stm.sciencemag.org/cgi/content/full/12/539/eaaz3833/DC1
Supplementary Materials for
Identification of tetranectin-targeting monoclonal antibodies to treat potentially
lethal sepsis
Weiqiang Chen, Xiaoling Qiang, Yongjun Wang, Shu Zhu, Jianhua Li, Ariella Babaev, Huan Yang, Jonathan Gong, Lance Becker, Ping Wang, Kevin J. Tracey, Haichao Wang*
*Corresponding author. Email: [email protected]
Published 15 April 2020, Sci. Transl. Med. 12, eaaz3833 (2020)
DOI: 10.1126/scitranslmed.aaz3833
The PDF file includes:
Materials and Methods Fig. 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.
Other Supplementary Material for this manuscript includes the following: (available at stm.sciencemag.org/cgi/content/full/12/539/eaaz3833/DC1)
Data file S1 (Microsoft Excel format). Primary data.
Materials and Methods
Genotyping
To verify the genotypes of wild-type (WT) and TN KO mice, tail biopsies was digested in
Direct-PCR lysis Reagent (Cat. No 102-T, Viagen Biotech, Inc.) containing 0.4 µg/ml proteinase
K (Cat. No EO0491, ThermoFisher Scientific), and lysate containing genomic DNA was
amplified by PCR reaction using the following primers: forward WT primer CAA AAA CCA
CAC ACT CCA TCT G; reverse WT primer CTT AGT ATC TAC CAC TCC TGT CTG AGG;
forward KO mutant primer CGG TCG CTA CCA TTA CCA GT; reverse KO mutant primer
TGT GTT GTA GTC CAG CAG AGG, under the following conditions: 95ºC 3 min; followed
by 37 cycles of 95ºC for 15 sec and 60ºC for 15 sec, 72ºC, 15 sec. The PCR products were
resolved on a 2% agarose gel and visualized by ethidium bromide staining.
Preparation of recombinant HMGB1 and TN proteins
The cDNA encoding for rat HMGB1 was cloned into a pCAL-n vector, and the recombinant
CBP-HMGB1 (rHMGB1) was expressed in E. coli BL21 (DE3) pLysS cells as previously
described (4). Recombinant HMGB1 containing a ~3 kDa calmodulin-binding peptide tag (CBP-
HMGB1 fusion protein, 33 kDa) was expressed in E. coli, and purified to remove contaminating
endotoxin by Triton X-114 extraction as previously described (50). Recombinant human TN
corresponding to amino acids 22-202 (without the 21-amio acid leader signal sequence) with a
C-terminal histidine tag was expressed in E. coli BL21 (DE3) pLysS cells, and purified by
histidine-affinity and Triton X-114 extraction to remove contaminating endotoxins. Recombinant
TN and HMGB1 proteins were tested for LPS content by the chromogenic Limulus amebocyte
lysate assay (Endochrome; Charles River), and the endotoxin content was less than 0.01 U per
microgram of recombinant proteins.
Cellular HMGB1 and TN uptake
Highly purified recombinant HMGB1 and TN proteins were labeled with either Alexa Fluor 555
or Alexa Fluor 488 using the appropriate labeling kits (Cat. # A30007 or Cat. # A30006, Thermo
Fisher Scientific) according to the manufacturer’s instructions. Murine macrophage-like RAW
264.7 cells on cover slips were incubated with unlabeled or Alexa Fluor 555-labeled HMGB1 in
the absence or presence of unlabeled or Alexa Fluor 488-labeled TN at 37⁰C for 2 h. An
endocytosis inhibitor, Dynasore (8.0 µM), or mAb8 (65 µg/ml) were added 30 min before the
addition of HMGB1 or TN. After extensive washing with 1 x PBS, cells were fixed with 4%
formalin for 20 min at room temperature and mounted on slides using Vectashield Mounting
Medium for Fluorescence with DAPI (Vector Laboratories, Inc., Cat. # H-1200) for nuclei
staining (blue). Endocytic uptake of Alexa Fluor 555-labeled HMGB1 (red) or Alexa Fluor 488-
labeled TN (green) was visualized under the Olympus IX51 Inverted Fluorescence & Phase
Contrast Tissue Culture Microscope. To quantitate cellular uptake of HMGB1 and TN,
macrophage cultures were incubated with recombinant HMGB1 and TN either individually or in
combination, at 37⁰C for 2 h. After extensive washing, cellular concentrations of HMGB1 and
TN were measured by Western blotting analysis with reference to a housekeeping protein, β-
actin.
