SEPSIS Copyright © 2020 Identification of tetranectin-targeting … · 2020-04-15 · higher...

Chen et al., Sci. Transl. Med. 12, eaaz3833 (2020) 15 April 2020

S C I E N C E T R A N S L A T I O N A L M E D I C I N E | R E S E A R C H A R T I C L E

1 of 14

S E P S I S

Identification of tetranectin-targeting monoclonal antibodies to treat potentially lethal sepsisWeiqiang Chen1,2*, Xiaoling Qiang1,2*, Yongjun Wang1*, Shu Zhu1,2*, Jianhua Li1, Ariella Babaev1, Huan Yang1,2, Jonathan Gong1,2, Lance Becker1,2, Ping Wang1,2, Kevin J. Tracey1,2, Haichao Wang1,2†

For the clinical management of sepsis, antibody-based strategies have only been attempted to antagonize pro-inflammatory cytokines but not yet been tried to target harmless proteins that may interact with these pathogenic mediators. Here, we report an antibody strategy to intervene in the harmful interaction between tetranectin (TN) and a late-acting sepsis mediator, high-mobility group box 1 (HMGB1), in preclinical settings. We found that TN could bind HMGB1 to reciprocally enhance their endocytosis, thereby inducing macrophage pyroptosis and con-sequent release of lactate dehydrogenase and apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain. The genetic depletion of TN expression or supplementation of exogenous TN protein at subphysiological doses distinctly affected the outcomes of potentially lethal sepsis, revealing a previously under-appreciated beneficial role of TN in sepsis. Furthermore, the administration of domain-specific polyclonal and monoclonal antibodies effectively inhibited TN/HMGB1 interaction and endocytosis and attenuated the sepsis- induced TN depletion and tissue injury, thereby rescuing animals from lethal sepsis. Our findings point to a possibility of developing antibody strategies to prevent harmful interactions between harmless proteins and pathogenic mediators of human diseases.

INTRODUCTIONSepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection that annually claims hundreds of thousands of victims in the United States alone (1, 2). Its complex pathogenesis is partly attributable to both dysregulated inflammatory responses and resultant immunosuppression (2, 3). The high-mobility group box-1 (HMGB1) protein is released by activated macrophages/monocytes and functions as a late mediator of lethal endotoxemia (4) and sepsis (5, 6). When initially secreted by innate immune cells in relatively low amounts, HMGB1 might still be proinflammatory during an early stage of sepsis (4). However, when it is passively re-leased by the liver (7) and other somatic cells in overwhelmingly higher quantities, HMGB1 could also induce immune tolerance (8, 9), macrophage pyroptosis (7, 10), and immunosuppression (11), thereby impairing the host’s ability to eradicate microbial infections (12, 13). It was previously unknown what other endogenous proteins could affect extracellular HMGB1 functions and could be pharmacologically modulated for treating inflammatory diseases.

In 1986, tetranectin (TN) was first characterized as an oligomeric plasminogen-binding protein (14) with an overall 76% amino acid sequence identity (87% similarity) between humans and rodents (15). It is expressed most abundantly in the lung (16, 17), and its blood concentrations in healthy humans range from moderate (~8 g/ml) in infants to high (10 to 12 g/ml) in adults (18). Structurally, TN has several distinct domains responsible for its extracellular secretion (residues 1 to 21, leader signal sequence), heparin binding (residues 22 to 37) (19), and oligomerization (residues 47 to 72, the -helical domain), as well as carbohydrate recognition (residues 73 to 202) of oligosaccharides in plasminogen (20, 21), apolipoprotein A1 (22),

hepatocyte growth factor, and tissue-type plasminogen activator (23). However, the specific roles of TN in physiology and pathology re-main poorly understood. Recent evidence revealed that enhanced expression or genetic depletion of TN caused abnormal osteogenesis (24), excessive curvature of the thoracic spine (25), deficient motor func-tion (such as limb rigidity) (26), or impaired wound healing (27, 28), implying the importance of maintaining physiological TN concen-trations in health.

Previously, it was unknown whether blood TN concentrations were altered during clinical and experimental sepsis and whether these could be pharmacologically modulated to fight against inflammatory diseases. In the present study, we sought to understand the role of TN in lethal sepsis by examining its dynamic changes in sepsis and possible interaction with HMGB1 and determine how alterations of TN concentrations (genetic depletion or pharmacological supplemen-tation) or activities (using domain-specific antibodies) affect the out-comes of lethal sepsis in preclinical settings.

RESULTSBlood TN was depleted in septic patientsTo search for endogenous proteins modulating HMGB1 functions, we characterized the dynamic changes of serum HMGB1 and other proteins in a group of septic patients admitted to the Northwell Health System. In a septic patient with elevated serum HMGB1 (Fig. 1A, S), the concentration of a 20-kDa protein (denoted as “P20”) was much lower than that of a normal healthy participant (Fig. 1A, N). This protein was identified as human TN by in-gel trypsin digestion and mass spectrometry analysis (Fig. 1A). To further verify its identity, we immunoblotted serum samples from two normal healthy controls (Fig. 1B, N) and two septic patients (“S”) who either survived (“L”) or died (“D”) of sepsis with a TN-specific rabbit monoclonal antibody (mAb) (table S1). As expected, this mAb specifically recognized a 20-kDa band in the serum of healthy humans (Fig. 1B) and animals (fig. S1A) but not in the serum or lungs of TN-deficient mice (fig. S1A).

1The Feinstein Institutes for Medical Research, Northwell Health, 350 Community Drive, Manhasset, NY 11030, USA. 2Donald and Barbara Zucker School of Medicine at Hofstra/Northwell, 500 Hofstra Blvd., Hempstead, NY 11549, USA.*These authors contributed equally to this work†Corresponding author. Email: [email protected]

Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

by guest on April 24, 2020

http://stm.sciencem

ag.org/D

ownloaded from

http://stm.sciencemag.org/



2 of 14

Moreover, immunoblotting assays confirmed a marked reduction of serum TN concentration in a sepsis survivor (“L”, Fig. 1B) and an almost complete TN depletion in a patient who died of septic shock within 24 hours of the initial diagnosis and blood sampling (“D”, Fig. 1B). Statistical analysis of a larger cohort of age-matched healthy controls and critically ill patients revealed a 62 to 67% reduction of plas-ma TN concentrations in patients with sepsis or septic shock (Fig. 1C),

confirming a marked loss of plasma TN in sepsis. These observed differences be-tween normal controls and septic patients were not likely skewed by occasionally imbalanced gender ratios (for example, 11:6 versus 6:11; table S2), because there was no significant difference in plasma TN concentrations between male and fe-male healthy controls (P = 0.67; Fig. 1D).

Genetic depletion of TN rendered mice more susceptible to lethal sepsisTo assess the role of TN in sepsis, we first determined how genetic TN depletion affects the sepsis-induced tissue injury and lethality in age-matched animals. The genotypes of wild-type (WT) and TN knockout (KO) mice were confirmed by immunoblotting (fig. S1A) and genotyp-ing (fig. S1B) analyses of serum, lung, and tail samples. TN KO mice exhibited a significantly higher mortality rate than that of age-matched WT littermate con-trols (P = 0.035; Fig. 2A), which was associated with an increased systemic release of lactate dehydrogenase (LDH), as well as liver alanine aminotransferase (ALT) and aspartate aminotransferase (AST; Fig. 2B). Histological analysis showed more severe inflammation and injury, as manifested by the increase in alveolar sep-tal wall thickening, leukocyte infiltration, and alveolar congestion in the lungs of TN KO mice (Fig. 2B). Correspondingly, RNA sequencing (RNA-seq) analysis re-vealed markedly increased gene expres-sion of several proinflammatory mediators (for example, Il1b, Il6, Lif, and Cox2) in the lungs of TN KO mice (Fig. 2C), in-dicating possible anti-inflammatory prop-erties of lung TN in sepsis.

Supplementation of exogenous TN conferred dose-dependent protection against lethal endotoxemia and sepsisIn healthy animals, TN was most abun-dantly expressed in the lung and also de-tected in the circulation (fig. S2, A and B). Assuming a 25-g mouse with an average blood volume of 1.5 ml and a mean cir-

culating TN concentration of 10.0 g/ml (fig. S2B), the physiological blood TN concentration is estimated to be around 0.6 mg/kg body weight. After experimental endotoxemia and sepsis (Fig. 3A), circu-lating TN concentrations were decreased in a time-dependent fashion, with a >70% reduction at 24 hours after the onset of these diseases—a time point when some endotoxemic or septic animals started to suc-cumb to death. Furthermore, the parallel reduction of TN content

B

72 --43 -

34 -

25 -

15 -

10 -

--

-

N SL DH1 H2

kDa

0

20

40

60

80

100

120

TN (A

U)

N SL DH1 H2

- TN

N SL DH1 H2

72 --43 -

34 -

25 -

15 -

10 -

--

-

kDa

SDS-PAGE Western blot

Tryp

sin

dig

estio

n

Mal

di-T

OF

mas

s sp

ectro

met

ry

Peptide (mass)

LDTLAQEVALLK (1313.7)

- P20 -

P20:

Hum

an te

trane

ctin

( TN

)

72 --43 -

34 -

25 -

15 -

10 -

--

-

N SAkDa

DIQR

1.5 IQR

Median

Mean

TN (µ

g/m

l)

Normal healthy

P = 0.67

02468

101214

C

TN (µ

g/m

l)

14−30

(15)

31−55

(17)

56−90

(12)

31−55

(14)

56−90

(17)

56−90

(14)

Normal healthy Sepsis Septicshock

P = 3.56E-16

P = 2.98E-14 P = 1.61E-13

02468

101214

Age

(n)

Sex

(n)

M

(24)

F

(20)

Fig. 1. Identification of TN as a serum protein depleted in septic patients. (A) Mass spectrometry analysis of a 20-kDa (P20) protein, which was abundant in a normal healthy participant (N) but depleted in a septic patient who died (S) of sepsis within 72 hours of the initial diagnosis and blood sampling. (B) SDS-PAGE and Western blotting analysis of serum TN in normal healthy controls (N) and septic patients who either survived (denoted as “L”) or died (“D”) of septic shock within 24 hours of the initial diagnosis and blood sampling. Bar graph indicates the relative TN concentrations in arbitrary units (AU) in the serum samples. (C) Box plot representation of plasma TN concentrations in normal healthy controls and patients with sepsis or septic shock. Data represent mean [interquartile range (IQR), 25 to 75%] value of plasma TN concentrations. One-way ANOVA was used to compare the means between different groups, and P values are indicated. (D) Box plot representation of plasma TN concentrations in 24 male and 20 female healthy controls. The nonparametric Kruskal-Wallis ANOVA test was used to calculate the P value.


http://stm.sciencem

ag.org/D

ownloaded from




3 of 14

in the serum (fig. S3A) and lung tissue (fig. S3B) of endotoxemic ani-mals supports the lung as a possible source of circulating TN (17).

