LSECtin attenuates hepatic Th17 expansion in a murine model of cirrhosis and signals through the LAG-3 receptor

LSECtin attenuates hepatic Th17 expansion in a murine model of cirrhosis and signals through the LAG-3 receptor Authors Sebastián Martínez-López, Oriol Juanola, Isabel Gómez-Hurtado, …, Amaya Puig-Kröger, Esther Caparrós, Rubén Francés Correspondence [email protected] (R. Francés), [email protected] (E. Caparrós). Graphical abstract Treg Th17 Th17 Treg Treg Th17 T-cell LSEC P PP P IL-6R STAT3 STAT3 PP MHC TCR COMPLEX LAG3 LSECtin T-cell AKT PERK P RORγt Th17 supression Hepatic sinusoid CMV LSECTinOE Wild type cirrhotic Knock in cirrhotic Treg Treg Th17 Th17 Treg Th17 Th17 Th17 expansion Th17 Treg LSECtin mediated supression Tolerogenic balance Treg Th17 Th17 Repressed LSECtin Upregulated LSECtin ZAP70 Clec4g Pro-inflammatory imbalance LSEC Highlights: •We have generated a murine model of LSECtin overexpression. •LSECtin modulates the Th profile and reduces proinflammatory cell death in cirrhosis. •LSECtin attenuates Th17 differentiation through LAG3 signaling. •Restoring LSECtin positively impacts on fibrosis and serum markers of liver function. Impact and implications: LSECtin downregulation is associated with the expansion of Th17 cell subpopulation during cirrhosis. Findings highlight the crucial role of LSECtin in regulating immune responses and mitigating liver damage in cirrhosis. Restoring LSECtin expression through targeted liver-directed molecular interventions may be of therapeutic relevance in cirrhosis. https://doi.org/10.1016/j.jhepr.2025.101482 © 2025 The Author(s). Published by Elsevier B.V. on behalf of European Association for the Study of the Liver (EASL). This is an open access article under the CC BYNC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). JHEP Reports, 2025, 7, 1–12 Research article LSECtin attenuates hepatic Th17 expansion in a murine model of cirrhosis and signals through the LAG-3 receptor Sebastián Martínez-López 1,2 , Oriol Juanola 1,2,3 , Isabel Gómez-Hurtado 2,3 , Enrique Ángel-Gomis 1,2 , Paula Boix 1,2 , Anabel Fernández-Iglesias 3,4 , Alex Gallardo-Cruz 1 , Francisco Javier Cubero 3,5 , Jordi Gracia-Sancho 3,4 , Amaya Puig-Kröger 6 , Esther Caparrós 1,2,3, *, Rubén Francés 1,2,3, * JHEP Reports 2025. vol. 7 | 1–12 Background & Aims: LSECtin downregulation during cirrhosis progression is associated with adaptive T-cell expansion. We analyzed the molecular mechanism for LSECtin modulation of Th17 proliferation in an experimental cirrhosis model. Methods: A transgenic mouse model of Clec4g/LSECtin overexpression (Clec4g KI) was subjected to CCl 4 -induced cirrhosis. Cell death, liver function, and inflammation markers (n = 6/group) were evaluated. LSECtin-LAG3 signaling in Th17-differentiated spleen cells (n = 5) and LAG3 expression in livers of human individuals with cirrhosis (n = 6) were also characterized. Results: Densitometred LSECtin expression was significantly downregulated during cirrhosis in wild-type but not Clec4g-KI mice (0.6 ± 0.2 vs. 2.3 ± 0.6 AU, p = 0.001). LSECtin overexpression in cirrhotic mice resulted in less histological damage and improved liver enzyme levels (alanine aminotransferase: 152.7 ± 66.3 vs. 76.33 ± 13.33 U/L, p = 0.004). Cell-death pathway analysis revealed no differences in apoptosis markers Casp3 and Casp8 but reduced levels of necroptotic intermediates Mlkl and Ripk3. LSECtin overexpression reduced hepatic leukocyte infiltration and enriched the differentiation from inflammatory Rorgt + / IL-17 + (2.4 ± 0.8 vs. 7.7 ± 3.5% CD4 + , p = 0.043) to regulatory Foxp3 + /IL-10 + cells (3.6 ± 0.9 vs. 1.2 ± 0.7% CD4 + , p = 0.047) in cirrhotic animals. LSECtin interacted with LAG-3 on polarized spleen-derived CD4 + T cells, inhibiting Th17 differentiation (10.5 ± 2.7 vs. 6.6 ± 2.1% CD4 + , p = 0.037) by suppressing Stat3 and Zap70 pathways. LSECtin did not restrain the Th17 subset expansion during LAG3 blockade (6.6 ± 2.1 vs. 14.2 ± 4.0% CD4 + , p = 0.005). Livers of human individuals with cirrhosis showed increased LAG-3-expressing Th17 cells compared with controls (3.2 ± 1.4 vs. 0.1 ± 0.1 cells/mm 2 , p = 0.001). Conclusions: We have established a valuable murine model of LSECtin overexpression to assess its impact on hepatic inflammation during cirrhosis. LAG-3 was identified as the molecular mechanism by which LSECtin attenuates Th17 differentiation. Given LSECtin capacity to modulate the Th profile and decrease proinflammatory cell death, liver-directed molecular approaches to restore its expression may be of therapeutic interest during cirrhosis. © 2025 The Author(s). Published by Elsevier B.V. on behalf of European Association for the Study of the Liver (EASL). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Introduction Hepatic inflammation is a hallmark of advanced chronic liver disease (aCLD). 1 Microbiota changes and gut barrier dysfunction during disease progression are important contributing factors to liver inflammation. 