• Eosinophils promote arterial thrombosis in hematopoietic LNK deficiency.

  • Eosinophil LNK is essential in suppression of arterial thrombosis.

Abstract

Increased eosinophil counts are associated with cardiovascular disease and may be an independent predictor of major cardiovascular events. However, the causality and underlying mechanisms are poorly understood. Genome-wide association studies have shown an association of a common LNK variant (R262W, T allele) with eosinophilia and atherothrombotic disorders. LNK(TT) reduces LNK function, and Lnk-deficient mice display accelerated atherosclerosis and thrombosis. This study was undertaken to assess the role of eosinophils in arterial thrombosis in mice with hematopoietic Lnk deficiency. Hematopoietic Lnk deficiency increased circulating and activated eosinophils, JAK/STAT signaling in eosinophils, and carotid arterial thrombosis with increased eosinophil abundance and extracellular trap formation (EETosis) in thrombi. Depletion of eosinophils by anti–Siglec-F antibody or by the ΔdbIGata1 mutation eliminated eosinophils in thrombi and markedly reduced thrombosis in mice with hematopoietic Lnk deficiency but not in control mice. Eosinophil depletion reduced neutrophil abundance and NETosis in thrombi without altering circulating neutrophil counts. To assess the role of Lnk specifically in eosinophils, we crossed Lnkf/f mice with eoCre mice. LnkΔeos mice displayed isolated eosinophilia, increased eosinophil activation, and accelerated arterial thrombosis associated with increased EETosis and NETosis in thrombi. DNase I infusion abolished EETs and neutrophil extracellular traps (NETs) in thrombi and reversed the accelerated thrombosis. Human induced pluripotent stem cell–derived LNK(TT) eosinophils showed increased activation and EETosis relative to isogenic LNK(CC) eosinophils, demonstrating human relevance. These studies show a direct link between eosinophilia, EETosis, and atherothrombosis in hematopoietic Lnk deficiency and an essential role of eosinophil LNK in suppression of arterial thrombosis.

1.
Ahmad
FB
,
Anderson
RN
.
The leading causes of death in the US for 2020
.
JAMA
.
2021
;
325
(
18
):
1829
-
1830
.
2.
Vallejo-Vaz
AJ
,
Fayyad
R
,
Boekholdt
SM
, et al
.
Triglyceride-rich lipoprotein cholesterol and risk of cardiovascular events among patients receiving statin therapy in the TNT trial
.
Circulation
.
2018
;
138
(
8
):
770
-
781
.
3.
Cannon
CP
,
Blazing
MA
,
Giugliano
RP
, et al
.
Ezetimibe added to statin therapy after acute coronary syndromes
.
N Engl J Med
.
2015
;
372
(
25
):
2387
-
2397
.
4.
Sabatine
MS
,
Giugliano
RP
,
Keech
AC
, et al;
FOURIER Steering Committee and Investigators and Investigators
.
Evolocumab and clinical outcomes in patients with cardiovascular disease
.
N Engl J Med
.
2017
;
376
(
18
):
1713
-
1722
.
5.
Ridker
PM
,
Everett
BM
,
Thuren
T
, et al
.
Antiinflammatory therapy with canakinumab for atherosclerotic disease
.
N Engl J Med
.
2017
;
377
(
12
):
1119
-
1131
.
6.
Tardif
JC
,
Kouz
S
,
Waters
DD
, et al
.
Efficacy and safety of low-dose colchicine after myocardial infarction
.
N Engl J Med
.
2019
;
381
(
26
):
2497
-
2505
.
7.
Welsh
C
,
Welsh
P
,
Mark
PB
, et al
.
Association of total and differential leukocyte counts with cardiovascular disease and mortality in the UK Biobank
.
Arterioscler Thromb Vasc Biol
.
2018
;
38
(
6
):
1415
-
1423
.
8.
Totani
L
,
Evangelista
V
.
Platelet-leukocyte interactions in cardiovascular disease and beyond
.
Arterioscler Thromb Vasc Biol
.
2010
;
30
(
12
):
2357
-
2361
.
9.
Niccoli
G
,
Sgueglia
GA
,
Conte
M
, et al
.
Eosinophil cationic protein and clinical outcome after bare metal stent implantation
.
Atherosclerosis
.
2011
;
215
(
1
):
166
-
169
.
10.
Gudbjartsson
DF
,
Bjornsdottir
US
,
Halapi
E
, et al
.
Sequence variants affecting eosinophil numbers associate with asthma and myocardial infarction
.
Nat Genet
.
2009
;
41
(
3
):
342
-
347
.
11.
Jacobsen
EA
,
Jackson
DJ
,
Heffler
E
, et al
.
Eosinophil knockout humans: uncovering the role of eosinophils through eosinophil-directed biological therapies
.
Annu Rev Immunol
.
2021
;
39
:
719
-
757
.
12.
Zhao
X
,
Jiang
L
,
Xu
L
, et al
.
