(PTx) and/or irradiation219, 220. Recent studies have addressed the recruitment and migration behavior of activated Th17 cells directly in situ by applying intravital two-photon microscopy (TPM) to either the brain or spinal cord tissues

34 in rodents221, 222, 223, 224, 225, 226. While these studies shed new light on how immune cells were recruited from the blood vessels into EAE lesions, these observations were generally made in the passive transfer model and during the

onset of peak clinical symptoms. As such, they do not address how peripherally

activated, myelin-specific T cells first access the CNS during the critical transition

from the initiation stage to the infiltration stage of EAE pathogenesis107, before

the arrival of late-stage effector immune cells which amplify local CNS inflammation, tissue destruction and further vessel disruption.

CNS vessel integrity disruption is a hallmark of MS; yet, it is not clear whether

vascular compromise is the initiator or the result of EAE pathology227, 228, 200, 229.

Furthermore, although the breakdown of the blood-CNS barrier has been

implicated, its contribution during the early, asymptomatic phase of active EAE

induction has not been directly characterized in situ. Current available data

regarding vessel integrity in neuroinflammation does not adequately describe the

nature and dynamics of the vessel “leakiness”. We hypothesize the vessel leak is

the initiating event of microglia activation and subsequent neuroinflammation.

In the current study, we use sequential intravital TPM through a cranial

observation window to examine cellular events at the tissue interface between

SAS and CNS parenchyma in the cortex beginning on day 0 of active EAE

induction to include the entire asymptomatic phase (first 12 days). We describe

35 early focal, intermittent and transient disruptions of post-capillary junctional blood

venules associated with PTx administration, which precede subsequent microglia

activation, immune cell accumulation and inflammation. The transient pial vessel

leaks initiate an inflammatory response in the parenchyma within hours to days

as evidenced first by microglial uptake of blood contents, followed by an influx of

both CD11c+ and CD11b+ APCs. The increasing numbers of activated CNS-

resident and blood-derived APCs on days 3-6 then facilitate the ingress and

retention of activated T cells in a myelin antigen-specific manner into the perivascular space and the brain parenchyma on days 6-12. These immune cellular infiltrations are found in close proximity to each other and to sites of early blood vessel leaks, forming cellular plaques similar to characterized cortical lesions in MS. In addition, we show that histamine H1 receptor antagonist acts to curtail the frequency of PTx-induced focal vessel leakage, reduce the degree of microglia activation, diminish subsequent recruitment of blood-derived immune cells, thus protecting mice from EAE incidence and disease severity.

2.3 RESULTS

2.3.1 Transient focal leaks in leptomeningeal vessels occur early during EAE induction Review of the literature reveals EAE induction efficiency and clinical presentation can often be variable. To reliably capture rare cellular events in the CNS by TPM, we introduced fluorescent-reporter naïve myelin-specific 2D2 transgenic CD4+ T

36 cells into recipient animals in order to increase the precursor frequency of myelin- specific T cells prior to disease induction. To induce disease while preventing mortality, a low dose of pertussis toxin (PTx) was administered daily on the first 3 days of induction without irradiation230. This resulted in 100% disease incidence

compared to mice without transferred 2D2 CD4+ T cells, with hind limb paralysis

achieved in all mice by day 21 (Fig. 5a). Previously, a non-invasive

bioluminescence study indicated that T cell infiltration first occurs in the brain

around day 7 after EAE induction, 3 days before spinal cord infiltration and 5 to 7

days before onset of clinical symptoms2. Therefore, we focused our intravital

observation in the cortex during the asymptomatic first 12 days of EAE induction.

A cranial observation window was surgically implanted 4 to 10 days prior to 2D2

cell transfer, ensuring limited residual inflammatory changes to underlying

leptomingeal structures and cellular response231. We have previously

demonstrated that, when carefully performed, cranial window implantation yields

similar observations as those obtained through a thinned-skull approach, and can

provide an experimental platform for longitudinal serial observation of meningeal

and parenchymal changes in the cortex of EAE mice231.

The role of PTx in EAE induction has been attributed, in part, to its ability to

disrupt the BBB integrity138, although the exact location, timing and dynamics of

such BBB breach have not been carefully characterized. Using TPM imaging and

fluorescently labeled vessel dye, we were able to directly observe focal transient

Figure 5: PTx administration causes transient and focal CNS vessel leak.

Figure 5 (continued): PTx administration causes transient and focal CNS vessel leak.

(a) Reliable and consistent clinical scores were achieved in mice pre-transferred with 3x106 naïve 2D2 T cells (filled squares) as compared to WT (non-transferred) controls (open squares) prior to EAE induction.

(b) TPM imaging of pial vessels (red) on day 3 after EAE induction revealed localized transient vessel leaks at post-capillary venule junctions that resolved over a few minutes (region of interest (ROI) 1, 2), while other vessels within the imaging field remain intact (ROI 3, 4). Scale bar = 50μm. (c) Dynamic mean fluorescent intensity (MFI) measurements of the indicated ROI’s in (b) over time revealed the transient and focal nature of the vessel leaks in ROI 1 and 2. (d) Frequency of cerebral vessel leaks was quantitated sequentially in a cohort of mice by TPM during the first 12 days of EAE induction. An increased frequency of vessel leaks was associated with PTx administration on day 0 to 2. A second wave of vessel leaks was observed on days

7 to 9 at a time when effector immune cells were shown to infiltrate the CNS232. Arrows: PTx injections. (e)

The duration of individual vessel leaks in (d) was measured dynamically by TPM. Arrows: PTx injections. (f)

ROI 5, a cross-sectional view of ROI 1 in (b) revealed a slower clearance of vessel dye under the pia surface in the parenchyma (P) as compared to the meninges (M). Scale bar = 25μm (g) The presence of vessel dyes (red) can be detected in the perivascular areas of the CNS parenchyma in fixed CX3CR1+/GFP brain tissue by IHC, both near the superficial vessels and the deep vessels away from the meningeal- parenchyma interface (*). Dash line: Imaging depth achievable by TPM. Scale bar = 100μm.

39 leakage of CNS vascular contents hours to days after PTx injections on days 0 to

2. These transient BBB breaches were visualized as blooms of dye leaking from the blood vessels (Fig. 5b; Supplemental movie 1). The effect of PTx on CNS vessel integrity disruption was neither uniform nor permanent, as certain post- capillary venule junctions were prone to intermittent vessel leaks while other vessels of similar caliber within the same imaging volume remained completely intact. Some vessels exhibited multiple leaks, while others exhibited the leak only once during the hour-long imaging session (Fig. 5c). As a control, mice with implanted cranial windows and 2D2 cell transfer without PTx or EAE induction exhibited little to no vessel leaks, indicating that these leaks were directly related to EAE induction and not damages associated with TPM or prior surgical procedures (Fig. 6). In addition, we also observed vessel leaks that originated from venule junctions that were outside of the imaging field, arguing against this phenomenon as a result of artifacts of intravital TPM imaging (Supplemental

movie 2).

When we characterized these vessel leaks in the same mouse cohort serially

through the first 12 days of EAE induction, we observed that the highest

frequency of vessel leaks occurred during the first 3 days, coincident with PTx

administration (Fig. 5d). The duration of individual leaks during this early period

was variable, with some lasting ~30 minutes before resolving (Fig. 5e).

Interestingly, the frequency and duration of vessel leaks subsided gradually on

Figure 6: Insignificant number of leaks detected in control mouse.

Control mice undergoing cranial window implantation but not full EAE induction exhibited a small number of vessel leaks only in the first 3 days following the surgical procedure. In addition, the duration of the leaks was smaller. There were no flares detected after day 3, and none of these mice, observed up to 21 days, showed any signs of clinical disease. Data obtained from 4 control mice.

41 subsequent days before increasing again on days 8-10 at a time when effector T cells were known to traffic to and accumulate in the CNS232.

Blood solutes from vessel breach persist within the CNS parenchyma

While signal from the vessel dye at the pial surface dissipated within a few

minutes where bulk CSF flow in the meningeal space can explain the clearance

of the dye, blood solutes released into the parenchyma took a longer time to

clear after the BBB breach had resolved (Fig. 5f). Evidence of dye persistence

can be found deep in the parenchyma, as fluorescently labeled dextran can be found on day 3 of induction in fixed brain tissue sections (Fig. 5g). We hypothesized that the presence of the blood contents may initiate CNS-resident

microglia activation201, 233, thereby starting a cellular cascade that results in the

subsequent infiltration and accumulation of immune cells from circulation.

Intermittent vessel leaks result in localized phagocytic uptake of blood vessel contents by microglia

The observed focal nature of the BBB breach led us to develop an assay to evaluate the overall BBB integrity by measuring the location and phagocytic activity of APCs that have taken up vessel dye throughout the course of EAE induction. We introduced TRITC-labeled dextran intravenously into recipient mice and measured the activity of TRITC-labeled phagocytic cells as an indirect

42 readout of the vessel integrity in the meningeal and parenchymal compartments

hours to days later219. Using this method, we observed patchy distribution of

TRITC-labeled phagocytes in the brain parenchyma 3 days after PTx and TRITC-

dextran administration, while other areas within the TPM scanning field have little to no detectable phagocytic cells (Fig. 7a). The phagocyte distribution in the

parenchyma is consistent with the distribution pattern of transient vessel leaks

observed during dynamic imaging (Fig. 5).

Toxin dependent phagocytic activation

To assess how the phagocyte distribution may be affected by neuroinflammatory

stimuli, we compared TRITC+ phagocyte distribution and numbers in the CNS

following systemic exposure to PTx and the TLR4 agonist, lipopolysaccharide

(LPS). Second harmonic generation (SHG) signals were used to precisely locate

the collagen-rich pia and to differentiate parenchyma from meninges (data not

shown)231. Interestingly, we observed a distinct distribution pattern of

parenchymal TRITC-positive phagocytic cells between the two stimuli. Systemic

LPS treatment resulted in an even distribution of phagocytic cells throughout the

entire parenchyma and meninges, whereas PTx exposure resulted in a patchy

phagocyte distribution (Fig. 7b) as seen with EAE induction (Fig. 7a). Both PTx

and LPS administration resulted in a significant increase in phagocytic cells in

both the meninges and parenchyma as compared to PBS control, with slightly

higher number of cells with phagocytic uptake in the meninges of mice exposed

Figure 7: Focal vessel leaks induce localized activation of endogenous CNS immune cells and increased DC accumulation.

Figure 7 (continued): Focal vessel leaks induce localized activation of endogenous CNS immune cells and increased DC accumulation.

(a) TPM vessel imaging of cortex on day 3 following PTx administration revealed patchy activation of CNS phagocytes (red) while other image areas were devoid of phagocytic activities. Numbers shown are phagocytic cell counts within the boxed parenchymal volume. Scale bar = 100μm. (b) PTx and LPS resulted in distinct distribution of phagocytic cells (yellow) in the CNS; LPS caused diffuse phagocytic activity while

PTx caused patchy phagocytic activity around vessels (red). Scale = 100μm. (c) Quantification of phagocytes in the meninges and parenchyma following systemic exposure to LPS and PTx. * p<0.01, ** p<0.001, *** p<0.0001. (d) Sequential TPM CNS imaging of the CD11c-GFP mice over the first 12 days of

EAE induction showed progressive changes in the accumulation of CD11c+ (green) and phagocytic CD11c+ cells (purple) in left panel and phagocytic cells (red) in right panel over time. Scale bar = 60μm. (e)

Quantification of CD11c+ (green), phagocytic CD11c+ cells (purple) and phagocytic (red) cells in the meninges and CNS parenchyma over a 12-day longitudinal imaging sequence as shown in (d).

45 to LPS (Figure 7c). This in vivo phagocytic assay allows us to sequentially discern the patterns of vessel leaks in the same hosts with finer time and space resolution than previously reported234, 235, 236, 237, 238.

+ + TRITC phagocytes are CX3CR1 microglia and macrophages in early EAE induction

CX3CR1 is a chemokine receptor that is constitutively expressed by microglia

and a sub-population of macrophages in the CNS4. To identify whether the

+ + TRITC phagocytes include CX3CR1 microglia / macrophages, we induced EAE

+/GFP in Cx3cr1 mice and observed phagocytic activity through a cranial window

following TRITC-dextran injection. 5 hours after PTx administration on day 0, we

found that the only TRITC-containing cells in the meninges and parenchyma

+ - + were exclusively CX3CR1 cells, as we did not detect any CX3CR1 TRITC phagocytes (Supplemental movie 3).

CD11c+ DCs infiltrate the CNS following phagocyte activation

Prior studies have shown that CD11c+ DCs are required in EAE disease

induction, although the exact timing of CD11c+ infiltration in the CNS during the

disease process is not clearly understood13, 85, 239. To investigate this, we imaged

CD11c-GFP+ mice daily in the first 12 days of active EAE induction to determine

the time course of infiltrating DCs and their phagocytic activities during disease

46 progression. We quantified the number of CD11c-GFP+ cells, TRITC+ phagocytic

cells, or both in the meninges and parenchyma over 12 days (Fig. 7e). We

observed an increased number of CD11c+TRITC- cells in the meninges starting

on days 3 through 12, with increasing phagocytic activities from days 6 to 12.

The entry of CD11c+TRITC- DCs into CNS parenchyma was much lower

compared to those in the meninges, with increasing numbers of DCs starting on

day 6. Their phagocytic capacity increases over the subsequent 6 days. In

contrast, the number of CD11c- TRITC+ phagocytes decreased from days 3 to 12

in the meninges, even as parenchymal CD11c- TRITC+ phagocytes remain

constant throughout this period.

Presentation of myelin antigens by CNS APCs enhances activated myelin-

specific T cell entry and retention

Following the characterization of CD11c+ and CD11c- APCs in the EAE brain, we investigated the timing and antigen requirement of 2D2 cell recruitment and behavior within the CNS. We co-injected equal number of differentially labeled naïve OTII and 2D2 cells into recipient mice followed by immunization with both

OVA323-339 and MOG35-55 peptides in the presence of CFA and PTx (Fig. 8a).

Significantly more 2D2 cells began to infiltrate the brain parenchyma as early as day 3 and day 6, even though both T cell populations were found in equivalent numbers within the peripheral lymph nodes (Fig. 9c). The majority of the OTII cells found within the CNS remain in the perivascular location near

Figure 8: Peripherally activated T cells accumulate and migrate near sites of vessel leak and DC aggregate in an antigen-dependent manner.

Figure 8 (continued): Peripherally activated T cells accumulate and migrate near sites of vessel leak and DC aggregate in an antigen-dependent manner.