We then determined how purified human or murine TN proteins expressed in human embryonic kidney (HEK) 293 kidney cells or Escherichia coli (fig. S4) affect the outcomes of lethal endotoxemia and sepsis. Recombinant murine TN migrated on SDS–polyacrylamide gel electrophoresis (SDS-PAGE) gel as a 17- and 24-kDa band in the absence of a reducing agent [dithiothreitol (DTT); fig. S4B] but mi-grated as 24-kDa band in the presence of DTT (fig. S4B), suggesting a possible variation of the redox status of different cysteines of TN

protein (fig. S4A). The supplementation of endotoxemic mice with recombinant human TN (2.0 mg/kg) partially restored blood TN concentrations at 2 hours after lipopolysaccharide (LPS), which were then gradually diminished by 24 hours (Fig. 3B). Moreover, the in-traperitoneal administration of either eukaryote-derived human TN (Fig. 3C) or prokaryote-derived murine TN (fig. S5) conferred a re-producible and dose-dependent protection against lethal endotoxemia (Fig. 3C, left) and sepsis (Fig. 3C, right, and fig. S5A), supporting a beneficial role of TN in lethal systemic inflammation. Correspondingly, supplementation of exogenous TN led to a marked attenuation of

ConditionIl6LifOsmAdmZfp36Nr4a1FosIghaIl1bKcne4Egr1Cox2Gm1138Fgf23Igkv5-43Igkv8-27Sectm1bRgs16Fbxw10PtprqXlr3b Cd5lIghg2cAlox15 Rgs1Egr2Tnfrsf9Aldh1a3Ccl22Oasl1

KO WTC

B

0

50

100

150

ALT

(U/li

ter)

0

100

200

300

400

500

AST

(U/li

ter)

0

400

800

1200

1600

2000

LDH

(U/li

ter)

WT

−CLP

−CLP

+CLP

+CLP

KO

*

*

*

*

*

#

#

#

Lung

sco

re

WT

−CLP

−CLP

+CLP

+CLP

KO

0.0

0.2

0.4

0.6

0.8

1.0

* *#

0 2 130

20406080

100

Surv

ival

(%)

Time (d post CLP)1 3 14

KO

WT

*P = 0.035

A

~ ~~ ~

~ ~

n = 21 (9 F + 12 M) KO WT

−CLP

+CLP

100 µm

Fig. 2. Genetic depletion of TN rendered animals more susceptible to lethal sepsis. (A) Age-matched wild-type (WT) C57BL/6J or TN KO mice were subjected to lethal sepsis, and animal survival was monitored for 2 weeks. n = 21 animals (9 females and 12 males) per group. (B) In parallel experiments, blood and lung tissue were harvested at 24 hours after CLP and assayed for tissue injury by measuring blood concentrations of tissue enzymes or lung histology. n = 6 to 12 animals per group. *P < 0.05 versus sham control (−CLP); #P < 0.05 versus WT CLP group (+CLP). (C) TN KO exacerbated sepsis-induced gene expression of proinflammatory cytokines in the lung. A biclustering heat map was used to visualize the expression profile of the top 30 differentially expressed genes sorted by their adjusted P value and log2 fold of changes. Each row represents a gene, and each column represents one sample from each animal.


http://stm.sciencem

ag.org/D

ownloaded from




4 of 14

sepsis-induced injury in the lung (Fig. 3D) and liver (Fig. 3E), further confirming a protective role of TN in lethal sepsis. Because there was only 79% (159 of 202) amino acid sequence identity [and 87% (174

of 202) similarity] between human and mouse, a lower dose of murine TN was needed to confer a reproducible protec-tion against lethal sepsis (fig. S5A). Al-though supplementation of septic mice with subphysiological doses of murine TN (0.1 mg/kg) conferred reproducible protection (n = 10, N = 2; fig. S5A), ad-ministration of murine TN at supraphys-iological doses (1.0 mg/kg) did not show any protective effect in sepsis (fig. S5B), suggesting a possibility that TN may exert divergent effects at different concentra-tions in sepsis.

Divergent effects of TN domain-specific polyclonal antibodies and mAbs on lethal sepsisTo further evaluate the role of TN in sep-sis, we generated polyclonal antibodies (pAbs) against murine TN in rabbits and examined their effects on septic lethality. The total immunoglobulin Gs (IgGs) pu-rified from two rabbits (pAb2 and pAb3) reproducibly increased animal survival rates in a murine model of lethal sepsis (Fig. 4A), even when the first dose was given at 22 hours after cecal ligation and puncture (CLP). To characterize these pAbs, we screened a library of peptides spanning the entire sequence of human TN (Fig. 4B, left) to determine the epitope profile of these protective pAbs (Fig. 4B, right) and found that both protective pAb2 and pAb3 recognized a specific pep-tide, P5 (Fig. 4B), which forms stable - helical epitopes either alone in syn-thetic peptides or being carried by TN proteins (Fig. 4C).

We thus immunized Balb/C mice with human TN antigen and generated a panel of hybridoma clones producing mAbs against P5 (four clones) and P2 (three clones) peptides (fig. S6A). Immuno-blotting analysis of human and murine serum samples confirmed the specificity of these P2- and P5-specific mAbs (fig. S6B). To further define the epitope sequences of the P5-reactive mAbs, we immunoblotted these mAbs with 10 smaller peptides (P5-1 to P5-10, fig. S7, A and B) and found that three of the four P5-binding mAbs reacted with P5-5 peptide (NDALYEYLRQ, fig. S7, A and B). This P5-5 epitope sequence shares 60 to 70% identity (but still 100% similarity) between humans and rodents

(fig. S8A), as the variant residues (E versus D, F versus Y, H versus Q, and A versus L) still exhibit similar biochemical properties. This NDALYEYLRQ epitope sequence is 100% identical between TN proteins

C

2

4

6

8

10

−1 0 1 2 3 4 5 13 14

Time (days post-LPS)

# o

f su

rviv

ors

0

90%*

50%40% Saline

TN (1.0)

TN (2.0)

(+2 hours)

−1 0 1 2 3 4 5 13 140

4

8

12

16

20

Time (days post-CLP)

# o

f su

rviv

ors

TN (2.0)

Saline

60%*

25%

(+2 hours)

B

E

AS

T (U

/lit

er)

0

100

200

300

400

*#

–TN

−CLP

+CLP

+CLP

+TN

0

40

80

120

160

ALT

(U/l

ite

r)

*#

–TN

−CLP

+CLP

+CLP

+TN

0.4

0.6

0.8

1.0

Lu

ng

sco

re

–TN

−CLP

+CLP

+CLP

+TN

*#

D –CLP +CLP +CLP + TN

Time (hours post-LPS)

Ser

um

TN

(%

)

0

20

40

60

80

100

120

0 12 24 36 48

A

0 2 6 12 24 48

0

40

80

120

Time (hours post-CLP)

20

60

100

0

20

40

60

80

100

120

Ser

um

TN

(%

)

2−−

22−LPS

TN

(hours)

(mg/kg)−

24

2

24

#

** *

* * ***

* *~ ~

~ ~~ ~

~ ~

~ ~~ ~

~ ~

100 µm

Fig. 3. Supplementation of exogenous TN conferred protection against lethal endotoxemia and sepsis. (A) Time- dependent reduction of circulating TN concentrations in murine models of lethal endotoxemia (LPS) and sepsis (CLP). Balb/C mice were subjected to lethal endotoxemia (LPS, i.p., 7.5 mg/kg) or CLP sepsis, and blood samples were harvested at various time points after LPS or CLP to measure TN concentrations by Western blotting analysis. n = 3 animals per group. *P < 0.05 versus “time 0.” (B) Balb/C mice were given LPS (i.p., 7.5 mg/kg) with or without human TN (i.p., 2.0 mg/kg), and animals were euthanized 2 or 24 hours later to harvest blood to measure serum TN concentrations. *P < 0.05 versus time 0 (“−LPS-TN”); #P < 0.05 versus “+LPS-TN” at the same time point, n = 4 animals per group. (C) Recombinant human TN was given at indicated doses (1.0 or 2.0 mg/kg, i.p.) at 2 hours after the onset of lethal endotoxemia or sepsis. Animal survival was monitored for 2 weeks to ensure long-lasting protection. *P < 0.05 versus saline control group. n = 10 animals per group for the LPS model; n = 20 animals per group for the CLP model. (D) Recombinant murine TN (0.1 mg/kg) was given at 2 and 24 hours after CLP, and animals were euthanized at 28 hours after CLP to harvest lung tissue for histological analysis. *P < 0.05 versus “−CLP” group; #P < 0.05 versus +CLP group. n = 5 to 6 animals per group. (E) At 28 hours after CLP, animals were euthanized to harvest blood to measure serum concentrations of ALT and AST. *P < 0.05 versus sham control (−CLP); #P < 0.05 versus saline group (+CLP). n = 5 to 9 animals per group.


http://stm.sciencem

ag.org/D

ownloaded from




5 of 14

in humans and many other mammalian species, including baboon, bear, bovine, buffalo, camel, cattle, cougar, elephant, goat, gorilla, hedgehog, horse, lemur, monkey, pig, rabbit, rhinoceros, seal, sheep, and tiger (fig. S8, A and B), suggesting that these P5-5–reacting mAbs could recognize TN protein in a wide spectrum of mammalian species.

We then explored the therapeutic potential of these mAbs by ad-ministering them to septic animals in a delayed fashion—starting at 24 hours after CLP. Administration of a P2-specific mAb (mAb9) re-

producibly worsened the outcome of lethal sepsis (Fig. 4D, bottom), confirming a beneficial role of TN in lethal inflammatory diseases. In a sharp contrast, delayed administration of three P5-reacting mAbs that could recognize both human and murine TN [mAb2 (IgG2a), mAb6 (IgG1), and mAb8 (IgG2b); figs. S6B and S9] similarly and partially rescued mice from lethal sepsis (Fig. 4D). As expected, a P5- reactive mAb5 (IgG1) incapable of binding murine TN (fig. S6B), along with several irrelevant IgG2a or IgG2b isotype controls, uniformly

A Rabbit pAbs

B

pAb3 (50%)

pAb4 (20%)

<

<

pAb1 (70%)

pAb2 (90%)

Peptide sequence Dot blots

D Murine mAbs

Conformation

54 (P2: coiled)

70

86 (P5: -helix)109

TN protein

Peptides

C

P2-r

eact

ive

02468

1012

0 2 4 6 14

SalinepAb 4

pAb 3

# of

sur

vivo

rs


~ ~~ ~

~ ~~ ~

(+22, +46 hours)

P5-r

eact

ive

Surv

ival

(%)

90%

60% Control

mAb6 (2.0)IgG1

n = 20 (M)P = 0.031

0 20 40 60 80 1000

20

40

60

80

100

1st 2nd

336

~ ~~ ~

~ ~

# of

sur

vivo

rs

02468

10

0 2 4 6 14

SalinepAb1pAb2


~ ~~ ~

~ ~

0 20 40 60 80 3360

20

40

60

80

100

Surv

ival

(%) 77%

22% Control

mAb8 (2.0)IgG2b

n = 9 (M)P = 0.022

1st 2nd

~ ~~ ~

~ ~

Surv

ival

(%)