Predictive value of in-hospital white blood cell count in Chinese patients with triple-vessel coronary disease
.
Eur J Prev Cardiol
.
2019
;
26
(
8
):
872
-
882
.
13.
Pongdee
T
,
Manemann
SM
,
Decker
PA
, et al
.
Rethinking blood eosinophil counts: epidemiology, associated chronic diseases, and increased risks of cardiovascular disease
.
J Allergy Clin Immunol Glob
.
2022
;
1
(
4
):
233
-
240
.
14.
Sundström
J
,
Söderholm
M
,
Borné
Y
, et al
.
Eosinophil cationic protein, carotid plaque, and incidence of stroke
.
Stroke
.
2017
;
48
(
10
):
2686
-
2692
.
15.
Ames
PR
,
Margaglione
M
,
Mackie
S
,
Alves
JD
.
Eosinophilia and thrombophilia in churg strauss syndrome: a clinical and pathogenetic overview
.
Clin Appl Thromb Hemost
.
2010
;
16
(
6
):
628
-
636
.
16.
Todd
S
,
Hemmaway
C
,
Nagy
Z
.
Catastrophic thrombosis in idiopathic hypereosinophilic syndrome
.
Br J Haematol
.
2014
;
165
(
4
):
425
.
17.
Marx
C
,
Novotny
J
,
Salbeck
D
, et al
.
Eosinophil-platelet interactions promote atherosclerosis and stabilize thrombosis with eosinophil extracellular traps
.
Blood
.
2019
;
134
(
21
):
1859
-
1872
.
18.
Hofheinz
K
,
Seibert
F
,
Ackermann
JA
, et al
.
Formation of atherosclerotic lesions is independent of eosinophils in male mice
.
Atherosclerosis
.
2020
;
311
:
67
-
72
.
19.
Meng
Z
,
Zhang
S
,
Li
W
, et al
.
Cationic proteins from eosinophils bind bone morphogenetic protein receptors promoting vascular calcification and atherogenesis
.
Eur Heart J
.
2023
;
44
(
29
):
2763
-
2783
.
20.
Thibord
F
,
Klarin
D
,
Brody
JA
, et al;
Global Biobank Meta-Analysis Initiative; Estonian Biobank Research Team; 23andMe Research Team; Biobank Japan; CHARGE Hemostasis Working Group
.
Cross-ancestry investigation of venous thromboembolism genomic predictors
.
Circulation
.
2022
;
146
(
16
):
1225
-
1242
.
21.
Bersenev
A
,
Wu
C
,
Balcerek
J
,
Tong
W
.
Lnk controls mouse hematopoietic stem cell self-renewal and quiescence through direct interactions with JAK2
.
J Clin Invest
.
2008
;
118
(
8
):
2832
-
2844
.
22.
Takaki
S
,
Morita
H
,
Tezuka
Y
,
Takatsu
K
.
Enhanced hematopoiesis by hematopoietic progenitor cells lacking intracellular adaptor protein, Lnk
.
J Exp Med
.
2002
;
195
(
2
):
151
-
160
.
23.
Velazquez
L
,
Cheng
AM
,
Fleming
HE
, et al
.
Cytokine signaling and hematopoietic homeostasis are disrupted in Lnk-deficient mice
.
J Exp Med
.
2002
;
195
(
12
):
1599
-
1611
.
24.
Wang
W
,
Tang
Y
,
Wang
Y
, et al
.
LNK/SH2B3 loss of function promotes atherosclerosis and thrombosis
.
Circ Res
.
2016
;
119
(
6
):
e91
-
e103
.
25.
Dou
H
,
Kotini
A
,
Liu
W
, et al
.
Oxidized phospholipids promote NETosis and arterial thrombosis in LNK(SH2B3) deficiency
.
Circulation
.
2021
;
144
(
24
):
1940
-
1954
.
26.
Doyle
AD
,
Jacobsen
EA
,
Ochkur
SI
, et al
.
Homologous recombination into the eosinophil peroxidase locus generates a strain of mice expressing Cre recombinase exclusively in eosinophils
.
J Leukoc Biol
.
2013
;
94
(
1
):
17
-
24
.
27.
Karcz
TP
,
Whitehead
GS
,
Nakano
K
, et al
.
UDP-glucose and P2Y14 receptor amplify allergen-induced airway eosinophilia
.
J Clin Invest
.
2021
;
131
(
7
):
131
.
28.
Germic
N
,
Hosseini
A
,
Stojkov
D
, et al
.
ATG5 promotes eosinopoiesis but inhibits eosinophil effector functions
.
Blood
.
2021
;
137
(
21
):
2958
-
2969
.
29.
Mack
EA
,
Stein
SJ
,
Rome
KS
, et al
.
Trib1 regulates eosinophil lineage commitment and identity by restraining the neutrophil program
.
Blood
.
2019
;
133
(
22
):
2413
-
2426
.
30.