(a) Equal numbers of naïve OTII (open circle) and 2D2 (closed circle) CD4+ T cells were transferred into WT recipients 24 hours before s.c. immunization with OVA323-339 and MOG35-55 peptides in the presence of

CFA and PTx. Direct longitudinal TPM observation of the same recipient mice revealed that 2D2 cells migrated to the CNS in greater numbers than the OTII cells at each time point in the first 12 days following immunization. (b) Similar experiments were conducted as in (a) except the recipient mice were immunized only against the OVA323-339 peptide. Relatively low numbers of both T cell populations were found in the

CNS on day 3 during the first wave of vessel leak but not seen at all on subsequent days. (c) Sites of focal vessel leaks early (day 4) in EAE induction resulted in phagocyte (red) and CD11c-GFP+ (green) cell accumulation (days 6-9), and localized 2D2 cell (blue) migration and accumulation (day 9). Colored tracks on day 9 highlight paths taken by individual 2D2 cells (blue) near areas of CD11c-GFP+ cell clusters (green) in the parenchyma (P) compared to the meninges (M). (d) Distribution of contact duration with CD11c-GFP+ cells by 2D2 T cells on day 9 showed predominantly short interactions (<5 mins) with the CD11c-GFP+ cells.

(e) 2D2 cells with short interactions (<2 mins) with the CD11c-GFP+ cells exhibited a higher overall velocity than 2D2 cells that maintain a longer interaction with DCs (average velocity: 14.8 ± 4.2 μm/min versus 12.5

± 3.6 μm/min, respectively). (f) Total number of unique 2D2 cells migrating towards CD11c+ rich regions

(GFP hi) are significantly higher than those migrating towards low CD11c-GFP (GFP lo) regions on days 9 and 12. (g) 2D2 cells exhibited similar overall contact time with phagocytic (TRITC+GFP+), non-phagocytic

CD11c+ (TRITC-GFP+) cells and phagocytic non-CD11c+ (TRITC+GFP-) cells on days 9 and 12. (h) More

2D2 cells were observed to egress (average 0.67%) from the blood vessels into the parenchyma than those

2D2 cells entering (average 0.30%) into the blood vessels from the CNS on days 9 and 12. ** p<0.001. n.s.: not significant.

Figure 9: Characterization of OTII and 2D2 cells when co-activated during induction.

(a) Distribution of OTII T cells (green) and 2D2 T cells (yellow) in the brain parenchyma on day 9 revealed that a few OTII cells are restricted to the perivascular spaces. Scale = 100μm (b) Quantification of OTII and

2D2 cells revealed a greater accumulation of 2D2 cells in the parenchyma. (c) OTII and 2D2 cells proliferate to similar numbers in the peripheral draining lymph nodes following injections of MOG35-55 / OVA323-339 /

CFA emulsions. (d) Comparison of the average speeds of OTII and 2D2 cells found in the brain revealed that 2D2 cells are more motile than OTII cells in the brain parenchyma.

50 sites of previous vessel leaks, migrated with a reduced speed as compared with

2D2 cells (Fig. 9b), and remain in the CNS only transiently (data not shown).

However, when mice infused with naïve OTII and 2D2 cells were immunized only

with OVA323-339, we saw a near complete absence of infiltrating 2D2 or OTII

cells in the meninges or the CNS parenchyma (Fig. 8b).

This absence of activated myelin-specific T cells (Fig. 8b) disrupts this intricate

cellular and structural collaboration, resulting in a complete absence of effector

cell infiltration and disease induction (data not shown), even though vessel leaks,

phagocytic APC accumulation and robust activation of non-CNS specific T cells were all present within the host. Taken together, our data imply that the

cooperation between PTx-induced leaky vessels, resident and infiltrating APCs, the availability of CNS-derived antigens and activated myelin-specific T cells are preconditions for MOG-specific T cell entry into the CNS, which in turn amplifies

other cellular infiltration later in the disease progression.

Localized blood vessel leak precedes phagocyte accumulation and the

infiltration of DCs and T cells

To further examine the cellular and structural cooperation during successful EAE

induction, we performed multiplex sequential imaging of the vessel leak and

cellular interaction among TRITC+ phagocytic microglia, CD11c-GFP+ DCs and

CFP+ 2D2 cells during the first 9 days of EAE induction. Following transient

vessel leaks (Fig. 8c, day 4), the vessel content was cleared with a concomitant

increase in the number of TRITC+ phagocytes and the arrival of CD11c+ DCs by

day 6 (Fig. 8c, days 6-9). This was followed on day 9 by a large number of

infiltrating 2D2 cells that congregated around locations of DC accumulation near

the sites of previous vessel leaks (Fig. 8c, day 9), eventually leading to extensive

inflammatory changes and tail and limb weakness beginning on days 10 to 12

(data not shown). When we analyzed the behavior of 2D2 cells found on day 9,

we observed that the activated 2D2 cells surveyed a large area of the CNS

parenchyma, with a particular focal concentration of movements surrounding previous vessel injury sites where clusters of CD11c+ cells were found (Fig. 8c,

day 9; Supplemental movie 4). This surveillance behavior toward CD11c+ cells by

2D2 cells was reminiscent of chemotactic guidance behavior of T cells by activated DCs in an inflamed LN240. The 2D2 cells exhibited both transient (< 2

min) and prolonged (≥ 2 min) contacts with CD11c+ DCs (Fig. 8d). Those 2D2

cells that interacted briefly with CD11c+ DCs (<2 mins) migrated at a faster

velocities (~16 μm/min), while those that exhibited prolonged interaction with

CD11c+ DCs traveled with reduced velocities (~12 μm/min) (Fig. 8e). In all, 17.7

± 11.2% of total 2D2 cells were stationary versus 83% motile and 6.1 ± 5.1% of

2D2 cells were found to be perivascular with limited motility.

2D2 cells preferentially congregate to CD11c+ DC-rich regions

To further assess whether 2D2 cells were preferentially drawn to sites of DC

aggregates in the CNS parenchyma, we analyzed the number of 2D2 cells that

enter a 50m radius of a CD11c+ cellular aggregate. Our analysis revealed a

significant increase in the number of unique 2D2 cells moving towards an area high in CD11c-GFP signal as compared to areas with low CD11c-GFP signal

within the same imaging field (Fig. 8f), suggesting that indeed the T cells were

attracted preferentially to CD11c+ cells. Interestingly, interaction duration analysis

of 2D2 cells with CD11c+ and CD11c- phagocytes showed a trend toward higher duration with CD11c+ cells, although the difference was not statistically significant

(Fig. 8g). When the number of 2D2 cells crossing the BBB was analyzed, we

found a greater number of 2D2 cells egressing from nearby blood vessels to

enter the CD11c+ rich regions as compared to 2D2 cells entering those vessels from the parenchyma (Fig. 8h).

Histamine H1 receptor blockade reduces microglia activation, DC recruitment, disease incidence and severity

As histamine release by mast cells has been postulated to mediate PTx-induced vessel leak138, we investigated whether blockade of the histamine H1 receptor

(H1R) may ameliorate observed vessel leak and subsequent phagocytic cell

activation, T cell recruitment and disease progression. We administered

hydroxyzine (HXYZ), a first-generation antagonist of H1R that readily passes

across the BBB, in the drinking water of mice undergoing EAE induction. In

53 contrast to 100% disease incidence in conventional EAE induction in naïve 2D2- transferred recipients (Fig. 5a), we observed a 60% overall reduction of EAE incidence in mice receiving HXYZ treatment, with 40% of mice developing delayed disease onset and reduced clinical scores, and another 20% of the mice never showed clinical symptoms (Figure 10a). Note that vessel leaks after PTx administration were observed despite HXYZ treatment via gavage (Figs. 10b,

10c), suggesting the regulation of CNS vessel integrity under this stimulation condition was partially controlled via a non-histamine H1R mediated mechanism.

Interestingly, however, HXYZ gavage did result in a reduction in the number of

TRITC+ phagocytes in the parenchyma following EAE induction (Figs. 10b;

Supplementary movie 5). This is not entirely unexpected, as microglia has been

shown to express H1R, and engagement of the receptor induces activation of

microglia241. Similarly, there was a significant reduction in the number of both

CD11b+CD11c+ DCs and CD11b+CD11c- macrophages on days 6-9 as assessed

by whole brain flow cytometry (Figs. 10d, 10e). These data were further

corroborated by direct TPM observation, showing reduction of phagocytic DCs

and microglia and well as non-phagocytic DCs in the brain as late as day 12 post

EAE induction (Fig. 10f). There was also an absence of detectable 2D2 cells by

TPM on day 12 of HXYZ-treated EAE mouse (data not shown). Flow analysis of

T cell infiltration revealed a similar reduction in the number of infiltrating CD4+ and CD8+ T cells in HXYZ-treated groups early in disease induction, with

Figure 10: Histamine receptor blockade inhibits early activation of CNS phagocytes, diminishes DC and 2D2 accumulation, and blunts EAE severity.

Figure 10 (continued): Histamine receptor blockade inhibits early activation of CNS phagocytes, diminishes DC and 2D2 accumulation, and blunts EAE severity.

(a) 60% of mice undergoing EAE induction displayed either absent or reduced disease severity when exposed to hydroxyzine (HXYZ) via drinking water during EAE induction, with 20% disease-free incidence and 40% with a delayed onset and reduced overall clinical severity. (b) Mice treated with PTx for 3 days and exposed to HXYZ showed a dramatic reduction in phagocytic cell number. Scale bar = 100um. (c)

Longitudinal TPM imaging revealed no statistical difference of vessel leaks in HXYZ-gavaged mice compared to control mice during EAE induction, except on day 6 when 2/3 of HXYZ-treated mice exhibited more vessel leaks compared with controls. (d) HXYZ was administered to mice via gavage and EAE was induced. After isolation, flow cytometry analysis revealed a significant decrease in the number of

CD11b+CD11c- cells in the CNS of HXYZ-treated mice on days 3 and 6 following EAE induction (e) The number of CD11b+CD11c+ cells in the CNS of HXYZ-treated mice was also significantly lower as compared to control on day 6 following EAE induction. (f). Longitudinal sequential TPM imaging analysis of HXYZ- treated and control mice confirmed the findings in (e) and (f).

Figure 11: Additional effects of HXYZ on immune cells during EAE induction.

Figure 11 (continued): Additional effects of HXYZ on immune cells during EAE induction.

HXYZ was administered via gavage from day -1 to day 12 while EAE was being induced. Whole-brain percoll isolations of individual cells from HXYZ-treated and control mice were performed. Flow cytometry analysis of the isolated cells showed, (a) a significant decrease in the number of CD8+ cells in the CNS with

HXYZ treatment on Day 3; (b) a significant decrease in the number of CD4+ cells in the CNS with HXYZ treatment on Day 3; (c) the total number of CD4+ T cells expressing IFN-γ, IL-17 and FoxP3 were not significantly different with HXYZ treatment in the spleen on day 9; (d) even though the total number of CD4+ cells in the CNS was similar between treatment and control groups, there was a significant decrease in the number of IFN-γ and IL-17 producing cells with the CNS on day 9; and (e) there is a significant increase in phagocytic cell number in both meninges and parenchyma with PTx treatment as compared to PBS controls, whereas the parenchymal phagocyte numbers in PTx + HXYZ treated animals were comparable to PBS controls.

58 reduced number of IFN-γ and IL-17 producing CD4+ T cells at later time points

(days 9; Fig. 11).

2.4 Discussion CNS blood vessel leak has been implicated as a trigger in neurodegenerative

disorders including epilepsy, Alzheimer, and Parkinson’s disease29, 54, 242.

Although changes in CNS vessel permeability have been documented previously

in vivo, the timing and kinetics of how this occurs at the tissue level during neuroinflammation has not been described25, 32, 225, 234, 243. In this study, we

provided dynamic evidence of the transient pulsatile nature of altered CNS

vascular permeability, supporting the hypothesis that BBB leakage is an initiating

event rather than a byproduct of effector immune cell aggregation and tissue

destruction near the perivascular EAE lesions. These leaks were focal, transient,

and they occurred during PTx treatment well in advance of observable EAE

symptoms. Our observations are in accordance with histologic characteristics

found in sub-pial demyelinating gray matter lesions6, 244 such as the accumulation

of macrophages91 and CD4+ T cells87, 245. The location of these clinical sub-pial

gray matter lesions further highlights the importance of our imaging at the meninges-parenchyma interface to understand sequential structural and cellular changes at this location.

Vessel leaks were found both in the meninges and throughout the parenchyma.

Fluorescent dextran released from vessels in the SAS was cleared quickly due to

CSF flow and phagocytes, whereas fluorescent dextran released into the CNS

parenchyma was not cleared as efficiently. Evidence of vessel leaks can be

detected by IHC beyond the TPM imaging depth away from leptomeningeal

areas. During the first 3 days of EAE induction, blood contents including plasma

proteins, PTx and fibrinogen25 are released into the parenchyma and initiate a

parenchymal inflammatory response, including activating the phagocytic capacity

+ 246 of resident CX3CR1 microglia . During the initial transient vessel leaks

following PTx administration, we did not visualize any influx of circulating

monocytes or T cells migrating across the blood vessel wall in the cortex,

suggesting that the opening of the BBB at this stage does not permit an influx of

circulating cells into the CNS. At later time point (days 4-6) when the vessel

leakage subsides and perivascular microglia had phagocytosed vessel contents,

we began to observe increasing immune cell infiltrate coincident with a second

wave of vessel leak (days 7-9), an observation which is consistent with the two-

step paradigm of EAE induction73.

Microglia act as neuronal support cells, continuously monitoring, phagocytizing or

endocytosing contents and interacting with their local tissue microenvironment4,

247. Not only are they important in pruning neuronal synapses248, they also

contribute to membrane turnover in oligodendrocytes249 and are the CNS resident immune cells capable of reacting to potential danger from internal and

60 external sources4. Microglial phagocytosis has been shown to be essential for

effective remyelination in injury models211. Upon exposure to toll-like receptor

stimuli, microglia respond as tissue-resident APCs, retracting dendritic processes

to assume an ameboid shape while upregulating MHCII and co-stimulatory

molecules to present potentially harmful antigens to T cells4, 89, 201, 250, 251. Mice with functionally deficient microglia exhibited delayed onset and reduced severity of EAE, suggesting the important contribution of microglia in EAE

210, 250 +/GFP pathogenesis . Using the CX3CR1 reporter mouse, we showed that microglia and macrophages constitute the main phagocyte populations that

absorb vessel contents in the first 3 days of EAE induction. These phagocytes

can internalize vessel contents in response to TLR agonist stimulation by one of two ways. First, microglia and macrophage act as scavengers, removing spilled blood solutes in the parenchyma as a direct result of transient vessel leakage.