2,3 Antigen derived from dysbiotic microbiota can reach the liver and are initially recognized by pattern recognition receptors such as Toll-like receptor (TLRs) or NOD-like receptors present in sinusoidal resident cells, namely Kupffer cells and liver sinusoidal endothelial cells (LSECs). 4 Following this recognition, different signaling pathways involved in transcription upregulation and proinflammatory cytokine secretion are activated, enabling further cellular recruitment and define the inflammatory tissue microenvironment. 5 This increased percentage of intrahepatic T helper (Th)17 cells has been associated with the progression from metabolic dysfunction-associated steatotic liver disease (MASLD) to metabolic dysfunction-associated steatohepatitis (MASH) in patients with MASLD, 6 and with the progression of chronic liver disease and the activation of profibrotic pathways in patients with HBV infection. 7 In the context of established cirrhosis, the progressive expansion of Th17 cells in the liver tissue of patients with compensated and decompensated cirrhosis has been recently been described. 8 LSECtin is an innate C-type lectin receptor expressed by LSECs that becomes crucial for modulating proinflammatory Tcell proliferation in the liver by interfering with CD44 and Lymphocyte-activation gene 3 (LAG-3)-mediated signaling. 9 The role of LSECtin in suppressing the hepatic adaptive immune response has been confirmed by the increased T-cell proliferation observed in LSECtin-deficient mice following acute liver injury. Conversely, administering recombinant LSECtin to these mice can mitigate liver damage during stages characterized by proinflammatory rather than tolerogenic responses. 10 During experimental chronic liver disease, the hepatic upregulation of proinflammatory cytokines such as tumor necrosis factor-alpha * Corresponding authors. Address: Hepatic and Intestinal Immunobiology Group, Department of Clinical Immunology, School of Medicine, Miguel Hernández University, San Juan de Alicante, Spain. Tel.: +34-965-233-796. E-mail addresses: [email protected] (R. Francés), [email protected] (E. Caparrós). https://doi.org/10.1016/j.jhepr.2025.101482 JHEP Reports, September 2025. vol. 7 | 101482 Research article or IL-6 is associated with a significantly downregulated transcriptional profile of LSECtin. 11 In fact, LSECtin expression is reduced upon stimulation of cultured primary murine LSECs with proinflammatory cytokines. 8 In this same study, we also confirmed that the signaling pathways activated by LSECtin influence the LSEC-induced secretome, modulating TLRinduced responses and contributing to a shift in the inflammatory microenvironment in the CCl 4 -cirrhotic murine model. Moreover, LSECtin is progressively downregulated in the liver of decompensated patients compared with compensated patients with cirrhosis and its loss is associated with a hepatic Th17 proinflammatory enrichment. 8 These results led us speculate that LSECtin directs Th17 downregulation and that this C-type lectin receptor retrieval may help restore LSEC immune function during cirrhosis. In fact, C-type lectin receptors have been involved in the control of Th differentiation. 12 To evaluate the mechanism for LSECtin modulation of Th17 expansion, we have generated a transgenic LSECtin overexpression mouse model. After confirming that LSECtin overexpression counterbalances the proinflammatory T cell associated immune response and attenuates liver damage during experimental cirrhosis, we explored the mechanism for LSECtin regulation of Th-cell differentiation through LAG-3 receptor engagement in vitro. Animals and methods Transgenic mice and genotyping The LSECtin overexpression (LSECtin OE ) mouse or knock-in (Ki) model was generated in the facilities of the Faculty of Health and Medical Sciences of Copenhagen University. In brief, Clec4g cDNA (ENSMUST00000062037) was cloned in the pcDNA3.1 plasmid under the control of the ubiquitous Cytomegalovirus (CMV) promoter. After releasing the CMVClec4G segment from the vector it was injected in the pronuclei of the C57BL/6NRj zygotes promoting its insertion in a random location in the mouse genome. The healthy pups positive for the insertion were selected and crossed to establish a colony. The offspring of these mice were rigorously genotyped for the inclusion of the intact transgene before being included it into the experiments (Fig. S1). No phenotypical differences were found between untreated Ki mice and untreated wild-type (Wt) mice. Genotyping was performed at weaning. DNA was extracted from ear samples and analyzed by PCR. Ear samples were digested using 25 mM NaOH and 0.2 mM EDTA. After 30 min at 98 ◦ C the digestion was neutralized with TRIS HCl 40 mM pH = 7.4. Then the DNA was isolated by vortexing and centrifugation at 13,000 rpm for 5 min at 22 ◦ C. DNA was added to the mix with the Taq PCR Master Mix Kit (201445, Qiagen, Hilden, Germany) and the primers. The result