Neutrophils promote Alzheimer’s disease-like pathology and ... · ! 1! Neutrophils promote...

1

Neutrophils promote Alzheimer’s disease-like pathology and

cognitive decline via LFA-1 integrin

Elena Zenaro1,*, Enrica Pietronigro1,*, Vittorina Della Bianca1, Gennj Piacentino1, Laura

Marongiu1, Simona Budui1, Ermanna Turano2, Barbara Rossi1, Stefano Angiari1, Silvia Dusi1,

Alessio Montresor1, 3, Tommaso Carlucci1, Sara Nanì1, Gabriele Tosadori1, 3, Lucia Calciano4,

Daniele Catalucci5, Giorgio Berton1, Bruno Bonetti2, Gabriela Constantin1, 3

1Department of Pathology and Diagnostics, University of Verona, 37134 Verona, Italy

2Department of Neurological and Movement Sciences, Neurology Section, University of

Verona, Verona, Italy

3The Center for Biomedical Computing (CBMC), University of Verona, 37134 Verona, Italy

4Department of Public Health and Community Medicine, University of Verona, 37134

Verona, Italy

5National Research Council (CNR), Institute of Genetic and Biomedical Research (IRGB), and

Humanitas Research Hospital, Milan, Italy

*These authors equally contributed to this work

Nature Medicine: doi:10.1038/nm.3913

2

SUPPLEMENTARY FIGURES

Supplementary Figure 1


3

Supplementary Figure 1 (continuation)


4

Supplementary Figure 1. Expression of vascular adhesion molecules in the brains of

5xFAD and 3xTg-AD mice and endothelial activation by oligomeric Aβ . Confocal

microscopy shows the expression of endothelial integrin ligands (ICAM-1 and VCAM-1) and

selectins (E-selectin and P-selectin) in 30-µm coronal brain sections from (a) healthy B6SJL

control and (b) 5xFAD at 4 months of age and (c) healthy C57BL/6 control and (d) 3xTg-AD

mice at 6 months of age. Nuclei are stained with DAPI (blue) and adhesion molecules are

stained with Avidin Texas Red (red). Scale bar = 20 µm (a-d). (e, f) Quantitative analysis of

adhesion molecules presented as mean vascular volume of fluorescent positive structures per

mm3 and mean fluorescence intensity (M.F.I.) of positive vessels per mm3. The analysis was

blinded and carried out as described at supplementary methods in the meningeal vessels of

5xFAD mice (n = 3) at 4 months of age (e) and in the hippocampal vessels of 3xTg-AD mice

(n = 3) at 6 months of age (f) compared with sex- and age-matched controls (n = 3). Values

represent the mean ± SEM of one representative experiment from a series of two independent

experiments with similar results (*P < 0.05 compared to the wild-type control). Statistical

analysis was carried out using the Mann-Whitney test. AU = arbitrary units. (g-j)

Representative confocal microscopy images show amyloid deposits and the expression of

vascular adhesion molecules ICAM-1 and VCAM-1 in 30-µm coronal brain sections of

5xFAD mouse at 4 months of age. (g) Sex- and age-matched wild-type controls show a

complete lack of amyloid deposits, whereas amyloid deposits are abundant (h left) in the deep

layer of the cortex (h middle) and in the hippocampus (h right) of 5xFAD mice, as detected

by Thioflavin S staining. ICAM-1 in the meningeal vessels (i) and VCAM-1 in the CA1

subfield of the hippocampus (j) are upregulated near Aβ deposits in 4-month-old 5xFAD mice.

Nuclei are stained with DAPI (blue) and adhesion molecules are stained with Avidin Texas

Red (red). Scale bar = 100 µm (g, h left), 50 µm (g and h middle and right), 30 µm (i, j). (k)

Immunofluorescence staining was carried out after cells were exposed to 10 µM Aβ42


5

oligomers for 18 h to investigate the effect of soluble Aβ42 on the expression of adhesion

molecules (ICAM-1 and VCAM-1) and selectins (E-selectin and P-selectin). Control bEend.3

cells (Ctrl) were treated with the vehicle alone, whereas TNF-α stimulation (25 U/ml), which

is known to upregulate the expression of vascular adhesion molecules, was used as a positive

control. ICAM-1 was constitutively expressed at low levels on endothelial cells (ICAM-1,