+ Second, perivascular CX3CR1 cells can sample blood contents directly within

intact blood vessels through their intravascular dendritic processes, as has

recently been reported231.

Bone marrow derived myeloid cells are important in the regulation of inflammatory response in the CNS. In particular, infiltrating CD11c+ DCs have

shown to be required for the induction of EAE13, 239, 252, 253, 254. In the adoptive

effector T cell transfer model, blood derived APCs with functional MHCII are required for T cell infiltration and for potentiating EAE61, 210, 250, 255. Depletion of

plasmacytoid DCs in the late inflammatory phase resulted in reduced disease86,

256, 257. To demonstrate that the infiltration and retention of activated 2D2 cells

require APCs to actively present myelin antigen in the CNS and not simply

through disrupted BBB, we either activated OTII cells alone or co-activated both

2D2 and OTII cells in the periphery and showed that activated OTII cells were

able to access the CNS only in the presence of co-activated 2D2 cells. On the

other hand, activated OTII cells with naïve 2D2 cells were incapable of accessing

CNS parenchyma, despite the presence of activated microglia and myeloid-

derived APCs. Our data confirmed the notion that APCs presenting endogenous

CNS peptides are critical to license T cell recruitment and retention in the EAE

brain. Our sequential imaging and FACS data are in good agreement with reports

showing that 5-10% of CD11b+ cells present myelin basic protein as early as day

1 after EAE induction, whereas CD11c+ DCs are not detected till day 5 and only

present myelin basic protein from day 7 onwards258.

T cell infiltration into the brain has traditionally thought to occur at the choroid

plexus, although recent data suggest an alternate route via post-capillary venules and the pial vessels from the SAS7, 8, 65. Our intravital TPM demonstrated T cells

migrating directly into the parenchyma from post-capillary venules. We also detected T cells that directly enter these vessels from the brain parenchyma (Fig.

8). Upon entering the brain, the T cells migrated in a pattern and exhibited APC

scanning behavior that were in contrast to published data showing non-directed T

cell behavior during peak EAE221, 222, 223, 224, 225, 226. A novel finding in our current

study is the focal nature of T cell accumulation and their repeated preferential

62 interaction with DCs during the asymptomatic phase of EAE progression, suggesting that there is intricate communication between newly arrived 2D2 cells and parenchymal DCs. We speculate that through these interactions, 2D2 cells are re-activated through myelin-derived peptide presentation by the DCs before

the T cells exert their pathogenic effector function that eventually leads to the

development of clinical symptoms.

PTx has been shown to increase the permeability of vessels ex vivo137, 138, 259,235,

236, 237, 238, 260, with direct effects on the choroid plexus and cerebral endothelial cells261, 262. In the brain, PTx is postulated to also act through the release of

histamine, which causes vasodilation and changes adhesion molecule

expression73, and alters both endothelial tight junctions and astrocyte end feet

barrier integrity. Histamine receptor 1 and 2 double knock-out mice have a

reduced EAE severity and BBB permeability190. One study has shown that the

CSF of MS patients contains elevated levels of histamine, and H1R antagonists

have shown clinical benefits in MS263. In our study, we used the first generation

histamine H1 receptor antagonist, hydroxyzine, which readily passes into the

CNS, to decrease vessel permeability. In a clinical trial, hydroxyzine was shown

to stabilize or improve clinical symptoms in 75% of MS patients197. Here, we

showed that hydroxyzine treatment reduced EAE disease incidence or severity in

60% of the mice. Hydroxyzine also decreased the total number of T cells found

within the CNS on days during PTx administration. Furthermore, hydroxyzine

reduced the extent of meningeal macrophage and microglia phagocytosis.

Second, it decreased the myeloid-derived APC infiltration on days 3-9. Third,

hydroxyzine decreased all APC subtypes and functional T cells found in the CNS on day 12 (Fig. 10; Fig. 11). Although these phenomena were observed in the brain, the number of IFN-γ, IL-17 and FoxP3 producing T cells in the spleen were similar to controls (Fig. 11). Preventing the early activation of microglia with hydroxyzine resulted in a reduction of both non-specific T cell infiltration on day 3 and antigen-specific T cell infiltration on day 12. These data provide further evidence supporting the view that microglia play a critical role in early EAE induction, and that blocking microglia activation during the inflammatory phase can lessen myelin and neuronal destruction217, 255, 264. Future work remains to

fully understand the regulation of focal and intermittent vessel disruption that was

only partially blocked by H1R blockade.

The exact kinetics of cellular inflammation in the brain has been hard to study,

especially during the very early stages of EAE induction, due to the low number

of relevant cells detected using conventional bulk cellular assays. Here, we

developed an approach to reproducibly induce EAE with low variability. This

method did not require irradiation or the use of chimeras that may disrupt BBB

integrity, and we employed different fluorescent reporter mice to provide dynamic

cellular data in an in situ disease model. Our study revealed the localized

sequential events involving early intermittent BBB disruption, local activation of

microglia, the recruitment of DCs, and the retention and licensing of activated T

cells in the CNS prior to disease manifestation.

Supplemental movie 1: Illustration of a transient vessel leak with vessel content persistence in parenchyma and of multiple transient vessel leaks from the same point of origin.

Region 1: At time 00:31:30, a focal transient leakage of TRITC vessel dye (red) starts and the fluorescence persists in the parenchyma until the end of the movie. Region 2: Starting at time 00:16:00, a focal transient leakage of TRITC vessel dye (red) initiates and resolves, then initiates again at time 00:38:00 and resolves with no residual fluorescence persisting in the parenchyma. Note: other vessels of similar caliber within the same imaging volume remained completely intact. Total time: 52 min 30 sec. Playback speed: 300X. Scale bar = 60 um. Time hh:mm:ss.

Supplemental movie 2: Transient vessel leaks originate from outside the dynamic field of imaging.

Maps of the whole cranial window were used to create a composite image on which a movie was overlaid.

Overlaid movie on the static composite image enables an approximation of the vessels that gave rise to vessel leaks seen in the dynamic movie. Total time: 60 min. Playback speed: 300X.

Supplemental movie 3: Only CX3CR1+/GFP cells phagocytose TRITC vessel dye.

Imaging on day 0 after EAE induction shows only CX3CR1+/GFP (green) cells take in 150kD TRITC vessel dye (red). Shown first is the TRITC-only channel where we observed the cells that take up vessel dye over time. At the end of the video is a two-channel overlay, showing TRITC+ cells were all within the

CX3CR1+/GFP population. Note: no TRITC+ GFP- cells were observed. Playback speed: 300X.

Supplemental movie 4: Infiltrating myelin specific T cells traffic to locations of previous vessel leak and CD11c+ accumulations.

Imaging on day 9 after EAE induction shows 2D2 T cells (blue) surveying the parenchyma concentrating around CD11c-GFP+ rich region. TRITC vessel dye also highlights phagocytic cells (red). Colored tracks

65 show the paths taken by 2D2 cells during the duration of the video, revealing high traffic around CD11c-

GFP+ rich region. Total time: 90 min. Playback speed: 300X. Scale bar = 100um. Time stamp: hh:mm:sec.

Supplemental movie 5: HXYZ treatment of EAE induced mice does not prevent vessel leak but does prevent phagocytosis.

Mice were given HXYZ via gavage over the course of EAE induction. The left panel shows the dynamic imaging of an full EAE-induced mouse while the right panel shows the dynamic imaging of a HXYZ-treated

EAE-induced mouse on day 3. At time 00:18:00 the EAE mouse exhibited a vessel leak (arrow) and phagocytic cells can be seen throughout the whole field. The HXYZ treatment mouse showed 2 locations of vessel leak (arrow) and no phagocytic cells. From time 00:01:00 – 00:14:00 one leak was observed. At a second location, the same vessel leaked 3 times during the imaging session, at times 00:03:30, 00:08:00 and 00:28:30. Total time: 59 min. Playback speed: 300X. Time hh:mm:ss.

3 Extravascular CX3CR1+ Cells Extend Intravascular Dendritic Processes into Intact CNS Vessel Lumen

3.1 Abstract Within the CNS, APCs play a critical role in orchestrating inflammatory responses

where they present CNS-derived antigens to immune cells that are recruited from

circulation to the CSF and SAS. Available data indicate that APCs do so

indirectly from outside of CNS blood vessels without direct contact with blood

luminal contents. Here, we applied high-resolution, dynamic intravital two-photon

laser scanning microscopy (2P-LSM) to directly visualize extravascular CX3CR1+

APC behavior deep within undisrupted CNS tissues in two distinct anatomical sites under three different inflammatory stimuli. Surprisingly, we observed that

CNS-resident APCs dynamically extend their cellular processes across intact vessel wall into the vascular lumen with preservation of vessel integrity. While only a small number of APCs displayed intravascular extensions in intact, non- inflamed vessels in the brain and the spinal cord, the frequency of projections increased over days in experimental autoimmune encephalomyelitis (EAE), whereas the number of projections remained stable compared to baseline days after tissue injury such as CNS tumor infiltration and aseptic spinal cord trauma.

Our observation of this unique behavior by parenchyma CX3CR1+ cells in the

CNS argues for further exploration into their functional role in antigen sampling

and immune cell recruitment.

3.2 Introduction In non-central nervous system (CNS) tissues, activation of antigen-presenting

cells (APCs) by pathogen- or damage-associated molecular patterns (PAMP or

DAMP) results in APC homing to sentinel lymph nodes (LNs) where they process

and present antigens to lymphocytes265. On the other hand, the pathway for

antigen surveillance and presentation in the CNS is unique for a variety of

reasons. First, the blood-brain barrier (BBB) poses a structural impediment for

circulating immune cells to freely move into and out of the CNS under steady-

state conditions. Therefore circulating lymphocytes have limited direct access to

CNS-resident APCs and associated antigens from inside the vessel lumen.

Second, whereas peripherally activated APCs and soluble antigens can drain

directly to the closest LN, in the CNS the cerebral spinal fluid (CSF) acts as the

lymphatic fluid that drains both soluble contents and myeloid-derived cells via the

cribriform plate into deep cervical LNs where antigen presentation can occur6, 8,

266, 267, 268, 269. Third, CNS APCs are both blood-derived and tissue-resident, with

each subset having distinct surveillance locations and functional roles61, 270, 271.

CX3CR1+ microglia, defined as Ly6Clo/CD45lo/Iba-1+, are tissue-resident and

constitute the main immune cell population in the CNS parenchyma under non-

inflamed conditions61, 64, 95. Microglia are present in both grey and white matter

and, although controversial, presumably possess different activation states and

numbers at different anatomic sites272, 273. For example, grey matter lesions in

multiple sclerosis (MS) display less microglia activation compared to that found in

white matter lesions274. Microglia function as tissue APCs in all anatomic sites

68 within the CNS, and infiltrating monocytes have been shown to migrate to the cervical LNs through the cribriform plate89, 268, 275.

On the other hand, CX3CR1+ perivascular and meningeal macrophages are

derived from circulating Ly6Chi/CD45hi/CCR2+ monocytes and can sample CSF

contents in the arachnoid and Virchow-Robin space3, 85. For these reasons,

perivascular macrophages have been postulated to play an important role in the

presentation of myelin-derived peptides to and re-activation of myelin-specific T

cells residing in the perivascular space during the induction of experimental

autoimmune encephalomyelitis (EAE). Activated effector T cells cross from blood

circulation into the CSF through the choroid plexus and Virchow-Robin space in a

CCR6-CCL20 dependent mechanism6, 65, 73, 221. Once in the CSF, primed myelin-

specific T cells encounter meningeal macrophages that present CNS tissue-

derived myelin peptides. The re-activation of T cells results in a local

inflammatory response, effector immune cell recruitment, tissue destruction and

BBB damage, eventually leading to the pathological hallmarks of EAE: immune cell accumulation, neuronal damage and conduction loss6, 200, 276.

Recently, histologic studies of static brain tissues suggest that parenchymal

CD11c+ APCs help to orchestrate immune responses by inserting dendritic extensions into the glial limitans, presumably to sample and present brain- derived antigens to passing immune cells in the Virchow-Robin space277. The observation that APCs can insert dendritic extensions through dense intact

69 tissues for antigen sampling in a separate anatomic compartment is not unique.

For example, dendritic cells have been shown to extend cellular processes across intact epithelial and endothelial tight junctions in the Peyer’s patches16,

mucosa of the small intestine17, the lungs18, the nasal mucosa19, cardiac

valves20, cornea21 and the lymphatic conduits in the LNs22, 23. In all these cases,

APCs extend their dendrites across the host-environment interface or another

tissue compartment and scan for potential foreign antigens for subsequent

presentation to immune cells on the same side of the tissue compartment as the

APCs.

In the current study, we utilized high-definition intravital two-photon laser

scanning microscopy (2P-LSM) to observe CX3CR1+ extravascular APC

behavior in the mouse brain and spinal cord under a variety of inflammatory

conditions. Using several surgical procedures to expose tissues for intravital

imaging including acute and chronic cranial window278, thinned-skull279, 280 and open laminectomy281, we observed in a dynamic fashion the ability of

extravascular APCs to display dendritic extensions into intact CNS vessel lumen.

Furthermore, we showed that the frequency of such intravascular extensions

increased during the progression of EAE and was greater within the cortical grey matter as compared with dorsal column white matter in the spinal cord. The frequency of extensions was unchanged during CNS tumor infiltration, and decreased immediately following spinal trauma before recovering within the first week following injury. In contrast with previous observations of APC extensions across intact tissues or the host-environment interface, we showed tissue-

70 resident APCs displaying dendrites directly into the blood vessel lumen without breaching vessel integrity. Hence, our novel observation adds another dimension to the ever-evolving understanding of immune cell behavior in the CNS.

3.1 Results In order to visualize the behavior of perivascular and parenchymal CX3CR1+ cells in the CNS parenchyma, we applied 2P-LSM to the parietal lobe of a

Cx3cr1+/GFP mouse through implanted cranial glass windows covering either an

open craniotomy or a thinned-skull preparation (Figs 12A-E). The CNS

parenchyma was visualized below the collagen-rich meningeal layers and the

pial surface was outlined by second-harmonic signal generation under 2-photon

excitation (Figs 13A-B). Under steady-state conditions, the majority of the

CX3CR1+ cells in the mouse CNS parenchyma consisted of Ly6Clo/CD45lo/Iba-1+ microglia, with very few Ly6Chi/CD45hi/CCR2hi blood-derived

monocytes/macrophages95. Indeed, dynamic intravital images revealed GFP+ cells with extensive ramified processes are evenly distributed throughout the

CNS parenchyma with a cellular morphology consistent with that of ramified non- activated microglia (Fig. 14A; Figs 12A-C). Sequential imaging showed that the cell bodies of these CX3CR1+ cells were stationary, with extensive dynamic

dendritic processes surveying surrounding tissues (Supplemental movie 6). Upon closer inspection, some of the ramified GFP+ CX3CR1+ cells were located near

CNS vessels, with their processes wrapped around the outer vessel wall (Figs

14B-D; Supplemental movie 7). While the larger-sized extravascular GFP+ cells had morphology consistent with that of microglia and macrophages, other smaller

Figure 12: Comparison of CX3CR1+ cellular morphology and baseline projection frequency using different tissue preparations.