was observed using electrophoresis in a 1% agarose gel prepared with 1 × Tris-Acetate-EDTA buffer and GelRed R staining (SCT123, Merck, Rahway, New Jersey, USA). Primers used for the genotyping can be found in Table S1. Experimental cirrhosis induction Wt (C57Bl/6J) (Harlan, Barcelona, Spain) and LSECtin OE male mice were included in a 12-week study protocol. Mice were caged at a constant room temperature of 21 ◦ C and exposed to a 12 h:12 h light/dark cycle. Adult mice weighing 20–22 g were fed with standard rodent chow and treated with 0.25 mmol/L phenobarbital in tap water during the study protocol. After 4 weeks, animals were subjected to experimental cirrhosis induction with two weight-controlled doses of CCl 4 per week (Sigma-Aldrich, Madrid, Spain) administered intragastrically as described previously. 13 Control animals received mineral oil for that period. Animals were sacrificed when severely ill, and death was suspected to be imminent. Laparotomies were performed under anesthesia with isofluorane at week 12 as described. During this process blood samples were collected by cardiac puncture before perfusion. After hepatic perfusion, livers were set aside either for molecular biology and histology experiments or flow cytometry experiments. Animals received care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals. The study was approved by the Animal Research Committee of Universidad Miguel Hernández (Alicante, Spain) with approval number 2024/VSC/PEA/0180. Patients and human samples Samples from patients with cirrhosis (n = 6) and from healthy donors (n = 4) were included in the study. Available human liver samples remnants from non-lesioned non-tumorous liver tissue resections were used from healthy controls and compared with cirrhotic liver tissue explants from patients who underwent liver transplantation. The study was performed in agreement with the Declaration of Helsinki and with local and national laws. The Human Ethics Committee of the hospital approved the study procedures (HCB/2015/0624), and all participants voluntarily signed an informed written consent before inclusion in the study. CD4 isolation, Th17 differentiation, and TCR and IL-6 downstream signaling To obtain CD4 + cells for in vitro experiments, spleens were extracted from the Wt control mice of the protocol. CD4 + were obtained by magnetic cell sorting with the CD4 + T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Then, purified CD4 + cells were cultured in α -CD3 (5 μ g/ml, BD Biosciences, Franklin Lakes, New Jersey, USA) coated plates, and then exposed to pro-Th17 conditions ( α -CD28 [2.5 μ g/ml, BD Biosciences], 5 ng/ml transforming growth factor beta [TGF-β, Peprotech, Cranbury, New Jersey, United States], and 30 ng/ ml IL-6 [Peprotech]) or pro-Treg conditions ( α -CD28 [2.5 μ g/ml, BD Biosciences], 5 ng/ml TGF-β [Peprotech] and 10 ng/ml IL-2 [Peprotech]) alone or in combination with 25 μ g/ml LSECtin-fc (NovusBio, Littleton, Colorado, United States) and 25 μ g/ml α - LAG-3 (eBioC9B7W, Thermo Fisher Scientific, Waltham, Massachusetts, USA) during 5 days. After that, cells were stained for flow cytometry. As controls, cells were stimulated with α -CD3 and α -CD28 alone. Both the controls and the proTh17 or pro-Treg conditions included a control IgG of the same isotype of the constant fraction (fc) of LSECtin-fc. In parallel, to study the molecular pathways involved in this process, a fraction of CD4 + cells were exposed to α -CD3 and α -CD28 for 48 h to induce LAG-3 expression as described previously. 14 After that, all the stimulants were removed for another 24 h. After serum starvation, CD4 + were stimulated again with α -CD3 (5 μ g/ml) or IL-6 (25 ng/ml, Peprotech) in the presence or absence of LSECtin-fc (25 μ g/ml) and α -LAG-3 (25 μ g/ml) for 30 min. Then, cells were collected for protein JHEP Reports, September 2025. vol. 7 | 101482 2 LSECtin modulatory mechanism through LAG-3 extraction and T cell receptor (TCR) or IL-6 signaling was assessed by Western blot. Flow cytometry Perfused livers were digested in vivo with collagenase A (Merck) as previously described. 15 Resultant digested livers were excised, and an in vitro digestion with the same buffer containing collagenase A was performed at 37 ◦ C for 30 min. The liver cell solution was then filtered by using 100 μ M nylon strainers and collected in cold Kreb’s solution containing 25 mM HEPES. The cell suspension was centrifuged at 50 × g for 5 min, and non-parenchymal cells were separated by collecting the supernatants and then centrifuged at 800 × g for 10 min. Resultant pellets were resuspended in 10 ml of Percoll 40% and non-parenchymal cells were enriched and isolated by differential centrifugation at 800 × g for 25 min. Cells from the pellet were collected, washed with PBS without Ca 2+ and Mg, 2 and resuspended in PBS supplemented with 2% FBS and 2 mM EDTA for cytometric staining. Lymphocytes from the differentiation protocols were also collected and