Ctrl). CD31 was used as a typical endothelial cell marker. Secondary antibody staining was

used to verify the lack of nonspecific fluorescence (data not shown). Nuclei are stained with

DAPI (blue) and adhesion molecules are stained with Avidin Texas Red (red). Scale bar = 20

µm. (l) WST-1 assay for cell viability, in which bEend.3 cells were treated with oligomeric

Aβ42 (10 µM) or TNF-α (25 U/ml) for 18 h, followed by 1 h incubation with WST-1. The

values are expressed as a percentage of WST-1 reduction compared to untreated cells

(designated as 100%). Data represent means ± SD of three experiments. (m) Relative

fluorescence quantification of CD31 and adhesion molecule expression in bEnd.3 cells treated

with Aβ42 or TNF-α, compared to untreated controls. Mean fluorescence intensity was

calculated by normalizing the summed pixels intensity from 3-5 images to mm2 (*P < 0.05;

**P < 0.01). Statistical analysis was carried out using the Mann-Whitney test. All the analyses

were performed in a blinded fashion.


6



7


Supplementary Figure 2. CD45+ cells and neutrophils accumulate in the brains of

5xFAD and 3xTg-AD mice. Confocal microscopy images show the presence of CD45+ cells

localized in meningeal vessels (left) and in deep cortical layers (right) of B6SJL control

animals (a) and 5xFAD mice (b) at 4 months of age, and in the plexi of lateral ventricles (left)

and the hippocampus (right) of C57BL/6 healthy controls (c) and 3xTg-AD mice (d) at 6

months of age. Nuclei are stained with DAPI (blue). Scale bar = 20 µm (a-d). Blinded

stereological quantification of CD45+ cells from 5xFAD mice (n = 4) (e) and 3xTg-AD mice

(n = 4) (f), each compared with wild-type controls (n = 4). Values represent means ± SEM of

one representative experiment from a series of two independent experiments with similar

results (*P < 0.05; Student's t-test). (g-i) Confocal microscopy images show the presence of

CD45+ (green cells) and CD18+ (red cells) colocalized in the plexi of lateral ventricles (upper

panels) and the hippocampus (lower panels) of C57BL/6 healthy controls (g) and 3xTg-AD

mice (h) at 6 months of age. Nuclei are stained with DAPI (blue). Scale bar = 20 µm (g, h).

Blinded stereological quantification of CD45+/CD18+ cells using 3xTg-AD mice (n = 4) (i)

compared with wild-type controls (n = 4). Values represent means ± SEM of one

representative experiment from a series of two independent experiments with similar results


8

(*P < 0.05; Student’s t-test). (j, l) Confocal microscopy images show the presence of Ly6G+

leukocytes localized in the meninges (left) and in deep cortical layers (righ) of (j) B6SJL

control animals and (l) 5xFAD mice at 4 months of age. (k, m) Ly6G + cells are shown in the

plexi of lateral ventricles (left) and hippocampal parenchyma (righ) of healthy C57BL/6

controls (k) and 3xTg-AD mice (m) at 6 months of age. (n, o) Data were quantified in

5XFAD (n) and 3xTg-AD mice (o) and in control wild-type animals in a blinded fashion as

described at Supplementary methods (n = 4 mice for each condition). Values represent means

± SEM of one representative experiment from a series of two independent experiments with

similar results (Student's t-test was used; ***P < 0.0005 and **P < 0.05 compared to WT).

Cell nuclei are labeled with DAPI (blue); Ly6G+ cells are labeled with Avidin Texas Red

(red); Scale bar = 20µm (j, k, l, m). (p) Representative histopathological images show the

presence of neutrophils in the cortex and hippocampus of 3xTg-AD mice at 6 months of age

compared with a wild-type control, each stained using the naphthol AS-D chloroacetate

esterase kit (n = 3). Intraparenchymal cells are highlighted using black arrows whereas

intravascular adherent cells are highlighted using a star symbol. Similar results were obtained

in 5xFAD mice (data not shown). Scale bar = 200 µm.


