Chitinase 3-like-1 contributes to acetaminophen-induced liver injury by promoting hepatic platelet recruitment

  1. Zhao Shan
  2. Leike Li
  3. Constance Lynn Atkins
  4. Meng Wang
  5. Yankai Wen
  6. Jongmin Jeong
  7. Nicolas F Moreno
  8. Dechun Feng
  9. Xun Gui
  10. Ningyan Zhang
  11. Chun Geun Lee
  12. Jack A Elias
  13. William M Lee
  14. Bin Gao
  15. Fong Wilson Lam
  16. Zhiqiang An  Is a corresponding author
  17. Cynthia Ju  Is a corresponding author
  1. Department of Anesthesiology, UTHealth McGovern Medical School, United States
  2. Center for Life Sciences, School of Life Sciences, Yunnan University, China
  3. Texas Therapeutics Institute, UTHealth McGovern Medical School, United States
  4. Laboratory of Liver Disease, National Institute on Alcohol Abuse and Alcoholism, NIH, United States
  5. Molecular Microbiology and Immunology, Brown University, United States
  6. Division of Medicine and Biological Sciences, Warren Alpert School of Medicine, Brown University, United States
  7. Division of Digestive and Liver Diseases, Department of Internal Medicine, University of Texas Southwestern Med School, United States
  8. Division of Pediatric Critical Care Medicine, Baylor College of Medicine, United States
  9. Center for Translation Research on Inflammatory Diseases, Michael E. DeBakey Veterans Affairs Medical Center, United States
8 figures, 2 tables and 3 additional files

Figures

Chitinase 3-like-1 (Chi3l1) is upregulated and plays a critical role in acetaminophen-induced liver injury (AILI).

(A) Immunohistochemical (IHC) staining for Chi3l1 in normal liver biopsies (Normal) and those from patients with AILI (Patient). Images shown are representative of 10 samples/group. Scale bar, 250 …

Figure 2 with 1 supplement
Chitinase 3-like-1 (Chi3l1) contributes to acetaminophen-induced liver injury (AILI) by promoting hepatic platelet recruitment.

(A) Immunohistochemical (IHC) staining to detect platelets (CD41+) in healthy liver biopsies (Normal) and those from patients with AILI (Patient). Scale bar, 250 μm (n = 10/group). (B) Male C57B/6 …

Figure 2—figure supplement 1
Depletion of platelets by anti-CD41 antibody reduces hepatic platelets recruitment.

Male C57B/6 mice were treated with control IgG (Ctrl IgG) or an anti-CD41 antibody (α-CD41 Ab) either 3 hr before (pre-) or 3 hr after (post-) APAP administration. Immunofluorescence (IF) staining …

Figure 3 with 3 supplements
Chitinase 3-like-1 (Chi3l1) functions through its receptor CD44.

(A) Immunoprecipitation with anti-CD44 antibody was performed using liver homogenates obtained from wild-type (WT) and Cd44-/- mice treated with acetaminophen (APAP) for 2 hr. Input proteins and …

Figure 3—figure supplement 1
Chitinase 3-like-1 (Chi3l1) promotes hepatic platelet recruitment and acetaminophen-induced liver injury (AILI) through CD44 expressing on Mɸs.

(A–C) Chil1-/- mice reconstituted with recombinant mouse Chi3l1 (rmChi3l1) were treated with either Ctrl IgG or α-CD44 Ab 30 min prior to acetaminophen (APAP) challenge. (A) Serum levels of ALT and …

Figure 3—figure supplement 2
Deletion of neither chitinase 3-like-1 (Chi3l1) nor CD44 affects acetaminophen (APAP) bio-activation.

Male wild-type (WT), Chil1-/-, Cd44-/- mice were treated with APAP (n = 3 mice/group). (A) glutathione (GSH) levels in the liver were measured at indicated time points by HPLC. (B) Hepatic protein …

Figure 3—figure supplement 3
Female Chil1-/- and Cd44-/- mice develop reduced liver injury compared to female wild-type (WT) mice.

Female WT, Chil1-/- and Cd44-/- mice were treated with acetaminophen (APAP). Serum ALT levels were measured at 6 and 24 hr after APAP treatment (n = 6–8 mice/group). One-way ANOVA was performed.