resuspended in the latter buffer. Then, isolated cells or treated lymphocytes were divided equally and incubated with the correspondent antibodies of each cytometry panel (Table S2). To evaluate the Th CD4 differentiation profile, cells were stimulated with phorbol myristate acetate/ionomycin (50 ng/ml and 1 μ g/ml, respectively) (Sigma-Aldrich) for 5 h, and Golgi traffic-blocked with monensin (BD Golgi STOP, BD Biosciences). Then, cells were then fixed, permeabilized, and stained intracellularly. Samples were analyzed in the Omics facility of the Instituto de Neurociencias de Alicante in a FACSAria II flow cytometer operated by FACSDiva software (BD Biosciences). Biochemical markers Serum levels of alkaline phosphatase (ALP), alanine transferase (ALT), aspartate transferase (AST), urea, albumin, total protein, cholesterol and total bile acids were determined in 200 μ l of total blood using an automatic liquid biochemistry analyzer (Skyla Vb1, CVM practice, Navarra, Spain) following manufactures instructions. Histological analysis Sirius red staining was performed using the Picro Sirus Red Stain Kit (ab150681, Abcam, Cambridge, United Kingdom) from Abcam in 5μ m sections of paraffin-embedded mouse liver according to the manufacturer’s instructions. In brief, after deparaffinization, sections were incubated in Picro Sirus red solution for 60 min followed by two quick washes in acetic acid solution and absolute alcohol. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining was performed using the TUNEL Assay Kit - HRP-DAB (ab206386) from Abcam in 5μ m sections of paraffin-embedded mouse liver according to the manufacturer’s instructions. In brief, after deparaffinization, sections were permeabilized with proteinase K and blocked with 3% hydrogen peroxide. Afterwards, the terminal deoxynucleotidyl transferase enzyme was added for 90 min. To determine the biotinylated DNA (dead cells), the sections were incubated with a streptavidin-HRP and developed with 3,3 ′ - diaminobenzidine (DAB). The slides were then counterstained with Harris hematoxylin (HH) (Leica Biosystems, Richmond Inc., Richmond, IL, USA). Immunohistochemical (IHC) and Immunofluorescence (IF) assays were carried out in 5μ m sections of paraffinembedded mouse or human liver tissue. Sections were handled following standard procedures. The slides were incubated with primary antibodies for key proteins involved in hepatic homeostasis and disease. As secondary antibodies, sections were incubated with the correspondent biotinylated antibodies (Palex Medical SA, Sant Cugat del Vallés, Spain) for IHC or with Alexa-647 modified antibodies for IF. Dilutions and antibody references can be found in Table S3. IHC slides were incubated with avidin–biotin–HRP complex ABC kit (Vector Laboratories Inc., Burlingame, CA, USA) and developed with DAB (Vector Laboratories Inc.). Nuclei were stained by incubating the sections in HH or DAPI (Thermo Fisher Scientific), respectively. As a negative control, staining was carried out in the absence of a primary antibody. Images were obtained in a camera-assisted optic Leica DMR microscope (Leica Biosystems). A semi-quantitative analysis of protein expression or a quantification of positive cells was performed using the ImageJ software (https:// rsbweb.nih.gov). Quantitative PCR analysis Total RNA was extracted using RNeasy Mini Kit (QIAgen) from liver samples. Quantitative PCRs were performed to evaluate the expression of LSECtin/Clec4g, as well as the key genes involved in hepatic disease. The reactions were performed in a 12.5 μ l PCR mixture using qScript One-Step SYBR Green RTqPCR (Quanta BioScience, Gaithesburg, MD, USA) In a CFX Connect (Bio-Rad, Hercules, CA, USA). β2-Microglobulin was used as a housekeeping gene in all gene expression analyses. Relative expression was calculated with the 2 -ΔΔCt method. Primer pairs used in the study can be found in Table S4. Western blot Liver samples or cell pellets from lymphocytes were lysed with radioimmunoprecipitation assay buffer containing cOmplete TM Protease Inhibitor Cocktail (Merck) and the phosphatases inhibitors sodium fluoride and sodium orthovanadate. Protein concentration was determined using the Bradford protein assay (EMD Millipore Corp., Billerica, MA). Thirty micrograms of protein extracts were resolved under reducing conditions on 6% to 15%, SDS-polyacrylamide gels depending on the size of the protein of interest. Then, gels were transferred to Immobilon-P membranes (Merck). After blocking, membranes were incubated with primary antibodies overnight. β-Actin (Sigma-Aldrich) was used as control. Finally, membranes were incubated with the appropriate horseradish peroxidase (HRP)- conjugated secondary antibody (Cell Signaling Technology, Leiden, The Netherlands). Immobilon Western Chemilum HRP Substrate (EMD Millipore Corp.) was used to detect the activity of the membrane-attached peroxidase. Images were obtained in ChemiDOC XRS+ operated by Image Lab software (BioRad). Protein bands were quantified by densitometry using ImageJ (https://rsbweb.nih.gov). Band densities were JHEP Reports, September 2025. vol. 7 | 101482 3 Research article A B E C D F G WT control WT cirrhosis KI cirrhosis KI control Relative expression 15 10 5 0 * *** ** Acta2 WT control WT cirrhosis KI cirrhosis KI control *** **** 0.055 Relative expression 15 10 5 0 Col1a1 WT control WT cirrhosis KI cirrhosis KI control Relative expression 35 30 25 20 15 10 5 0 **** *** * Mmp2 WT control WT cirrhosis KiI cirrhosis KI control Timp1 Relative expression 2.5 2.0 1.5 1.0 0.5 0.0 ** !"