Western blotting
The concentration of TN in human or murine serum was determined by Western blotting analysis
using commercial rabbit mAb against the C-terminus of human TN (Abcam) or our homemade
murine mAb (mAb8) against recombinant human TN. The cellular and extracellular
concentrations of TN and HMGB1 in murine macrophage and human monocyte cultures were
determined by Western blotting analysis using rabbit polyclonal or monoclonal antibodies. Equal
amounts of total cellular protein or equivalent volumes of cell-conditioned culture medium were
resolved on sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to polyvinylidene
difluoride (PVDF) membranes. After blocking with 5% nonfat milk, the membranes were
incubated with the appropriate antibodies (anti-TN, 1:1000; anti-ASC, 1:1000; anti-β-actin,
1:5000; anti-HMGB1, 1:1000) overnight. Subsequently, the membranes were incubated with the
appropriate secondary antibodies, and the immune-reactive bands were visualized by
chemiluminescence. The relative band intensity was quantified using the UN-SCAN-IT Gel
Analysis Software Version 7.1 (Silk Scientific Inc.).
Fig. S1. Immunoblotting and genotyping analysis of TN KO mice. (A) Western blotting
analysis of serum and lung TN from wild-type (TN+/+
), heterozygous (TN+/
), and homozygous
(TN-/-
) knockout mice. SDS-PAGE gel analysis confirmed equivalent sample loading. (B)
Genotyping of wild-type and TN knockout mice by PCR. Genomic DNA samples were isolated
from mouse tail biopsies and amplified by PCR. The PCR products of wild-type (300 bp) and
KO alleles (422 bp) were resolved on a 2% agarose gel and visualized after ethidium bromide
staining.
+/+ +/- -/- +/+ +/- -/-
Serum Lung
SDS-PAGE
35 -
25 -
15 -
Western blotting
TN +/+ +/- -/- +/+ +/- -/-
Serum Lung
- TN
Genotyping
- KO PCR product (422 bp)- WT PCR product (300 bp)
100 -200 -300 -400 -500 -600 -
A
B
TN +/+ +/- -/-
bp
Fig. S2. Survey of TN protein abundance in various tissues. (A) Blood samples and various
tissues were harvested from normal healthy Balb/C mice and assayed for TN concentrations by
Western blotting analysis using a commercial rabbit mAb (Cat. No. ab108999, Abcam). SDS-
PAGE gel of corresponding tissue or serum proteins as sample loading controls for estimating
relative TN content per total protein. (B) Blood samples were collected from five normal healthy
Balb/C mice (7-8 weeks, 20-25 g), and serum TN content was estimated by Western blotting
analysis with reference to purified recombinant TN at various dilutions.
TN
/ P
rote
in
(AU
)
0
4
8
12
16
Bra
in
He
art
Kid
ne
y
Liv
er
Lu
ng
Se
rum
Sp
lee
n
35 -
25 -
15 -
- TN
Bra
in
He
art
Kid
ne
y
Liv
er
Lu
ng
Se
rum
Sp
lee
n
A SDS-PAGEWestern blotting
B
Se
rum
TN
(µg
/ml)
0
2
4
6
8
10
12 IQR
1.5 IQR
Median
Mean
Fig. S3. Parallel reduction of lung and serum TN content during lethal endotoxemia.
Balb/C mice were subjected to lethal endotoxemia and serum (A) and lung tissue (B) were
harvested at different time points after endotoxemia to measure TN content by Western blotting
analysis. n = 3 animals per group.
