Mindin orchestrates the macrophage-mediated resolution of liver fibrosis in mice

Mindin orchestrates the macrophage-mediated resolution of liver fibrosis in mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mindin orchestrates the macrophage-mediated resolution of liver fibrosis in mice Yong dong Huang, Xian ling Zhao, Xiao mei Ou yang, Ying Lin, Xiao shen Cheng, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5155041/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Apr, 2025 Read the published version in Hepatology International → Version 1 posted 5 You are reading this latest preprint version Abstract Background & Aims: Liver disease that progresses to cirrhosis is an enormous health problem worldwide. The extracellular matrix protein Mindin is known to have immune functions, but its role in liver homeostasis remains largely unexplored. We aimed to characterize the role of Mindin in the regulation of liver fibrosis. Approach & Results: Mindin was upregulated in mice with carbon tetrachloride (CCl 4 ) or thioacetamide (TAA)-induced liver fibrosis, and was primarily expressed in hepatocytes. Global Mindin knockout mice were generated, which were susceptible to liver fibrosis. Notably, Mindin failed to activate hepatic stellate cells directly; however, it played a role in promoting the recruitment and phagocytosis of macrophages, and caused a phenotypic switch toward restorative macrophages during liver fibrosis. Furthermore, Mindin was found to bind to the αM-I domain of CD11b/CD18 heterodimeric receptors. To further explore this mechanism, we created Mindin and CD11b double-knockout (DKO) mice. In DKO mice, phagocytosis was further reduced, and liver fibrosis was markedly exacerbated. Conclusions Mindin promotes the resolution of liver fibrosis and the Mindin/CD11b axis might represent a novel target for the macrophage-mediated regression of liver fibrosis. Graphical abstract: extracellular matrix hepatic stellate cell flow cytometry migration Ly6C MMP phagocytosis phenotypic switch restorative macrophages CD11b DKO Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Liver fibrosis, which is characterized by the accumulation of extracellular matrix (ECM), represents a reversible wound-healing response to acute or chronic injury ( 1 ). This can progress to cirrhosis and hepatocellular carcinoma, which are accompanied by severe impaired liver function. Therefore, a better understanding of the physiologic and pathologic processes involved in fibrosis should assist with the development of new therapies for liver fibrosis. The ECM is a highly dynamic network that plays essential roles in maintaining normal homeostasis. The dysregulation of ECM composition and structure results in the development and progression of multiple diseases, including liver fibrosis ( 2 ). The activation of hepatic stellate cells (HSCs) enhances the migration and deposition of ECM components ( 3 ). The expression of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) in the liver plays crucial roles in both fibrogenesis and fibrinolysis( 4 ). The liver is a central immunological organ that contains large numbers of innate immune cells, and particularly macrophages. Macrophages play a crucial role in liver homeostasis and have been proposed to be central in the pathogenesis of chronic liver injury( 5 ). They play dual roles in the process of liver fibrosis, because they can promote fibrogenesis, but also support the regression of fibrosis ( 6 ). According to the expression of a surface marker, lymphocyte antigen 6 complex locus C (Ly6C), hepatic macrophages can be classified as Ly6C-high and Ly6C-low types( 7 ). Ly6C hi macrophages are recruited from the circulation and are critical for inflammation and fibrogenesis, whereas Ly6C lo macrophages are responsible for the suppression of inflammation and mediating the resolution of fibrosis ( 8 ). Specific molecular signals in pathological microenvironment promote the regression of liver fibrosis by triggering the transition of macrophages from an inflammatory to a restorative phenotype. A reduction in damage-associated molecular patterns expression ( 8 ), phagocytosis ( 9 , 10 ), and autophagy ( 11 ) regulate the phenotypic switch of the macrophages. Restorative macrophages are the primary source of MMPs, which support both the degradation of ECM and the resolution of fibrosis ( 8 , 12 ). The ECM protein Mindin/SPON2, serves as a novel integrin ligand and is critical for the recruitment of inflammatory cells, especially neutrophils and macrophages ( 13 ). Mice lacking Mindin exhibit impaired resolution of bacterial infections, and Mindin-deficient macrophages fail to respond to substantial microbial stimulation. In addition, Mindin plays an important role in multiple fibrotic diseases, such as cardiac fibrosis ( 14 ), renal fibrosis ( 15 ), and cutaneous fibrogenesis ( 16 ). However, there have been few studies of the role of Mindin in liver homeostasis, and therefore its role in liver fibrosis has not been characterized. In this study, we aimed to characterize the role of Mindin in liver fibrosis using two mouse models: repeated CCl 4 and TAA administration. We found that hepatocyte-derived Mindin causes macrophage migration and promotes the phenotypic switching of macrophages by binding to CD11b receptors. This upregulates the production of the collagenase MMP-9, which promotes the