9



10


Supplementary Figure 3. Neutrophil detection in brain homogenates from mice with

AD-like disease. (a) Representative FACS analysis of infiltrating CD45+ cells from the brains

of 3xTg-AD mice compared to wild-type controls, using simultaneous labeling with

antibodies recognizing neutrophil markers (anti-CD45-Vioblue, anti-CD11b-FITC, anti-Gr1-

PE and anti-Ly6G-APC). CD45+ cells were sub-gated as CD11b+/Gr-1+ or Gr-1+/Ly6G+ cells.

In the representative image for both 3xTg-AD and wild-type control cells, the first panel


11

shows the entire population of cells from brain homogenates, the second panel shows the gate

on CD45+ cells, the third panel shows gated CD11b+/Gr-1+ cells and the fourth panel shows

gated on Gr-1+ and Ly6G+ cells. (b, c) Representative FACS analysis of infiltrating CD45+

and CD18+ cells from the brains of 3xTg-AD mice (c) compared to wild-type controls (b),

using simultaneous labeling with antibodies recognizing neutrophil markers (anti-CD45-

Vioblue, anti-CD18-Alexa647, anti-CD11b-APC-Cy7, anti-Gr1-PE and anti-Ly6G-FITC).

CD45+ or CD18+ cells were sub-gated as CD11b+/Gr-1+ or Gr-1+/Ly6G+ cells. In the

representative images for both 3xTg-AD (n = 6) and wild-type control cells (n = 5), the first

panel shows the entire population of cells from brain homogenates, the second panel shows

gated CD45+ cells or CD18+ cells, the third panel shows in red CD11b+/Gr-1Low/+ and in blue

CD11bHigh/Gr-1High cells, and the fourth panel shows gated Gr-1+/Ly6G+ cells. (d)

Neutrophils were isolated from bone marrow and labeled with the CMTMR fluorescent cell

tracker and then injected intravenously into 3xTg-AD mice (n = 4) (red line) at 6 months of

age or sex- and age-matched wild-type controls (n = 4) (blue line). The percentage of

infiltrated neutrophils was determined at 0, 12, 24, and 36 h post-injection. The mice were

perfused and leukocytes were isolated from brain homogenates as described in the Methods

section. The leukocytes were then labeled with anti-CD45-Vioblue and anti-Ly6G-APC

antibodies and acquired by flow cytometry. CD45+/Ly6G+/CMTMR+ neutrophils were gated

from the living cells. The results are from one representative experiment from a series of two

with similar results.


12



13



14

Supplementary Figure 4. Soluble Aβ1-42 induces rapid adhesion of neutrophils and

respiratory burst in vitro and neutrophil depletion by the anti-Ly6G antibody in vivo.

(a) 18-well glass slides were coated with human fibrinogen or human ICAM-1. Mouse

neutrophils were incubated for 2 min in buffer (Ctrl) or the stated concentrations of

oligomeric Aβ (Aβ1-42). We used 1 µM fMLP as a positive control (fMLP). (b) Fibrillar

Aβ1-42 (Fib Aβ) efficiently triggered the rapid adhesion of human neutrophils, but the

oligomeric soluble form Aβ1-42 (sol Aβ) was twice as effective. Values represent mean

counts of bound cells in a 0.2-mm2 field in one representative experiment from a series of

three independent experiments with similar results. Error bars represent the SEM (*P < 0.05;

**P < 0.01; ***P < 0.001). Statistical analysis was carried out using the Mann-Whitney test. (c-

f) Human (c) or murine (d) neutrophils were plated on fibrinogen-coated wells and stimulated

with 10 µM soluble oligomeric Aβ1-42 (Aβ1-42), with 1 µM or 5 µM fMLP used as a

positive control for the human and murine neutrophils, respectively. The neutrophils were

incubated with 5 µM DPI for 5 min prior to stimulation with Aβ (Aβ1-42 + DPI) or fMLP