0 6 12 18 240
20
40
60
80
100
120
Lu
ng
TN
(%)
Time (h post LPS)
0 6 12 18 240
20
40
60
80
100
120S
eru
m T
N (
%)
Time (h post LPS)
A B
Fig. S4. Expression and purification of recombinant TN. (A) Amino acid sequence and
functional domains of human and murine TN. Three exons [1: 1-16; 2: 17-49, and 3: 50-181],
three predicted disulfide bonds (50-60, 77-176, and 152-168), and some key residues for each
functional domain are indicated. (B) Murine TN corresponding to amino acids 22-202 (or 1-181
excluding the 21-residue leader signal sequence) with a C-terminal histidine tag was expressed in
E. coli BL21 (DE3) pLysS cells and purified by histidine-affinity and Triton X-114 extraction to
remove contaminating endotoxins.
kDa
250 –
130 –
100 –
15 –
10 –
70 –
55 –
40 –
35 –
25 – - TN
+ D
TT
-D
TT
1 10 20 30 40 50 60 70 80 90 100
EPPTQKPKKIVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTVCLKGTKVHMKCFLAFTQTKTFHEASEDCISRGGTLGTPQTGSENDALYEYL
|.||.|:||::|||||:|.:|||||||.|.|.||||||||||.|||||||||||||.:||.|||||.||||||||||||.||||||||:..||:||:||:
ESPTPKAKKAANAKKDLVSSKMFEELKNRMDVLAQEVALLKEKQALQTVCLKGTKVNLKCLLAFTQPKTFHEASEDCISQGGTLGTPQSELENEALFEYA
← Exon 1 →← Exon 2 →
50 60 77
176
101 110 120 130 140 150 160 170 180
RQSVGNEAEIWLGLNDMAAEGTWVDMTGARIAYKNWETEITAQPDGGKTENCAVLSGAANGKWFDKRCRDQLPYICQFGIV (human)
|.||||:|:||||||||||||.||||||:.|||||||||||.||||||.|||||||||||||||||||||||||||||:||
RHSVGNDANIWLGLNDMAAEGAWVDMTGGLLAYKNWETEITTQPDGGKAENCAALSGAANGKWFDKRCRDQLPYICQFAIV (murine)
Heparin Binding α-helix Trimerization
- - - S – S - - -
- - - - - - S – S - - - - - -
S – S
152 168
Carbohydrate
Recognition Domain, CRD
B
A Amino acid sequence of mature human and murine tetranectin proteins
(without the 21- amino acid leader signal sequence)
Fig. S5. Recombinant murine TN conferred a dose-dependent protection against lethal
sepsis. Balb/C mice were subjected to lethal sepsis, then intraperitoneally administered
recombinant murine TN at sub-physiological (0.1 mg/kg, A) or supra-physiological (1.0 mg/kg,
B) doses at indicated time points. Animal survival was monitored for two weeks. n = 9 - 10
animals for each experiment, but some experiments were repeated (N = 2; A) to ensure
reproducibility.
-1 0 1 2 3 4 13 140
2
4
6
8
10
Time (days post CLP)
# o
f S
urv
ivo
rs
TN (1.0 mg/kg)
Saline
22%
20%
(+2, +24 h)
-1 0 1 2 3 4 13 140
4
8
12
16
20
Time (days post CLP)
# o
f S
urv
ivo
rs
TN (0.1 mg/kg)
Saline
60%
35%
(+2, +24 h)
A B
~ ~~ ~
~ ~~ ~
~ ~~ ~
~ ~~ ~
Fig. S6. Epitope mapping and specificity of representative mAbs raised against
recombinant human TN. (A) Dot blotting analysis of polyclonal antibodies (“pAb”) and
monoclonal antibodies (“mAbs”) using synthetic peptides corresponding to sequences shown in
Fig. 4B. Most purified mAbs specifically recognized an epitope on a particular peptide, such as
P2, P5, or P7. (B) Normal healthy human (“H”) and murine (“M”) serum proteins were resolved
by SDS-PAGE, and Western blotted with the different mAbs. Most mAbs recognized a specific
band with the predicted molecular weight of TN in the serum of normal healthy human or mouse.