Hepatic Mɸs promote platelet recruitment.

(A) Immunohistochemical (IHC) staining for macrophages (CD68+) and platelets (CD41+) in normal liver biopsies (Normal) and those from patients with AILI (Patient) (n = 10/group). Scale bar, 25 μm. (B

Figure 5 with 1 supplement
Chitinase 3-like-1 (Chi3l1)/CD44 signaling in Mɸs upregulates podoplanin expression and platelet adhesion.

(A) Male WT, Chil1-/-, Cd44-/- mice were treated with acetaminophen (APAP) (n = 4 mice/group). After 3 hr, mice were sacrificed and Mɸs were isolated to measure mRNA levels of various adhesion …

Figure 5—figure supplement 1
Podolanin expressing on Mɸs mediates interactions with platelets.

Mɸs were isolated from wild-type (WT) mice treated with acetaminophen (APAP) for 3 hr. The cells were treated in vitro with either control IgG (Ctrl IgG) or an anti-podoplanin antibody (α-podoplanin …

Evaluation of the therapeutic potential of targeting chitinase 3-like-1 (Chi3l1) in the treatment of acetaminophen-induced liver injury (AILI).

(A–C) Male C57B/6 mice were treated with acetaminophen (APAP) for 3 hr, followed by intraperitoneally (i.p.) injection of either a control IgG (Ctrl IgG) or an anti-mouse Chi3l1 Ab (α-mChi3l1 Ab, …

Schematic summary of the main findings.

Acetaminophen (APAP) overdose induces chitinase 3-like-1 (Chi3l1) expression, which binds CD44 on Mɸs and promotes Mɸs-mediated platelets recruitment through podoplanin/Clec-2 (C-type lectin-like …

Author response image 1
Deletion of Chi3l1- or CD44 does not affect liver recovery after APAP-induced injury.

Male WT, Chil1-/- and Cd44-/- mice were treated with APAP. Serum ALT levels were measured at 6hrs, 24hrs, 48hrs and 72hrs after APAP treatment (n=5-8 mice/group). One-way ANOVA was performed.