(fMLP + DPI). We then evaluated ROS production stimulated by fMLP or Aβ in human (e)

and murine neutrophils (f) in the presence or absence of 10 ng/ml TNFα, which was added to

the cells 10 min before Aβ (Aβ1-42 + TNFα) or fMLP (fMLP + TNFα). Chemiluminescence

was recorded every minute until the indicated time. Values represent means ± SD of three

independent experiments (*P < 0.05 compared to the control in (c) and (d) and *P < 0.05,

fMLP + TNFα versus fMLP and Aβ1-42 + TNFα versus Aβ1-42 in (e) and (f)). Statistical

analysis was carried out using the Mann-Whitney test. DPI = diphenyleneiodonium chloride;

CPS = counts per second. More details are provided in the Supplementary Methods. (g) 18-

well glass slides were coated with human ICAM-1. Human neutrophils were incubated for 2

min with buffer (Blank) or 10 µM soluble oligomeric Aβ1-42 (Aβ). We used 0.1 µM fMLP as

a positive control (fMLP). Values represent mean counts of bound cells in a 0.2-mm2 field in


15

one representative experiment from a series of three independent experiments with similar

results. BocMLF, an inhibitor of FPR1 (left panel), and PTx (right panel) significantly

inhibited neutrophil adhesion. Error bars represent the SD (***P < 0.0005 for: fMLP control

compared to fMLP + bocMLF; Aβ1-42 control compared to Aβ1-42 + bocMLF; fMLP

control compared to fMLP + PTx; Aβ1-42 control compared to Aβ1-42 + PTx). Statistical

analysis was carried out using the Mann-Whitney test. (h, i) Representative FACS analysis of

the blood from 3xTg-AD mouse before (h) and after (i) the injection of 0.5 mg of anti-Ly6G

antibody, 1A8 clone (n = 4). (h) FACS analysis of blood cells one day before the injection of

anti-Ly6G antibody. The first panel shows the gating of living cells (forward scatter versus

side scatter), the second panel shows the gating of CD45+ cells, and the third panel shows the

gating of CD11bhigh/Gr-1high (neutrophils) and CD11blow/Gr-1low (monocytes). The neutrophil

and monocyte populations were also characterized by measuring the expression of Ly6G

versus Ly6C in the fourth panel. CD11bhigh/Gr1high neutrophils (red) are also

Ly6Ghigh/Ly6Chigh whereas CD11blow/Gr1low monocytes (green) can be Ly6Gneg/Ly6Chigh or

Ly6Clow, representing inflammatory or residential monocytes, respectively. (i) FACS analysis

of blood cells 20 h after the injection of anti-Ly6G antibody using the same gating strategy

described above. (j, k) The isotype control antibody does not deplete neutrophils or

monocytes. Representative FACS analysis of the blood from 3xTg-AD mouse before (j) and

20 h after (k) the injection of 0.5 mg of the isotype control antibody (n = 4). The analysis is as

described for h and i. Shown is a representative experiment from a series of two with similar

results.


16



17



18

Supplementary Figure 5. Neutrophil depletion during early-stage of disease has a long-

term beneficial effect on cognition in 3xTg-AD mice. (a-d) Arm entry during the Y-maze

test was comparable among groups of mice. The 5xFAD (a, b) and 3xTg-AD (c, d) mice were

treated with antibodies and tested against sex- and age-matched wild-type control mice (as

described in Figure 4). All groups showed comparable arm-choice values once the treatment

was completed, indicating that AD-like mice had similar motor functions and exploratory

activity compared to their wild-type counterparts. Values represent the mean ± SEM of the

total number of arm entries during the 8-min test in each group of mice based on one

representative experiment from a series of three independent experiments with similar results.

(P > 0.05; Statistical analysis was carried out using the Mann-Whitney test). The analysis was

performed in a blind fashion. (e) Neutrophil depletion was carried out in 3xTg-AD and wild-

type control mice at 6 months of age. The anti-Gr-1 antibody was used to deplete neutrophils

from the peripheral circulation and an isotype-matched antibody was used as a control. Left

panels show the results from the contextual fear-conditioning test (CFC), and right panels

show the percentage of spontaneous alternation performance in the Y-maze test. Values

represent the mean ± SEM from a representative experiment with 10-12 mice per condition

from a series of three experiments with similar results. The analysis was performed in a blind

manner. (f) Blinded quantitative stereological analysis was performed as described at

Methods section (n = 3 mice/condition) to determine the total number of Iba-1+ cells and the

area occupied by the cell soma. Error bars indicate the SEM (*P < 0.05 and **P< 0.01). All

statistical analysis was carried out using the Mann-Whitney test. (g, h) Neutrophil depletion

was carried out for 4 weeks in 5xFAD mice starting at 4 months of age. Anti-Ly6G (g) and

anti-Gr-1 (h) antibodies were used to deplete neutrophils from the peripheral circulation.

