Chitinase 3-like-1 Contributes to Acetaminophen-induced Liver Injury by Promoting Hepatic Platelet Recruitment

Hepatic platelet accumulation contributes to acetaminophen (APAP)-induced liver injury (AILI). However, little is known about the molecular pathways involved in platelet recruitment to the liver and whether targeting such pathways could attenuate AILI. The present study unveiled a critical role of chitinase 3-like-1 (Chi3l1) in hepatic platelet recruitment during AILI. Increased Chi3l1 and platelets in the liver were observed in patients and mice overdosed with APAP. Compared to wild-type (WT) mice, Chi3l1-/- mice developed attenuated AILI with markedly reduced hepatic platelet accumulation. Mechanistic studies revealed that Chi3l1 signaled through CD44 on macrophages to induce podoplanin expression, which mediated platelet recruitment through C-type lectin-like receptor 2. Moreover, APAP treatment of CD44-/- mice resulted in much lower numbers of hepatic platelets and liver injury than WT mice, a phenotype similar to that in Chi3l1-/- mice. Recombinant Chi3l1 could restore hepatic platelet accumulation and AILI in Chi3l1-/- mice, but not in CD44-/- mice. Importantly, we generated anti-Chi3l1 monoclonal antibodies and demonstrated that they could effectively inhibit hepatic platelet accumulation and AILI. Overall, we uncovered the Chi3l1/CD44 axis as a critical pathway mediating APAP-induced hepatic platelet recruitment and tissue injury. We demonstrated the feasibility and potential of targeting Chi3l1 to treat AILI.

5 Chi3l1 is upregulated and plays a critical role in AILI. Although elevated serum levels of 120 Chi3l1 has been observed in chronic liver diseases, [13,[17][18][19] modulations of Chi3l1 levels 121 during acute liver injury have not been reported. Our data demonstrated, for the first time, that 122 compared with healthy individuals, patients with AILI displayed higher levels of Chi3l1 in the 123 liver and serum ( Figures 1A, B). Similarly, in mice treated with APAP, hepatic mRNA and serum 124 protein levels of Chi3l1 were upregulated ( Figures 1C, D). To determine the role of Chi3l1 in 125 AILI, we treated wild-type (WT) mice and Chi3l1-knockout (Chi3l1 -/-) mice with APAP. Compared 126 with WT mice, serum ALT levels and the extent of liver necrosis were dramatically lower in 127 Chi3l1 -/mice ( Figures 1E, F). Moreover, administration of recombinant mouse Chi3l1 protein 128 (rmChi3l1) to Chi3l1 -/mice enhanced liver injury to a similar degree observed in APAP-treated 129 WT mice (Figures 1E,F). These data strongly suggest that Chi3l1 contributes to AILI.

131
Chi3l1 contributes to AILI by promoting hepatic platelet recruitment. Thrombocytopenia is 132 often observed in patients with APAP overdose. [6,7,21] We hypothesized that this 133 phenomenon may be attributed to the recruitment of platelets into the liver. We performed 134 immunohistochemical (IHC) staining of liver biopsies from patients with APAP-induced liver 135 failure and found markedly increased numbers of platelets compared with normal liver tissues 136 ( Figure 2A). Similarly, in mice treated with APAP, a marked increase of platelets in the liver was 137 observed by intravital microscopy ( Figure 2B). It is reported that depletion of platelets prior to 138 APAP treatment can prevent liver injury in mice. [11] Our data demonstrated that even after that Chi3l1 might be involved in platelet recruitment to the liver during AILI. To examine this 143 hypothesis, we detected platelets in the liver by IHC using anti-CD41 antibody. Comparing with 144 WT mice, we observed much fewer platelets in the liver after APAP treatment ( Figure 2E).

6
Moreover, administration of rmChi3l1 to Chi3l1 -/mice restored hepatic platelet accumulation 146 similar to APAP-treated WT mice ( Figure 2E). These data suggest that Chi3l1 plays a critical 147 role in promoting hepatic platelet accumulation, thereby contributing to AILI.

148
Chi3l1 functions through its receptor CD44. To further understand how Chi3l1 is involved in 149 platelet recruitment, we set out to identify its receptor. We isolated non-parenchymal cells 150 (NPCs) from WT mice at 3h after APAP treatment and incubated the cells with His-tagged 151 rmChi3l1. The cell lysate was subjected to immunoprecipitation using an anti-His antibody. The 152 "pulled down" fraction was subjected to LC/MS analyses, and a partial list of proteins identified 153 is shown in Supplementary Table 1. Among the potential binding proteins, we decide to further 154 investigate CD44, which is a cell surface receptor expressed on diverse mammalian cell types,

165
To investigate the role of CD44 in mediating the function of Chi3l1, we treated CD44 -/mice with 166 rmChi3l1 simultaneously with APAP challenge. We found that rmChi3l1 had no effect on platelet 167 recruitment or AILI in CD44 -/mice ( Figures 3D-F). This is in stark contrast to restoring platelet 168 accumulation and increasing AILI by rmChi3l1 treatment in Chi3l1 -/mice ( Figure 1E, F; 2E).