pAb mAb1, 3, 9
P2 P2
P5
mAb2, 5, 6, 8
P5
mAb10
P7
P6 P7
P10
A
H M
100 -
55 -
35 -25 -
15 -
10 -
kDa
130 -
170 -
70 -
SDS-PAGE
35 -
15 -
35 -
15 -
35 -
15 -
- TN35 -
15 -
-35 -
15 -
Dot blotting analysis of mAbs using synthetic peptides
B Western blotting analysis of mAbs using human and mouse serum
mAb2 mAb5 mAb6 mAb8 mAb9
H M H M H M H M H M
Fig. S7. Cross-reactivity and epitope mapping of a panel of P5-reacting mAbs. (A) Sequence
of ten P5-overlapping peptides used for antibody epitope mapping. (B) Epitope mapping of a
panel of three P5-reacting monoclonal antibodies.
23F6 (mAb5) 25B2 (mAb6)27B12 (mAb8)
P5-overlapping peptide sequencesA
B Epitope mapping of P5-reacting MAbs by peptide dot blot
Fig. S8. Epitope sequence homology between different mammalian species. (A) Mammals
sharing similar or identical epitope sequence for P5-5-reacting mAbs. (B) Mammalian species
sharing 100% amino acid sequence identity in the epitope sequence (NDALYEYLRQ) of their
respective TN proteins.
Baboon Bear Bovine Buffalo Camel Cattle Cougar
Elephant Goat Gorilla Hedgehog Horse Human Lemur
Monkey Pig Rabbit Rhinoceros Seal Sheep Tiger
Mammals sharing identical epitope sequence (NDALYEYLRQ) B
NEALFEYARQ (Rat, 70% identity, 100% similarity)
|:||:||:||
NEALFEYARH (Murine, 60% identity, 100% similarity)
|:||:||:|:
NDALYEYLRQ (Human)
||||||||||
NDALYEYLRQ (Other mammals listed below in Panel B, 100% identity)
Epitope sequence homology between human and other mammaliar speciesA
Fig. S9. Characteristics of a panel of human TN-specific mAbs. The isotype of each mAb was
determined using the Rapid ELISA Mouse Isotyping kit (Cat. # 37503, ThermoFisher Scientific)
as per the manufacturer’s instruction. The epitope and equilibrium dissociation constant (KD)
was determined as described in the materials and methods. There are three CDRs (CDR1, CDR2,
and CDR3) on each of the heavy and light chains that collectively formulate a pocket that comes
into contact with the antigen. The antigen contact structure was generated using on-line ProABC
Prediction of Antibody Contacts Software (http://circe.med.uniroma1.it/proABC/) after inputting
respective CDR sequences of each mAb.
mAb8
IgG2b
P5-5
2.02e-9
1.48e-8
Protection
mAb6
IgG1
P5-5
9.15e-10
1.00e-7
Protection
mAb5
IgG1
P5-5
8.03e-8
weak
No Effect
mAb2
IgG2a
P5
1.14e-10
2.31e-8
Protection
Name:
Isotype:
Epitope mapping:
KD for human TN:
KD for murine TN:
Effect on Sepsis:
Prediction of antigen
contact structure
(H-bond) :
Fig. S10. IgG isotype controls did not affect sepsis lethality. Balb/C mice were subjected to
lethal sepsis and then intraperitoneally administered various IgG isotype controls at the indicated
dose (2.0 mg/kg) and time points (24 and 48 h after CLP). Animals were monitored for two
weeks.
IgG Isotype Controls (2.0 mg/kg)
60%
66% Saline
IgG2b (irrelevant IgG26)
Time (h post CLP)
0
20
40
60
80
100
Su
rviv
al (%
)
1st2nd
0 20 40 60 80 336
~ ~~ ~
~ ~~ ~
70%
80% Saline
IgG1 (murine TN-non-reactive mAb5)
n = 10; P = 0.63
0
20
40
60
80
100
Su
rviv
al (%
)
1st2nd
0 20 40 60 80 336
~ ~~ ~
~ ~
70% Saline
n = 10; P = 0.98
IgG2a (irrelevant IgG19)70%
0
20
40
60
80
100
Su
rviv
al (%
)
1st2nd
0 20 40 60 80 336
~ ~~ ~~ ~
80%
77% Saline
IgG2b (anti-P7 mAb10)
n = 9-10; P = 0.91
n = 9-10; P = 0.76
0
20
40
60
80
100
Su
rviv
al (%
)
1st2nd
0 20 40 60 80 336
~ ~~ ~~ ~
Fig. S11. Distinct effects of P2- and P5-reacting mAbs on sepsis-induced TN depletion. Male
Balb/C mice were subjected to lethal sepsis, then intraperitoneally administered a P5-reacting
mAb8 (2.0 mg/kg) or a P2-reacting mAb9 (2.0 mg/kg) at 2 and 24 h after CLP. At 28 h after
CLP, animals were euthanized to harvest blood, and serum TN concentrations were determined
by Western blotting analysis. Equivalent sample loading was verified by SDS-PAGE analysis of
serum proteins in parallel.