Results from the contextual fear conditioning test and Y-maze test. Values represent the mean

± SEM of mice in each group. Data are derived from representative experiments with 12–14


19

mice per condition from a series of three with similar results (*P < 0.05; ** P< 0.005 and ***P

< 0.0005). Statistical analysis was carried out using the Mann-Whitney test. (i-l) Neutrophil

depletion was carried out in 6-month-old 3xTg-AD mice for 4 weeks, as described in the

Methods section. An isotype-matched antibody was used as a control. Blinded quantitative

analysis of the amyloid area (stained with 6E10 antibody) was carried out as described at

methods section (main text) on sections of cortex and hippocampus from 3xTg-AD mice

treated at the early stage of disease (n = 3) (i) or at 6 months of age and then tested at 13

months age (n = 3) (k). Error bars indicate the SEM of one representative experiment from a

series of three independent experiments with similar results (**P < 0.005). Statistical analysis

was carried out using the Mann-Whitney test. Representative images (5x magnification) of

6E10 staining in 3xTg-AD mice treated with the control or anti-Gr-1 antibody during the

early stage of disease (j) or at 6 months of age, followed by testing at 13 months (l). Scale bar

= 500 μm (j, l). (m) The production of Aβ1−42 was analyzed by sandwich ELISA in the

brains of 9-month-old 3xTg-AD mice by three-step or four-step extraction procedures as

described in the methods section and in the supplementary information. Values represent the

mean ± SEM of four mice from one representative experiment from a series of two with

similar results (P > 0.05; Mann-Whitney test). (n-q) Neutrophil depletion was achieved by

treating mice with anti-Gr-1 antibody for 4 weeks starting at 6 months of age in 3xTg-AD

mice or with an isotype-matched antibody administered as a control, and C57BL/6 mice used

as wild-type controls (WT ctrl). Behavior was tested 6 months after completion of the

treatment, at 13 months of age. (n) In the contextual fear conditioning (CFC) test, the values

represent the mean ± SEM of the data obtained from a representative experiment with 10–12

mice per group from a series of two experiments with similar results (**P < 0.005). Statistical

analysis was carried out using the Mann-Whitney test. (o) Unbiased and blinded quantitative

stereological analysis was carried out on the brains of antibody-treated 3xTg-AD mice to


20

calculate the total number of Iba-1+ cells. Error bars indicate the SEM (*P < 0.05 and **P <

0.005). Statistical analysis was carried out using Student's t-test. Representative images show

Iba-1+ microglia in the CA1 (p) and dentate gyrus (q) of 3xTg-AD mice. Scale bar=10µm

(left panels p,q) and 25µm (righ panels p,q). (r, s) Cognitive tests and microglial analysis

were carried out using 129/C57BL6 and C57BL/6 mice at 6 months of age. (r) The results

shown are from the contextual fear-conditioning (CFC) tests performed in a blinded fashion.

Values represent mean ± SEM from a representative experiment with 10-15 mice per

condition from a series of three experiments with similar results (*P < 0.05). (s) Blinded

quantitative stereological analysis of microglial cells for each condition to determine the total

number of Iba-1+ cells and the area occupied by cell soma (n = 4 mice). Error bars indicate

SEM (*P < 0.05; **P < 0.005 and ***P < 0.0005). All statistical analyses were carried out

using the Mann-Whitney test.


21



22



23

Supplementary Figure 6. Distribution of neutrophils in the AD brain and graphical

abstract. Distribution of neutrophils in paraffin sections of human cortex from AD brains. (a)

Detailed images confirm the presence of MPO+ cells (green) detected in AD cerebral

parenchyma. Nuclei are stained with DAPI (blue). (b) Representative confocal images show

spreading MPO+ cells (green) inside a cortical vessel with Aβ angiopathy (red). The AD

blood vessels surrounded by a white square are shown at higher magnification in the second

panel to emphasize the presence of cells spreading inside the vessel with Aβ deposits. (c, d)

Representative images of perivascular migrating cells (arrows) with polysegmented nuclei.