0 20 40 60 80 1000

20

40

60

80

100

1st 2nd

50% Control

n = 10 (F)

mAb2 (0.5)IgG2a

80%

336

~ ~~ ~

~ ~

0 20 40 60 80 3360

20

40

60

80

100

Surv

ival

(%)

50%

80% Control

mAb9 (2.0 )IgG1

n = 30 (20 F + 10 M)P = 0.039

1st 2nd

Time (h post CLP)

~ ~~ ~

~ ~

(+22, +46 hours)

~ ~

n = 10 (M) n = 10 (M)

Fig. 4. Divergent effects of TN domain-specific polyclonal and monoclonal antibodies on septic lethality. (A) Male (“M”) Balb/C mice (7 to 10 weeks, 20 to 25 g) were subjected to CLP-induced sepsis and intraperitoneally administered total IgGs (40 mg/kg) from each TN-immunized rabbit (#1 to #4) at 22 and 46 hours after CLP (arrows). Animal survival was monitored for 2 weeks. n = 10 animals per group. (B) Sequences of 10 peptides spanning different regions of human TN for antibody epitope mapping of four different rabbit pAbs. The text underlined in red denotes the defined epitope sequence for several P5-reactive protective mAbs. Note that the two protective rabbit antibodies (pAb2 and pAb3) recognized a distinct peptide, P5. (C) Tertiary structure of human TN protein (top) and its two peptide domains: P2 and P5 (bottom). (D) Divergent effects of P5- and P2-reactive mAbs on lethal sepsis. Male (M) or female (“F”) Balb/C mice were subjected to lethal sepsis and intraperitoneally administered different mAbs at indicated doses (0.5 or 2.0 mg/kg) and time points (24 and 48 hours after CLP). Animals were monitored for 2 weeks to ensure long-lasting effects.


http://stm.sciencem

ag.org/D

ownloaded from




6 of 14

failed to confer any protection (fig. S10), confirming that the pro-tective effects of these P5-reactive mAbs were entirely dependent on their murine TN-binding capacities.

Protective mAbs attenuated the sepsis-induced TN depletion, bacterial infection, and tissue injuryTo understand how P5-reacting mAbs confer protection against le-thal sepsis, we administered TN P5-specific mAb8 and P2-specific mAb9 at 2 and 24 hours after CLP and then determined serum con-centrations of TN, as well as 62 other cytokines/chemokines at 28 hours after CLP. Unexpectedly, repeated administration of mAb8, but not mAb9, significantly attenuated the sepsis-induced TN de-pletion in both male (P = 0.0000287; Fig. 5A, left, and fig. S11) and female animals (P = 0.0000127; Fig. 5A, right). Similarly, the systemic accumulation of interleukin-6 (IL-6) and keratinocyte chemoattractant (KC), two surrogate markers of experimental sepsis (29, 30), was also markedly inhibited by mAb8, but not mAb9, in septic animals (fig. S12). Moreover, repetitive administration of mAb8 significantly attenuated the sepsis-induced lung injury (P = 0.0000459; Fig. 5B), as well as systemic release of liver ALT and AST enzymes (P = 0.000255 and 0.000167, respectively; Fig. 5B), suggesting that TN-specific mAb8 conferred protection partly by attenuating sepsis-induced tissue injury. mAb8 also markedly reduced blood bacterial count [colony- forming units(CFU)] at 28 hours after CLP (Fig. 5B), indicating that TN-specific protective mAb8 is capable of facilitating pathogen elimination in experimental sepsis.

TN selectively inhibited the LPS- and serum amyloid A (SAA)–induced HMGB1 release by capturing and facilitating its endocytosisTo understand the mechanisms underlying the dose-dependent divergent effects of TN in sepsis, we evaluated the possible anti- and proinflammatory properties of the recombinant TN proteins in vitro. Highly purified TN protein expressed in either eukaryotes (HEK293 cells) or prokaryotes (E. coli) dose-dependently inhibited the LPS- and SAA-induced HMGB1 release in both murine macro-phages (Fig. 6A) and human monocytes (Fig. 6A). This inhibi-tion was specific because TN did not inhibit the parallel release of other cytokines [including granulocyte colony-stimulating factor (G-CSF), IL-6, and IL-12; Fig. 6B] and chemokines [including KC, lipopolysaccharide-induced CXC chemokine (LIX), macrophage inflammatory protein– 1 (MIP-1), MIP-2, and regulated upon activation normal T cell expressed and secreted (RANTES); Fig. 6B], even when given at supraphysiological concentrations (20 g/ml). In primary human monocytes, TN reproducibly and specifically induced the release of growth-regulated oncogene (GRO) (CXCL1 or KC; Fig. 6C)—a surrogate marker of experimental sepsis (29, 30), as well as a potentially beneficial neutrophilic chemokine, epithelial cell-derived neutrophil-activating protein-78 (ENA-78), CXCL5, LIX; Fig. 6C (31).

To elucidate the mechanism by which TN selectively inhibited HMGB1 release, we first examined the possible TN/HMGB1 inter-action using the Nicoya Lifesciences Open surface plasmon resonance (OpenSPR) technology (Fig. 7A). Regardless of whether TN or HMGB1 was conjugated to the Sensor Chip via His-tag or carboxyl groups, there was a dose-dependent SPR response between TN and HMGB1, with an estimated equilibrium dissociation constant (Kd) in the range of 1.21 to 2.88 nM (Fig. 7A), indicating a strong interaction between these two proteins.

To understand how P5-reacting mAbs confer protection against lethal sepsis, we tested whether they interfere with TN/HMGB1 in-teraction in vitro. When TN was conjugated to the sensor chip, mAb8 exhibited a dose-dependent binding to TN with an estimated Kd of ~2.02 nM (Fig. 7B, top). However, when the TN-conjugated sensor chip was pretreated with mAb8 (29.6 nM), the SPR response signal for subsequent HMGB1 (200 nM) application was reduced by >85% from ~150 arbitrary units (AU) (Fig. 7A, bottom) to ~35 AU (Fig. 7B, bottom), which was paralleled by an almost sixfold increase of Kd

A

−CLP CLP+

Veh

CLP+

mAb8

CLP+

mAb9

020406080

100

Blo

od T

N (%

) 120

Male

−CLP +CLP +CLP + mAb8B

020406080

100120

ALT

(U/li

ter)

*#

−CLP CLP+

Veh

CLP+

mAb8

020406080

100120

Blo

od T

N (%

)

−CLP CLP+

Veh

CLP+

mAb8

CLP+

mAb9

Female

0

200

400

600

800

1000

AST

(U/L

)−CLP CLP

+Veh

CLP+

mAb8

* #

MedianMean

0.0

0.2

0.4

0.6

0.8

1.0

Lung

sco

re *#

−CLP CLP+

Veh

CLP+

mAb8

CLP+

Veh

CFU

(ml−1

)

−CLP CLP+

mAb8

0

5000

10,000

15,000

20,000

25,000 *

#

MedianMean

100 µm

Fig. 5. TN-specific mAb8 attenuated sepsis-induced TN depletion, bacterial infection, and tissue injury. (A) Male or female Balb/C mice were subjected to lethal sepsis and intraperitoneally administered a P5-reacting mAb8 (2.0 mg/kg) or a P2-reacting mAb9 (2.0 mg/kg) at 2 and 24 hours after CLP. At 28 hours after CLP, animals were euthanized to harvest blood, and serum TN concentrations were determined by Western blotting analysis. *P < 0.05 versus sham control (−CLP); #P < 0.05 versus vehicle control (“CLP + Veh”) group; &P < 0.05 versus “+CLP + mAb9” group. n = 2 to 4 animals per group. (B) TN-specific mAb8 (2.0 mg/kg) was given at 2 and 24 hours after CLP, and animals were euthanized at 28 hours after CLP to harvest blood and lung tissue for histological analysis, bacterial count, and liver enzyme assays. *P < 0.05 versus sham −CLP group; #P < 0.05 versus +CLP group. n = 6 to 10 animals per group.


http://stm.sciencem

ag.org/D

ownloaded from




7 of 14

(µg/ml)

(µg/ml)

A

0

20

40

60

80

100

SAA

TN

- HMGB1

HM

GB

1 (%

) **

* *

− 2.0 2.0 2.0 2.0 2.0

− − 5.0 10 20 30

Murine macrophage

(µg/ml)

(µg/ml)

0

20

40

60

80

100

HM

GB

1 (%

)

LPS

TN

- HMGB1

*

*

− 0.2 0.2 0.2 −

− − 20 30 30

Murine macrophage

SAA

TN

- HMGB1

HM

GB

1 (%

)

0

20

40

60

80

100

*

(µg/ml)

(µg/ml)− 2.0 2.0

− − 20

Human monocyte

−Control +LPS +LPS + TN (20)

+SAA+HMGB1 + TN (20) +SAA + TN (20)+HMGB1

B

+TN (E. coli)+TN (HEK293)−TNC

Murine macrophage

Human monocyte

Fig. 6. TN specifically inhibited the LPS- or SAA-induced HMGB1 release. (A) Murine peritoneal macrophages or human blood monocytes were stimulated for 16 hours with LPS or SAA in the absence or presence of TN at the indicated concentrations. The extracellular HMGB1 concentrations were determined by Western blotting and expressed as percentage of maximal stimulation in the presence of LPS or SAA alone. n = 3 per group. *P < 0.05 versus “+SAA alone” or “+LPS alone”. (B) Thioglycolate-elicited peritoneal macrophages were stimulated with LPS (0.2 g/ml), HMGB1 (1.0 g/ml), or SAA (2.0 g/ml) in the absence or presence of murine TN (20 g/ml) for 16 hours, and extracellular concentrations of 62 cytokines and chemokines were measured by Cytokine Antibody Arrays. (C) Human PBMCs were stimulated with recombinant human TN or murine TN (10 g/ml) for 16 hours, and extracellular concentrations of cytokines and chemokines were determined by Cytokine Antibody Arrays. TNF, tumor necrosis factor; VCAM-1, vascular cell adhesion molecule–1; VEGF-1, vascular endothelial growth factor–1; EGF, epidermal growth factor; MDC, macrophage-derived chemokine; TGF-1, transforming growth factor–1; IFN-, interferon-; SDF-1, stromal cell-derived factor 1; TARC, thymus and activation-related chemokine; TCA-3, T cell activation gene 3; TECK, thymus-expressed chemokine; TIMP-1, tissue inhibitors of metalloproteinase-1; sTNF RI, soluble TNF receptor-1; TPO, thrombopoietin; MCP, monocyte chemoattractant protein; LPT, lymphotactin; MIG, monokine induced by gamma interferon; PF-4, platelet factor 4; SCF, stem cell factor; IGFBP-3, insulin-like growth factor binding protein 3; BLC, B lymphocyte chemoattractant; Ang, angiotensin; OSM, oncostatin M; THPO, thrombopoietin; PGDF BB, platelet-derived growth factor containing two B subunits.