The morphology of CX3CR1+ cells (green) in control Cx3cr1+/GFP mice are shown using: (A) thinned-skull approach, where the images were obtained with an intact bone thickness of 25–50 mm (D, E; c: cranial bone); (B) cranial window implanted 4 days prior to imaging; and (C) acute open craniotomy through a glass window on the day of imaging. CX3CR1+ cellular morphology was similar in all cases, with some differences in the overall imaging properties as imaging depth and structural resolution was greatest with open craniotomy and least with thinned-skull preparation. Parenchymal vessels were labeled with TRITC-dextran

(red). Scale bar = 100 mm.(F) Baseline number of intravascular projections was quantified from four individual Cx3cr1+/GFP mice using 4-day implantation window and six individual Cx3cr1+/GFP mice using thinned-skull preparations, showing an average of 172.5 ± 96.8 projections/mm2 and 171.5 ± 82.3 projections/mm2, respectively. n.s. = not significant. Only CX3CR1+ cells in the parenchyma were analyzed

(Fig. 3).

spherical and highly mobile GFP+ cells could also be seen crawling within the

vessel lumen, which most likely represent other CX3CR1+ cell populations including NK cells and monocytes in the systemic circulation102 (Supplemental movie 8). High-resolution fluorescence and 3-dimensional reconstruction images revealed a surprising finding that on occasion, extravascular, stationary GFP+

CX3CR1+ cells were capable of extending their cellular projections into intact

CNS vessel lumen (Fig. 14B). These intraluminal cellular processes persisted

throughout a 45-minute imaging session, with the intravascular processes of two

nearby extravascular CX3CR1+ cells making contact with each other inside the

vessel lumen (Fig. 14C; Supplemental movie 9). Furthermore, these cells

seemed capable of inserting their cellular processes into the vessel lumen

without breaching the vascular integrity, as evidenced by the lack of intravascular

fluorescent dye leakage into CNS parenchyma.

To interrogate whether insertion of intravascular dendritic processes by

extravascular CX3CR1+ cells is only a behavior exhibited by extravascular CNS

APCs under steady state or a more general behavior found in CX3CR1+ cells near CNS vessels under inflammatory conditions, we induced a Cx3cr1+/GFP mouse to undergo experimental autoimmune encephalomyelitis (EAE).

Consistent with our previously published work282, Cx3cr1+/GFP mice developed

worsening clinical scores starting around day 12 following induction of disease.

Using similar intravital imaging techniques, we were able to easily visualize the presence of intraluminal dendritic processes extending from the main bodies of

Figure 13: Visualization, identification, and analysis of parenchymal CX3CR1+ cells with Intravital imaging methods.

Three-dimensional (xy, xz, yz) display views of the maximum intensity projection images of the spinal cord

(A) and the brain (B) of a control Thy-1-YFP-H+ Cx3cr1+/GFP mouse acquired through an open laminectomy

(A) and a cranial window (B), showing the relative positions of individual Thy-1-YFP-H axons (yellow),

CX3CR1+ cells (green), intact TRITC-dextran labeled blood vessels (red), and dura (blue, second harmonic signals). All of our quantitative analyses took place in the layers where Thy-1-YFP is present below the meninges and pial surface (dotted line). (C) Immunofluorescence histology of fixed tissue sections from Fig.

13 confirmed that the majority of CX3CR1+/GFP (green) cells in the visualized field also co-stained (E) for Iba-

1 (D, red), further identifying them as belonging to the activated monocytic lineage283, 284.

Figure 14: Intravital microscopy reveals persistent intravascular CX3CR1+ dendritic projections into intact CNS vessels.

Figure 14 (continued): Intravital microscopy reveals persistent intravascular CX3CR1+ dendritic projections into intact CNS vessels.

(A-C) A low-power snapshot from Supplemental movie 6 is shown in (A), demonstrating the overall distribution and ramified cellular morphology of CX3CR1+ cells (green) in the CNS parenchyma of a

Cx3cr1+/GFP mouse through a cranial window. Scale bar = 100 um. A few CX3CR1+ cells are found in close proximity to the blood vessel (red) (B-D; Supplemental movie 7). (B) Fluorescence and 3-D surface rendering of a snapshot from inset in (A) at time = 0 min of Supplemental movie 7, demonstrating intravascular dendritic insertion (white arrows) of extravascular CX3CR1+ cells (green) through an intact

CNS vessel (red). Scale bar = 15 um. (C) Fluorescence and 3-D surface rendering of the same cells as in

(B) at time = 45 min, showing persistence of intraluminal dendritic insertions (white arrows) over the duration of the imaging session. Projections from two extravascular CX3CR1 cells are seen touching each other inside the blood lumen. Scale bar = 15 um. (D) Extravascular CX3CR1+ cells with ramified morphology displayed projections (white arrows) into the vessels (red) on Day 12 after EAE induction, while a non- ramified, elongated perivascular CX3CR1+ cell nearby did not (asterisk) and were not included in the final analysis. Scale bar = 10 um. (E) The number of intraluminal projections (white arrows) by extracellular

CX3CR1+ cells increases in the CNS of Cx3cr1+/GFP mouse on day 5 after EAE induction. Scale bar = 10 um.

Only CX3CR1+ cells in the parenchyma were analyzed in A-E (Fig. 13).

77 extravascular CX3CR1+ cells near intact blood vessels on day 5 during EAE

induction (Fig. 14E), demonstrating that the ability of the extravascular CX3CR1+ cell to protrude intravascular dendritic processes was not unique to steady-state cells. To provide corroborating evidence for how the parenchymal CX3CR1+ cells

managed to display their dendritic processes through multicellular perivascular structures of CNS vessels, we analyzed naïve, nonmanipulated brain (Figs. 15A,

15B) as well as EAE-induced tissue sections (Figs. 15C,15D) by

immunohistochemical (IHC) analysis. We confirmed that the intravascular

dendritic processes within a vessel lumen in Fig. 14 were derived from the

extravascular CX3CR1+ cells with dendritic morphology and were not a result of

imaging artifact or blood-derived CX3CR1+ cells in the process of transmigration

across the vessel lumen. We also confirmed the juxtaposition of extravascular

CX3CR1+ cells and the extent of the reconstructed blood vessel lumen by two different intravital labeling methods (Fig. 16). In addition, electron micrographs

(EM) of fixed brain sections from naïve, unmanipulated mice confirmed the presence of intravascular dendritic processes displayed by a CX3CR1+ cell through the basement membrane and endothelium, with a cell body beyond astrocyte end feet and in close proximity to surrounding neurons in the CNS parenchyma (Figs. 15E, 15F). Within one thin section, we found 176 vessels in

38,556 mm3 of tissue and one projection.

Figure 15: Immuno-histology and TEM confirm intravascular CX3CR1+ dendritic projections into intact CNS vessels.

(A-B) Immunofluorescence microscopy of fixed tissue sections on day 30 after EAE induction confirmed the presence of dendritic extensions (arrows) by extracellular parenchyma (P) CX3CR1+ cells (green) into the vessel lumen (L) as outlined by anti-CD31 (F; red) and tomato-lectin (G; red) staining. (C-D)

Immunofluorescence microscopy of fixed tissue sections of a non-inflamed Cx3cr1+/GFP mouse confirmed the presence of dendritic extensions (green) flanking by GFAP+ astrocytic end-feet (pink) next to vessel lumen as outlined by laminin (blue). (E) TEM of fixed tissue sections of a non-inflamed Cx3cr1+/GFP mouse confirmed the presence of dendritic processes (blue) with intact endothelial cells and basement membrane.

Astrocytic processes (pink) and axons (green) are visualized next to the dendritic extension. Magnification =

10 000X.

We then assessed changes in the frequency of intravascular dendritic processes during the course of EAE induction. To accomplish this, we conducted sequential

imaging of CNS parenchymal CX3CR1+ cells on the same animal cohort for 12

days through either acute imaging of different mice or chronic cranial

implantation windows in the same animal (Fig. 17A-C). To further facilitate the

visualization of brain parenchyma versus meninges for subsequent analysis, we utilized double transgenic mice in which YFP is expressed under a neuron- restricted Thy1 promoter while one of the Cx3cr1 alleles is replaced by GFP (Fig.

13). Thy-1 YFP marker allowed us to identify the parenchyma within the imaging field. Under non-inflamed conditions, CX3CR1+ cells were uniformly present

throughout the parenchyma with a ramified morphology in both white and grey

matter (Fig. 13A-B). CX3CR1+ cells within the cortical grey matter had a more ramified and delicate pattern than those in the spinal cord white matter.

Intravascular dendritic processes from extravascular CX3CR1+ cells were again

detected (Fig. 17D-E). As EAE developed, parenchymal CX3CR1+ cell density

increased with concomitant increasing number of extravascular GFP+ cells

positioned next to CNS vessels (Fig. 17C). Next, we enumerated the number of intraluminal dendritic processes (“projections”) by extravascular stationary

CX3CR1+ cells throughout the first 12 days of EAE induction (Fig. 17F). In order

to ensure that we did not count as intraluminal dendritic processes GFP+ cells

that were within the vascular lumen caught in the process of extravasation, we

excluded cells with migration speed of > 3 um/min in the dynamic imaging

dataset, as well as small, spherical and motile GFP+ cells or elongated,

Figure 16: Vessel boundary outlined by different in vivo labeling techniques.

CNS vessel lumen boundary measured similarly during intravital imaging of the same region by sequentially injecting (A) TRITC-dextran (700 ug / mouse); and (B) tomato-lectin (16 ug / mouse) i.v. on two consecutive imaging days. Scale bar = 10 um.

Figure 17: Increasing number of extravascular CX3CR1+ cells with intravascular dendritic projections in the brain during early EAE induction.

(A) A snapshot of vessel (red) within the CNS of a Thy-1-YFP-H x Cx3cr1+/GFP mouse taken 4 days after cranial window implantation shows the steady-state distribution of CX3CR1+ cells (green) and intact neurons (yellow) within the CNS parenchyma, with the CX3CR1+ cells remaining in a ramified state. Scale bar = 50 um. (B, C) Snapshots of the mouse brain on days 0 (B) and 12 (C) after EAE induction show morphologic changes and an increase in the number of CX3CR1+ microglia (green). The blood vessels are outlined by the TRITC-dextran dye (red). Intraluminal portions of the CX3CR1+ cells are highlighted in grey.

Projections (arrows) occur in both the large and small vessels, but are more common in the smaller vessels.

YFP axon signal is removed for ease of visualizing microglia Scale bar = 50 um. (D, E) Coronal fluorescence

(D) and surface rendering (E) view of a blood vessel (red) in (C) demonstrates an intraluminal dendritic projection (white arrows) by an extravascular CX3CR1+ cell (green). Scale bar = 15um. (F) The number of intraluminal projections are quantified and normalized to total vessel surface area (#Projections / mm2) over the course of EAE induction, showing a 2-fold increase in projection frequency over the first 12 days. Only

CX3CR1+ cells in the parenchyma were analyzed (Fig. 13).

82 perivascular GFP+ cells that had >30% of the cell volume inside the vessel

lumen. At baseline, we found that CX3CR1+ cells exhibited 172.50 ± 24.2

projections per mm2 of vessel wall surface (Fig. 17F). The number of dendritic

projections more than doubled (not statistically significant, p>0.05) that found in

the non-inflamed control over the first 9 days following EAE induction, with the

frequency of projections plateauing between days 9 and 12 (Fig. 17F).

To compare number and characteristics of intravascular dendritic projections at

different anatomical sites containing grey matter or white matter, we also

examined the behavior of extravascular CX3CR1+ cells in the dorsal column

white matter next to spinal vessels during EAE. Under steady-state conditions,

CX3CR1+ cells in the spine were uniformly distributed throughout the

parenchyma of the spinal cord, with some cells closely associated with both large

and small caliber vessels (Fig. 18A-B). Similar to the brain, GFP+ dendritic

projections were again visualized to insert into both large and smaller spinal

vessels (Fig. 18D-E). Again, parenchymal CX3CR1+ cells in the spine appeared in a ramified, non-activated state (Fig. 18A-B), with an average of 74.6 ± 11.03 projections per mm2 of visualized vessel surfaces (Fig. 18F). Upon EAE induction, the number of intravascular projections doubled (not statistically

significant, p>0.05) to an average of 159.97 ± 27.26 projections per mm2 of vessel surface by day 12, with cells appearing in an activated, less ramified morphology (Fig. 18C). Although intravascular projections could be seen in the large central venous vessel, they were more commonly observed in close association with the smaller venous vessels (Fig. 18C).

Figure 18: Increasing number of extravascular CX3CR1+ cells with intravascular dendritic projections in the spine during early EAE induction.

(A) A snapshot of the central dorsal spinal vein of a Thy-1-YFP-H x Cx3cr1+/GFP mouse taken immediately after T10 laminectomy shows the steady-state distribution of CX3CR1+ cells (green) and intact axons in the spinal parenchyma (yellow), with the CX3CR1+ cells remaining in a ramified state. Scale bar = 50 um. (B, C)

Snapshots of the spine on days 0 (B) and 12 (C) after EAE induction show morphologic changes and an increase in the number of CX3CR1+ cells (green). The blood vessels are outlined by TRITC-dextran dye

(red). Intraluminal portions of the CX3CR1+ cells are highlighted in grey. Projections (arrows) occur in both the large and small vessels, but are more common in the smaller vessels. YFP axon signal is removed for ease of visualizing microglia. Scale bar = 50 um. (D, E) Coronal fluorescence (D) and surface rendering (E) view of a blood vessel (red) in (C) demonstrates intravascular dendritic projections (white arrows) by extravascular CX3CR1+ cells (green). Scale bar = 15um. (F) The number of intraluminal projections are quantified and normalized to total vessel surface area (# projections / mm2) over the course of EAE induction, showing a 2-fold increase in projection frequency over the first 12 days. Only CX3CR1+ cells in the parenchyma were analyzed (Fig. 13).