169
However, these effects of rmChi3l1 in Chi3l1 -/mice were abrogated when CD44 was blocked 170 by using an anti-CD44 antibody ( Supplementary Figures 2A-C). Together, these data 7 demonstrate a critical role of CD44 in mediating Chi3l1-induced hepatic platelet accumulation 172 and AILI. 173 174 CYP2E1-mediated APAP bio-activation to form N-acetyl para quinoneimine (NAPQI) and the 175 detoxification of NAPQI by glutathione (GSH) are important in determining the degrees of AILI. [5] 176 Although unlikely, there is a possibility that the phenotypes observed in Chi3l1 -/and CD44 -/-177 mice were due to the effects of gene deletion on APAP bio-activation. To address this concern, 178 we compared the levels of GSH, liver CYP2E1 protein expression, and NAPQI-protein adducts 179 among WT, Chi3l1 -/and CD44 -/mice (Supplementary Figures 3A-C). However, we did not 180 observe any difference, suggesting that Chi3l1 or CD44 deletion does not affect APAP bio-181 activation and its direct toxicity to hepatocytes.

183
Hepatic Mɸs promote platelet recruitment. To further identify the cell type on which Chi3l1 184 binds to CD44, we incubated liver NPCs with His-tagged rmChi3l1. We found that almost all 185 CD44 + Chi3l1 + cells were F4/80 + Mɸs (Supplemental Figure 2D). This finding suggested the 186 possible involvement of hepatic Mɸs in platelet recruitment. We performed IHC staining of liver 187 biopsies from AILI patients and observed co-localization of Mɸs (CD68+) and platelets (CD41+) 188 ( Figure 4A). In the livers of APAP-treated mice, adherence of platelets to Mɸs was also 189 observed by IHC ( Figure 4B) and intravital microscopy ( Figure 2B). Quantification of the staining 190 confirmed that there were higher numbers of platelets adherent to Mɸs than to LSECs after 191 APAP challenge ( Figure 4B).

192
To further investigate the role of hepatic Mɸs in platelet recruitment during AILI, we performed

241
Although NAC greatly reduces morbidity and mortality from ALF due to APAP overdose, the 242 death rate and need for liver transplantation remain unacceptably high. While elucidating the 243 underlining biology of Chi3l1 in AILI, we also generated monoclonal antibodies specifically 244 recognizing either mouse or human Chi3l1. We screened a panel of anti-mouse Chi3l1 245 monoclonal antibodies (α-mChi3l1 mAb) to determine their efficacies in attenuating AILI. We 246 injected WT mice with an α-mChi3l1 mAb or IgG at 3h after APAP challenge. Our data showed To evaluate the potential of targeting Chi3l1 as a treatment for AILI in humans, we screened all 251 of the α-hChi3l1 mAb we generated by IHC staining of patients' liver biopsies (data not shown) 252 and selected the best clone for in vivo functional studies. Because the amino acid sequence 253 homology between human and mouse Chi3l1 is quite high (76%), we treated Chi3l1 -/mice with 254 rhChi3l1. We found that rhChi3l1 was as effective as rmChi3l1 in promoting platelet recruitment

257
Together, these data indicate that monoclonal antibody-based blocking of Chi3l1 may be an 258 effective therapeutic strategy to treat AILI, and potentially other acute liver injuries.

272
The elevation of serum levels of Chi3l1 has been observed in various liver diseases, [13,[17][18][19] 273 but studies of its involvement in liver diseases have only begun to emerge. There are several 274 reports describing a role of Chi3l1 in models of chronic liver injuries caused by alcohol,

293
We identified hepatic Mɸs as a key player in promoting platelet recruitment to the liver during 294 AILI. Given the involvement of platelets in AILI, this finding would suggest that hepatic Mɸs also 295 contribute to liver injury. The role of hepatic Mɸs in AILI has been a topic of debate and the 296 current understanding is confined by the limitation of the methods used to deplete these cells.

414
Intravital confocal microscopy. Mice were prepared for intravital microscopy as previously 415 described. [48] Briefly, mice were anesthetized using pentobarbital and underwent tracheostomy 416 (to facilitate breathing) and internal jugular catheterization (for antibody administration) followed 417 by liver exteriorization as described by Marques et al,[49]

431
Image analysis of intravital microscopy experiments. The images were then analyzed by a 432 blinded investigator to assess platelet area. Eleven to fifteen 1-minute fields of view (1X optical 433 zoom) were analyzed per mouse using FIJI/ImageJ software. Background noise was removed 434 using a Guassian filter (1 pixel) for all channels prior to analysis. Vascular area was measured 435 in each field using the region of interest (ROI) selection brush in the TRITC (albumin) channel.

436
The platelet area within the vascular ROI was then determined using threshold of the DyLight