regression of hepatic fibrosis. The findings demonstrate that Mindin facilitates the regression of liver fibrosis, which may inform the search for therapies for chronic liver disease. Materials and methods Mice Mindin −/− mice were generated using the CRISPR-Cas9 system in collaboration with the State Key Laboratory of Cellular Stress Biology at Xiamen University, China. CD11b −/− mice were purchased from the Jackson Laboratory, USA (B6.129S4-Itgamtm1Myd/J, Strain ID: B005273). Mindin −/− mice were crossed and bred with CD11b −/− mice to obtain homozygous DKO mice. Models of liver fibrosis For the CCl 4 -induced model of liver fibrosis, 6–8-week-old mice underwent intraperitoneal (i.p.) injection with carbon tetrachloride (diluted 1:4 in olive oil) or vehicle (olive oil) at a dose of 2.5 mL/kg twice a week for 7 weeks. For the TAA model, liver fibrosis was induced by i.p. injections of TAA in saline (200 mg/kg) or vehicle (saline) twice weekly for 6 weeks. The mice were euthanized 72 hours after the final i.p. injection. Statistical analysis Statistical analysis was performed using Prism v.9 software (GraphPad, Boston, MA, USA). One- and two-way ANOVA were used for comparisons of multiple groups and Student’s unpaired t -test was used for comparisons of two groups. All data are expressed as the mean ± SD. p < 0.05 was considered to represent statistical significance. More detailed information is provided in the Supplemental Digital Content. Results Mindin expression is upregulated in fibrotic liver To investigate the relationship between Mindin and liver fibrosis, we initially established a mouse model of liver fibrosis through repeated injections of CCl 4 and characterized its expression (Figure S1 a). The successful induction of liver fibrosis was first confirmed using H&E, Sirius red, and Masson’s trichrome staining of liver sections (Figure S1 b). Next, we performed transcriptome sequencing analysis of liver tissue samples from the fixation locus. Volcano plot analysis showed that the expression of 565 genes was downregulated and that of 1,670 genes was upregulated in fibrotic livers (Figure S1 c). Given that a key component of the pathology of liver fibrosis is the excessive production of ECM proteins, and the top 50 differentially expressed genes were screened for a connection with the ECM. Heat maps show that Mindin expression was high in fibrotic liver tissue (Fig. 1 a). Furthermore, both the mRNA and protein expression levels of Mindin were high in the CCl 4 - and TAA-induced mouse models of liver fibrosis (Fig. 1 b and c). To characterize the distribution of Mindin in the fibrotic liver, we used RNAscope in situ hybridization (ISH), and found Mindin RNA expression principally in hepatocytes (Fig. 1 d and S1d). Furthermore, hepatocytes and non-parenchymal cells (NPCs) were isolated from the livers of mice with CCl 4 - and TAA-induced fibrosis. Consistent with the above finding, both the mRNA and protein expression of Mindin was principally in hepatocytes, rather than NPCs (Fig. 1 e and f). Mindin deficiency aggravates liver fibrosis To define the role of Mindin in liver fibrosis, we used the CRISPR-Cas9 system to generate Mindin −/− mice ( 17 ) and confirmed their genotype by sequencing (Figure S2a). Next, liver fibrosis was induced using CCl 4 and TAA injection in WT and Mindin-deficient mice (Figure S1 A). As shown in Fig. 2 a and b, H&E, Sirius red, and Masson’s trichrome staining revealed that the area of fibrosis was significantly larger in the Mindin −/− mice. Moreover, collagen type detection in Sirius red-stained sections using a polarizing microscope revealed that there were significantly more type I collagen fibers in Mindin −/− mice with CCl 4 -induced liver fibrosis than in the WT mice, whereas there were significantly more type I and type Ⅲ collagen fibers in Mindin −/− mice with TAA-induced liver fibrosis (Fig. 2 c). In addition, assessment of the hepatic hydroxyproline content showed significantly more collagen deposition in Mindin −/− mice than in WT mice following the induction of liver fibrosis by CCl 4 or TAA administration (Fig. 2 d). Consistent with this, the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were higher in Mindin −/− mice with fibrosis induced by CCl 4 or TAA than in WT mice (Fig. 2 e and f). The mice with liver fibrosis showed more significant weight loss than the control mice. However, there were no significant differences in the body masses of the WT and Mindin −/− mice, whether they were controls or had liver fibrosis (Figure S2b and c). Similarly, there were no significant differences in the liver or spleen indices between the two groups (Figure S2d and e). Taking these findings together, Mindin deficiency exacerbates liver fibrosis in mice. Mindin does not play a direct role in the activation of hepatic stellate cells Because persistently activated HSCs synthesize the ECM that is involved in the progression of liver fibrosis, we hypothesized that Mindin might slow this process. To test this hypothesis, we established transient Mindin-overexpressing HSC LX2 cells. However, we found no significant differences in the mRNA expression of collagen 1a1, α-sma, or TGF-β in the Mindin-overexpressing and control cells (Fig. 3 a). To further evaluate the effect of Mindin in HSCs, we isolated activated HSCs from WT and Mindin −/− mice with CCl 4 -induced fibrosis and