Kotini
AG
,
Chang
CJ
,
Chow
A
, et al
.
Stage-specific human induced pluripotent stem cells map the progression of myeloid transformation to transplantable leukemia
.
Cell Stem Cell
.
2017
;
20
(
3
):
315
-
328.e7
.
31.
Lai
W
,
Xie
H
,
Liu
Y
, et al
.
Human pluripotent stem cell-derived eosinophils reveal potent cytotoxicity against solid tumors
.
Stem Cell Reports
.
2021
;
16
(
7
):
1697
-
1704
.
32.
Pazdrak
K
,
Young
TW
,
Straub
C
,
Stafford
S
,
Kurosky
A
.
Priming of eosinophils by GM-CSF is mediated by protein kinase CbetaII-phosphorylated L-plastin
.
J Immunol
.
2011
;
186
(
11
):
6485
-
6496
.
33.
Thorne
KJ
,
Richardson
BA
,
Mazza
G
,
Butterworth
AE
.
A new method for measuring eosinophil activating factors, based on the increased expression of CR3 alpha chain (CD11b) on the surface of activated eosinophils
.
J Immunol Methods
.
1990
;
133
(
1
):
47
-
54
.
34.
Carmo
LA
,
Bonjour
K
,
Ueki
S
, et al
.
CD63 is tightly associated with intracellular, secretory events chaperoning piecemeal degranulation and compound exocytosis in human eosinophils
.
J Leukoc Biol
.
2016
;
100
(
2
):
391
-
401
.
35.
Reichman
H
,
Rozenberg
P
,
Munitz
A
.
Mouse eosinophils: identification, isolation, and functional analysis
.
Curr Protoc Immunol
.
2017
;
119
:
14.43.1
-
14.43.22
.
36.
Zimmermann
N
,
McBride
ML
,
Yamada
Y
, et al
.
Siglec-F antibody administration to mice selectively reduces blood and tissue eosinophils
.
Allergy
.
2008
;
63
(
9
):
1156
-
1163
.
37.
Uderhardt
S
,
Ackermann
JA
,
Fillep
T
, et al
.
Enzymatic lipid oxidation by eosinophils propagates coagulation, hemostasis, and thrombotic disease
.
J Exp Med
.
2017
;
214
(
7
):
2121
-
2138
.
38.
Yu
C
,
Cantor
AB
,
Yang
H
, et al
.
Targeted deletion of a high-affinity GATA-binding site in the GATA-1 promoter leads to selective loss of the eosinophil lineage in vivo
.
J Exp Med
.
2002
;
195
(
11
):
1387
-
1395
.
39.
Liu
Y
,
Carmona-Rivera
C
,
Moore
E
, et al
.
Myeloid-specific deletion of peptidylarginine deiminase 4 mitigates atherosclerosis
.
Front Immunol
.
2018
;
9
:
1680
.
40.
Fuchs
TA
,
Brill
A
,
Duerschmied
D
, et al
.
Extracellular DNA traps promote thrombosis
.
Proc Natl Acad Sci U S A
.
2010
;
107
(
36
):
15880
-
15885
.
41.
Carolan
EJ
,
Casale
TB
.
Degree of platelet activating factor-induced neutrophil migration is dependent upon the molecular species
.
J Immunol
.
1990
;
145
(
8
):
2561
-
2565
.
42.
Wardlaw
AJ
,
Moqbel
R
,
Cromwell
O
,
Kay
AB
.
Platelet-activating factor. A potent chemotactic and chemokinetic factor for human eosinophils
.
J Clin Invest
.
1986
;
78
(
6
):
1701
-
1706
.
43.
Ueki
S
,
Melo
RC
,
Ghiran
I
,
Spencer
LA
,
Dvorak
AM
,
Weller
PF
.
Eosinophil extracellular DNA trap cell death mediates lytic release of free secretion-competent eosinophil granules in humans
.
Blood
.
2013
;
121
(
11
):
2074
-
2083
.
44.
Ueki
S
,
Tokunaga
T
,
Melo
RCN
, et al
.
Charcot-Leyden crystal formation is closely associated with eosinophil extracellular trap cell death
.
Blood
.
2018
;
132
(
20
):
2183
-
2187
.
45.
Adachi
T
,
Alam
R
.
The mechanism of IL-5 signal transduction
.
Am J Physiol
.
1998
;
275
(
3
):
C623
-
C633
.
46.
Mui
AL
,
Wakao
H
,
Harada
N
,
O'Farrell
AM
,
Miyajima
A
.
Interleukin-3, granulocyte-macrophage colony-stimulating factor, and interleukin-5 transduce signals through two forms of STAT5
.
J Leukoc Biol
.
1995
;
57
(
5
):
799
-
803
.
47.
Dellon
ES
,
Peterson
KA
,
Murray
JA
, et al
.
Anti-siglec-8 antibody for eosinophilic gastritis and duodenitis
.
N Engl J Med
.
2020
;
383
(
17
):
1624
-
1634
.
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