http://stm.sciencem

ag.org/D

ownloaded from




8 of 14

−HMGB1 +HMGB1 +HMGB1 + TN +HMGB1 + TN + DYN

10 µm

−100−100 0 100 200 300 400 500 600

0

100

200

300

400

Res

pons

e un

its

Time (s)

TN HMGB1400

20010025

Kd = 2.88 nM

−100

0

200

400

600

800

−100 0 100 200 300 400 500 600

Res

pons

e un

its

HMGB1 TN280

933110

Kd = 1.21 nM

−Control +Alexa 555–HMGB1 +Alexa 488–TN +Alexa 555–HMGB1 +Alexa 488–TN

10 µm

−100

0

10

20

30

40

−100 0 100 200 300 400 500 600

Res

pons

e un

its

Time (s)

TN + mAb8 HMGB1

20050.025.012.5

Kd = 18.5 nM

−250−100 0 100 200 300 400 500 600

0

150

300

450

600

Res

pons

e un

its

TN mAb829.6

11.8

5.92.90.9

Kd = 2.02 nM

0

20

40

60

80

100

120

TN(A

U)

*

# #

HMGB1TN

mAb8DYN

−−−

+−−

−+−

−+−

+++

+++

−−− +−−

HM

GB

1 (A

U)

*#

0

20

40

60

80

#

A TN/HMGB1 SPR TN/mAb8/HMGB1 SPRB C Cellular TN/HMGB1

D Effect of TN and dynasore on cellular uptake of Alexa 555-HMGB1

E Colocalization of Alexa 488–TN and Alexa 555–HMGB1

DAPI Alexa 555

Fluo

resc

ence

Ove

rlay

of fl

uore

scen

ce&

phas

e co

ntra

st

Phase contrast

DAPI A 555

Fluo

resc

ence

Ove

rlay

A 488

Phase contrast

A 555 A 488

Alexa 555

Overlay

Fig. 7. TN interacted with HMGB1 and reciprocally enhanced the uptake of TN/HMGB1 complexes. (A) SPR Assay was used to assess TN/HMGB1 interaction by immobilizing highly purified HMGB1 (top) or TN (bottom) protein on the sensor chip and then applying TN or HMGB1 at different concentrations. The response units were recorded over time to estimate the equilibrium dissociation constant (Kd). (B) Highly purified recombinant TN was immobilized on the sensor chip, and mAb8 was applied at indicated concentrations to assess the Kd for TN-mAb8 interaction (top). Alternatively, TN-conjugated sensor chip was first pretreated with mAb8 at 29.6 nM, and then, HMGB1 was applied at various concentrations (bottom). (C) Murine macrophage-like RAW264.7 cells were incubated with HMGB1 (0.5 g/ml) in the absence or presence of TN (10.0 g/ml), mAb8 (65.0 g/ml), or dynasore (DYN, 8.0 M) for 2 hours. Cellular content of HMGB1 or TN was determined by Western blotting analysis and expressed as a ratio to -actin. Bar graph represented average of three samples (n = 3) from two independent experiments (N = 2). *P < 0.05 versus positive control (“+HMGB1” or “+TN” alone); #P < 0.05 versus “+HMGB1 + TN” group. (D) Murine macrophage-like RAW264.7 cells were incubated with Alexa 555–labeled HMGB1 (100 ng/ml) in the absence or presence of recombinant TN (10.0 g/ml) for 2 hours. After extensive washing and fixation, images were acquired. Scale bar, 10 m. Arrows point to Alexa 555 HMGB1–containing cytoplasmic vesicles. (E) Fluorescent Alexa 555–labeled HMGB1 (100 ng/ml) and Alexa 488–labeled TN (500 ng/ml) were added to RAW264.7 cell cultures separately or together and incubated at 37° for 2 hours. After extensive washing and fixation, images were acquired. Scale bar, 10 m.


http://stm.sciencem

ag.org/D

ownloaded from




9 of 14

(from 2.88 to 18.5 nM), indicating that mAb8 effectively interrupted TN/HMGB1 interaction. Furthermore, mAb8 markedly prevented the reciprocal enhancement of cellular uptake of HMGB1 (Fig. 7C, top) and TN (Fig. 7C, bottom), indicating that the protective mAbs confer protection possibly through inhibiting TN/HMGB1 interac-tion and endocytosis.

Consistent with previous reports (7, 10), we observed a basal amount of HMGB1 endocytosis in murine macrophage cultures (Fig. 7, C to E). However, at physiological concentrations, TN markedly enhanced HMGB1 cellular uptake (Fig. 7, C and D), which was prevented by an endocytosis inhibitor, dynasore (Fig. 7, C and D), implying that TN may have enhanced HMGB1 uptake via endocytosis. Conversely, HMGB1 also enhanced the cellular uptake of TN [Fig. 7C (bottom) and E], which was similarly attenuated by dynasore (Fig. 7C), indi-cating that TN and HMGB1 might be endocytosed simultaneously as a protein complex.

To test this possibility, we labeled HMGB1 and TN with two dif-ferent fluorescent dyes and added them simultaneously to macrophage cultures. When they were coadded to macrophage cultures, HMGB1- positive cytoplasmic (red) vesicles almost completely colocalized with TN-positive (green) particles (Fig. 7E), confirming that TN and HMGB1 were likely endocytosed by macrophages as protein complexes. Immunoblotting of cellular proteins with HMGB1- or TN-specific anti-bodies confirmed the TN-mediated enhancement of HMGB1 cellular uptake, as well as the appearance of additional lower molecular weight bands (marked by empty arrowheads in fig. S13, A and B) that might be indicative of possible degradation of endocytosed HMGB1 and TN. The high molecular bands (marked by solid arrowheads, fig. S13B) may indicate possible oligomerization of endocytosed TN protein.

TN-specific protective mAb8 inhibited TN/HMGB1-induced macrophage pyroptosisAs a proinflammatory form of programmed necrosis, pyroptosis is morphologically characterized by the oligomerization of the apoptosis- associated speck-like protein containing a C-terminal caspase recruit-ment domain (ASC) and the resultant integration of a large inflammasome complex (pyroptosome) that eventually triggers the disruption of cy-toplasmic membranes (32). Because HMGB1 endocytosis could trigger macrophage pyroptosis (7, 10), we examined whether TN and HMGB1 increased the uptake of trypan blue dye and release of LDH or ASC, a marker for macrophage pyroptosis (33). Consistent with previous findings (7, 10), HMGB1 itself did not significantly increase cell death when it was given at a relatively low concentration (0.5 g/ml, P = 1.0 and 0.08, respectively; Fig. 8A and fig. S14). However, in the presence of TN, HMGB1 induced a significant increase of trypan blue dye uptake (P = 0.00175; Fig. 8A and fig. S14) and LDH release (P = 0.000066; Fig. 8A), which were significantly inhibited by dynasore and mAb8 (P = 0.0027 and 0.00175 and P = 0.00002 and 0.0045, respectively; Fig. 8A and fig. S14). Similarly, the coaddition of both proteins trig-gered an additive enhancement of ASC release (Fig. 8B), suggesting that TN/HMGB1 interacts to facilitate their endocytosis to trigger macrophage pyroptosis. When TN and HMGB1 were coadded to human macrophage cultures, they induced translocation of nuclear ASC to cytoplasmic regions, where ASC either aggregated into minute puncta that appeared to be secreted through microvesicle shedding (Fig. 8C, narrow arrows) or aggregated into a larger focus or speck (pyroptosome) that would trigger pyroptosis (Fig. 8C, wide arrow). Pretreatment with dynasore (20 M) or mAb8 (40 g/ml) prevented the TN/HMGB1- induced cytoplasmic ASC translocation

or aggregation into large ASC specks in 10 randomly selected micro-scopic fields of three separate samples (Fig. 8C).

DISCUSSIONThroughout evolution, mammals have developed multiple mecha-nisms to regulate innate immune functions. In the present study, we report a role for TN in capturing HMGB1 and facilitating its cellu-lar uptake via possible endocytosis of TN/HMGB1 complexes. The reciprocal enhancement of HMGB1/TN endocytosis may promote macrophage pyroptosis and possible immunosuppression that may compromise the host’s ability to eradicate microbial infections (12, 13). Moreover, we have found a panel of TN domain-specific monoclonal antibodies that effectively prevented TN/HMGB1 interaction and their cellular uptake, thereby attenuating the sepsis-induced TN de-pletion and animal lethality in preclinical settings. Because these protective mAbs recognize a distinct amino acid sequence with 100% identity between humans and many other mammalian species, they hold promising potential for the clinical management of inflammatory diseases. Moreover, our current findings revealed an antibody strategy to preserve a beneficial protein by preventing its harmful interaction with HMGB1, a pathogenic mediator of lethal sepsis.

Upon active secretion by innate immune cells or passive release by somatic cells such as hepatocytes, extracellular HMGB1 binds a family of cell surface receptors including the Toll-like receptor 4 (TLR4) (34) and the receptor for advanced glycation end products (RAGE) (35) to induce the expression and production of various cytokines and chemokines or to trigger macrophage pyroptosis if HMGB1 is internalized via RAGE receptor–mediated endocytosis (7, 10). As a highly charged protein, HMGB1 could bind to many negatively charged pathogen-associated molecular patterns (PAMPs; such as CpG-DNA or LPS) to facilitate their cellular uptake via RAGE receptor–mediated endocytosis. Upon reaching acidic endosomal and lysosomal com-partments (pH 5.4 to 6.2) near HMGB1’s isoelectric pH (pI = pH 5.6), HMGB1 becomes neutrally charged and, thus, sets free its cargos (7) to their cytoplasmic TLR9 (35) and Caspase-11 receptors (7). Conse-quently, HMGB1 not only augments the PAMP-induced inflammation (35) but also promotes the PAMP-induced pyroptosis (7), resulting in a dysregulated inflammatory response, as well as macrophage de-pletion and possible immunosuppression during sepsis (fig. S15).