kDa
250 –
100 –
15 –
70 –
55 –
40 –
35 –
25 –
10 –
N CLP
+
Veh
CLP
+
mAb8
CLP
+
mAb9
SDS-PAGE
- TN
25 –
15 –
Fig. S12. Divergent effects of mAb8 and mAb9 on sepsis-induced systemic KC
accumulation. Male Balb/C mice were subjected to lethal sepsis, then intraperitoneally
administered a P5-reacting mAb8 (2.0 mg/kg) or a P2-reacting mAb9 (2.0 mg/kg) at 2 and 24 h
after CLP. At 28 h after CLP, animals were euthanized to harvest blood, and serum
concentrations of 62 different cytokines and chemokines were determined by Cytokine Antibody
Arrays. KC is the murine homolog of human GRO.
+ CLP
SDFSDF--11aa
TARCTARC
TCATCA --33
TECKTECK
TIMPTIMP--11
TNFTNF
sTNFsTNF RIRI
sTNFsTNF RIIRII
TPOTPO
VCAMVCAM --11
VEGFVEGF --11
+ +
ILIL--44
ILIL--55
ILIL--66
ILIL--99
ILIL--1010
ILIL--1212((p40/p70p40/p70))ILIL--1212((p70p70))ILIL--1313
ILIL--1717
KCKC
LeptinLeptin RR
LeptinLeptin
LIXLIX
LL--selectinselectin
LPTLPT
MCP1MCP1
MCPMCP--55
MM--CSFCSF
MIGMIG
MIPMIP--11aa
MIPMIP--11gg
MIPMIP--22
MIPMIP--33bb
MIPMIP--33aa
PFPF-
-55
ILIL--66
ILIL--99
ILIL--1010
ILIL--1212((p40/p70p40/p70))ILIL--1212((p70p70))ILIL--1313
ILIL--1717
KCKC
LeptinLeptin RR
LeptinLeptin
LIXLIX
LL--selectinselectin
LPTLPT
MCP1MCP1
MCPMCP--55
MM--CSFCSF
MIGMIG
MIPMIP--11aa
MIPMIP--11gg
MIPMIP--22
MIPMIP--33bb
MIPMIP--33aa
PFPF--44
PP--SelectinSelectin
RANTESRANTES
SCFSCF
++
-
--
--
AxlAxl
BLCBLC
CD30 LCD30 L
CD30 TCD30 T
CD40CD40
CRGCRG--22
CTACKCTACK
CXCL16CXCL16
EotaxinEotaxin
EotaxinEotaxin--22
FasFas LL
FractalkineFractalkine
GCSFGCSF
GMGM --CSFCSF
IFNIFN--gg
IGFBPIGFBP--33
IGFBPIGFBP--55
IGFBPIGFBP--66
ILIL--11aa
ILIL--11bb
ILIL--22
ILIL--33
ILIL--3 3 RbRb
-
SDFSDF--11aa
TARCTARC
TCATCA --33
TECKTECK
TIMPTIMP--11
TNFTNF
sTNFsTNF RIRI
sTNFsTNF RIIRII
TPOTPO
VCAMVCAM --11
VEGFVEGF --11
+ +
ILIL--44
ILIL--55
ILIL--66
ILIL--99
ILIL--1010
ILIL--1212((p40/p70p40/p70))ILIL--1212((p70p70))ILIL--1313
ILIL--1717
KCKC
LeptinLeptin RR
LeptinLeptin
LIXLIX
LL--selectinselectin
LPTLPT
MCP1MCP1
MCPMCP--55
MM--CSFCSF
MIGMIG
MIPMIP--11aa
MIPMIP--11gg
MIPMIP--22
MIPMIP--33bb
MIPMIP--33aa
PFPF-
-55
ILIL--66
ILIL--99
ILIL--1010
ILIL--1212((p40/p70p40/p70))ILIL--1212((p70p70))ILIL--1313
ILIL--1717
KCKC
LeptinLeptin RR
LeptinLeptin
LIXLIX
LL--selectinselectin
LPTLPT
MCP1MCP1
MCPMCP--55
MM--CSFCSF
MIGMIG
MIPMIP--11aa
MIPMIP--11gg
MIPMIP--22
MIPMIP--33bb
MIPMIP--33aa
PFPF--44
PP--SelectinSelectin
RANTESRANTES
SCFSCF
+
-+
--
--
AxlAxl
BLCBLC
CD30 LCD30 L
CD30 TCD30 T
CD40CD40
CRGCRG--22
