Key Points

  • Neutrophil NADPH oxidase limits LTB4 production by controlling calcium influx.

  • LTB4 is a major driver in promoting neutrophilic inflammation in CGD mice in response to fungal cell walls.

Abstract

Leukocyte reduced NADP (NADPH) oxidase plays a key role in host defense and immune regulation. Genetic defects in NADPH oxidase result in chronic granulomatous disease (CGD), characterized by recurrent bacterial and fungal infections and aberrant inflammation. Key drivers of hyperinflammation induced by fungal cell walls in CGD are still incompletely defined. In this study, we found that CGD (CYBB) neutrophils produced higher amounts of leukotriene B4 (LTB4) in vitro after activation with zymosan or immune complexes, compared with wild-type (WT) neutrophils. This finding correlated with increased calcium influx in CGD neutrophils, which was restrained in WT neutrophils by the electrogenic activity of NADPH oxidase. Increased LTB4 generation by CGD neutrophils was also augmented by paracrine cross talk with the LTB4 receptor BLT1. CGD neutrophils formed more numerous and larger clusters in the presence of zymosan in vitro compared with WT cells, and the effect was also LTB4- and BLT1-dependent. In zymosan-induced lung inflammation, focal neutrophil infiltrates were increased in CGD compared with WT mice and associated with higher LTB4 levels. Inhibiting LTB4 synthesis or antagonizing the BLT1 receptor after zymosan challenge reduced lung neutrophil recruitment in CGD to WT levels. Thus, LTB4 was the major driver of excessive neutrophilic lung inflammation in CGD mice in the early response to fungal cell walls, likely by a dysregulated feed-forward loop involving amplified neutrophil production of LTB4. This study identifies neutrophil LTB4 generation as a target of NADPH oxidase regulation, which could potentially be exploited therapeutically to reduce excessive inflammation in CGD.

REFERENCES

REFERENCES
1.
Dinauer
MC
.
Inflammatory consequences of inherited disorders affecting neutrophil function
.
Blood
.
2019
;
133
(
20
):
2130
-
2139
.
2.
Morgenstern
DE
,
Gifford
MA
,
Li
LL
,
Doerschuk
CM
,
Dinauer
MC
.
Absence of respiratory burst in X-linked chronic granulomatous disease mice leads to abnormalities in both host defense and inflammatory response to Aspergillus fumigatus
.
J Exp Med
.
1997
;
185
(
2
):
207
-
218
.
3.
Segal
BH
,
Han
W
,
Bushey
JJ
, et al
.
NADPH oxidase limits innate immune responses in the lungs in mice
.
PLoS One
.
2010
;
5
(
3
):
e9631
.
4.
Petersen
JE
,
Hiran
TS
,
Goebel
WS
, et al
.
Enhanced cutaneous inflammatory reactions to Aspergillus fumigatus in a murine model of chronic granulomatous disease
.
J Invest Dermatol
.
2002
;
118
(
3
):
424
-
429
.
5.
Schäppi
M
,
Deffert
C
,
Fiette
L
, et al
.
Branched fungal beta-glucan causes hyperinflammation and necrosis in phagocyte NADPH oxidase-deficient mice
.
J Pathol
.
2008
;
214
(
4
):
434
-
444
.
6.
Warnatsch
A
,
Tsourouktsoglou
TD
,
Branzk
N
, et al
.
Reactive Oxygen Species Localization Programs Inflammation to Clear Microbes of Different Size
.
Immunity
.
2017
;
46
(
3
):
421
-
432
.
7.
Endo
D
,
Fujimoto
K
,
Hirose
R
, et al
.
Genetic Phagocyte NADPH Oxidase Deficiency Enhances Nonviable Candida albicans-Induced Inflammation in Mouse Lungs
.
Inflammation
.
2017
;
40
(
1
):
123
-
135
.
8.
Singel
KL
,
Segal
BH
.
NOX2-dependent regulation of inflammation
.