The sections were stained with hematoxylin and eosin and Congo Red (Electron Microscopy

Sciences, Hatfield, PA) to reveal Aβ deposits. (d) A cell with a polysegmented nucleus is

shown near an Aβ deposit. White arrows indicate cells with polysegmented nuclei that have

migrated perivascularly (c) and intraparenchymally (d). (e, f) Neutrophils in the human AD

brain (e) and age-matched control (f) stained with the naphthol AS-D chloroacetate esterase

kit and counterstained with hematoxylin. White arrows indicate intraparenchymally migrating

cells. A cell with polysegmented nucleus is shown in e (arrow, right panel). (g, h)

Representative images of CD66b+ cells counterstained with hematoxylin in the cerebral

parenchyma of an AD patient (g) and an age-matched control (h). White arrows indicate

intraparenchymally migrating neutrophils (g). Cells were predominantly intravascular in

control subjects (f, h). Scale bar = 20 µm (a, d), 50 µm (b right panel), 10 µm (b and c left

panels), 20 µm (c right panel, e, f, g, h). (i-m) Proximity between MPO+ cells and amyloid

plaques. (i) Representative image of paraffin-embedded section form a patient with

Alzheimer’s disease in which amyloid deposits were stained in red with antibody 6F/3D, and

MPO+ cells were stained in green with an anti-MPO antibody. Nuclei were stained in blue

with DAPI. (j) The Imaris software (Bitplane) surface rendering tool was used to create a

volume based on the fluorescence intensity. The area surrounded by a white square is shown


24

in panel j. (k) The distance between an MPO+ cell and the adjacent plaque is shown by the

white line, which connects the center of the amyloid plaque (red) and the center of the MPO+

cell (green). (l) Distribution of distances between MPO+ cells (n = 105) and adjacent plaques

in 11 AD patients (bar graphs) compared to the distribution of random distances between

MPO+ cells and plaques generated using Monte Carlo simulations (Gaussian curve in black)

(P < 0.00001; Anderson-Darling test). (m) Distances between MPO+ cells and adjacent

plaques in AD patients (experimental) included in the distance classes (≤50, >50 to ≤100,

>100 µm) and compared to the distance classes from the random data generated with Monte

Carlo simulations (random). The experimental frequency distribution (59%, 19%, 22%) is

different from the random distribution (7%, 8%, 85%) across the three distance classes (P <

0.00001; Pearson’s Chi-squared test). (n) Graphical abstract. Aβ or danger signals released

from brain cells lead to the activation of the endothelium, inducing the expression of adhesion

molecules and chemoattractants. Vascular inflammation favors neutrophil endothelium

adhesion interactions mediated by LFA-1, β2 integrin, ICAM-1 and potentially other adhesion

molecules. Greater Aβ levels in the blood may prime circulating neutrophils through formyl

peptide receptors (FPRs). Chemoattractants and Aβ on the endothelial surface may activate

the LFA-1 high-affinity state and trigger rapid intravascular adhesion through FPRs and other

G-protein-coupled receptors. Neutrophils that have adhered and spread inside the blood

vessels may release reactive oxygen species (ROS), cytokines, chemokines and enzymes

contributing to the destruction of the blood-brain barrier. Cells adhering within the

vasculature release neutrophil extracellular traps (NETs), comprising decondensed chromatin

and active proteases, including neutrophil elastase and myeloperoxidase. Extravasated

neutrophil movement inside the parenchyma is controlled by LFA-1 and potentially by

Aβ/FPRs and other chemoattractants/GPCRs and may harm neural cells by releasing NETs,

enzymes, ROS, nitric oxide (NO), cytokines and chemokines. Intraparenchymal Aβ may


25

activate neutrophil integrins by binding to FPRs. The neutrophil–microglial interaction may

create several feedback loops that amplify and sustain their activation, potentially

representing a negative driving force in the pathogenesis of Alzheimer’s disease. Neutrophils

migrating within the parenchyma also contribute to the formation of Aβ deposits, tau

phosphorylation and synaptic dysfunction, suggesting they play a pivotal role in the cognitive

decline related to Alzheimer’s disease.