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Genetic reagent (Mus musculus)C57BL/6JJackson Laboratory,
PMID:14759567
Stock #:000664
MGI Cat# 3849035, RRID:MGI:3849035
Genetic reagent (Mus musculus)Cd44-/- mice (also called Cd44tm1Hbg/Cd44tm1Hbg)Jackson LaboratoryStock #:005878
MGI Cat# 4941902, RRID:MGI:4941902
Genetic reagent (Mus musculus)Chil1-/-micePMID:19414556MGI #:3846223
RRID:MGI:3846223
Dr Jack A. Elias (Brown University)
Chemical compound, drugAcetaminophenSigma-AldrichA7085210 mg/kg for male mice, 325 mg/kg for female mice
Peptide, recombinant proteinRecombinant mouse Chi3l1Sino Biological50929-M08H500 ng/mouse in 100 μl PBS
Commercial assay or kitALT diagnostic assay kitTeco Diagnostics,A526-120
AntibodySyrian hamster polyclonal IgG Ctrl IgG for anti-podoplanin antibodyBioxcell InvivoMabBE0087,
RRID:AB_1107782
100 μg/mouse
AntibodySyrian hamster monoclonal anti-mouse podoplanin antibodyBioxcell InvivoMabBE0236,
RRID:AB_2687718
100 μg/mouse
AntibodyRat monoclonal Ctrl IgG for anti-mouse CD41 antibodyBD Biosciences553922,
Clone R334,
RRID:AB_479672
2 mg/kg
AntibodyRat monoclonal anti-mouse CD41 antibodyBD Biosciences553847, Clone MWReg 30,
RRID:AB_395084
2 mg/kg, 1:200 for IF
Peptide, recombinant proteinRecombinant human Chi3l1Sino Biological11227-H08H1 μg/mouse in 100 μl
AntibodyRabbit polyclonal anti-human CD41Proteintech24552–1-AP,
RRID:AB_2879604
1:200 for IHC
AntibodyMouse monoclonal anti-human CD68Thermo FisherMA5-13324,
RRID:AB_10987212
1:100 for IHC
AntibodyRabbit polyclonal anti-human Chi3l1Proteintech12036–1-AP,
RRID:AB_2877819
1:100 for IHC
AntibodyRat monoclonal anti-mouse F4/80, Alexa 647 conjugatedBiolegend123122,
RRID:AB_893480
1:100 for IF
AntibodyRat monoclonal anti-mouse CD44abcamab112178, clone KM81,
RRID:AB_10864553
1:200 for IF
AntibodyGolden Syrian Hamster monoclonal anti-mouse podoplaninNovus, biologicalNB600-1015,
RRID:AB_2161937
1:100 for IF
AntibodyRabbit polyclonal anti-mouse Clec-2Biorbytorb312182,
RRID:AB_2891123
1:100 for IF
AntibodyDonkey anti-rat polyclonal immunoglobulin, Alexa 488-conjugatedInvitrogenA-21208,
RRID:AB_141709
1:1000 for IF
AntibodyGoat anti-rabbit polyclonal immunoglobulin, Alexa 488-conjugatedInvitrogenA-11034,
RRID:AB_141709
1:1000 for IF
AntibodyGoat anti-rabbit polyclonal immunoglobulin, Alexa 594-conjugatedInvitrogenA-11012,
RRID:AB_141359
1:1000 for IF
AntibodyGoat anti-hamster polyclonal immunoglobulin, Alexa 594-conjugatedInvitrogenA-21113,
RRID:AB_2535762
1:1000 for IF
AntibodyHoechstInvitrogenH3570,
RRID:AB_10626776
1:10000 for IF
Peptide, recombinant proteinTRITC-labeled AlbuminSigma-AldrichA2289-10MG
RRID:AB_2891111
10 μl/mouse for intravital microscopy
AntibodyRat monoclonal anti-mouse anti-F4/80 antibody, BV421-labeledBiolegend123132,
RRID:AB_11203717
15 μl/mouse for intravital microscopy
AntibodyRat monoclonal anti-mouse CD41 antibody, DyLight 649-labeledemfret ANALYTICSX649,
RRID:AB_2861336
30 μl/mouse for intravital microscopy
Table 1
Real-time PCR primers used.
GeneForward (F)/reverse (R) primerPrimer sequences
PdpnFACCGTGCCAGTGTTGTTCTG
RAGCACCTGTGGTTGTTATTTTGT
Psgl-1FGAAAGGGCTGATTGTGACCCC
RAGTAGTTCCGCACTGGGTACA
Cd40FTGTCATCTGTGAAAAGGTGGTC
RACTGGAGCAGCGGTGTTATG
McamFGTGGCGTTGACATCGTTGG
RCTATGTACTTCGTATGCAGGTCG
Icam-1FGTGATGCTCAGGTATCCATCCA
RCACAGTTCTCAAAGCACAGCG
FcrFAGGGCCTCCATCTGGACTG
RGTGGTTCTGGTAATCATGCTCTG
Lfa1FCCAGACTTTTGCTACTGGGAC
RGCTTGTTCGGCAGTGATAGAG
VwfFCTCTTTGGGGACGACTTCATC
RTCCCGAGAATGGAGAAGGAAC

Additional files

Source data 1

Source data for all numerical bar graph shown in the manuscript including figure supplements.

https://cdn.elifesciences.org/articles/68571/elife-68571-data1-v2.xlsx
Supplementary file 1

Representative list of potential chitinase 3-like-1 (Chi3l1)-interacting proteins detected by mass spectrometry.

Non-parenchymal cells were isolated from C57B/6 mice treated with acetaminophen (APAP) for 3 hr and the cell lysate was incubated with rmChi3l1 overnight. Proteins potentially bound to rmChi3l1 were immune-precipitated with an anti-His antibody and subjected to mass spectrometry analyses.

https://cdn.elifesciences.org/articles/68571/elife-68571-supp1-v2.xlsx
Transparent reporting form
https://cdn.elifesciences.org/articles/68571/elife-68571-transrepform-v2.docx

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