#$ % &"'% (# !"#$ )' ' * % +)+ ,)# $% &"'% (# ,)# $)''*% +)+ - .- - ./ 0.- 0./ 1.- 1./ ! "# $% 2 3(4")53#367' 3++)%& !! WT control WT cirrhosis KI cirrhosis KI control 6 4 2 0 Relative expression *** **** Tgfb1 WT control WT cirrhosis KI control Ki cirrhosis WT control WT cirrhosis KI cirrhosis KI control LSECtin 70 60 50 40 30 20 10 0 Positive area (%) **** *** *** Control Cirrhosis Control Cirrhosis Wild type KI (LSECtin OE ) LSECtin WT control WT cirrhosis KI cirrhosis KI control Positive area (%) 25 20 15 10 5 0 **** **** * Collagen WT control WT cirrhosis KI cirrhosis KI control **** *** * Positive area (%) 20 15 10 5 0 Sirus red WT control WT cirrhosis KI cirrhosis KI control 35 30 25 20 15 10 5 0 Positive area (%) **** * ** Vimentin WT control WT cirrhosis KI cirrhosis KI control **** **** ** Positive area (%) 15 10 5 0 αSMA Control Cirrhosis Control Cirrhosis Wild type KI (LSECtin OE ) n.s. WT control WT cirrhosis KI cirrhosis KI control 1 4 0 5 3 2 Relative mRNA expression *** *** * WT control WT cirrhosis KI cirrhosis KI control 1 4 0 5 3 2 Relative protein expression ** *** *** 45 40 LSECtin Actin Control Cirrhosis Control Cirrhosis Wild type KI (LSECtinOE) 4 weeks CCl 4 (2 weekly doses)Phenobarbital 12 weeks Laparatomies CMV LSECTinOE Wild type (WT) Knock-in (KI) ● Histology ● Molecular biology ● Flow cytometry Clec4g LSECtin Clec4g kDa Sirius redαSMA Col1A1 Vim Fig. 1. LSECtin overexpression in Clec4g-KI mice attenuates liver damage in experimental cirrhosis. (A) After the induction of cirrhosis by CCl 4 for 12 weeks, livers from Wt and Clec4g-KI mice (overexpression of LSECtin under the CMV promoter) were collected for different analysis. (B–D) mRNA and protein relative expression of LSECtin in total liver homogenates. Mean ± standard deviation is represented (n = 5). (E) Representative LSECtin staining in the liver and their respective quantifications. (F) Fibrosis-related staining in the liver. Representative liver images stained with Sirius Red, α -SMA, Collagen I or Vimentin and their respective JHEP Reports, September 2025. vol. 7 | 101482 4 LSECtin modulatory mechanism through LAG-3 expressed relative to total β-actin protein. Dilutions and antibody references can be found in Table S5. Statistical analysis Categorical variables were represented as frequency or percentages and continuous variables as mean ± standard deviation or median and percentile values. To verify the normality of continuous variables the Kolmogorov–Smirnov test (p <0.05) was used. Differences between groups were analyzed using the Mann–Whitney U test for continuous variables. Multiple comparisons were analyzed according to Bonferroni correction. All reported p-values were two-sided, and p-values <0.05 were considered significant. GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, USA) was used for statistical analysis and graph design. Results LSECtin overexpression attenuates liver damage during cirrhosis We induced cirrhosis in Wt and Clec4g-KI generated mice (Fig. 1A). LSECtin overexpression was confirmed at gene, protein, and tissue levels in Clec4g-KI mice both in control and in CCl 4 -induced cirrhosis (Fig. 1B–E). LSECtin expression was significantly downregulated in cirrhosis, both in Wt and Clec4g-KI generated mice. Biochemical parameters of liver function are represented in Table 1. As observed, the increment in ALT levels and total bile acids were significantly lower in Clec4g-KI cirrhotic vs. control mice than in Wt cirrhotic vs. control mice. The histological damage observed in Wt cirrhotic mice was attenuated in our Clec4g-KI cirrhotic model as shown in Fig. 1F. Gene expression of profibrogenic markers Acta2, Col1a1 and Mmp2 were reduced in the Clec4g-KI vs. Wt cirrhotic animals, whereas Timp1 and Tgfb1 remained unchanged (Fig. 1G). Cell-death related TUNEL analyses showed that apoptosis was increased during cirrhosis in Clec4g-KI and Wt cirrhotic mice compared with their controls, although it was significantly reduced in Clec4g-KI vs. Wt cirrhotic mice (Fig. 2A). Necroptosis intermediaries receptor-interacting serine/threonineprotein kinase 1 and 3 (Ripk3) and mixed lineage kinase domain like pseudokinase (Mlkl) expression were also downregulated in Clec4g-KI cirrhotic mice, reaching similar levels to those observed in control mice. Apoptotic substrates Casp3 (Caspase 3) and Casp8 (Caspase 8) showed a significant increase