26

Supplementary Table I. Presence of neutrophils in subjects with AD.

Detailed neuropathology of each subject included in the study, along with age, sex and

duration of disease. All material was collected from donors for or from whom a written

informed consent for a brain autopsy and the use of the material and clinical information for

research purposes had been obtained by the MRC London Brain Bank for Neurodegenerative

Patient Neuropathology Age Sex Duration of disease

Total MPO+ cells inside vessels

Total MPO+ cells inside parenchyma

Mean nr. MPO+ cells per 1.5 mm2 inside vessels

Mean nr. MPO+ cells per 1.5 mm2

inside parenchyma

Sporadic AD

A027/11 AD Braak 6 BNE6

61 F 5 y 157 488 5.2 ± 1.4 16.3 ± 1.7

A312/10 AD HP-tau stage 6 with MAA

63 F 5 y

198 398 6.6 ± 0.9 13.3 ± 3.8

A310/07 AD Braak 6 94 F 7 y 78 153 2.6 ± 0.9 5.1 ± 1.5 A125/09 AD Braak 6 with

MAA 58 F 15 y 206 249 6.9 ± 1.3 8.3 ± 3.1

A282/10 AD HP-tau stage 6 with MAA

68

M

5 y

166 387 5.5 ± 1.5 12.9 ± 2.7


70 M 10 y 111 210 3.7 ± 0.9 7.0 ± 1.5

A171/09 AD Braak 5 with MAA

88 M 17 y 69 250 2.3 ± 1.0 8.3 ± 0.8


82 M 6 y 182 395 6.1 ± 2.0 13.2 ± 2.0

A031/09 AD Braak 6 80 M 7 y 37 189 1.2 ± 0.4 6.3 ± 1.9 A75/09 AD Braak 6 89 M 7 y 87 180 2.9 ± 1.4 6.0 ± 1.4 AX AD 77 M 8 y 290 321 9.7 ± 1.3 10.7 ± 2.5 Controls

A049/10 Tau pathology Braak/BNE 3

75 F NCSD 102 38 3.4 ± 0.9 1.3 ± 0.5

A085/11 AD-type pathology, BNE 3

86 F NCSD 48 3 1.6 ± 1.0 0.1 ± 0.3

A310/09 Braak 2 consistent with normal aging

84 F NCSD 28 6 0.9 ± 0.6 0.2 ± 0.4

0908 Normal autopsy 68 F 71 40 2.4 ± 0.8 1.3 ± 0.7 1003 Normal autopsy 75 F 72 3 2.4 ± 1.5 0.1 ± 0.3 3984 Depression 71 F 44 27 1.5 ± 0.9 0.9 ± 0.8 A276/05 Mild AD-type

changes due to ageing Braak 3

92 M NCSD 27 12 0.9 ± 0.5 0.4 ±0.5

1500 Normal autopsy 78 M 18 0 0.6 ± 0.7 0.0 ± 0.0 4003 Normal autopsy 65 M 57 12 1.9 ± 0.7 0.4 ±0.5 16003 Normal autopsy 72 M 18 1 0.6 ± 0.7 0.0 ± 0.2 1294 Prostatic

carcinoma 73 M 54 11 1.8 ± 0.6 0.4 ±0.5


27

Diseases, which belongs to the BrainNet Europe (http://www.brainnet-europe.org). The

average age is 74.5 ± 10.4 for AD subjects (n = 11) and 76.2 ± 7.7 for age-matched controls

(n = 11). Columns 6 and 7 show the total number of cells for each subject derived from the

counting of 10 acquisition fields for each section (n = 3) as described in the materials and

methods section. Columns 8 and 9 show the mean value ± SD of the absolute number of

MPO+ cells per acquisition field (1.5 mm2) for each subject. AD = Alzheimer’s disease; HP-

tau = hyperphosphorylated tau; BNE = Brain Net Europe; MAA = Mild Amyloid

Angiopathy; NCSD = No Clinical Symptoms of Dementia.