Clin Sci (Lond)
.
2016
;
130
(
7
):
479
-
490
.
9.
de Luca
A
,
Smeekens
SP
,
Casagrande
A
, et al
.
IL-1 receptor blockade restores autophagy and reduces inflammation in chronic granulomatous disease in mice and in humans
.
Proc Natl Acad Sci USA
.
2014
;
111
(
9
):
3526
-
3531
.
10.
Busse
WW
.
Leukotrienes and inflammation
.
Am J Respir Crit Care Med
.
1998
;
157
(
6 pt 1
):
S210
-
S213
.
11.
Miyabe
Y
,
Miyabe
C
,
Luster
AD
.
LTB4 and BLT1 in inflammatory arthritis
.
Semin Immunol
.
2017
;
33
:
52
-
57
.
12.
Okunishi
K
,
Peters-Golden
M
.
Leukotrienes and airway inflammation
.
Biochim Biophys Acta
.
2011
;
1810
(
11
):
1096
-
1102
.
13.
Németh
T
,
Mócsai
A
.
Feedback Amplification of Neutrophil Function
.
Trends Immunol
.
2016
;
37
(
6
):
412
-
424
.
14.
Kim
ND
,
Chou
RC
,
Seung
E
,
Tager
AM
,
Luster
AD
.
A unique requirement for the leukotriene B4 receptor BLT1 for neutrophil recruitment in inflammatory arthritis
.
J Exp Med
.
2006
;
203
(
4
):
829
-
835
.
15.
Chou
RC
,
Kim
ND
,
Sadik
CD
, et al
.
Lipid-cytokine-chemokine cascade drives neutrophil recruitment in a murine model of inflammatory arthritis
.
Immunity
.
2010
;
33
(
2
):
266
-
278
.
16.
Lämmermann
T
,
Afonso
PV
,
Angermann
BR
, et al
.
Neutrophil swarms require LTB4 and integrins at sites of cell death in vivo
.
Nature
.
2013
;
498
(
7454
):
371
-
375
.
17.
Kienle
K
,
Lämmermann
T
.
Neutrophil swarming: an essential process of the neutrophil tissue response
.
Immunol Rev
.
2016
;
273
(
1
):
76
-
93
.
18.
Tan
SY
,
Weninger
W
.
Neutrophil migration in inflammation: intercellular signal relay and crosstalk
.
Curr Opin Immunol
.
2017
;
44
:
34
-
42
.
19.
Afonso
PV
,
Janka-Junttila
M
,
Lee
YJ
, et al
.
LTB4 is a signal-relay molecule during neutrophil chemotaxis
.
Dev Cell
.
2012
;
22
(
5
):
1079
-
1091
.
20.
Subramanian
BC
,
Majumdar
R
,
Parent
CA
.
The role of the LTB4-BLT1 axis in chemotactic gradient sensing and directed leukocyte migration
.
Semin Immunol
.
2017
;
33
:
16
-
29
.
21.
Rådmark
O
,
Werz
O
,
Steinhilber
D
,
Samuelsson
B
.
5-Lipoxygenase, a key enzyme for leukotriene biosynthesis in health and disease
.
Biochim Biophys Acta
.
2015
;
1851
(
4
):
331
-
339
.
22.
Wan
M
,
Tang
X
,
Stsiapanava
A
,
Haeggström
JZ
.
Biosynthesis of leukotriene B4
.
Semin Immunol
.
2017
;
33
:
3
-
15
.
23.
Clemens
RA
,
Lowell
CA
.
CRAC channel regulation of innate immune cells in health and disease
.
Cell Calcium
.
2019
;
78
:
56
-
65
.
24.
Leslie
CC
.
Cytosolic phospholipase A2: physiological function and role in disease
.
J Lipid Res
.
2015
;
56
(
8
):
1386
-
1402
.
25.
Geiszt
M
,
Kapus
A
,
Német
K
,
Farkas
L
,
Ligeti
E
.