during cirrhosis, without differences in gene expression between both models (Fig 2B). End apoptoticand necroptotic-route products Casp3 and Mlkl were also evaluated at protein level. We observed a significant reduction of pMlkl as a validated intermediary of necroptosis pathway activation, whereas apoptosis levels, as measured by cleaved Casp 3, remained unaltered in Clec4g-KI vs. Wt mice during cirrhosis (Fig. 2C). T cell immune response is counterbalanced by LSECtin overexpression during cirrhosis Next, we set out to explore the profile of the cellular immune system infiltrating the liver during experimental cirrhosis. We observed a reduction in leukocyte infiltration in liver tissue as shown by CD45 staining (Fig. 3A) in the Clec4g-KI vs. Wt cirrhotic mice. In the T cell subpopulation characterization during cirrhosis, an increase in T CD8+ cell population was observed only in Wt mice. The CD4+ population in cirrhosis was significantly decreased in both Wt and Clec4g-KI mice. This reduction is supported by the significant reduction in gene expression levels of Ccl5 and Ccl20 in the Clec4g-KI cirrhotic mice (Fig. S2A). However, when comparing T CD4+ cells between cirrhotic models, Clec4g-KI mice showed a significant increase in this cell subset compared with Wt mice (Fig. 3B). The functional characterization of the T cell subset revealed a significant shift of their phenotype from IL-17 to IL-10expressing T cell subpopulations in the Clec4g-KI vs. Wt cirrhotic animals (Fig. 3C–E). We did not observe any changes in the CD4 + IFN-γ + T cell subpopulation between groups (Fig. S3). Accompanying the change in the CD4 + T-cell polarization, an increase in the functionally active state marked by Table 1. Median and percentile values of liver function parameters between study groups. Wild-type control (n = 6) Wild-type cirrhosis (n = 6) Knock-in control (n = 6) Knock-in cirrhosis (n = 6) Median (P25–P75) Median (P25–P75) Median (P25–P75) Median (P25–P75) Albumin (g/dl) 3.60 (3.43–3.78) 3.90 (3.83–3.98) 3.30 (3.13–3.55) 3.85 (3.80–3.90) † Total protein (g/dl) 5.05 (4.65–5.45) 5.90 (5.43–6.23)*4.70 (4.63–4.70) 5.85 (5.65–6.05) † ALP (U/L) 116 (102.25–122.25) 99.50 (96.25–103.50) 93 (91.25–94)*92 (89.50–93.75) ALT (U/L) 34 (29.75–42.75) 155 (119.75–216.50)*40 (32.75–48.75) 82 (67.50–89) ‡ AST (U/L) 106 (91–112) 152 (140.25–180)*121 (80.25–128) 137 (125–146) Cholesterol (mg/dl) 152 (139.50–156.25) 162 (142.50–212.25) 146 (142–159) 162 (146.50–173.75) Total bile acids (mmol/L) 0.50 (0.00–1.00) 41.70 (38.45–87.10)*0.50 (0.00-1.00) 13.05 (10.90–14.75) †,‡ Urea (mg/dl) 46.55 (42.48–47.85) 51.15 (49.43–52.35) 51.50 (47.63–55.35) 58.20 (56.20–62.68) Mann–Whitney U test was performed. ALP, alkaline phosphatase; ALT, alanine transaminase; AST, aspartate transferase. *p <0.05 compared with wild-type control. † p <0.05 compared with knock-in control. ‡ p <0.05 compared with wild-type cirrhosis. ◀ quantifications. (G) mRNA relative expression of Acta2, Col1a1, Mmp2, Timp1 and Tgfb1 in total liver homogenates. Mean ± standard deviation is represented (n = 5). The signal from the histological experiments was blindly measured in user-specified regions of interest (ROIs) as brown or red area percentage in respect to the total area of the ROI using ImageJ software. Mean ± standard deviation is represented (n = 6). Scale bar = 100 μ m. Mann–Whitney U test was performed. Values of p are indicated as follows: *p <0.05; **p <0.01; ***p <0.001; and ****p <0.0001. α -SMA and Acta2, Alpha smooth muscle actin; Col1a1, Collagen 1; Ki, Clec4g-KI; Mmp2, Matrix metalloproteinase-2; Tgfb1, Transforming growth factor beta 1; Timp1, tissue inhibitor of metalloproteinases 1; Vim, vimentin; Wt, wild-type. JHEP Reports, September 2025. vol. 7 | 101482 5 Research article CD69 expression and a sustained regulatory phenotype defined by the expression of PD-1 are observed in Clec4g-KI vs. Wt cirrhotic animals (Fig. 3F and G). The functional active state of CD8 + T cells is represented in Fig. 3H and I, showing a reduced percentage of CD69 + CD8 + T cells in the Clec4g-KI vs. Wt cirrhotic animals, while the sustained regulatory phenotype observed for CD4 + T cells is also present in this CD8 + T cell subpopulation. LSECtin restrains hepatic Th17 expansion in cirrhosis through LAG-3 signaling To ascertain the mechanism for LSECtin to modulate Th17 expansion in vitro, we first isolated spleen-derived CD4 + T cells from control mice and stimulated them with a differentiation cocktail with or without LSECtin-Fc and anti-LAG-3, as described in the Animals and methods section. As observed in Fig. 4A, the proinflammatory differentiation cocktail induced a significant proliferation of CD4 + IL-17 + T cells in vitro. This expansion was significantly reduced in the presence of LSECtin-Fc, although it was restored in the presence of antiLAG-3. Accordingly, supernatant levels of secreted IL-17 were significantly decreased with LSECtin and significantly increased in