To test whether the increase in intravascular dendritic processes was simply due

to activation of brain and spinal CX3CR1+ cells in response to non-specific tissue

injury, we examined intravascular dendritic projections in other models of CNS

inflammation. First, we investigated an aseptic traumatic spinal cord injury model

in which a traumatic crush injury was created in the dorsal column without

breaking the major blood vessels. We observed that CX3CR1+ cells accumulated

at the injury site over a course of 8 days post injury while the vessel integrity to

large molecular weight solutes remained intact throughout the healing process,

as evidenced by the lack of vessel dye uptake by surrounding tissue phagocytes

(Fig. 19A-B). Similar to the findings in EAE, extravascular CX3CR1+ cells can

insert their GFP+ dendritic processes through intact vessel walls into the blood lumen (Fig. 19C-D). Contrary to the findings in EAE, however, CX3CR1+ cells accumulated around both the large and small vessels at the spinal injury site

(Fig. 18C, 19B). We observed an average of 46.59 ± 10.1 intravascular projections per mm2 of intact vessel wall at baseline (Fig. 19E). Upon

enumeration of intravascular dendritic projections during an 8-day span of tissue

recovery, we observed an immediate 40.5 % reduction (p = 0.048) in projections

at the site of injury, and then a slow recovery to an average intravascular

projection frequency of 39.46 ± 7.91 per mm2 in the lesion (not statistically

significant, p>0.05), a frequency that was 84.7 % of a healthy spinal cord (Fig.

18F, 19E).

Figure 19: Intravascular projections decrease and then recover in aseptic traumatic spinal cord injury and do not increase in the CNS tumor microenvironment.

Figure 19 (continued): Intravascular projections decrease and then recover in aseptic traumatic spinal cord injury and do not increase in the CNS tumor microenvironment.

(A, B) Snapshots from tile scan of the spinal cord dorsal columns at T10 on days 0 (A) and 8 (B) after crush injury (dashed box) shows intact dorsal vein (red) and CX3CR1+ cell (green) distribution. Intraluminal portions of the CX3CR1+ cells are highlighted in grey. The morphology of CX3CR1+ cells on Day 8 appeared to be more rounded and less ramified. (C, D) Coronal fluorescence (C) and surface rendering (D) view of a blood vessel (red) in (B) demonstrates intravascular dendritic projections (white arrows) by extravascular CX3CR1+ cell (green). (E) Quantification of intravascular projections post crush injury showing a near-full recovery in projection numbers in 8 days. (F) A snapshot of the CNS tumor microenvironment 7 days after inoculation of MM1-DsRed into a Cx3cr1+/GFP mouse shows local accumulation of CX3CR1+ cells and development of neo-vasculature. Intraluminal portions of the CX3CR1+ cells are highlighted in grey. MM1-DsRed signals are removed in F-H for ease of visualizing CX3CR1+ cells. (G, H) Fluorescence

(G) and surface rendering (H) view of a blood vessel (red) in (F) demonstrates intravascular dendritic projections (white arrows) by extravascular CX3CR1+ cells (green). (I) The number of intravascular projections in the CNS tumor microenvironment are quantified from 3 tumor-bearing mice and normalized to total vessel surface area, showing an average number of 72.9 ± 6.3 projections / mm2 (same as baseline in

Figs 12F and 13F). Only CX3CR1+ cells in the parenchyma were analyzed (Fig. 13).

A second tissue injury model was a syngeneic CNS tumor model. To determine if intraluminal projections by extravascular CX3CR1+ cells could also be detected

in the vessels within a CNS tumor microenvironment, we inoculated i.c. MM1-

DsRed2, a fluorescent syngeneic mouse medulloblastoma cell line derived from

Patch+/-/p53-/- mice, into three Cx3cr1+/GFP mice and recorded the responses of

CX3CR1+ cells in the CNS tumor-associated neo-vasculature. We observed local

accumulation of CX3CR1+ cells in the CNS tumor microenvironment with extravascular CX3CR1+ cells extending intravascular dendritic projections into

the tumor neo-vasculature (Fig. 19F-H). This was similar to that found in the

spine and brain parenchyma in EAE and spinal trauma. Distinct from the EAE

model, the average frequency of intraluminal dendritic processes per mm2 of

intact tumor-associated vessel wall was only 72.9 ± 6.3, a number that was

consistent across tumor lesions in three different experimental animals and in

good agreement with that observed in the control brain and spinal cord (Fig. 17F,

18F, 19E). Our findings from EAE, aseptic traumatic spinal cord injury and CNS

tumor models suggest that, while there is a baseline frequency of intravascular

dendritic projections by extravascular CX3CR1+ cells in vessels of the brain and

the spine, there exists discernable differences in regulating the number of

intravascular processes by CX3CR1+ cells in response to the inflammatory signals associated with EAE induction compared to that in response to the inflammatory signals in association with tissue injury and CNS tumor growth.

3.2 Discussion At baseline, we observed an average of 60 and 173 intravascular projections by

extravascular parenchymal CX3CR1+ cells per mm2 of vessels found within the

white matter of the dorsal columns of the spinal cord and the grey matter in the

cortex, respectively (Fig. 18F, 17F). The number of projections doubled in the

spine and the brain on day 12 following EAE induction, at a time when mice first

began to exhibit neurologic deterioration. The differences in projection

frequencies suggest that there may be differences in these two tissue types with

respect to CX3CR1+ cellular response that inversely correlate with the

abundance of myelin found at each site. Interestingly, our observation of

projection frequencies during steady state and inflammatory conditions mirrored

that seen on peri-epithelial APCs in the lamina propria of Cx3cr1+/GFP mice16. The consistent observation of CX3CR1+ APC behavior in multiple tissue types under

steady state as well as distinct inflammatory inducers highly suggest that the

extra-compartmental dendritic protrusions through intact endothelia and epithelia

tight junctions are a fundamental feature of the immune surveillance strategy

employed by CX3CR1+ APCs. Such important cellular features may have

previously been under-appreciated, in the current study, however, we have been

able to successfully preserve these processes by transcardial PFA tissue fixation

procedures (Fig. 15).

All three CNS inflammatory models examined in the current report entail different

states of vascular activation. In EAE there is an acute induction of systemic

89 inflammation by Toll-like receptor (TLR) signals and pertussis toxin used in the

immunization cocktail. In aseptic spinal cord injury or CNS tumor model, the vessel endothelium and CNS APCs are spared from exposure to such signals from bacterial products as contained in the EAE immunization cocktails. How differential states of endothelial activation contribute to the observed frequency of intravascular dendritic projections will be a subject of future investigation, specifically with respect to potential signaling crosstalk between the endothelium and extravascular CX3CR1+ APCs.

It has been argued that, in contrast to thin skull procedures, the cranial window

technique engenders too much local trauma and inflammation to the imaged

brain tissue such that the observed APC populations in the meninges and

parenchyma could be artificially activated225, 285, 286. However, the thin-skull

approach, while avoiding direct contact between the surgical instruments with the

meninges, requires the breach of bone marrow integrity and therefore could elicit systemic inflammation affecting underlying meningeal and parenchymal tissues.

We performed acute and chronic craniotomy windows as well as thinned-skull

surgical approach in the current study. In our hands, we found that the relative

frequencies of intravascular extensions are comparable when these procedures

were executed carefully (Fig. 12). In all of our intravital experiments, we paid

special attention during intravital surgical procedures and post-operatively, and

excluded mice from imaging analyses that had undergone a sub-optimal surgery

procedure or endured visible vessel trauma. Both cranial window and thinned-

90 skull approaches revealed ramified parenchymal CX3CR1+ cells at baseline, consistent with CNS-resident APCs in a non-activated state (Figs. 14, 15A-B,

17A-B; Fig. 12). It is possible that both surgical methodologies designed to visualize the resident brain parenchymal cells induce some degree of injury.

Regardless of the basal level of inflammation due to surgical trauma, the frequency of intravascular extension by parenchyma CX3CR1+ cells was in good

agreement with that seen in the gut16, 17, and increased with additional tissue

inflammation in EAE but not in spinal trauma or tumor inoculation (Figs 17F, 18F,

19E, 19I). More importantly, we were able to capture intraluminal extension of

CX3CR1+ cells in naïve, noninflamed brain tissues by IHC and EM (Fig. 15).

Using these methods, we observed the entrance of CX3CR1+ cells into the vessel lumen through the basement membrane and endothelial layer of the CNS vessel. Furthermore, the astrocyte end feet were on either side of the dendrite entry point, indicating that they have shifted positions to allow for the CX3CR1+ projections into the vessel lumen.

Intravenous administration of both fluorescent dextran and tomato-lectin provided clear fluorescent signals to consistently delineate the interface between the blood content and the vessel wall throughout the prolonged duration of intravital imaging (Fig. 16). We used a combination of 3-dimensional static images and time-resolved sequential datasets in our analysis of dendritic projection to verify that the identified intraluminal GFP signals came from stationary CX3CR1+ cells whose cell body was largely outside of the vessel wall, and not from smaller,

91 spherical mobile CX3CR1+ cells such as NK cells and T cell subsets that

attached transiently to the luminal wall72, 102 or from Ly6Clo/CX3CR1+ vessel-

patrolling monocytes that were in the process of transmigrating from the blood

lumen to the perivascular space. Other than by morphology and Iba-1 staining

(Fig. 13C)283, 284, the Cx3cr1+/GFP mice used in our current study do not allow for

precise identification of the specific CX3CR1+ cell populations in the captured 2P-

LSM images (i.e. microglia versus perivascular monocyte/macrophages or

dendritic cells). This shortcoming is especially relevant in later stages of CNS

inflammatory processes where both tissue-resident and blood-derived APCs are

present and express CX3CR1. To overcome this shortfall, future experiments will

require the use of bi-phenotypic fluorescent reporter mice such as the

Cx3cr1+/GFP / Ccr2+/RFP mice95 or crossing CD11c-mCherry mice287 to Cx3cr1+/GFP mice. Furthermore, the extent of intravascular dendritic projections in the brain of

Cx3cr1+/GFP mice can be compared with that in Cx3cr1GFP/GFP mice to further

delineate whether functional CX3CR1 is required for the dendritic protrusion.

Recent years have seen a paradigm shift in our understanding of the immune compartment in the CNS288, 289. While at steady state few circulating immune

cells are found in the meninges and brain parenchyma, the CNS allows

orchestrated infiltration of multiple immune cell subtypes in conditions such as

infections, trauma and autoimmune diseases. These observations suggest that

the BBB is a barrier that can be manipulated to regulate traffic of immune cells in-

and-out of the CNS. Work in models of viral infection and EAE have thus far

92 focused on the role of inflamed CNS vascular endothelium in recruiting circulating immune cells into the CNS via adhesion molecules and inflammatory

chemokines289, 290, 291. In these models, both blood-derived (i.e. monocytes /

macrophages / dendritic cells) and CNS-resident (microglia) APCs are implicated

in the re-activation of infiltrated lymphocytes in the perivascular CSF space65, 73.

This suggests APCs play an ancillary role in the recruitment of initial immune cell

invasion since cognate antigen recognition is postulated to only occur after the

immune cells have been actively recruited to the CNS tissue with the aid of the

inflamed endothelium. In this light, our current observation that CX3CR1+ APCs

extend intravascular dendritic projections directly into intact CNS vessel lumen is

highly significant, as it suggests an opportunity for: 1) antigen surveillance

directly in the blood lumen by CNS APCs through intact CNS vessels; and 2)

direct CNS antigen presentation to and recruitment of circulating immune cells by

CNS APCs. Further explorations will be required to test these possibilities. If true, this hypothesis could explain how circulating lymphocytes were able to extravasate to CNS parenchyma in an antigen specific manner in early stages of pathological conditions such as EAE, where the BBB is presumably intact.

In summary, we used dynamic high-resolution optical fluorescent microscopy with 2-photon excitation, coupled with cell lineage-specific fluorescent reporter mice and multiple surgical techniques that allowed for longitudinal monitoring to study cellular events in the CNS. We observed that extravascular CX3CR1+ cells

possess the capacity of inserting part of their cellular extensions through intact

CNS endothelium. How these cells accomplish this through multiple cell layers that comprise the BBB on a cellular and molecular level remains to be explored.

Furthermore, the functional role such biological process may have in regulating

immune responses in the CNS needs to be carefully tested. Our discovery

highlights the important role that high-definition intravital dynamic optical

fluorescence imaging plays in uncovering this novel cellular process in the CNS.

In addition, our observations may offer opportunities for potential targeted

therapeutic strategies in CNS-related diseases including infection, cancer and autoimmunity by interfering with the regulation of intravascular dendritic extensions by extravascular CNS APCs through intact CNS vascular endothelium.

Supplemental movie 6: Microglia morphology and distribution in non-inflamed CNS parenchyma of a

Cx3cr1+/GFP mouse.

Sequential imaging of the parietal lobe of a Cx3cr1+/GFP mouse was captured through a cranial window implanted 4 days prior to imaging. CNS vessels are highlighted by TRITC-dextran. Total imaging time: 60 min. Playback speed: 300X.

Supplemental movie 7: Dynamic dendritic motility of stationary extravascular CX3CR1+ cells.

A zoomed-in view of a GFP+ CX3CR1+ cell (green) next to an intact CNS blood vessel (red) illustrates the highly dynamic motility of dendritic extensions probing the extravascular space. Other smaller, spherical

CX3CR1+ cells can be seen crawling in the blood vessel lumen, which most likely represents circulating

CX3CR1+ NK cells or monocytes72. Total imaging time: 45 min. Playback speed: 300X.

Supplemental movie 8: Extravascular CX3CR1+ cells project dendrites into CNS vessels.

Dendritic projections of extravascular CX3CR1+ cells are vividly visualized within the CNS vessel lumen.

The extravascular microglia body projects stably into the vessel lumen for at least 30 minutes. Dendritic projections from two CX3CR1+ cells can be seen contacting each other within the vessel lumen. Note the absence of TRITC-dextran dye in the surrounding parenchyma at the site of intravascular dendritic insertions. Total time: 45 min. Playback speed = 300X.

Supplemental movie 9: Three-dimensional view of the intravascular dendritic extension by extravascular CX3CR1+ cells.

A snapshot from Supplemental movie 7 (at time stamp = 36 min 30 sec) is shown in a 3-dimensional rendering view, demonstrating the relative position of the green dendritic body with respect to intact CNS blood vessel wall. Total time: 22 min. Playback speed: 450X. Scale bar = 15 um.