28

SUPPLEMENTARY VIDEO LEGENDS

Supplementary Video 1. Neutrophils migrate into the brain parenchyma of 5xFAD mice.

Neutrophils were labeled in red with fluorescent cell tracker CMTPX and blood vessels were

labeled in green using 525-nm non-targeted Qdots. The two-photon time-lapse video shows

neutrophils migrating into the brain cortex of 4-month-old 5xFAD mice. The motile cells

displayed tails representing the cell tracking visualized with IMARIS software.

Supplementary Video 2. Neutrophils interact with the vascular endothelium in 5xFAD

mice. Neutrophils were labeled in red using fluorescent cell tracker CMTPX and blood

vessels were labeled in green using 525-nm non-targeted Qdots. The two-photon time-lapse

video shows crawling neutrophils within the cortical blood vessels of 4-month-old 5xFAD

mice.

Supplementary Video 3. Neutrophils show strong directional movement in the brain

parenchyma of 5xFAD mice. Neutrophils labeled in red using fluorescent cell tracker

CMTPX were injected into 5xFAD-YFPH mice (fluorescent neurons are shown in green).

Cell tracking was visualized with the IMARIS software package. The trajectory of cell

movement during intraparenchymal migration (cortex) showed a strong directional bias. The

vessel edge was tracked artificially and is shown as a dashed white line to delimit the vessel

bed.

Supplementary Video 4. Neutrophils show non-directional movement inside the brain

parenchyma. Neutrophils were labeled in red using fluorescent cell tracker CMTPX and

blood vessels were labeled in green using 525-nm non-targeted Qdots. The two-photon time-

lapse video shows the non-directional movement of neutrophils in the brain cortex of 4-


29

month-old 5xFAD mice. Neutrophils accumulated in small clusters in discrete areas where the

cells appeared to swarm around unidentified brain structures.

Supplementary Video 5. Neutrophil infiltration into the brain parenchyma occurs

preferentially in Aβ-rich areas. Neutrophils labeled in red using fluorescent cell tracker

CMTPX were injected into 5xFAD-YFPH mice (fluorescent neurons are shown in green). Aβ

was labeled in blue using MeO-X04. The vessel edge was tracked artificially and is shown as

a dashed white line to delimit the vessel bed. Neutrophils migrated inside the parenchyma

preferentially in cortical areas with Aβ plaques.

Supplementary Video 6. Vascular Aβ deposition promotes neutrophil adhesion in

cortical vessels of 5xFAD mice. Neutrophils labeled in red using fluorescent cell tracker

CMTPX were injected into 5xFAD-YFPH mice (fluorescent neurons are shown in green). Aβ

was labeled in blue using MeO-X04. The vessel edge was tracked artificially and is shown as

a dashed white line to delimit the vessel bed. Neutrophils interacted strongly with the vascular

endothelium in cortical vessels containing Aβ deposits and migrated inside the parenchyma.

Supplementary Video 7. LFA-1 integrin is necessary for neutrophil infiltration into the

brain parenchyma of 5xFAD mice. Blood vessels were labeled in green using 525-nm non-

targeted Qdots. Wild-type neutrophils labeled in blue using fluorescent cell tracker CMAC

invaded the brain parenchyma of 5xFAD mice, whereas neutrophils from LFA1-deficient

mice labeled in red using fluorescent cell tracker CMTPX did not interact with brain vessels

and were not able to infiltrate the brain parenchyma.


30

Supplementary Video 8. Blocking LFA-1 integrin inhibits the motility of neutrophils

within the brain parenchyma of 5xFAD mice. Neutrophils labeled in blue using fluorescent

cell tracker CMAC were injected into 5xFAD mice. Blood vessels were labeled in green

using 525-nm non-targeted Qdots. The blocking of integrin LFA-1 with a monoclonal

antibody reduced the motility of neutrophils inside the brain parenchyma (cortex) and a

smaller area was covered.


Neutrophils promote Alzheimer’s disease-like pathology and ... · ! 1! Neutrophils promote...

Documents

Transcript of Neutrophils promote Alzheimer’s disease-like pathology and ... · ! 1! Neutrophils promote...