CTACKCTACK
CXCL16CXCL16
EotaxinEotaxin
EotaxinEotaxin--22
FasFas LL
FractalkineFractalkine
GCSFGCSF
GMGM --CSFCSF
IFNIFN--gg
IGFBPIGFBP--33
IGFBPIGFBP--55
IGFBPIGFBP--66
ILIL--11aa
ILIL--11bb
ILIL--22
ILIL--33
ILIL--3 3 RbRb
+
+
-
-
-
+
-
+
- CLP
+ CLP + Ab8 + CLP + Ab9
Fig. S13. TN enhanced HMGB1 uptake and possible degradation by macrophage cultures.
Macrophage cultures were incubated with HMGB1 in the absence or presence of TN or
Dynasore at indicated concentrations for 2 h, and cellular content of HMGB1 (A) or TN (B) was
determined by Western blotting analysis, with reference to housekeeping protein β-actin.
Increased cellular HMGB1 (A) or TN (B) content was associated with the appearance of lower
molecular bands (marked by empty arrowheads) that may be indicative of possible degradation.
The high molecular bands (marked by solid arrowheads) may indicate possible TN
oligomerization.
---
+--
++-HMGB1
TN
DYN
(0.5 mg/ml)
(10.0 mg/ml)
(8.0 mM)+
+
+
100 -70 -55 -
35 -25 -
15 -
10 -
kDa
130 -
250 -
- HMGB1
- β-actin - β-actin
- TN
100 -
55 -
35 -25 -
15 -
10 -
130 -
250 -
70 -
---
+--
++-HMGB1
TN
DYN
(0.5 mg/ml)
(10.0 mg/ml)
(8.0 mM)+
+
+
A B
Fig. S14. mAb8 and dynasore inhibited the TN/HMGB1-induced increase of trypan blue
dye uptake in macrophage cultures. Murine peritoneal macrophages were stimulated with TN
(10 µg/ml) or HMGB1 (0.5 µg/ml) either alone or together in the absence or presence of mAb8
(65 µg/ml) or dynasore (10.0 µM) for 16 h, then stained with trypan blue dye. Shown are
representative fields with trypan blue-stained cells (blue). Scale bars, 50 µm.
- Control + TN + HMGB1
+ TN + HMGB1 + TN + HMGB1 + mAb8 + TN + HMGB1 + Dyn
Fig. S15. Proposed model for the mAb8-mediated protection against lethal sepsis.
Endotoxemic and septic insults cause systemic accumulation of HMGB1 and concurrent
depletion of TN, partly because circulating HMGB1 binds to TN to facilitate the endocytosis and
possible degradation of TN/HMGB1 complexes. The endocytosis of TN/HMGB1 complex may
trigger ASC oligomerization and release, resulting in possible macrophage pyroptosis and
immunosuppression that may compromise effective pathogen elimination and contribute to
septic lethality. The P5-reactive mAbs, such as mAb8, could bind to TN to interrupt its
interaction with HMGB1, thereby impairing HMGB1 endocytosis and macrophage pyroptosis,
and possibly reversing the sepsis-induced immunosuppression to confer protection against lethal
sepsis.