4 Image Analysis of Dynamic Intravital Multi-photon Imaging.

4.1 Introduction With the advent of two-photon laser scanning microscopy it became possible to perform dynamic, intravital, immuno-imaging at the cellular level292, 293, 294, 295.

Cell-cell contact has been shown to be important in adaptive immunity296, 297 for information sharing in the partnership of antigen presenting cell and T cell within the lymph node298, 299. High resolution, real-time fluorescence imaging has permitted visualization of single cell interactions and therefore insights into immune cell interaction, the immunologic synapse and interactions with the environment, rather than bulk population behavior300. First generation analyses of

T cells in lymph nodes and their interactions with other APC populations such as dendritic cells, macrophages and B cells, have shown that there are a limited set of motility variables that can be examined301, 302, 303 (Fig. 20) and from these measurements, general interpretations can be made about cellular behaviors. In our process of image analysis, we utilize a software (Imaris, Bitplane Inc.) that can segment fluorescent images and determine the tracks of cells. From there, we import the fluorescent information, cell and track position data into another software (Matlab, Mathworks Inc.) for further interaction analysis. In this chapter, we will describe analysis strategies and the algorithms used in these programs.

Figure 20: From Matheu. Two-photon multidimensional data analysis parameters302.

Path length in μm is the entire distance the cell moved. Greater path length

generally signifies cell scanning behavior. However, cells engaged in productive

interactions can scan the surface of the target cell generating long path lengths

within a confined region.

Displacement in μm is the distance from the first point to the last point the cell

moved and is mainly used to determine whether a cell is stationary or engaged in

a productive interaction. When used in conjunction with distance, it can indicate

whether the cell took an efficient path. This measure is termed chemotactic index

which is defined as distance / displacement. The closer the chemotactic index is

to 1, the straighter the path to the target and thus the chemoattractant signal is

strong. To understand the implications of chemotactic index one needs to

understand the concept of chemokines and chemokine gradients. The other

parameters of cell motility in Fig. 20 can be clearly described within the context of

chemokine signaling.

4.2 Chemokine Influence on Motile Cells In contrast to the neural system that is tightly packed with neurons and accessory

cells fixed in place, the immune system is comprised of multiple subsets of highly

mobile cells. This constant motion and surveillance of all tissues in the body

enables rapid deployment to sites of pathogen challenge. Complex T cell, B cell

and DC cell-cell interactions rely on motility in order to communicate and

98 coordinate responses for tolerance to self and effector responses to various

pathogens. The pathogen threat that we face is very diverse, thus our adaptive

immune system has evolved the ability to recognize the entire gamut of the

antigenic universe. They do this by undergoing a form of genetic rearrangement

called somatic hypermutation. By recombining the genes that encodes the alpha

and beta chains of the T cell receptor, T cells are able to generate a trillion

different unique sequences capable of identifying 25 million different specificities.

Because of the different specificities, only 1 in 105 – 106 T cell recognize cognate

antigen in a primary immune response304. Despite these incredible odds, our

immune system has no problem mounting a timely and efficient response under

normal circumstances. This is largely due to chemoattractants directing immune

cells via chemokine receptors on their surface.

Interestingly, the first experiments to understand chemokine-mediated signaling

showed that treatment with PTx inhibited the mobilization of neutrophils by

CXCL8. It was determined from this that chemokine receptors had to be a G-

coupled protein305. It is now known that chemokine receptors are seven-

transmembrane G protein–coupled receptors that can have multiple ligand

binding partners and can signal through multiple pathways to induce differential

responses. The single signaling pathway common to all chemokine receptors is cellular migration.

Chemokines are cytokines that cause the directed movement of immune cells.

Even though chemokines are secreted they are not free floating but are

immobilized on cells or extracellular matrix surrounding the cell producing the

chemokine, thus effectively reducing the total amount of chemokine produced by

the cell while maintaining a gradient306. A population assessment of chemotactic

index, especially if large number cells in a region are close to 1, coupled with this knowledge of the extracellular matrix can also be an indicator of the structures within the microenvironment allowing this movement. For example, it has been shown that fibroblastic reticular cells within the lymph node create an immune cell highway network to allow cells to efficiently traverse and encounter APCs with a cognate antigen307 308.

Motility coefficient309 was devised to characterize Brownian motion, where the

closer the mean displacement / square root of time is to 1, the closer the cell is to

random Brownian motion. Initial two-photon microscopy reports of immune cell

motility within the lymph node used naïve T cells under homeostatic conditions

which resulted in no discerning pattern of movement310. Under these conditions

and in the context of a burgeoning chemokine field, it was logical to use this

measure to describe a population of cells within an imaging field and concluded

that movement within a lymph node had a motility coefficient close to 1 and was

therefore described random motion. This measure ignores the direction vector of

these cells, and subsequently parameters have been devised to more fully

describe the motion of a cell within the context of a chemokine gradient.

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Recently, motility coefficient has been used to compare across different

conditions, such as the effect of a pharmaceutical therapy or motility within a

chemokine knockout mouse. It has been determined the closer the motility

coefficient is to 0 there is confined migration, if it is greater than 1 there is

directed migration.

4.3 Point of Reference Before the idea of chemokine directed motility, the first attempts to describe cell

movement was based on the assumption of Brownian motion and that the

movement of cells were random, resulting in the motility coefficient. Graphing of

this process resulted in taking track data and referencing the beginning of the

track to 0,0 of an XY-plot. We programed a plot generator to plot each track

beginning of the track to 0,0, and as depicted in literature, the result looks like

there is no directional trend and can be determined as a random walk. Each track

gets assigned a different line style and color, where the initial position is removed

from all samples along the path. This latter step ‘resets” the origin of each track,

where this allows different path lengths and shapes to be visually discriminated.

However, in the case of chemokine directed cell movement, the interesting part

of the cell path is the end, whether the cell ends at the target of interest. A related

plot generator as described above was created, however, the final position is reassigned to 0,0 of an XY-plot by removing this position from all samples along

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the path. This ‘resets” the destination of each track, where cells that are being

influenced by a chemokine gradient will take a directed path as opposed to non-

interacting cells that should follow a Brownian path to their final destination

during the imaging experiment. The resulting graph often shows a directional

trend towards the target of interest. This data is also represented in a 3D plot

(Note: the net output of this code resembles the appearance of the Deathstar

from the movie “Star Wars.”)

4.4 Angle of Approach For T cells, a change in direction towards a target in the vicinity of an APC is an

important indicator of chemokines creating a path towards a productive

interaction. Turning angle is the angle of a portion of the path relative to the previous portion as a measure of a directional change made by a cell. A wider turning angle implies that the cell has deviated greatly from initial course and has changed course to follow a chemotactic signal. Of the hundreds to thousands of tracks in the position record of a typical experiment, most will start far away from the user-defined target location. Thus, the analysis of turning angle has caveats.

First, the diameter for the volume of influence about an APC has a substantial impact on the reported value: a diameter that is too large will lead to many “false positive” tracks, whereas a diameter that is too small does not accurately reflect the physical action of chemokines at a distance from the APC. Second, there is a presumption that the spatial distribution and gradient for the chemokine signal

follows unrestricted diffusion, but the physical tissue microstructure that is not

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visualized, might alter the cues for the T cell to change direction. Third, the angle

of approach and the time at which the cell detects the chemokine gradient

impacts the measurement. For example, a cell travelling tangentially to the

volume of influence will deviate from the initial path and produce a wide angle to

move towards a target, but a cell already travelling directly towards a target from

a distance, will not deviate much from the initial path when it enters the volume of

influence even if it engages in a productive interaction with the target. From this

example, it can be seen that population averages of turning angle generally do

not result in a representative measure. The complementary measurement of

turning angle is directional persistence and is dictated by intracellular actin

rearrangement311. Directional persistence is the length of time a cell moves in a

particular direction with small angular deviations from the mean300 and is also an

indication of a chemokine gradient.

Some strong chemoattractants expressed by a single cell or a cohort of cells can

have far reaching effect, and is visually astounding in dynamic data. A program

was developed to identify the flux of T cells to a cell of interest. The code first centers the volume of interest (VOI) at a user-defined location and then excludes tracks that are never inside the VOI throughout the duration of the experiment.

Also, tracks that originate within the VOI, or have less than three timeframes before or after entering the VOI are also excluded because these tracks are usually at the beginning or the end of the imaging session and conclusions cannot be made about these tracks. For the remaining set of tracks, a linear

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model of motion based on location in three timeframes prior to crossing the

defined threshold for interaction is used to construct an equation for a line in 3D.

Similarly, a linear model is constructed using the three points after the track

crosses the threshold of the VOI. The angle between these two lines is

calculated and compared against the 3D angle to the target at the timeframe just

prior to entering the VOI. For example, if the cell in tangential to the VOI and the

required angle change to go straight to the target is +90º and the actual change

is +80º, the scenario indicates a strong response to the cues for the T cell to

move towards the target APC. Likewise, an actual change of -5º would

correspond to a weak negative response to the chemotactic cues for moving

towards the APC. Thus, there needs to be a categorization of tracks based on

the angle of approach to the VOI. As shown in Fig. 21, a representative selection

of track paths from an actual experiment demonstrate a subset of features that

can be quickly inferred from this analysis framework.

4.5 Other Motility Parameters Average velocity in μm/min is the total distance / total time and instantaneous

velocity change in distance / change in time. Average velocity can be used to

describe a population of cells and the state of the microenvironment, for example

the average speed of a T cell in an inflamed lymph node is 10-12μm/min whereas microglia within a non-inflamed CNS will have an average speed of

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Figure 21: Types of T Cell tracks

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Figure 21 (continued): Types of T Cell tracks.

Only tracks that cross into a spherical volume of influence (VOI) with diameter 50um (gray circle) about an

APC (gray dot) during the imaging experiment are considered for analysis. Paths in real data have indicated:

(a) no interaction, (b) a direct approach with minimal interaction while inside the VOI, (c) a tangential approach followed by a direction change towards the target and some surveying before escaping the VOI

(i.e. path of black dots), (d) a productive interaction at reduced velocity followed by escape from the VOI, or

(e) a productive interaction with a substantial number of interaction points. This only shows the top-view of the vicinity of the target (i.e. the focal plane runs into and out of the page), where linear extrapolation of position three timeframes before (blue line) and after (red line) were used to compare changes in the T cell track direction.

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0.5μm/min. Instantaneous velocity of a cell can be linked to chemokine gradients

where a decrease implies interaction with chemokine gradient and presumably

the cell is close to the chemoattractant source.

Contact time is the total time two cells are in contact, min. A longer contact time

implies a more productive cell-cell interaction. Hit rate analysis was developed to

quantify the frequency of a T cell touching a target DC and to show differences in

T cell-DC interactions under different inflammatory chemokine guidance312. Kon

is the calculated hit rate (mm6. hr-1) of the T cell touching a DC, but could be generalized to any cell type of interest.

Kon= A / [T Cells] [DC]• t.

A is the total observed number of DC-T cell contacts, [T cells] and [DC] are densities of T cells and DC in a data collection volume, while t represents the duration of imaging session. Hit rate ratio is a way to normalize a population of interest to a control and is determined as the ratio between the Kon for T cells interacting with activate DCs and the Kon for interactions involving a control set of DCs within the same imaging region.

The interesting finding from the analysis of Kon is that stable productive interactions result in better T cell activation and memory generation312. As an extension of that work it was determined that T cells encounter many DCs within

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the lymph node and that at different inflammatory phases, T cells interact

differently with DCs313, 314(Fig. 22). It was subsequently determined that the

information or signals from sequential T cell-DC encounters are integrated by T

cells in antigen specific activation315. These types of cellular behavior findings

can only be accomplished by intravital two-photon microscopy.

The use of high resolution confocal imaging of cell motility in vitro discovered

many key aspects of the effects of chemokines, the most important being that

chemokine receptors polarize on the leading edge of the cell and this localization

is essential to signaling for motility and cell activation316, 317. This localization of

chemokine receptors also plays a role in the actin cytoskeleton remodeling and

thus the shape of the cell as it moves either across endothelium or through the

extracellular matrix. Shape index is a measure of the longest axis / the shortest axis of a cell. Stationary cells are usually spherical and have a shape index close to 1. However, motile cells have a larger shape index due to elongation as a result of taxis. In addition, cell activation generally results in enlargement of the cell thus cell volume can be used as a measurement of cell activation and in some instances proliferation, however there are more reliable ways than imaging to determine activation and proliferation.

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Figure 22: Three phases of naıve T-cell priming by dendritic cells in peripheral lymph nodes, as revealed by multiphoton intravital microscopy from Mempel318

“During the first 8 h after entering from the blood, T cells underwent multiple short encounters with DCs, progressively decreased their motility, and upregulated activation markers. During the subsequent 12 h T cells formed long-lasting stable conjugates with DCs and began to secrete interleukin-2 and interferon-g. On the second day, coinciding with the onset of proliferation, T cells resumed their rapid migration and short DC contacts. Thus, T-cell priming by DCs occurs in three successive stages: transient serial encounters during the first activation phase are followed by a second phase of stable contacts culminating in cytokine production, which makes a transition into a third phase of high motility and rapid proliferation.”318

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Recently there have been developments in functional reporter mice319 and

functional reporter constructs that can be introduced into the cell ex vivo. Ex vivo

calcium flux dyes have been used for years as a functional indicator of T cell

activation320 however, the applications were mainly in vitro as these dyes

exhausted within hours. In vivo functional imaging based on a calcium flux

fluorescent reporter construct where the functional response of T cells within the

CNS during EAE infiltration has recently been described and T cells in the CNS

were shown to have very different kinetics than those seen during the T cell activation phase in the lymph node226. In vivo calcium flux fluorescence imaging

cannot be normalized to a known concentration of calcium thus a ratio was

developed to report increases in calcium within a cell321.

∆F/F = (F-Fo)/Fo

where F is the maximum fluorescent signal detected in the cell whereas, Fo is

the baseline fluorescent signal detected during the imaging session. This can be

correlated to events such as cell-cell interactions and recently a productive

interaction was defined as one where the velocity was low and the fluorescent

ratio was high226.

4.6 Conclusion TPM technology allows sequential high-sensitivity and high-resolution detection

of individual fluorescently labeled cells and structures within the undisturbed

microenvironment of a live, anesthetized animal. Our unique imaging approach

110 offers serial high-definition dynamic real-time visualization of developing inflammation and invading cells within the structural context of the CNS in an anesthetized, live mouse during the entire inflammatory process. These parameters help us understand the migration and interaction behavior of immune cells within different microenvironments such as inflammation. Intravital imaging of reporter mice also serves as a potentially powerful experimental platform for future in vivo therapeutic efficacy monitoring.