In contrast to exogenous PAMPs, HMGB1 also binds endogenous proteins such as haptoglobin and C1q but triggers anti-inflammatory responses via distinct signaling pathways (36, 37). Here, we have un-covered an important role for another endogenous protein, TN, in capturing HMGB1 to enhance the endocytosis of TN/HMGB1 com-plexes without impairing HMGB1’s cytokine/chemokine-inducing capacities. The reciprocal enhancement of TN/HMGB1 endocytosis was associated with an increase of macrophage cell death and release of ASC, a marker of macrophage pyroptosis (33). TN was capable of stimulating human monocytes to release: (i) GRO/CXCL1/KC, a surrogate marker of experimental sepsis (29, 30) associated with in-flammasome activation and pyroptosis (38); and (ii) ENA-78/CXCL5/LIX, a neutrophilic chemokine possibly beneficial in sepsis (31). Thus, TN likely plays two seemingly conflicting roles in sepsis. On the one hand, TN promoted HMGB1 endocytosis and macrophage pyropto-sis, which likely contributes to immunosuppression in sepsis (fig. S15). On the other hand, TN selectively attenuated the release of a patho-genic sepsis mediator (HMGB1) and induced the secretion of a po-tentially beneficial chemokine (ENA-78/CXCL5/LIX) (31).


http://stm.sciencem

ag.org/D

ownloaded from




10 of 14

The mechanism by which TN-specific mAbs rescue animals from lethal sepsis remains an exciting subject for future investigation. At least in part, it might be attributable to the effective attenuation of sepsis-induced TN depletion, which was likely pathogenic in sepsis

for several reasons. First, genetic disruption of TN expression ren-dered animals more susceptible to septic insults. Second, circulating TN was depleted under pathological conditions during experimental and clinical sepsis. Third, supplementation of septic animals with

10 µm

DAPI

Overlay

ASC

−Control +TN/HMGB1C

A B

+−−−+−TN

HMGB1 ++

100 -

55 -35 -25 -15 -

10 -

kDa

130 -250 -

SDS-PAGE

- ASC

100 -70 -55 -35 -25 -15 -

10 -

130 -250 -

+−−−+−

++

Western blot

+−−−+−

++

0

5

10

15

20

25

30

35

40

45

* *

#

ASC (AU)

+TN/HMGB1 + Dynasore

Cel

l dea

th (

%)

+−−−+−TN

HMGB1mAb8

Dyn

++

++++

−−−−−−

−−

+−−+

#

&

0

3

6

9

12

15

* &

Trypan blue uptakeMeanMedian

0

5

10

15

20

25LDH release

#

&

* &

+−−−+−

++

++++

−−−−−−

−−

−−++

TN/HMGB1 + mAb8

PericellularASC puncta

PerinuclearASC puncta

PerinuclearASC speck

Fig. 8. TN and HMGB1 cooperate to induce macrophage cell death. (A) Thioglycolate-elicited murine peritoneal macrophages were treated with TN (10 g/ml) in the absence or presence of HMGB1 (0.5 g/ml), TN-specific mAb8 (65.0 g/ml), or dynasore (10.0 M) for 16 hours, and cell viability was assessed by trypan blue dye exclusion or LDH release assay. *P < 0.05 versus negative control; #P < 0.05 versus +TN or +HMGB1 alone; &P < 0.05 versus positive control “+TN + HMGB1” group, n = 6 to 10 per group. (B) Murine peritoneal macrophages were stimulated with TN (10 g/ml) in the absence or presence of HMGB1 (1.0 g/ml) for 16 hours, and the cell-conditioned medium was assayed for ASC release by Western blotting analysis. SDS-PAGE gel indicated equivalent sampling loading. Bar graph represented average of three samples (n = 3) from two independent experiments (N = 2). *P < 0.05 versus negative controls (“−HMGB1 − TN”); #P < 0.05 versus positive control (+HMGB1 or +TN alone). (C) Differentiated human macrophages were stimulated with HMGB1 (1.0 g/ml) and TN (10.0 g/ml) in the absence or presence of TN-specific mAb (40 g/ml) or dynasore (20.0 M) for 16 hours. Subsequently, cells were im-munostained with Alexa Fluor 594–conjugated anti-ASC IgGs. Scale bars, 10 m. Narrow arrows point to minute ASC puncta; the wide arrow points to a larger ASC speck.


http://stm.sciencem

ag.org/D

ownloaded from




11 of 14

exogenous TN at subphysiological doses conferred marked protec-tion. Last, a panel of P5-reacting mAbs capable of rescuing animals from lethal sepsis uniformly attenuated the sepsis-induced TN de-pletion. These TN-specific protective mAbs prevented the sepsis- induced TN depletion, possibly through disrupting TN/HMGB1 interaction and thereby preventing their endocytotic degradation. Our current findings echoed with a recent report that an HMGB1- neutralizing mAb (2G7) similarly inhibited HMGB1 endocytosis (39), thereby conferring protection against lethal sepsis possibly by atten-uating cellular HMGB1 uptake.

There are a number of limitations to the present study: (i) We have not yet obtained sufficient numbers of age-matched critically ill pa-tients who died of severe sepsis or septic shock at comparable ages and could, thus, not perform a statistical comparison of plasma TN concentrations between age-matched sepsis survivors and nonsurvivors; (ii) it remains elusive whether TN domain-specific mAbs confer pro-tection by altering ENA-78/CXCL5/LIX expression in sepsis; (iii) it is not yet known what exact macrophage cell receptors are involved in the endocytosis of TN/HMGB1 complexes; (iv) it is unclear whether TN-specific mAbs similarly affect TN interaction with other proteins (for example, plasminogen) that may affect sepsis-induced dysregulated coagulopathy; (v) although TN/HMGB1 complexes might be readily engulfed by innate immune cells, it should still be possible and im-portant to characterize the TN/HMGB1 complexes in patients’ plas-ma samples. However, the discovery of mAbs capable of disrupting TN/HMGB1 interaction and endocytosis has suggested an exciting possibility of exploring antibody strategies to fight against inflam-matory diseases. It would be exciting to translate these preclinical findings into clinical applications through the use of humanized TN-specific mAbs capable of preventing its undesired interaction with pathogenic mediators that could cause macrophage pyroptosis and immunosuppression.

MATERIALS AND METHODSStudy designThe aim of this study was to assess the pathogenic changes of plas-ma TN concentrations in critically ill patients with sepsis or septic shock and use TN domain-specific mAbs to prevent TN depletion in the preclinical setting. For the clinical investigation, blood sam-ples were obtained from normal healthy controls and patients with sepsis or septic shock recruited to the Northwell Health System, and their plasma TN concentrations were assessed by immunoassays. Sample sizes were purely based on availability, and no blinding or random-ization was applied for this noninterventional observation. For the preclinical study, animals were randomly assigned to different ex-perimental groups and treated with recombinant TN or specific an-tibodies at the indicated dosing regiments. The outcomes included animal survival rates, blood bacterial counts, lung histology scores, and blood concentrations of liver-derived enzymes. Lung histology scores were collected under blinded experimental conditions. Study design and sample sizes used for each experiment are provided in the figure legends. No data, including outlier values, were excluded. Primary data are reported in data file S1. All reagent sources are listed in table S1.

Cell culturePrimary peritoneal macrophages were isolated from male Balb/c mice (7 to 8 weeks, 20 to 25 g) at 3 days after intraperitoneal injection of

2-ml thioglycolate broth (4%) as previously described (40, 41, 42). Human blood was purchased from the New York Blood Center (Long Island City, NY, USA), and human peripheral blood mononuclear cells (HuPBMCs) were isolated by density gradient centrifugation through Ficoll (Ficoll-Paque PLUS) as previously described (43–45). To differentiate into macrophages, HuPBMCs were cultured in the presence of human macrophage colony-stimulating factor (M-CSF; 20 ng/ml) for 5 to 6 days. Murine macrophages, human monocytes (HuBPMCs), and differentiated human macrophages were cultured in Dulbecco’s modified Eagle’s medium supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum or 10% human serum. When they reached 70 to 80% confluence, adherent cells were gently washed with, and immediately cultured in, Opti-MEM I before stimulating with crude LPS, purified SAA, HMGB1, in the absence or presence of human TN. The intracellular and extracellular concentrations of HMGB1, TN, or various other cytokines/chemokines were determined by Western blotting analysis or Cytokine Antibody Arrays as previ-ously described (40, 46–48). Alternatively, murine or human mac-rophages were stimulated with HMGB1 (0.5 to 1.0 g/ml) and TN (10 g/ml) either alone or concurrently in the absence or presence of TN-specific mAb8 (40.0 or 65.0 g/ml) or dynasore (10.0 M) for 16 hours, and cell viability or the formation of ASC speck was exam-ined 16 hours later.

Cell viabilityCell viability was evaluated by the trypan blue exclusion method, which distinguished the unstained viable cells from nonviable cells that taken up the dye to exhibit a distinctive blue color. Phase contrast images of multiple fields were randomly captured, and the percentage of trypan blue-stained cells was calculated. The released LDH in the culture medium was measured using an LDH Assay Kit (catalog no. L7572, Pointe Scientific Inc.) according to the manufacturer’s instructions. The optical density was measured at 340 nm using the ELISA plate reader, and the LDH content was expressed as the percentage of the maximal LDH release in the presence of 2% Triton X-100.

ImmunofluorescenceAfter inflammasome activation, ASC polymerizes to form a large sin-gular structure termed the ASC “speck” (32), which could be visualized by immunofluorescence of endogenous ASC using fluorescence- labeled ASC antibodies. Briefly, after stimulation with TN and HMGB1 in the absence or presence of TN-specific mAb8 or dynasore for 16 hours, differentiated human macrophages were fixed with 4% formaldehyde and permeabilized with 0.5% Triton X-100 for 15 min. After blocking with 5% albumin in 0.1% Triton X-100 [in 1× phosphate-buffered saline (PBS)] for 30 min, cells were incubated with Alexa Fluor 594–conjugated ASC Antibody (1.5:1000) for 1 hour and then washed three times with 0.1% Triton X-100 (in 1× PBS). Afterward, coverslips were mounted upside down on microscope slides, and images were ac-quired using the Olympus IX51 Inverted Fluorescence & Phase Contrast Tissue Culture Microscope.

Clinical characterization of septic patientsThis study was approved by the institutional review board of the Feinstein Institutes for Medical Research (FIMR) and endorsed by written informed consent from all participants providing blood sam-ples. Blood samples (5.0 ml) were collected from 33 healthy control participants, 31 patients with sepsis, and 14 patients with septic shock. Patients were diagnosed with sepsis or septic shock as per the American


http://stm.sciencem

ag.org/D

ownloaded from




12 of 14

College of Chest Physicians/Society of Critical Care Medicine Con-sensus Conference definitions of Sepsis and Septic Shock (Sepsis-2 definition) (49). The first cohort of 31 patients with sepsis was recruited to the North Shore University Hospital (NSUH) between 2010 and 2011 (listed as the “NS-P” in data file S1). The second cohort of 14 patients with septic shock was recruited to NSUH and the Long Island Jewish Medical Center between 2018 and 2019 (listed as “LIQ/H-P” in data file S1). In addition, we obtained 11 healthy control plasma samples from the Discovery Life Science Open Access Biorepository. The plasma TN concentrations were determined by Western blotting and enzyme-linked immunosorbent assay (ELISA) using a human CLEC3B/TN ELISA kit (catalog no. ELH-CLEC#B-1, RayBiotech) with reference to purified recombinant human TN at various dilutions.