Regulation of capacitative Ca2+ influx in human neutrophil granulocytes. Alterations in chronic granulomatous disease
.
J Biol Chem
.
1997
;
272
(
42
):
26471
-
26478
.
26.
Tintinger
GR
,
Theron
AJ
,
Steel
HC
,
Anderson
R
.
Accelerated calcium influx and hyperactivation of neutrophils in chronic granulomatous disease
.
Clin Exp Immunol
.
2001
;
123
(
2
):
254
-
263
.
27.
Geiszt
M
,
Kapus
A
,
Ligeti
E
.
Chronic granulomatous disease: more than the lack of superoxide?
J Leukoc Biol
.
2001
;
69
(
2
):
191
-
196
.
28.
Rada
BK
,
Geiszt
M
,
Hably
C
,
Ligeti
E
.
Consequences of the electrogenic function of the phagocytic NADPH oxidase
.
Philos Trans R Soc Lond B Biol Sci
.
2005
;
360
(
1464
):
2293
-
2300
.
29.
Tintinger
GR
,
Steel
HC
,
Theron
AJ
,
Anderson
R
.
Pharmacological control of neutrophil-mediated inflammation: strategies targeting calcium handling by activated polymorphonuclear leukocytes
.
Drug Des Devel Ther
.
2009
;
2
:
95
-
104
.
30.
Pollock
JD
,
Williams
DA
,
Gifford
MA
, et al
.
Mouse model of X-linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production
.
Nat Genet
.
1995
;
9
(
2
):
202
-
209
.
31.
Xue
X
,
Pech
NK
,
Shelley
WC
,
Srour
EF
,
Yoder
MC
,
Dinauer
MC
.
Antibody targeting KIT as pretransplantation conditioning in immunocompetent mice
.
Blood
.
2010
;
116
(
24
):
5419
-
5422
.
32.
Clemens
RA
,
Chong
J
,
Grimes
D
,
Hu
Y
,
Lowell
CA
.
STIM1 and STIM2 cooperatively regulate mouse neutrophil store-operated calcium entry and cytokine production
.
Blood
.
2017
;
130
(
13
):
1565
-
1577
.
33.
Carter
GW
,
Young
PR
,
Albert
DH
, et al
.
5-lipoxygenase inhibitory activity of zileuton
.
J Pharmacol Exp Ther
.
1991
;
256
(
3
):
929
-
937
.
34.
Lv
J
,
Xiong
Y
,
Li
W
,
Yang
W
,
Zhao
L
,
He
R
.
BLT1 Mediates Bleomycin-Induced Lung Fibrosis Independently of Neutrophils and CD4+ T Cells
.
J Immunol
.
2017
;
198
(
4
):
1673
-
1684
.
35.
Faget
J
,
Boivin
G
,
Ancey
P-B
, et al
.
Efficient and specific Ly6G+ cell depletion: A change in the current practices toward more relevant functional analyses of neutrophils
.
bioRxiv
.
2018
.
36.
Li
X
,
Utomo
A
,
Cullere
X
, et al
.
The β-glucan receptor Dectin-1 activates the integrin Mac-1 in neutrophils via Vav protein signaling to promote Candida albicans clearance
.
Cell Host Microbe
.
2011
;
10
(
6
):
603
-
615
.
37.
Sun
D
,
Shi
M
.
Neutrophil swarming toward Cryptococcus neoformans is mediated by complement and leukotriene B4
.
Biochem Biophys Res Commun
.
2016
;
477
(
4
):
945
-
951
.
38.
Reátegui
E
,
Jalali
F
,
Khankhel
AH
, et al
.
Microscale arrays for the profiling of start and stop signals coordinating human-neutrophil swarming
.
Nat Biomed Eng
.
2017
;
2017
:
0094
.
39.
Bartemes
KR
,
Kita
H
.
Innate and adaptive immune responses to fungi in the airway
.
J Allergy Clin Immunol
.