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5. Conclusion and Future Direction

5.1. Conclusion

The literature on the role of resident immune cells in autoimmunity and infection

within the CNS has conflicting evidence partly because it has been difficult to

extract cells from the tissue and probe individual function due to the intimate

connection microglia have with other glia accessory cells, microglia in different

parts of the brain have different responses, and until recently, the integral

inhibitory relationship of microglia and neurons had not been fully realized.

Preservation of an organ critical for high level mammalian functions has led to

fine regulation by immune cells that localize the inflammation so as to minimize detrimental effects on the CNS. This work elucidates localized mechanisms that account for the patchy, immune cell filled lesions seen in EAE and MS, while

complementing and confirming previous seminal works in the field.

We showed that during inflammation, there are transient blood leaks that result in activation of the microglia surrounding the blood vessel. This activation of microglia from the first day of neurotoxic stimuli up to day 4 is followed by

infiltration of peripheral DCs on day 6, and subsequently, T cell infiltration

beginning on day 9. The activation of microglia can be affected by the

administration of histamine receptor antagonist, hydroxyzine thus affecting the

successive infiltration of peripheral immune cells.

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We were also the first to find that during this inflammation, an increased number

of microglia are able to interface with other cells of the BBB to project extensions

into the blood vessel through an intact BBB. This work advanced our knowledge

about the initiating events of EAE and deepened our understanding of microglia

behavior with the blood vessel under inflammatory conditions. Here I will set forth

a line of investigation that will further our knowledge of the interface between the

resident immune cells of the CNS and the peripheral immune cells.

5.2. Imaging to Detect Vessel Leaks in a Relapsing-Remitting Model

The model used in this thesis took advantage of C57BL/6J mice that allows the

use of syngeneic myelin-specific 2D2 transgenic T cells and syngeneic APC

fluorescent reporter mice for dendritic cells, microglia and macrophages to

dissect the temporal role of each cell. The use of MOG 35-55 to induce EAE results in a chronic-progressive form of disease49. More akin to human disease,

proteolipid protein used in the induction of SJL/J mice results in a relapsing-

remitting form of disease50 but there are no T cell transgenic mice nor fluorescent

reporter mice due to the genetic strain of the SJL/J mice. However, the leakiness of the blood vessel and phagocytic uptake of cells can still be studied. In fact, repeating the blood vessel integrity studies in this mouse model would provide superior insight into whether the vessel leak is initiating inflammation because the relapsing portion of the disease does not require further induction or manipulation, and has fairly consistent timing of recurrence.

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5.3. Imaging of CX3CR1 Knock-out Mice

Chapter 2 focused on the role and timing of CD11c-GFP+ cells infiltrating the

brain parenchyma and eluded to the role of microglia in the first 4 days of induction using phagocytosis as a marker of resident cells. Chapter 3 showed data from the CX3CR1GFP/+ mouse model where microglia and its interaction with

the blood vessel could be directly visualize during EAE induction. In future

studies, it would be advantageous to use a report mouse where CX3CR1GFP/+

was crossed with a CD11c-mCherry mouse into which 2D2-CFP cells were

transferred resulting in the ability to visualize GFP microglia, mCherry red CD11c and CFP MOG T cells simultaneously during induction. This would illuminate the important interplay between these cells within the CNS using TPM.

In addition, it has been shown that CX3CR1GFP/GFP mice induce with EAE show

increased disease severity9. It would be helpful to understand whether CX3CR1 expression on microglia plays a role in the recruitment or retention of cells in the

CNS. Thus I would create a CX3CR1GFP/GFP CD11c-mCherry reporter mouse and

transfer 2D2-CFP cells for TPM analysis.

5.4. Signaling of Vessel Projections

The novel finding of microglia projecting across the intact BBB into the blood

stream invokes intriguing, lingering questions: why do we see extensions in

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steady state? Can the projections during non-inflammed conditions be the

gateways that allow vessel leak and/or recruit immune cells to that particular

location? Is there a distribution difference of microglia projections throughout the

CNS? What is on the surface of the microglia extension in the blood vessel?

Could it be upregulating adhesion molecules on the endothelial cells, releasing protein fragments, micelles, cytokines into the blood stream? What is the

activation state of microglia when they are projecting processes across the

endothelium? Could microglia extension be plugging a vessel leak?

To be able to probe these questions about microglia projections, one would have

to start with the correct mouse model. Identifying the cell that is projecting across

into the blood vessel expressing the peptide of interest would be important in

assessing function. This functional reporter would be advantageous to quickly

identify cells, and to identify similarly behaving cells so that molecular analysis is

not diluted by a cohort. In the liMOG transgenic mouse322, MOG35-55 peptide is

expressed as a CLIP replacement peptide upon tamoxifen injection. Similar to published experiments323, one could cross the liMOG mouse to a tamoxifen

inducible CX3CR1-CreERT mouse to have microglia that express MOG in the context of MHCII. The efficiency of the liMOG when crossed to a CD11c-CreERT

resulted in only 5% of the total CD11c population expressing the correct

peptide323. We also know that CX3CR1 is expressed on both microglia and

macrophages and this distinction is particularly important in the EAE model. So

we would develop a P2ry12-CreERT mouse because it has been determined that

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P2ry12 is expressed on microglia but not macrophages83. In addition, we want a

way to identify cells only when the MOG35-55 peptide is presented on the MHCII

on the microglia. To do this, we would need to create a liMOG-iRES-mCherry-lox

inversion mouse, thus when this mouse is crossed with the P2ry12-CreERT

mouse, the mCherry fluorescent reporter protein would only get translated when tamoxifen is administered. Then when a microglia cell is projecting into the blood

vessel and expressing mCherry the researcher would be reassured that microglia is presenting MOG35-55 and thus interactions with fluorescently labelled 2D2 T cells would be an antigen specific interaction. It may be important to still cross this mouse to the CX3CR1GFP/+ mouse because it would also be interesting if none of the projections into the blood vessel ever expressed the peptide.

Once the P2ry12-CreERT liMOG-mCherry-Lox CX3CR1GFP/+ mouse has been created, create a cranial window observation to see if microglia extensions can be detected in the blood stream, with and without PTx administration, with naïve or activated fluorescently labelled 2D2 T cells. It would be important to know the expression level of MHCII in the blood vessel projection and this could be answered via IHC. If these preliminary experiments prove to be positive, one would perform a full induction by creating a cranial window observation, introducing fluorescently labelled 2D2 T cells and inducing EAE with an emulsion of peptide and PTx. To definitively determine if the interaction with the projection is productive, we could retrovirally transduce the 2D2 T cells with TN-

XXLCD∆neoR, a real-time calcium flux indicator226. Thus if a T cell contacts a

116 microglia expressing MOG peptide and if the T cell produces a calcium flux, this is a functional read-out of a productive interaction.

The most comprehensive assay after developing this mouse would be to use laser microdissection to isolate the specific cell that projecting into the vessel, presenting antigen, followed by RNA chip sequencing and possibly microRNA analysis. To adequately determine the activation state of microglia with active vessel projections, we would also isolate microglia that are not near the vessel, in both non-inflamed and inflamed models. It would be useful in this system to capture the astrocytes adjacent to the projection site, oligodendrocytes, and any infiltrating immune cells. Often research is focused on the status of the immune cells infiltrating or microglia without determining the status of other accessory cells and neurons adjacent to the cells of interest, but by collecting this information concurrently we can better determine the environmental status around this phenomenon.

The questions of what is being expressed on the surface can be pursued via IHC of most likely upregulated candidates from the RNA analysis. Upregulation of adhesion molecules on endothelial cells around projections can also be addressed by IHC.

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The recruitment of immune cells into the brain parenchyma could only be

effectively proven by dynamic intravital microscopy, using methods previously

described in this manuscript. Detecting the origin of proteins and cytokines in the

blood stream would be impossible to determine from the rare events of microglia

projecting into the blood stream. It would be possible to determine blood cytokine

levels by ELISA. Even a reporter mouse that expressed fluorescent protein

driven by the promoter of a particular cytokine would only report that the cell

produces that cytokine, not that the cytokine is released from the projection into

the blood.

Activated macrophages release microvesicles containing polarized M1 or M2 mRNAs324. It has been recently published that a common antigen from many

mouse tumor types can be detected with high sensitivity from blood samples by

using a nested RT-PCR method325. This could be a method by which we could

detect possible protein fragments and micelles released from microglia

extensions into the blood stream.

5.5. Determining the Role of Microglia in the Balance of T cell

Regulation versus Autoimmune Destruction of the CNS.

Our work elucidated a set of cellular events that showed that microglia are

activated in the first 3 days and that this activation state causes recruitment of

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peripheral APCs. With the treatment of HXYZ, the activation of microglia is

decreased which in turn reduces the recruitment of peripheral APCs. We had

postulated that microglia become overwhelmed during the initial stages PTx administration, with activation and phagocytosis functions thus recruiting peripheral cells to help resolve the inflammation, and it was published that in fact,

TREM2 signaling in microglia is initiated by apoptotic neurons to help with phagocytosis212 and overexpression of TREM2 on myeloid precursor cells can

help clear debris that microglia were unable to eliminate and resulted in recovery

in EAE246, 326. In addition, it has been shown that CX3CR1-/- mice develop more

severe disease9. We also know that CX3CL1 is expressed on neurons to keep

microglia in a less inflammatory state4. When this regulatory signaling is removed in the CX3CR1-/- mice, microglia become hyperactivated and I propose it is this

activation state of microglia that detrimental to tissue integrity327.

Histamine vesicle degranulation is used all over the body to cause the blood

vessel to dilate and allow cells to get into the target tissue during and

inflammatory event. Efforts to alleviate vessel leaks in our model via this

histamine mechanism did not work at doses indicated in literature, and warrants

a dose curve study. An intriguing finding not reported in literature before was the

non-specific infiltration of T cells on day 3 of EAE induction as determined by

FACs which we presume is due to the action of PTx. The administration of HXYZ

did have an effect on the non-specific infiltration of T cells on day 3 and

phagocytic capacity of microglia on day 6, thus further investigation on the

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activation state of these cells in response to HXYZ would be informative. Lastly,

EAE induced mast cell-deficient mice exhibited a lower frequency of IFN-γ

positive cells in the draining lymph node compared to induced WT mice which is consistent with our findings of cells isolated from brains on day 9328, 329.

Regulatory T cells (Treg) are CD4+CD25+FoxP3+ cells that have immune

suppressive effects and an association of T cell plasticity in MS and EAE has

been made due to the commonality of inducing cytokines330. It has been

documented that Treg cells can provide some protection during induction of

relapsing-remitting disease and that Treg cells administered after onset of

disease could reduce disease burden331, 332. It would be interesting if the timed

use of anti-histamine to reduce the number of IFN-γ positive cells followed by

transfer of myelin-specific Treg cells could further reduce CNS inflammation and provide a better therapeutic outcome after the onset of clinical disease.

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APPENDIX I Material and Methods

Mice GFP/GFP Six- to 12-week old syngeneic female C57BL/6, B6.129P Cx3cr1 (stock

b #005582) and B6.Cg-Tg Thy-1-YFP-H (stock #003782) mice (H-2 ), B6.FVB-Tg

CD11c-DTR/GFP mice (stock #004509), C57BL/6-Tg 2D2 mice (stock #006912),

B6.129 (ICR)-Tg actin beta CFP mice (stock #004218), and C57BL/6-Tg ubiquitin

GFP mice (#004353) were obtained from the Jackson Laboratory (Bar Harbor,

ME). B6.129S6 OTII mice (stock #1896) were obtained from Taconic. 2D2 mice

expressing the TCR transgene specific for MOG35-55 peptide / I-Ab were

crossed with actin beta CFP mice to derive mice expressing 2D2-CFP cells for in

vivo tracking by TPM. Similarly, OTII mice expressing the TCR transgene specific

for OVA323–339 peptide / I-Ab were crossed with ubiquitin GFP mice to derive

+/GFP OTII-GFP cells for in vivo TPM tracking. Cx3cr1 reporter mice have one

Cx3cr1 allele replaced with the gene encoding GFP, and are derived by crossing

GFP/GFP Cx3cr1 with C57BL/672. CX3CR1 (fractalkine receptor) is almost exclusively expressed in the microglia population in the CNS of these mice, while

NK cells, activated CD8+ T cells, dendritic cells and a subset of monocytes also

express the GFP marker in the peripheral tissues72. Thy-1-YFP-H mice harbor

yellow fluorescent protein (YFP) expression in a subset of neurons in the dorsal

root ganglion and cortical layers333. Thy-1-YFP-H mice were crossed with

GFP/GFP +/GFP Cx3cr1 mice to obtain double transgenic mice, Thy-1-YFP-H x Cx3cr1 .

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Animals were housed, bred and handled in the Animal Resource Center facilities

at Case Western Reserve University according to approved protocols. Similarly,

all animal experiments were executed with strict adherence to active

experimental animal protocols approved by Case Western Reserve University

Institutional Animal Care and Use Committee.

EAE Induction 191, 192 Mice were induced to develop EAE using established protocols . Briefly,

3x106 naïve splenic 2D2 CFP CD4 T cells, isolated by negative depletion using

Dynal beads (Life Technologies, Grand Island, NY, USA), were injected into recipient mice. 24 hours later, an emulsion with 200 µg myelin oligodendrocyte glycoprotein (MOG) peptide 35–55 (MOG35–55; Anaspec, San Jose, CA, USA) with 8 mg/ml H37RA and incomplete Freuds adjuvant was injected subcutaneously (s.c.) bilaterally on the lower back of the recipient mouse (Hooke

Laboratories, Lawrence, MA, USA). Control mice were injected s.c. bilaterally with an emulsion of PBS, 8 mg/ml H37RA and incomplete Freund’s adjuvant. 100 ng Pertussis toxin was administered intraperitoneal (i.p.) on Days 0, 1 and 2.

Clinical scores were assigned as follows334, 335: 0, no motor deficits; 1, tail

weakness; 2, hind limb weakness; 3, hind limb hemiplegia; 4, total hind limb

paralysis; 5, moribund. This induction protocol results in limp tail, hind leg

paralysis and progressive neurological dysfunction in recipient mice beginning

191 around days 12-14 post induction .