MALDI-TOF mass spectrometryTo identify the 20-kDa band that was depleted in septic patients, serum samples of healthy controls and septic patients were resolved by SDS- PAGE gel electrophoresis, and the corresponding 20-kDa band was subjected to matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) mass spectrometry analysis as previously described (47). Briefly, the 20-kDa band was excised from the SDS-PAGE gel and subjected to in-gel trypsin digestion. The mass of the tryptic pep-tides was measured by MALDI-TOF mass spectrometry and then sub-jected to peptide mass fingerprinting database analysis to identify the 20-kDa protein (P20).

Open surface plasmon resonanceWe used the Nicoya Lifesciences gold-nanoparticle-based OpenSPR technology to characterize protein-protein interactions following the manufacturer’s instructions. For instance, highly purified recombinant HMGB1 or TN protein was immobilized on the amine sensor chip (catalog no. SEN-Au-100-10-AMINE) or NTA sensor chip (catalog no. SEN-Au-100-10-NTA), respectively, and TN, mAb, or HMGB1 was applied at different concentrations. To determine the binding affinities of mAbs to human or murine TN, highly purified human or murine TN was immobilized on the NTA sensor chip (catalog no. SEN-Au-100-10-NTA), and various mAbs were applied at various concentrations. The response units were recorded over time, and the binding affinity was estimated as the equilibrium Kd using the Trace Drawer Kinetic Data Analysis v.1.6.1 (Nicoya Lifesciences).

Cytokine antibody arrayMurine Cytokine Antibody Arrays (catalog no. M0308003, RayBiotech), which simultaneously detect 62 cytokines on one membrane, were used to measure relative cytokine concentrations in macrophage- conditioned culture medium or animal serum as described previously (40, 50). Human Cytokine Antibody C3 Arrays (catalog no. AAH-CYT-3-4), which detect 42 cytokines on one membrane, were used to determine cytokine concentrations in human monocyte-conditioned culture medium as previously described (41, 44).

Generation of anti-TN pAbs and mAbspAbs were generated in Female New Zealand White Rabbits at the Covance Inc. (Princeton, NJ, USA) using recombinant murine TN in combination with Freund’s complete adjuvant following standard procedures. Blood samples were collected in 3-week cycles of im-munization and bleeding, and the antibody titers were determined by indirect TN ELISA. Total IgGs were purified from the serum using Protein A affinity column. Briefly, rabbit serum was prebuffered with

PBS and slowly loaded onto the Protein A column to allow sufficient binding of IgGs. After washing with 1× PBS to remove unbound serum components, the IgGs were eluted with acidic buffer [0.1 M glycine- HCl (pH 2.8)] and then immediately dialyzed into 1× PBS buffer at 4°C overnight.

The monoclonal antibodies were generated in Balb/C and C57BL/6 mice at the GenScript (Piscataway, NJ, USA) using highly purified human TN following standard procedures. Blood samples were col-lected in 2-week cycles of immunization and bleeding, and serum titers were assessed by indirect ELISA. After four immunizations, mouse splenocytes were harvested, fused with mouse Sp2/0 myeloma cell line, and screened for antibody-producing hybridomas by indirect ELISA, dot blotting, and Western blotting analysis. After limiting dilution, pu-rified hybridoma clones were generated to produce mAbs following standard procedures. For V-region sequencing, five independent hy-bridoma preparations for each clone were used to isolate total RNA, reverse transcribed into complementary DNA. The heavy and light chain variable regions were amplified by polymerase chain reaction and sub-cloned into a selectable bacterial shuttle vector for DNA sequencing analysis of the complementarity-determining regions of each mAb.

Animal model of lethal endotoxemia and sepsisThis study was conducted in accordance with policies of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of the FIMR. To evaluate the role of TN in lethal sepsis, Balb/C mice (male or female, 7 to 8 weeks old, 20 to 25 g) were subjected to lethal endotoxemia or sepsis induced by CLP as previously described (51, 52). Briefly, the cecum of Balb/C mice was ligated at 5.0 mm from the cecal tip and then punctured once with a 22-gauge needle. At 30 min after CLP, all animals were given a subcutaneous dose of imipenem/cilastatin (0.5 mg per mouse; Primaxin, Merck & Co. Inc.) and re-suscitation with normal sterile saline solution (20 ml/kg). Recombinant TN or anti-TN polyclonal or monoclonal IgGs were intraperitoneally administered to endotoxemic or septic mice at the indicated doses and time points, and animal survival rates were monitored for up to 2 weeks. To evaluate the role of TN in lethal sepsis, breeding pairs of the heterozygous TN (also called “CLCE3B”) KO mice (on C57BL/6J genetic background) were obtained from the Jackson Laboratory (stock no. 027554) and bred to produce homozygous TN KO and WT littermates. Age- and sex-matched WT or TN KO C57BL/6J mice were then subjected to CLP sepsis, and animal survival rates were compared between WT and TN KO mice for up to 2 weeks.

Tissue injuryLung tissues were collected at 24 or 28 hours after the onset of sepsis and stored in 10% formalin before fixation in paraffin. The fixed tissue was then sectioned (5 m) and stained with hematoxylin and eosin. Tissue injury was assessed in a blinded fashion using a semiquantitative scoring system developed by the American Thoracic Society. Briefly, histological lung injury was scored on the basis of the presence of infiltrated inflammatory cells in the alveolar and interstitial spaces, the presence of hyaline membranes and proteinaceous debris within airspaces, and alveolar septal thickening, according to the following definition: 0, no injury; 1, moderate injury; 2, severe injury. Using a weighted equation with a maximum score of 100 per field, the pa-rameter scores were calculated and then averaged as the final lung injury score in each experimental group. At 28 hours after CLP, animals were euthanized to harvest blood to measure serum concentrations


http://stm.sciencem

ag.org/D

ownloaded from




13 of 14

of hepatic injury markers such as ALT and AST using commercial kits (catalog nos. A7561 and A7526, Pointe Scientific Inc.) as per the manufacturer’s instructions.

RNA-seq analysisAt 24 hours after the onset of sepsis, various tissues were harvested to isolate total RNA, and the expression of all transcripts in WT or TN KO mice was assessed by RNA-seq (GENEWIZ). Gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis were applied to analyze the differentially expressed genes by using String online tools (https://string-db.org/cgi/input.pl). Dif-ferential expression analysis was performed using the Wald test (DESeq2) to generate P values and log2 fold changes. Genes with an adjusted P value of <0.05 and an absolute log2 fold change of >2 were defined as differentially expressed.

Colony-forming unitsBacterial counts were performed on aseptically harvested blood samples after serial dilution in sterile PBS and cultured on tryptic soy agar plates supplemented with 5% sheep blood (Becton Dickinson). After incubation at 37°C for 24 to 48 hours, the CFUs were counted.

Peptide dot blottingA library of synthetic peptides corresponding to different regions of human TN sequence were synthesized and spotted (1.0 g in 2.5 l) onto nitrocellulose membrane (catalog no. 88013, Thermo Fisher Scientific). Subsequently, the membrane was probed with IgGs from different rabbits or murine hybridomas following a standard protocol.

Statistical analysisAll data were assessed for normality by the Shapiro-Wilk test before conducting appropriate statistical tests between groups. The com-parison of two independent samples was assessed by the Student’s t test and the Mann-Whitney test for Gaussian and non-Gaussian distributed samples, respectively. For comparison among multiple groups with normal data distribution, the differences were analyzed by one-way analysis of variance (ANOVA) followed by the Fisher’s least significant difference test. For comparison among multiple groups with non-normal (skewed) distribution, the statistical differences were evaluated with the nonparametric Kruskal-Wallis ANOVA test. For survival studies, the Kaplan-Meier method was used to compare the differences in mortality rates between groups with the nonparametric log-rank post hoc test. A P value of <0.05 was considered statistically significant.

SUPPLEMENTARY MATERIALSstm.sciencemag.org/cgi/content/full/12/539/eaaz3833/DC1Materials and MethodsFig. S1. Immunoblotting and genotyping analysis of TN KO mice.Fig. S2. Survey of TN protein abundance in various tissues.Fig. S3. Parallel reduction of lung and serum TN content during lethal endotoxemia.Fig. S4. Expression and purification of recombinant TN.Fig. S5. Recombinant murine TN conferred a dose-dependent protection against lethal sepsis.Fig. S6. Epitope mapping and specificity of representative mAbs raised against recombinant human TN.Fig. S7. Cross-reactivity and epitope mapping of a panel of P5-reacting mAbs.Fig. S8. Epitope sequence homology between different mammalian species.Fig. S9. Characteristics of a panel of human TN-specific mAbs.Fig. S10. IgG isotype controls did not affect sepsis lethality.Fig. S11. Distinct effects of P2- and P5-reacting mAbs on sepsis-induced TN depletion.Fig. S12. Divergent effects of mAb8 and mAb9 on sepsis-induced systemic KC accumulation.

Fig. S13. TN enhanced HMGB1 uptake and possible degradation by macrophage cultures.Fig. S14. mAb8 and dynasore inhibited the TN/HMGB1-induced increase of trypan blue dye uptake in macrophage cultures.Fig. S15. Proposed model for the mAb8-mediated protection against lethal sepsis.Table S1. Reagent sources.Table S2. Demographics of 44 normal healthy controls and 45 septic patients.Data file S1. Primary data.

View/request a protocol for this paper from Bio-protocol.

REFERENCES AND NOTES 1. M. Singer, C. S. Deutschman, C. W. Seymour, M. Shankar-Hari, D. Annane, M. Bauer,

R. Bellomo, G. R. Bernard, J. D. Chiche, C. M. Coopersmith, R. S. Hotchkiss, M. M. Levy, J. C. Marshall, G. S. Martin, S. M. Opal, G. D. Rubenfeld, T. van der Poll, J. L. Vincent, D. C. Angus, The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA 315, 801–810 (2016).

2. J. Cohen, J. L. Vincent, N. K. Adhikari, F. R. Machado, D. C. Angus, T. Calandra, K. Jaton, S. Giulieri, J. Delaloye, S. Opal, K. Tracey, T. van der Poll, E. Pelfrene, Sepsis: A roadmap for future research. Lancet Infect. Dis. 15, 581–614 (2015).

3. R. S. Hotchkiss, I. E. Karl, The pathophysiology and treatment of sepsis. N. Engl. J. Med. 348, 138–150 (2003).

4. H. Wang, O. Bloom, M. Zhang, J. M. Vishnubhakat, M. Ombrellino, J. Che, A. Frazier, H. Yang, S. Ivanova, L. Borovikova, K. R. Manogue, E. Faist, E. Abraham, J. Andersson, U. Andersson, P. E. Molina, N. N. Abumrad, A. Sama, K. J. Tracey, HMG-1 as a late mediator of endotoxin lethality in mice. Science 285, 248–251 (1999).

5. H. Yang, M. Ochani, J. Li, X. Qiang, M. Tanovic, H. E. Harris, S. M. Susarla, L. Ulloa, H. Wang, R. DiRaimo, C. J. Czura, H. Wang, J. Roth, H. S. Warren, M. P. Fink, M. J. Fenton, U. Andersson, K. J. Tracey, Reversing established sepsis with antagonists of endogenous high-mobility group box 1. Proc. Natl. Acad. Sci. U.S.A. 101, 296–301 (2004).