the presence of anti-LAG-3 (Fig. 4B). To check that LAG-3 remained expressed in T cells under experimental conditions, we confirmed its expression by flow cytometry (Fig. 4C). Ex vivo flow cytometry analysis on murine cirrhotic liver T lymphocytes confirmed the increased LAG-3 expression in CD8 + and CD4 + cells (Fig. 4D). This increment is also specifically observed in the IL-17 + subpopulation among CD4 + T cells (Fig. 4E). While LAG3 expression on CD4 + IL17 + T cells is very reduced in control mice, there is a significant increase in cirrhotic animals (Fig. 4E). However, CD4 + IL10 + T cells show a relatively high expression of LAG-3, which further increases in cirrhosis (Fig. 4F). Fig. S4 shows that in vitro polarized Tregs are further differentiated in the presence of LSECtin-Fc, and they were restrained in the presence of anti-LAG-3. Fig. S5 shows an increase in the percentage of LAG-3 + CD69+ and LAG-3 + PD-1+ in CD4 + T cells. We validated LAG-3 as a functional T cell ligand interacting with LSECtin, we evaluated relevant LAG-3 downstream signaling pathways activated in T cells. Early signaling A B C TUNEL Control Cirrhosis Control Cirrhosis Wild type KI (LSECtinOE) 55 kDa 19-17 kDa 45 kDa Control Cirrhosis Control Cirrhosis Wild type KI (LSECtin OE ) P-Mlkl Casp3 Cleaved Actin Ripk1 Relative expression 1.5 1.0 0.5 0.0 Ripk3 3 2 1 0 Relative expression ** ** Mlkl Relative expression 3 2 1 0 ** Casp3 Relative expression 3 2 1 0 ** *** Casp8 Relative expression 1.5 1.0 0.5 0.0 2.0 2.5 ** WT control WT cirrhosis KI cirrhosis WT control WT cirrhosis KI cirrhosis WT control WT cirrhosis KI cirrhosis WT control WT cirrhosis KI cirrhosis WT control WT cirrhosis KI cirrhosis TUNEL stainnig Positive cells per mm2 25 20 15 10 5 0 *** *** ** WT control WT cirrhosis KI cirrhosis 2.0 WT control WT cirrhosis KI cirrhosis 1.5 1.0 0.5 0.0 2.5 Relative expression *** ** 0.053 WT control WT cirrhosis KI cirrhosis 3 2 1 0 4 5 Relative expression ***** Casp3 cleaved P-Mlkl KI control KI control KI control KI control KI control KI control KI control KI control Fig. 2. LSECtin overexpression in Clec4g-KI mice attenuates cell death by necroptosis in the liver. (A) Representative TUNEL staining in the liver and their respective quantifications. The signal was blindly measured in user-specified ROIs as brown positive cells per area using ImageJ software. Mean ± standard deviation is represented (n = 6). Scale bar = 100 μ m. (B) mRNA relative expression of Ripk1, Ripk3, Mlkl, Casp8, and Casp3 in total liver homogenates. Mean ± standard deviation is represented (n = 5). (C) Representative relative protein expression of p-Mlkl and Casp3-cleaved by Western blot in liver tissue homogenates. Band densitometry is shown by the image as relative to actin levels. Mean ± standard deviation is represented (n = 5). Mann–Whitney U test was performed. Values of p are indicated as follows: *p <0.05; **p <0.01; and ***p <0.001. Casp3/8, Caspase 3 and 8; Ki, Clec4g-KI; Mlkl, Mixed lineage kinase domain like pseudokinase; Ripk1/3, Receptor-interacting serine/threonine-protein kinase 1 and 3, ROI, region of interest; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; Wt, wild-type. JHEP Reports, September 2025. vol. 7 | 101482 6 LSECtin modulatory mechanism through LAG-3 A B FG H I C D E RORgT FoxP3 Control Cirrhosis Control Cirrhosis Wild type KI (LSECtin OE ) 0 50K 100K 150K SSC Control 10 5 10 3 10 4 0 0 50K 100K 150K Comp-PE-Cy7-A 10 5 10 310 4 0 0 50K 100K 150K Cirrhosis 10 5 10 310 4 0 0 50K 100K 150K 10 5 10 310 4 0 IL-10-PECy7 KI WT 0 50K 100K 150K Control 10 6 10 3 10 4 0 0 50K 100K 150K 10 6 10 3 10 4 0 0 50K 100K 150K Cirrhosis 10 6 10 3 10 4 0 0 50K 100K 150K 10 6 10 3 10 4 0 SSC IL-17-APC KI WT CD45 Wt control Wt cirrhosis Ki control Ki cirrhosis Positive area (%) 25 20 15 10 5 0 WT control WT cirrhosis KI cirrhosis KI control ** * * WT control WT cirrhosis KI cirrhosis KI control 30 20 10 0 Out of singlets cells (%) CD8+ * WT control WT cirrhosis KI cirrhosis KI control CD4+ 50 40 30 0 20 10 Out of singlets cells (%) **** **** *** CD45 Composite Control Cirrhosis Control Cirrhosis Wild type KI (LSECtinOE) WT control WT cirrhosis KI cirrhosis KI control Out of CD4 + cells (%) IL-10 + 5 10 15 0 * WT control WT cirrhosis KI cirrhosis KI control Out of CD4 + cells (%) IL-17 + 1 2 5 0 3 4 * * ** Positive cells per mm 2 25 30 45 20 35 40 15 5 10 0 Foxp3 *** *** WT control WT cirrhosis KI cirrhosis KI control Rorγt * ** *** Positive cells per mm 2 15 20 35 10 25 30 5 0 WT control WT cirrhosis KI control KI cirrhosis WT control WT cirrhosis KI cirrhosis KI control 10 5 10 3 10 4 0 CD69 APC Count 59.7 13.5 57.6 33.4 ** * **** CD69 + 30 45 90 15 60 75 0 Out of CD4 + cells (%) WT control WT cirrhosis KI cirrhosis KI control 10 5 10 3 10 4 0 17.7 40.4 19.1 18.9 CD69 APC Count WT control WT cirrhosis KI cirrhosis KI control 30 45 90 15 60 75 0 Out of CD8 + cells (%) CD69 + **** ** * 10 5 10 3 10 4 0 PD-1 PE -10 3 Count 06.4 21.3 38.2 39.5 WT