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Priming of Antigen-specific T cells To determine whether myelin specific T cells require antigen-specific APCs for

recruitment to the CNS, EAE was actively induced using a combination of two antigenic peptides in 8-12 week old female recipient mice. 3x106 naïve 2D2-CFP

and 3x106 naïve OTII-GFP CD4+ T cells were isolated separately by negative

depletion using Dynal beads and co-injected into naive recipient mice. 24 hours

later, an emulsion of 200 µg MOG35-55 only, 10 µg OVA323-339 only, or 200 µg

MOG35-55 and 10 µg OVA323-339 combined (Anaspec, San Jose, CA, USA)

with 8 mg/mL H37RA (Difco Laboratories, Detroit, MI, USA) and incomplete

Freund’s adjuvant (Difco Laboratories, Detroit, MI, USA) was injected s.c.

bilaterally on the lower back of the recipient mice. Control mice were injected

s.c. bilaterally with an emulsion of PBS, 8 mg/mL H37RA and incomplete

Freund’s adjuvant. 100 ng Pertussis toxin (List Biological Laboratories, Inc,

Campbell, CA, USA) was administered i.p. on days 0, 1 and 2 to induce EAE.

Spinal Trauma Model We modified published protocols to create a dorsal column crush injury281.

Briefly, mice were anesthetized with inhaled 1-2% isoflurane and a laminectomy

was performed aseptically to expose a spinal cord segment at the T10 level,

allowing for microscopic examination in both EAE and spinal cord trauma

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models. To create the spinal trauma model, small dural openings were made at

0.5 mm lateral to the midline with a 30 gauge needle, and a dorsal column crush

lesion was made by inserting and squeezing a pair of Dumont # 4 forceps 1 mm

into the dorsal spinal cord and holding pressure for 10 seconds three times. The

laminectomy site was covered with saline-soaked Gelfoam (Pfizer), and the para-

spinal muscles and skin were closed with sutures. For post-operative pain

control, mice received a single dose of Marcaine (1.0 mg/kg) s.c. at the incision

site and buprenorphine (0.1 mg/kg) intramuscularly (i.m.) daily for 3 days.

Tumor Cell Preparation and Injection Mouse medulloblastoma (MB) cell line, MM1, was derived from Patch+/-/p53-/- mice

(H-2b) as a spontaneous tumor, and was a generous gift from Dr. Gregory Plautz

at the Cleveland Clinic Foundation. MM1 was transfected with a plasmid carrying

the red fluorescent reporter, DsRed2 (pDsRed2-N1), with a neomycin selection

marker using JetPEI (Polyplus Transfection). Transfected cells (MM1-DsRed2)

were selected with G418 (300 μg/ml) and enriched for red fluorescence

expression by fluorescence-activated cell sorting (FACS). Female Cx3cr1+/GFP mice were anesthetized with inhaled 1-2% isoflurane (Aerrane; Baxter) and placed in a stereotactic holder. After removing a circular scalp flap, 3x104 MM1-DsRed2 cells

were injected intracranially (i.c.) at a depth of 1.5 mm into the center of the left

parietal cortex with a Hamilton syringe. The left parietal region of the skull was

then removed and a cranial observation window was installed as previously

124

187 described . Intracranial MM1-DsRed2 tumor development was then imaged

serially with 2P-LSM on days 7, 10, and 14 after tumor inoculation.

Mouse Surgery and Preparation for Intravital Imaging In all intravital experiments involving imaging the mouse brain, mice were

187 implanted with cranial windows according to published protocol and imaging

sessions were carried out immediately for acute analysis or at least 4 days

following the implantation procedure. Briefly, 5 mg/kg carprofen (Butler-Schein,

Dublin, OH, USA) was used given s.c. as an analgesic, and 0.2 mg/kg dexamethasone (Butler-Schein, Dublin, OH, USA) was used given s.c. as a

general anti-inflammatory. The scalp was removed to expose the skull. A

craniotomy was performed using a dental drill and the bone was replaced with a

sterile 5 mm #1 circular glass coverslip. The glass coverslip was glued in place

with Vetbond, and dental acrylic (Butler-Schein, Dublin, OH, USA) was used to

create a permanent well for imaging. Alternatively, thinned-skull preparations

188, 189 were utilized according to published protocol , leaving an intact skull of 25-

50 um thickness (Fig. 12). Mice were anesthetized with nebulized isoflurane (2%

induction, 1.5% maintenance) in 30% O2 / 70% air, with body temperatures

maintained at 37oC via a temperature-controlled environmental chamber and heating pads throughout the entire mouse preparation and imaging session.

Breath rate and animal responsiveness were used to monitor adequate levels of anesthesia, with breath rate maintained at ~60-100 breaths per minute and animal responsiveness assessed by foot and tail pinch. Mice were placed onto a

125 stereotactic holder, and the entire assembly was placed in a temperature- controlled environmental chamber. The body temperature was monitored and maintained between 36.5 to 38oC using a combination of an environmental temperature probe and a rectal probe. Ten to 30 minutes prior to imaging, fluorescent dye markers were injected intravenously (i.v.) to allow blood vessel visualization. The vessel dyes used in the experiments included 700 μg of

TRITC-Dextran (150KD; Sigma, Inc.) or 100 ul of 0.1 μM non-targeted QTracker-

655 (Invitrogen, Inc.). Tomato-lectin (Vector Laboratories; 16 ug/mouse)336 was also used to visualize the endothelial cells337. Large molecular weight dextran markers are selected to study BBB integrity as previously described338.

Spinal Imaging For sequential spinal cord imaging of traumatic injury, skin and muscle flaps covering the previous T10 laminectomy site were re-opened aseptically with the animal under anesthesia. The site of injury was exposed by carefully dissecting the muscles and previously implanted Gelfoam (Pfizer) with the aid of a dissection scope. In preparation for spinal imaging in EAE mice, a laminectomy was performed at the S1 level. In both cases, para-spinal muscle was removed from the lateral part of the vertebrae one level above and one level below the site of laminectomy to allow for attachment of Narshinge STS-A spinal clamps mounted on a custom aluminum base. The clamps were covered with Parafilm and a well for immersion fluid (Dulbecco’s aCSF) was created around the spinal clamps with OrthoJet dental acrylic (Lang Dental).

126

Two-Photon Imaging Equipment, Data Acquisition Upon completion of tissue preparation for intravital imaging, the entire mouse

imaging assembly, including the stereotactic holder, was placed on the

microscope stage enclosed within a custom-made temperature-controlled

environmental chamber. The tissues were imaged using a Leica SP5 fitted with a

DM6000 stage, a 20X water immersion lens (N.A. 1.0; Leica HCX-APO-L), and a

16W Ti/Sapphire IR laser (Chameleon, Coherent) tuned to excitation

wavelengths between 800nm and 880nm. Imaging planes (760 x 760 μm)

collected at 1-5 μm z-intervals were repeated at 20-60 second intervals for up to

6 hours to yield xyzt data sets collected through a four channel non-descanned

external detector using a filter set separating 400-455, 467-499, 500-550, 565-

605 for brain and spine imaging, and, a filter set separating 467-499, 500-550,

565-605, 625-675 for tumor imaging. This raw data set was then used for

processing and analysis. The imaging platform also included a motorized stage

with Tile Scan capabilities to allow for broad-field survey and high-resolution

voxel (0.75 x 0.75 x 1 μm) image collection of the tissues (see below).

Image Analysis High-resolution fluorescent 4D imaging data sets collected from intravital 2P-LSM

experiments were analyzed using Imaris (BitPlane, Inc). Mosaic broad-field

survey images were compiled using Xuv Stitch software (Xuv Tools). Volumes of

1550x1550x150 um3 up to 1550x2025x150 um3 were analyzed for EAE imaging in

127

the brain. For imaging of the spine during EAE, spinal cord crush injury and CNS

3 tumor models, a volume of 775x775x150-300 um was analyzed. For spinal

crush models, only areas within the lesion site containing damaged axons were analyzed. Projection numbers were normalized to surface area of vessels found

within the lesion. Image processing included Gaussian smoothing and creating a

surface rendering of vessel walls as defined by the extent of the intravenous

dyes. GFP signals within the vessel lumen were then segmented from the total

images, and surface rendering of GFP+ projections were created. Only cells

residing in the parenchyma were chosen for projection frequency analysis,

excluding those in the meninges (Fig. 12). When choosing relevant extravascular

GFP+ APC populations to analyze for intravascular dendrites, and to avoid including GFP+ cells that may be perivascular or circulating cells in the process of

extravasation from vessels, we applied the following three criteria to exclude

GFP+ cells from analyses: (1) Cells with migration speed >3 um/min or were visualized to be in an active process of transmigration across the vessel wall

from vascular lumen in the 4D dynamic imaging data sets; (2) Surfaces with a

sphericity of 0.9-1 as possible rolling or circulating cells within the vessel; (3)

Surfaces that only extended within the perivascular space and did not cross the vessel wall surface; and (4) Surfaces that had more than 30% of the cell volume within the vessel as possible perivascular cells extending processes into the parenchyma178. The total number of dendritic projections that met the above

criteria was then normalized to the calculated surface area of the vessel wall in

order to derive a frequency of dendritic protrusions per unit vessel surface area.

128

Based on the dynamic 4D data in which the direction of vascular flow can be

determined, the projections were from CX3CR1+ cells outside of post-capillary

venules and larger size veins.

For Chapter 3 and cranial window imaging, second harmonic generation (SHG)

signal produced by high collagen content structures was used to identify the pial

layer and to discriminate parenchyma from meninges. Relative fluorescent

intensity region of interest (ROI) measurements of dextran leaking into the

parenchyma were made using the LAS-AF software (Leica Microsystems Inc,

Buffalo Grove, IL, USA). Timing and duration of vessel leaks were manually

processed. Phagocytic cells and CD11c-GFP were identified by fluorescent

intensity. Co-localization of TRITC and GFP signals and volume data were

calculated using the Imaris software (BitPlane Inc, Zurich, Switzerland). T cell motility data was also analyzed using the Imaris software, which allows cell

identification, as well as 3D tracking over time by producing information on

individual positions, time and track length. T cell interactions with APCs were grouped into short contacts (< 2 min) and prolonged interactions (≥ 2 min).

Individual T cell migration tracking was further analyzed for track length, velocity,

mean fluorescent intensity and cell flux using MATLAB software (Mathworks Inc,

Natick, MA, USA). Cell flux was calculated by determining the vector between the

beginning of the T cell track and the point at which the T cell came within a 50

μm radius from the center of an APC cluster. Contact time between APC and T

cells, and T cell movement to and from the blood vessel were analyzed manually

129 by visual inspection of individual image slices over time using the Imaris software.

Electron Microscopy Normal, non-manipulated CX3CR1 GFP/+ mice were fixed by transcardial perfusion with 0.1% glutaraldehyde and 4% PFA and postfixed overnight.

Samples were stained en bloc with anti-GFP primary antibody (Life

Technologies) and goat anti-rabbit 2nm gold conjugated secondary (Ted Pella,

Inc., Redding, CA, USA). Gold particles were then enhanced using the silver enhancer kit (Sigma) until darkening of the sample was seen. Osmication with

1% osmium (EM Sciences), dehydration in ethanol (Sigma) and then propylene oxide (EM Sciences) and eponate-12 resin (Ted Pella) infiltration were performed using the corresponding Pelco BioWave Pro microwave protocols. Samples were embedded in resin and polymerized at 60 for 48 hours in BEEM capsules in a traditional polymerization oven. Blocks were trimmed and 85nm ultrathin sections cut on an ultramicrotome and placed on copper formvar support grids (EM

Sciences). Tissue was negative stained with lead acetate (EM Sciences) and uranyl acetate (EM Sciences). Grids were imaged using the STEM mode on a

FEI Helios Nanolab 650 for rapid scanning of large areas (Swagelock Center,

Case Western Reserve University). EM micrographs were captured at 10,000X.

Blood brain barrier leakiness analysis

130

To compare the pattern of vessel leakiness induced by different inflammatory

stimulants, 100 ng LPS (Sigma-Aldrich, St. Louis, MO, USA), 100 ng Pertussis

toxin or PBS were injected i.p. into C57BL/6 mice on days 0, 1 and 2. 700 ng of

150 kDa TRITC dextran (Sigma-Aldrich, St. Louis, MO, USA) vessel dye was

injected i.v. on day 1 and day 2. On day 3, QTracker-655 (Life Technologies,

Grand Island, NY, USA) were injected to highlight the vessels. The mice were

sacrificed and whole brain tissues were imaged immediately by TPM.

Immunofluorescence Histology +/GFP Naive or inflamed Cx3cr1 mice were sacrificed and subjected to transcardial

perfusion with 4% PFA. The brain and spinal tissues were harvested, and 12um

cryosections were stained with DyLight 594 tomato-lectin336 (Vector Laboratories)

or antibodies against CD31 (MEC13.3; BD Biosciences), laminin (Biotrend,

Germany), Iba-1 (Wako Chemicals, USA), or GFAP (Z0334; Dako, Glostrup,

Denmark) followed by appropriate secondary antibodies conjugated to Alexa

fluor (Life Technologies). The samples were then subjected to confocal

microscopy to obtain fluorescent micrographs.

Flow cytometry analysis For single-cell APC and T cell characterization, brain tissues were harvested on

days 0 through 12 of EAE induction. Tissues were dissociated by collagenase D

(1 mg/mL) and DNAse (250 units/mL) (Sigma-Aldrich, St. Louis, MO, USA) to

131 form a single cell suspension after gentle dissociation with a dounce homogenizer and subjected to a 70%/37%/30% Percoll gradient339. Isolated cells were stained for CD45, CD11c, CD11b, CD4, CD8, IL-17, IFN-γ and

FoxP3. Absolute numbers of cells per brain were determined by multiplying the number of events on FACS per collection volume by the total volume of cell samples. Spleen and LN were isolated from mice at the end of imaging on Day

12. Organs were crushed with the ends of 1 ml syringes through a 40 µm cell strainer into single cell suspensions and were stained for Vα2 (OTII

TCRchain), Vβ11 (2D2 TCR chain), CD4, CD25, CD44, and CD69. Samples were collected and analyzed using the Accuri flow cytometer (BD Biosciences

Co, San Jose, CA, USA).

Statistical consideration In Chapter 2, the number of mice and projections quantified in the imaging experiments are as follows: Fig. 15F – 3-4 mice per group, 7092 total projection events; Fig. 17F - 3 mice per group, 503 total projection events; Fig. 18E - 3 mice per group, 216 total projection events; Fig. 18I - 3 mice total, 196 total projection events; Fig. 16F - 4-6 mice per group, 2213 total projection events.

Statistical analyses were performed using GraphPad Prism 5 software

(GraphPad Software Inc, San Diego, CA). To test for statistical significance the

Student's T-test and two-way analysis of variance was used to compare between treatment groups. For all analyses P < 0.05 was considered significant.

132

Results are collective data from 2 to 6 repeat experiments with minimum of 3 mice per experiment.

133

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