6. S. Qin, H. Wang, R. Yuan, H. Li, M. Ochani, K. Ochani, M. Rosas-Ballina, C. J. Czura, J. M. Huston, E. Miller, X. Lin, B. Sherry, A. Kumar, G. Larosa, W. Newman, K. J. Tracey, H. Yang, Role of HMGB1 in apoptosis-mediated sepsis lethality. J. Exp. Med. 203, 1637–1642 (2006).

7. M. Deng, Y. Tang, W. Li, X. Wang, R. Zhang, X. Zhang, X. Zhao, J. Liu, C. Tang, Z. Liu, Y. Huang, H. Peng, L. Xiao, D. Tang, M. J. Scott, Q. Wang, J. Liu, X. Xiao, S. Watkins, J. Li, H. Yang, H. Wang, F. Chen, K. J. Tracey, T. R. Billiar, B. Lu, The endotoxin delivery protein HMGB1 mediates caspase-11-dependent lethality in sepsis. Immunity 49, 740–753.e7 (2018).

8. S. M. Robert, H. Sjodin, M. P. Fink, R. K. Aneja, Preconditioning with high mobility group box 1 (HMGB1) induces lipoteichoic acid (LTA) tolerance. J. Immunother. 33, 663–671 (2010).

9. R. K. Aneja, A. Tsung, H. Sjodin, J. V. Gefter, R. L. Delude, T. R. Billiar, M. P. Fink, Preconditioning with high mobility group box 1 (HMGB1) induces lipopolysaccharide (LPS) tolerance. J. Leukoc. Biol. 84, 1326–1334 (2008).

10. J. Xu, Y. Jiang, J. Wang, X. Shi, Q. Liu, Z. Liu, Y. Li, M. J. Scott, G. Xiao, S. Li, L. Fan, T. R. Billiar, M. A. Wilson, J. Fan, Macrophage endocytosis of high-mobility group box 1 triggers pyroptosis. Cell Death Differ. 21, 1229–1239 (2014).

11. M. Gregoire, J. M. Tadié, F. Uhel, A. Gacouin, C. Piau, N. Bone, T. Y. Le, E. Abraham, K. Tarte, J. W. Zmijewski, Frontline Science: HMGB1 induces neutrophil dysfunction in experimental sepsis and in patients who survive septic shock. J. Leukoc. Biol. 101, 1281–1287 (2017).

12. C. A. Wild, C. Bergmann, G. Fritz, P. Schuler, T. K. Hoffmann, R. Lotfi, A. Westendorf, S. Brandau, S. Lang, HMGB1 conveys immunosuppressive characteristics on regulatory and conventional T cells. Int. Immunol. 24, 485–494 (2012).

13. V. S. Patel, R. A. Sitapara, A. Gore, B. Phan, L. Sharma, V. Sampat, J. Li, H. Yang, S. S. Chavan, H. Wang, K. J. Tracey, L. L. Mantell, High mobility group box-1 mediates hyperoxia-induced impairment of pseudomonas aeruginosa clearance and inflammatory lung injury in mice. Am. J. Respir. Cell Mol. Biol. 48, 280–287 (2013).

14. I. Clemmensen, L. C. Petersen, C. Kluft, Purification and characterization of a novel, oligomeric, plasminogen kringle 4 binding protein from human plasma: Tetranectin. Eur. J. Biochem. 156, 327–333 (1986).

15. C. B. Sorensen, L. Berglund, T. E. Petersen, Cloning of a cDNA encoding murine tetranectin. Gene 152, 243–245 (1995).

16. L. Berglund, T. E. Petersen, The gene structure of tetranectin, a plasminogen binding protein. FEBS Lett. 309, 15–19 (1992).

17. U. M. Wewer, K. Iba, M. E. Durkin, F. C. Nielsen, F. Loechel, B. J. Gilpin, W. Kuang, E. Engvall, R. Albrechtsen, Tetranectin is a novel marker for myogenesis during embryonic development, muscle regeneration, muscle cell differentiation in vitro. Dev. Biol. 200, 247–259 (1998).

18. B. A. Jensen, P. McNair, L. Hyldstrup, I. Clemmensen, Plasma tetranectin in healthy male and female individuals, measured by enzyme-linked immunosorbent assay. J. Lab. Clin. Med. 110, 612–617 (1987).


http://stm.sciencem

ag.org/D

ownloaded from

https://string-db.org/cgi/input.pl

http://stm.sciencemag.org/cgi/content/full/12/539/eaaz3833/DC1

https://en.bio-protocol.org/cjrap.aspx?eid=10.1126/scitranslmed.aaz3833




14 of 14

19. R. H. Lorentsen, J. H. Graversen, N. R. Caterer, H. C. Thogersen, M. Etzerodt, The heparin-binding site in tetranectin is located in the N-terminal region and binding does not involve the carbohydrate recognition domain. Biochem. J. 347 (pt 1), 83–87 (2000).

20. J. H. Graversen, R. H. Lorentsen, C. Jacobsen, S. K. Moestrup, B. W. Sigurskjold, H. C. Thøgersen, M. Etzerodt, The plasminogen binding site of the C-type lectin tetranectin is located in the carbohydrate recognition domain, binding is sensitive to both calcium and lysine. J. Biol. Chem. 273, 29241–29246 (1998).

21. J. H. Graversen, B. W. Sigurskjold, H. C. Thøgersen, M. Etzerodt, Tetranectin-binding site on plasminogen kringle 4 involves the lysine-binding pocket and at least one additional amino acid residue. Biochemistry 39, 7414–7419 (2000).

22. C. Kluft, A. F. Jie, P. Los, E. de Wit, L. Havekes, Functional analogy between lipoprotein(a) and plasminogen in the binding to the kringle 4 binding protein, tetranectin. Biochem. Biophys. Res. Commun. 161, 427–433 (1989).

23. U. B. Westergaard, M. H. Andersen, C. W. Heegaard, S. N. Fedosov, T. E. Petersen, Tetranectin binds hepatocyte growth factor and tissue-type plasminogen activator. Eur. J. Biochem. 270, 1850–1854 (2003).

24. U. M. Wewer, K. Ibaraki, P. Schjorring, M. E. Durkin, M. F. Young, R. Albrechtsen, A potential role for tetranectin in mineralization during osteogenesis. J. Cell Biol. 127, 1767–1775 (1994).

25. K. Iba, M. E. Durkin, L. Johnsen, E. Hunziker, K. Damgaard-Pedersen, H. Zhang, E. Engvall, R. Albrechtsen, U. M. Wewer, Mice with a targeted deletion of the tetranectin gene exhibit a spinal deformity. Mol. Cell. Biol. 21, 7817–7825 (2001).

26. E.-S. Wang, X.-P. Zhang, H.-B. Yao, G. Wang, S.-W. Chen, W.-W. Gao, H.-J. Yao, Y.-R. Sun, C.-H. Xi, Y. D. Ji, Tetranectin knockout mice develop features of Parkinson disease. Cell. Physiol. Biochem. 34, 277–287 (2014).

27. K. Iba, N. Hatakeyama, T. Kojima, M. Murata, T. Matsumura, U. M. Wewer, T. Wada, N. Sawada, T. Yamashita, Impaired cutaneous wound healing in mice lacking tetranectin. Wound Repair Regen. 17, 108–112 (2009).

28. K. Iba, Y. Abe, T. Chikenji, K. Kanaya, H. Chiba, K. Sasaki, T. Dohke, T. Wada, T. Yamashita, Delayed fracture healing in tetranectin-deficient mice. J. Bone Miner. Metab. 31, 399–408 (2013).

29. M. F. Osuchowski, K. Welch, J. Siddiqui, D. G. Remick, Circulating cytokine/inhibitor profiles reshape the understanding of the SIRS/CARS continuum in sepsis and predict mortality. J. Immunol. 177, 1967–1974 (2006).

30. J. G. Heuer, G. R. Sharma, B. Gerlitz, T. Zhang, D. L. Bailey, C. Ding, D. T. Berg, D. Perkins, E. J. Stephens, K. C. Holmes, R. L. Grubbs, K. A. Fynboe, Y.-F. Chen, B. Grinnell, J. A. Jakubowski, Evaluation of protein C and other biomarkers as predictors of mortality in a rat cecal ligation and puncture model of sepsis. Crit. Care Med. 32, 1570–1578 (2004).

31. J. Mei, Y. Liu, N. Dai, M. Favara, T. Greene, S. Jeyaseelan, M. Poncz, J. S. Lee, G. S. Worthen, CXCL5 regulates chemokine scavenging and pulmonary host defense to bacterial infection. Immunity 33, 106–117 (2010).

32. M. S. Dick, L. Sborgi, S. Ruhl, S. Hiller, P. Broz, ASC filament formation serves as a signal amplification mechanism for inflammasomes. Nat. Commun. 7, 11929 (2016).

33. B. S. Franklin, L. Bossaller, N. D. De, J. M. Ratter, A. Stutz, G. Engels, C. Brenker, M. Nordhoff, S. R. Mirandola, A. Al-Amoudi, M. S. Mangan, S. Zimmer, B. G. Monks, M. Fricke, R. E. Schmidt, T. Espevik, B. Jones, A. G. Jarnicki, P. M. Hansbro, P. Busto, A. Marshak-Rothstein, S. Hornemann, A. Aguzzi, W. Kastenmuller, E. Latz, The adaptor ASC has extracellular and 'prionoid' activities that propagate inflammation. Nat. Immunol. 15, 727–737 (2014).

34. M. Yu, H. Wang, A. Ding, D. T. Golenbock, E. Latz, C. J. Czura, M. J. Fenton, K. J. Tracey, H. Yang, HMGB1 signals through toll-like receptor (TLR) 4 and TLR2. Shock 26, 174–179 (2006).

35. J. Tian, A. M. Avalos, S. Y. Mao, B. Chen, K. Senthil, H. Wu, P. Parroche, S. Drabic, D. Golenbock, C. Sirois, J. Hua, L. L. An, L. Audoly, G. La Rosa, A. Bierhaus, P. Naworth, A. Marshak-Rothstein, M. K. Crow, K. A. Fitzgerald, E. Latz, P. A. Kiener, A. J. Coyle, Toll-like receptor 9-dependent activation by DNA-containing immune complexes is mediated by HMGB1 and RAGE. Nat. Immunol. 8, 487–496 (2007).

36. H. Yang, H. Wang, Y. A. Levine, M. K. Gunasekaran, Y. Wang, M. Addorisio, S. Zhu, W. Li, J. Li, D. P. de Kleijn, P. S. Olofsson, H. S. Warren, M. He, Y. Al-Abed, J. Roth, D. J. Antoine, S. S. Chavan, U. Andersson, K. J. Tracey, Identification of CD163 as an antiinflammatory receptor for HMGB1-haptoglobin complexes. JCI Insight 1, e85375 (2016).