Endotoxemia
Sepsis
HMGB1 TN
HMGB1 / TN
Pyroptosis
mAb8
Immunosuppression
Ineffective
Pathogen Elimination
Table S1. Reagent sources.
SOURCE IDENTIFIER
Antibodies
Mouse anti-β-actin antibody Sigma-Aldrich Cat. # A1978
Rabbit mAb against the C-terminal
region (residue 150 to the C-terminus)
of human TN
Abcam Cat. # ab108999
HRP conjugated donkey anti-rabbit IgG GE Healthcare Cat. # NA934
Rabbit polyclonal IgGs against murine
ASC
Santa Cruz
Biotechnology Cat. # sc-22514
Monoclonal IgG1 against human ASC Santa Cruz
Biotechnology Cat. # sc-271054
Chemicals, Peptides, and
Recombinant Proteins
Crude bacterial endotoxin
(lipopolysaccharide, LPS) Sigma-Aldrich E. coli 0111:B4
Human serum Sigma-Aldrich Cat. # H3667
Recombinant human SAA (also termed
Apo-SAA PeproTech Cat. # 300-13
Recombinant human TN ACROBiosystems Cat. # CLB-
H5226
Human macrophage colony-stimulating
factor (M-CSF) PeproTech Cat. # SRP-3110
Dulbecco’s modified Eagle medium
(DMEM)
Invitrogen/Life
Technologies Cat. # 11995-065
OPTI-MEM I Reduced-Serum Medium ThermoFisher Scientific Cat. # 31985062
Penicillin/streptomycin Invitrogen/Life
Technologies Cat. # 15140-122
Trypan blue Invitrogen/Life
Technologies Cat. # 15250-061
Alexa Fluor 555 labeling kit A ThermoFisher Scientific Cat. # A30007
Alexa Fluor 488 labeling kit A ThermoFisher Scientific Cat. # A30006
DAPI Vector Laboratories, Inc Cat. # H-1200
Chemicals, Peptides, and
Recombinant Proteins
Human CLEC3B/tetranectin ELISA kit RayBiotech Cat. # ELH-
CLEC3B-1
LDH Assay Kit Pointe Scientific Inc Cat. # L7572
Amine sensor chip Nicoya Lifesciences Cat. # SEN-Au-
100-10-AMINE
NTA sensor chip Nicoya Lifesciences Cat. # SEN-Au-
100-10-NTA
Murine Cytokine Antibody Arrays RayBiotech Inc Cat. #. M0308003
Human Cytokine Antibody C3 Arrays RayBiotech Inc Cat. # AAH-
CYT-3-4
AST assay kit Pointe Scientific Inc. Cat. # A7561
ALT assay kit Pointe Scientific Inc. Cat. # A7526
Mice
Balb/C mice Jackson Laboratory Stock # 000651
Heterozygous TN (“CLCE3B”)-KO
mice Jackson Laboratory Stock # 027554
Software and Algorithms
ProABC Prediction of Antibody
Contacts Software
http://circe.med.uniroma
1.it/proABC/
Table S2. Demographics of 44 normal healthy controls and 45 septic patients.
Normal Sepsis
Septic
Shock
Age Group
(14-30 )
(31-55)
(56-90)
(31-55)
(56-90)
(56-90)
Sample Size (n) 15
17 12 14 17 14
Age
Range 14 - 30
33 - 55 57 - 90 31 - 55 58 - 83 62 - 87
Mean
±
SD
21
±
5
45
±
8
66
±
9
42
±
11
71
±
8
72
±
8
Gender ratio
(M/F)
7/8
11/6
6/6
5/9
6/11
4/10
Plasma [TN]
Mean
±
SD
(SEM)
10.1
±
1.8
(0.5)
10.1
±
1.4
(0.3)
9.2
±
2.2
(0.6)
3.8
±
1.7
(0.4)
3.5
±
2.1
(0.5)
3.0
±
1.2
(0.3)