2018
;
142
(
2
):
353
-
363
.
40.
Steele
C
,
Rapaka
RR
,
Metz
A
, et al
.
The beta-glucan receptor dectin-1 recognizes specific morphologies of Aspergillus fumigatus
.
PLoS Pathog
.
2005
;
1
(
4
):
e42
.
41.
Bagaitkar
J
,
Pech
NK
,
Ivanov
S
, et al
.
NADPH oxidase controls neutrophilic response to sterile inflammation in mice by regulating the IL-1α/G-CSF axis
.
Blood
.
2015
;
126
(
25
):
2724
-
2733
.
42.
Segal
BH
,
Kuhns
DB
,
Ding
L
,
Gallin
JI
,
Holland
SM
.
Thioglycollate peritonitis in mice lacking C5, 5-lipoxygenase, or p47(phox): complement, leukotrienes, and reactive oxidants in acute inflammation
.
J Leukoc Biol
.
2002
;
71
(
3
):
410
-
416
.
43.
Rada
BK
,
Geiszt
M
,
Van Bruggen
R
,
Nemet
K
,
Roos
D
,
Ligeti
E
.
Calcium signalling is altered in myeloid cells with a deficiency in NADPH oxidase activity
.
Clin Exp Immunol
.
2003
;
132
(
1
):
53
-
60
.
44.
Henderson
WR
,
Klebanoff
SJ
.
Leukotriene production and inactivation by normal, chronic granulomatous disease and myeloperoxidase-deficient neutrophils
.
J Biol Chem
.
1983
;
258
(
22
):
13522
-
13527
.
45.
Hamasaki
T
,
Sakano
T
,
Kobayashi
M
,
Sakura
N
,
Ueda
K
,
Usui
T
.
Leukotriene B4 metabolism in neutrophils of patients with chronic granulomatous disease: phorbol myristate acetate decreases endogenous leukotriene B4 via NADPH oxidase-dependent mechanism
.
Eur J Clin Invest
.
1989
;
19
(
4
):
404
-
411
.
46.
Feinmark
SJ
,
Udén
AM
,
Palmblad
J
,
Malmsten
C
.
Leukotriene biosynthesis by polymorphonuclear leukocytes from two patients with chronic granulomatous disease
.
J Clin Invest
.
1983
;
72
(
5
):
1839
-
1843
.
47.
Henderson
WR
,
Klebanoff
SJ
.
Leukotriene B4, C4, D4 and E4 inactivation by hydroxyl radicals
.
Biochem Biophys Res Commun
.
1983
;
110
(
1
):
266
-
272
.
48.
Moskaluk
CA
,
Pogrebniak
HW
,
Pass
HI
,
Gallin
JI
,
Travis
WD
.
Surgical pathology of the lung in chronic granulomatous disease
.
Am J Clin Pathol
.
1994
;
102
(
5
):
684
-
691
.
49.
Sherif
R
,
Segal
BH
.
Pulmonary aspergillosis: clinical presentation, diagnostic tests, management and complications
.
Curr Opin Pulm Med
.
2010
;
16
(
3
):
242
-
250
.
50.
Falcone
EL
,
Holland
SM
.
Invasive fungal infection in chronic granulomatous disease: insights into pathogenesis and management
.
Curr Opin Infect Dis
.
2012
;
25
(
6
):
658
-
669
.
51.
Secatto
A
,
Soares
EM
,
Locachevic
GA
, et al
.
The leukotriene B4/BLT1 axis is a key determinant in susceptibility and resistance to histoplasmosis
.
PLoS One
.
2014
;
9
(
1
):
e85083
.
52.
Secatto
A
,
Rodrigues
LC
,
Serezani
CH
, et al
.
5-Lipoxygenase deficiency impairs innate and adaptive immune responses during fungal infection
.
PLoS One
.
2012
;
7
(
3
):
e31701
.
53.
Santos
PC
,
Santos
DA
,
Ribeiro
LS
, et al
.