control WT cirrhosis KI cirrhosis KI control PD-1 + 0 20 80 40 60 Out of CD4 + cells (%) **** *** ** 105 10 3104 0 -10 3 PD-1 PE Count 09.1 39.9 24.2 28.5 WT control WT cirrhosis KI control KI cirrhosis PD-1 + 20 40 60 80 0 Out of CD8 + cells (%) **** WT control WT cirrhosis KI cirrhosis KI control 3.58 5.11 4.89 11.02 0.78 2.93 0.32 1.19 Fig. 3. LSECtin overexpression in Clec4g-KI mice attenuates T cell response in experimental cirrhosis. (A) Representative CD45 fluorescent staining in the liver and their respective quantifications. Mean ± standard deviation is represented (n = 6). (B) Flow cytometry analysis of T CD8 + and CD4 + cells from liver tissue. Mean ± standard deviation is represented (n = 5). (C, D) Representative dot plot images from flow cytometry analysis of T CD4 + cells expressing either IL-10 + or IL-17 + , respectively. Mean ± standard deviation is represented (n = 5). (E) Representative RorγT and Foxp3 staining in the liver and their respective quantifications. Mean ± standard deviation is represented (n = 6). (F, G) Representative histogram images from flow cytometry analysis of T CD4 + expressing CD69 + or PD-1 + , respectively. Mean ± standard deviation is represented (n = 5). (H, I) Representative histogram images from flow cytometry analysis of T CD8 + expressing CD69 + or PD-1 + , respectively. Mean ± standard deviation is represented (n = 5). Signal in histological staining was blindly measured in user-specified ROIs as positive area percentage or positive cells in respect to the total area of the ROI using ImageJ software. Scale bar = 100 μ m. For flow cytometry, data are expressed as percentage of positive cells of the previous gate. Mann–Whitney U test was performed. Values of p are indicated as follows: *p <0.05; **p <0.01; ***p <0.001; and ****p <0.0001. CD, Cluster of differentiation; Foxp3, Forkhead box P3; Ki, Clec4g-KI; Pd1, Programmed Death-1; Rorγt, RAR-related orphan receptor gamma; Wt, wild-type. JHEP Reports, September 2025. vol. 7|101482 7 Research article A D BC 1,500 Supernatant levels (pg/ml) 500 1,000 0 ** ** *** αCD3 + αCD28 Diff. C Diff. C + LSECtin Diff. C + LSECtin + αLAG3 IL-17 IL-17 + 0.39 0 500 1,000 10 2 10 3 10 4 10 1 10 0 FSC-A IL-17-APC αCD3 + αCD28 10 2 10 3 10 4 10 1 10 0 0 500 1,000 IL-17 + 11.0 Diff. cocktail IL-17 + 6.88 0 500 1,000 10 2 10 3 10 4 10 1 10 0 Diff. cocktail + LSECtin Diff. cocktail + LSECtin + αLAG3 0 500 1,000 10 2 10 3 10 4 10 1 10 0 IL-17 + 15.2 0 5 10 15 20 25 αCD3 + αCD28 Diff. C Diff. C + LSECtin Diff. C + LSECtin + αLAG3 Out of CD4 + cells (%) ** * IL-17+ E F 30 40 50 60 70 80 Out of CD8 + cells (%) *** 0 10 20 LAG3 + Control Cirrhosis Control Cirrhosis 30 40 50 60 70 Out of CD4 + cells (%) 0 10 20 ** LAG3 + 0.17 75.2 CD8 8.16 10 2 10 3 10 4 10 1 10 0 10 2 10 3 10 4 10 1 10 0 CD8-BV510 CD4 16.5 CD4-FITC Control 0.46 73.6 10 2 10 3 10 4 10 1 10 0 10 2 10 3 10 4 10 1 10 0 CD8 16.6 CD4 9.35 Cirrhosis LAG3-BV421 Count 19.4 47.9 10 2 10 3 10 4 10 1 10 0 CD8 LAG3-BV421 Count 10 2 10 3 10 1 10 0 14.4 39.6 0 10 3 10 4 10 5 Cell count LAG3-APC Control CD3 + CD28 Post-starving 0 10 3 10 4 10 5 Cell count LAG3-APC Control CD3 + CD28 Diff. cocktail 0.040 53.8 44.2 il17 1.95 10 2 10 3 10 4 10 1 0 10 2 10 3 10 4 10 1 -10 1 IL-17-APC Control 0 0.13 38.5 56.2 il17 5.19 Cirrhosis 10 2 10 3 10 4 10 1 -10 1 0 10 2 10 3 10 4 10 1 0 CD4-FITC IL-17 + 5 10 15 20 0 Out of CD4 + cells (%) * Control Cirrhosis LAG3 + Control Cirrhosis 45 60 75 90 0 15 30 Out of CD4 IL-17 + cells (%) ** LAG3-BV421 Count 10 2 10 3 10 4 10 1 10 0 11.8 54.7 Th17 0.27 46.7 50.4 IL10 2.54 Control 10 2 10 3 10 4 10 1 10 0 10 2 10 3 10 4 0 0.44 50.2 37.3 IL10 12.1 Cirrhosis 10 2 10 3 10 4 0 10 2 10 3 10 4 10 1 10 0 IL-10-PECy7 CD4-FITC Count LAG3-BV421 10 2 10 3 10 4 10 1 10 0 Treg 82.3 98.7 Control Cirrhosis Control Cirrhosis 5 10 15 20 0 Out of CD4 + cells (%) IL-10 + *** LAG3 + 50 100 150 0 Out of CD4 IL-10 + cells (%) **** Fig. 4. LSECtin restrains hepatic Th17 expansion in cirrhosis through LAG-3. (A) Representative dot plot images from flow cytometry analysis of T CD4 + cells expressing IL-17 after different conditions in the differentiation protocol. Data are expressed as percentage of positive cells of the previous gate. Mean ± standard deviation is represented (n = 5). (B) IL-17 levels in lymphocyte culture supernatants. Mean ± standard deviation is represented (n = 5). (C) Representative histogram from flow cytometry analysis of T CD4 + expressing LAG-3 + indicating that cells maintain LAG3 expression throughout the experiment. (D) Percentage of LAG-3 expression out of hepatic CD8 + and CD4 + T cells from control (n = 5) and cirrhotic animals (n = 5). Mean ± standard deviation is represented. (E) Percentage of IL-17+ out of hepatic CD4+ T cells and percentage of LAG-3+ out of hepatic CD4+ IL-17+ T cells from control (n = 5) and cirrhotic animals (n = 5). Mean ± standard deviation JHEP Reports, September 2025. vol. 7 | 101482 8 LSECtin modulatory mechanism through LAG-3