37. M. Son, A. Porat, M. He, J. Suurmond, F. Santiago-Schwarz, U. Andersson, T. R. Coleman, B. T. Volpe, K. J. Tracey, Y. Al-Abed, B. Diamond, C1q and HMGB1 reciprocally regulate human macrophage polarization. Blood 128, 2218–2228 (2016).

38. M. Kovarova, P. R. Hesker, L. Jania, M. Nguyen, J. N. Snouwaert, Z. Xiang, S. E. Lommatzsch, M. T. Huang, J. P.-Y. Ting, B. H. Koller, NLRP1-dependent pyroptosis leads to acute lung injury and morbidity in mice. J. Immunol. 189, 2006–2016 (2012).

39. H. Yang, H. Liu, Q. Zeng, G. H. Imperato, M. E. Addorisio, J. Li, M. He, K. F. Cheng, Y. Al-Abed, H. E. Harris, S. S. Chavan, U. Andersson, K. J. Tracey, Inhibition of HMGB1/RAGE-mediated endocytosis by HMGB1 antagonist box A, anti-HMGB1 antibodies, cholinergic agonists suppresses inflammation. Mol. Med. 25, 13 (2019).

40. W. Li, G. Bao, W. Chen, X. Qiang, S. Zhu, S. Wang, M. He, G. Ma, M. Ochani, Y. Al-Abed, H. Yang, K. J. Tracey, P. Wang, J. D'Angelo, H. Wang, Connexin 43 hemichannel as a novel mediator of sterile and infectious inflammatory diseases. Sci. Rep. 8, 166 (2018).

41. W. Li, M. Ashok, J. Li, H. Yang, A. E. Sama, H. Wang, A major ingredient of green tea rescues mice from lethal sepsis partly by inhibiting HMGB1. PLOS ONE 2, e1153 (2007).

42. Y. Zhang, W. Li, S. Zhu, A. Jundoria, J. Li, H. Yang, S. Fan, P. Wang, K. J. Tracey, A. E. Sama, H. Wang, Tanshinone IIA sodium sulfonate facilitates endocytic HMGB1 uptake. Biochem. Pharmacol. 84, 1492–1500 (2012).

43. G. Chen, J. Li, M. Ochani, B. Rendon-Mitchell, X. Qiang, S. Susarla, L. Ulloa, H. Yang, S. Fan, S. M. Goyert, P. Wang, K. J. Tracey, A. E. Sama, H. Wang, Bacterial endotoxin stimulates macrophages to release HMGB1 partly through CD14- and TNF-dependent mechanisms. J. Leukoc. Biol. 76, 994–1001 (2004).

44. W. Li, J. Li, M. Ashok, R. Wu, D. Chen, L. Yang, H. Yang, K. J. Tracey, P. Wang, A. E. Sama, H. Wang, A cardiovascular drug rescues mice from lethal sepsis by selectively attenuating a late-acting proinflammatory mediator, high mobility group box 1. J. Immunol. 178, 3856–3864 (2007).

45. B. Rendon-Mitchell, M. Ochani, J. Li, J. Han, H. Wang, H. Yang, S. Susarla, C. Czura, R. A. Mitchell, G. Chen, A. E. Sama, K. J. Tracey, H. Wang, IFN-gamma induces high mobility group box 1 protein release partly through a TNF-dependent mechanism. J. Immunol. 170, 3890–3897 (2003).

46. S. Zhu, Y. Wang, W. Chen, W. Li, A. Wang, S. Wong, G. Bao, J. Li, H. Yang, K. J. Tracey, J. D'Angelo, H. Wang, High-density lipoprotein (HDL) counter-regulates serum amyloid A (SAA)-induced sPLA2-IIE and sPLA2-V expression in macrophages. PLOS ONE 11, e0167468 (2016).

47. W. Li, S. Zhu, J. Li, J. D'Amore, J. D'Angelo, H. Yang, P. Wang, K. J. Tracey, H. Wang, Serum amyloid A stimulates PKR expression and HMGB1 release possibly through TLR4/RAGE receptors. Mol. Med. 21, 515–525 (2015).

48. W. Chen, S. Zhu, Y. Wang, J. Li, X. Qiang, X. Zhao, H. Yang, J. D'Angelo, L. Becker, P. Wang, K. J. Tracey, H. Wang, Enhanced macrophage pannexin 1 expression and hemichannel activation exacerbates lethal experimental sepsis. Sci. Rep. 9, 160 (2019).

49. M. M. Levy, M. P. Fink, J. C. Marshall, E. Abraham, D. Angus, D. Cook, J. Cohen, S. M. Opal, J. L. Vincent, G. Ramsay, 2001 SCCM/ESICM/ACCP/ATS/SIS international sepsis definitions conference. Crit. Care Med. 31, 1250–1256 (2003).

50. S. Zhu, M. Ashok, J. Li, W. Li, H. Yang, P. Wang, K. J. Tracey, A. E. Sama, H. Wang, Spermine protects mice against lethal sepsis partly by attenuating surrogate inflammatory markers. Mol. Med. 15, 275–282 (2009).

51. W. Li, S. Zhu, J. Li, A. Assa, A. Jundoria, J. Xu, S. Fan, N. T. Eissa, K. J. Tracey, A. E. Sama, H. Wang, EGCG stimulates autophagy and reduces cytoplasmic HMGB1 levels in endotoxin-stimulated macrophages. Biochem. Pharmacol. 81, 1152–1163 (2011).

52. W. Li, S. Zhu, J. Li, Y. Huang, R. Zhou, X. Fan, H. Yang, X. Gong, N. T. Eissa, W. Jahnen-Dechent, P. Wang, K. J. Tracey, A. E. Sama, H. Wang, A hepatic protein, fetuin-A, occupies a protective role in lethal systemic inflammation. PLOS ONE 6, e16945 (2011).

Acknowledgments: We thank a former lab member, W. Li, for the initial characterization of TN as a protein depleted in septic patients who died of sepsis, the subsequent epitope mapping of rabbit pAbs against murine TN, and the finding of ASC release by TN-stimulated peritoneal macrophages. We also thank G. Bao for testing the protective efficacy of recombinant human TN in animal model of sepsis and J. Kim for providing 16 plasma samples to the healthy control group. Funding: This work was supported by the NIH grants R01GM063075 and R01AT005076 (to H.W.). Author contributions: H.W. supervised the study, interpreted the results, generated the figures, and wrote the manuscript. W.C., X.Q., Y.W., S.Z., J.L., A.B., and H.Y. performed experiments and generated data. J.G. and L.B. recruited some septic patients and provided plasma samples for the study. P.W. and K.J.T. provided important input to the experimental design and manuscript revision. Competing interests: H.W., J.L., K.J.T., and W.C. are co-inventors of patent applications entitled “Use of tetranectin and peptide agonists to treat inflammatory diseases” (application no. 16/617,811) and “Tetranectin-targeting monoclonal antibodies to fight against lethal sepsis and other pathologies” (application 62/885,890). All other authors declare that they have no competing interests. Data and materials availability: All data associated with this study are present in the paper or the Supplementary Materials.

Submitted 4 September 2019Accepted 9 March 2020Published 15 April 202010.1126/scitranslmed.aaz3833

Citation: W. Chen, X. Qiang, Y. Wang, S. Zhu, J. Li, A. Babaev, H. Yang, J. Gong, L. Becker, P. Wang, K. J. Tracey, H. Wang, Identification of tetranectin-targeting monoclonal antibodies to treat potentially lethal sepsis. Sci. Transl. Med. 12, eaaz3833 (2020).


http://stm.sciencem

ag.org/D

ownloaded from


sepsisIdentification of tetranectin-targeting monoclonal antibodies to treat potentially lethal

Lance Becker, Ping Wang, Kevin J. Tracey and Haichao WangWeiqiang Chen, Xiaoling Qiang, Yongjun Wang, Shu Zhu, Jianhua Li, Ariella Babaev, Huan Yang, Jonathan Gong,

DOI: 10.1126/scitranslmed.aaz3833, eaaz3833.12Sci Transl Med

discusses the implications of these findings.et al.Paterson worsening inflammation, whereas an antibody blocking this interaction can be protective. A related Focus byof sepsis. The authors found that HMGB1 binds tetranectin, resulting in endocytosis of both proteins and with sepsis and associated with better survival in mouse models. The other protein, HMGB1, is a known mediatorbetween two proteins may provide a useful intervention. One of these proteins, tetranectin, is depleted in patients

determined that an antibody blocking the interactionet al.the mechanisms of inflammation in sepsis, Chen Sepsis, potentially lethal organ dysfunction caused by infection, remains a major problem. By investigating

Got to keep them separated

ARTICLE TOOLS http://stm.sciencemag.org/content/12/539/eaaz3833

MATERIALSSUPPLEMENTARY http://stm.sciencemag.org/content/suppl/2020/04/13/12.539.eaaz3833.DC1

CONTENTRELATED

http://stm.sciencemag.org/content/scitransmed/7/287/287ra71.fullhttp://stm.sciencemag.org/content/scitransmed/8/335/335ra55.fullhttp://stm.sciencemag.org/content/scitransmed/9/412/eaan5689.fullhttp://stm.sciencemag.org/content/scitransmed/11/518/eaax9000.fullhttp://stm.sciencemag.org/content/scitransmed/12/539/eabb2575.full

REFERENCES

http://stm.sciencemag.org/content/12/539/eaaz3833#BIBLThis article cites 52 articles, 12 of which you can access for free

PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions

Terms of ServiceUse of this article is subject to the

registered trademark of AAAS. is aScience Translational MedicineScience, 1200 New York Avenue NW, Washington, DC 20005. The title

(ISSN 1946-6242) is published by the American Association for the Advancement ofScience Translational Medicine

of Science. No claim to original U.S. Government WorksCopyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement


http://stm.sciencem

ag.org/D

ownloaded from

http://stm.sciencemag.org/content/12/539/eaaz3833

http://stm.sciencemag.org/content/suppl/2020/04/13/12.539.eaaz3833.DC1

http://stm.sciencemag.org/content/scitransmed/12/539/eabb2575.full

http://stm.sciencemag.org/content/scitransmed/11/518/eaax9000.full

http://stm.sciencemag.org/content/scitransmed/9/412/eaan5689.full

http://stm.sciencemag.org/content/scitransmed/8/335/335ra55.full

http://stm.sciencemag.org/content/scitransmed/7/287/287ra71.full

http://stm.sciencemag.org/content/12/539/eaaz3833#BIBL

http://www.sciencemag.org/help/reprints-and-permissions

http://www.sciencemag.org/about/terms-service


SEPSIS Copyright © 2020 Identification of tetranectin-targeting … · 2020-04-15 · higher...

Documents

Transcript of SEPSIS Copyright © 2020 Identification of tetranectin-targeting … · 2020-04-15 · higher...