The pivotal role of 5-lipoxygenase-derived LTB4 in controlling pulmonary paracoccidioidomycosis
.
PLoS Negl Trop Dis
.
2013
;
7
(
8
):
e2390
.
54.
Balderramas
HA
,
Ribeiro
OG
,
Soares
AM
,
Oliveira
SL
.
The role of leukotriene B4 in early stages of experimental paracoccidioidomycosis induced in phenotypically selected mouse strains
.
Med Mycol
.
2013
;
51
(
6
):
625
-
634
.
55.
Caffrey-Carr
AK
,
Hilmer
KM
,
Kowalski
CH
, et al
.
Host-Derived Leukotriene B4 Is Critical for Resistance against Invasive Pulmonary Aspergillosis
.
Front Immunol
.
2018
;
8
:
1984
.
56.
Lee
EKS
,
Gillrie
MR
,
Li
L
, et al
.
Leukotriene B4-Mediated Neutrophil Recruitment Causes Pulmonary Capillaritis during Lethal Fungal Sepsis
.
Cell Host Microbe
.
2018
;
23
(
1
):
121
-
133.e124
.
57.
Sadik
CD
,
Luster
AD
.
Lipid-cytokine-chemokine cascades orchestrate leukocyte recruitment in inflammation
.
J Leukoc Biol
.
2012
;
91
(
2
):
207
-
215
.
58.
Haeggström
JZ
.
Leukotriene biosynthetic enzymes as therapeutic targets
.
J Clin Invest
.
2018
;
128
(
7
):
2680
-
2690
.
59.
Yokomizo
T
,
Nakamura
M
,
Shimizu
T
.
Leukotriene receptors as potential therapeutic targets
.
J Clin Invest
.
2018
;
128
(
7
):
2691
-
2701
.
60.
Mestas
J
,
Hughes
CC
.
Of mice and not men: differences between mouse and human immunology
.
J Immunol
.
2004
;
172
(
5
):
2731
-
2738
.
61.
Bhatt
L
,
Roinestad
K
,
Van
T
,
Springman
EB
.
Recent advances in clinical development of leukotriene B4 pathway drugs
.
Semin Immunol
.
2017
;
33
:
65
-
73
.
62.
Martin
TR
,
Pistorese
BP
,
Chi
EY
,
Goodman
RB
,
Matthay
MA
.
Effects of leukotriene B4 in the human lung. Recruitment of neutrophils into the alveolar spaces without a change in protein permeability
.
J Clin Invest
.
1989
;
84
(
5
):
1609
-
1619
.
63.
Tager
AM
,
Luster
AD
.
BLT1 and BLT2: the leukotriene B(4) receptors
.
Prostaglandins Leukot Essent Fatty Acids
.
2003
;
69
(
2-3
):
123
-
134
.
64.
Turner
CR
,
Breslow
R
,
Conklyn
MJ
, et al
.
In vitro and in vivo effects of leukotriene B4 antagonism in a primate model of asthma
.
J Clin Invest
.
1996
;
97
(
2
):
381
-
387
.
65.
Zhang
H
,
Clemens
RA
,
Liu
F
, et al
.
STIM1 calcium sensor is required for activation of the phagocyte oxidase during inflammation and host defense
.
Blood
.
2014
;
123
(
14
):
2238
-
2249
.
66.
Stauderman
KA
.
CRAC channels as targets for drug discovery and development
.
Cell Calcium
.
2018
;
74
:
147
-
159
.
67.
Immler
R
,
Simon
SI
,
Sperandio
M
.
Calcium signalling and related ion channels in neutrophil recruitment and function
.
Eur J Clin Invest
.
2018
;
48
(
Suppl 2
):
e12964
.
68.
Tian
C
,
Du
L
,
Zhou
Y
,
Li
M
.
Store-operated CRAC channel inhibitors: opportunities and challenges
.
Future Med Chem
.
2016
;
8
(
7
):
817
-
832
.
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