characterized them using desmin immunofluorescence staining (Fig. 3 b) and flow cytometry analysis (Figure S3a and b). The activation of the HSCs was stimulated by recombinant Mindin (rMindin) in a time- and dose-dependent manner. Nevertheless, the collagen concentrations of the conditioned medium, assessed by measuring the hydroxyproline content, did not show significant differences after incubation in the presence or absence of rMindin (Fig. 3 c–e). These results indicate that Mindin does not play a role in the direct activation of HSCs. Mindin promotes the migration of macrophages into the liver during fibrosis Inflammation plays a pivotal role in liver fibrosis and the interactions between immune cells and activated HSCs can promote fibrogenesis or the regression of fibrosis. To determine whether Mindin is involved in the progression of liver fibrosis through the orchestration of the immune status, we isolated immune cells from the NPC fraction of the digested mouse livers and analyzed these using flow cytometry. The results showed that the percentages and cell counts of the total Live/Dead-CD45 + CD11b + F4/80 + macrophages were significantly higher in the livers of mice with CCl 4 -induced liver fibrosis than in controls, whereas there were significantly fewer macrophages in Mindin −/− mice than in WT mice with fibrosis (Fig. 4 a). Consistent with this, the percentages and numbers of macrophages in Mindin −/− mice were significantly lower than in WT mice with TAA-induced liver fibrosis (Fig. 4 b). Subsequently, we analyzed CD4 + T cells, CD8 + T cells, B cells, natural killer cells, and dendritic cells in the livers of mice with CCl 4 -induced fibrosis and controls using flow cytometry. The results showed no significant differences in the percentages or numbers of these cells between WT and Mindin −/− mice (Figure S4A and B). Because inflammatory responses drive the activation of HSCs and promote survival ( 18 , 19 ), we conducted α-sma IHC staining of the mouse livers, which is a marker of the activation of HSCs. Data have shown significantly greater HSC infiltration in mice administered CCl 4 or TAA. However, no notable difference was observed in the livers of WT mice, compared to Mindin −/− mice (Figure S5a and b). We next counted the viable CD45 + Ly6G − CD11b + F4/80 + bone marrow-derived macrophages and did not find significant differences between WT or Mindin −/− mice, irrespective of whether they had CCl 4 -induced fibrosis (Figs. 4 c and S5C). These data indicate that the knockout of Mindin in mice does not affect the differentiation of bone marrow-derived macrophages. In addition, we identified circulating live CD45 + CD11b + Ly6G − F4/80 + cells using flow cytometry (Figure S5D), and found a significantly higher proportion of these macrophages in Mindin −/− mice than in WT mice in the presence of CCl 4 -induced liver fibrosis (Fig. 4 d). Therefore, we hypothesized that the retention of macrophages in the bloodstream results in less entry into the liver, which may be attributable to an impairment in macrophage migration in Mindin-deficient mice, and is consistent with previous findings ( 13 ). In addition, GO analysis of the mouse livers illustrated the pivotal role of Mindin in the orchestration of macrophage function (Fig. 4 e). We conducted an in vitro experiment to further explore the effect of Mindin on macrophage migration using Transwell culture. We found that the migration of RAW264.7 cells was significantly greater when they were incubated in medium containing rMindin than in medium lacking this (Fig. 4 f and g). Thus, Mindin promotes the migration of macrophages. Mindin promotes the phenotypic switch to restorative macrophages and promotes phagocytosis in liver fibrosis Having shown that Mindin regulates immunity in liver fibrosis, we next explored the potential mechanisms involved. Hepatic macrophages are a remarkably heterogeneous population of immune cells that play a dual role in liver fibrosis ( 19 , 20 ). Therefore, we analyzed the differential macrophages in more detail, and found that Mindin −/− mice had larger number of Ly6C hi macrophages and fewer Ly6C lo macrophages in their livers than WT mice in the presence of CCl 4 -induced fibrosis (Fig. 5 a). Ly6C lo macrophages have an important role in matrix degradation by secreting MMPs, which are anti-fibrotic. We employed RT-qPCR analysis to measure the expression of relevant MMPs in the livers of mice with CCl 4 -induced fibrosis. We found significantly lower MMP-9 and MMP-12 expression in Mindin-deficient mice than in WT mice (Fig. 5 b). To confirm these findings, we injected mice with CCl 4 -induced fibrosis with MMPSense 680, a fluorescent pan-MMP substrate, to enable us to assess the activity of liver MMPs in mice by live animal imaging. This showed lower MMP activity in Mindin −/− mice than in WT mice (Fig. 5 c). We then depleted the macrophages using intraperitoneal injections of scavengers during the progression of liver fibrosis and assessed the efficiency of clearance by flow cytometry (Fig. 5 d and S6a). Depletion of the macrophages caused the surfaces of the livers of the mice to become rough and irregular, consistent with worsening cirrhosis (Fig. 5 e). Moreover, Sirius red staining confirmed greater fibrosis in the livers after the clearance of the macrophages. There was more severe fibrosis in Mindin −/− mice than in WT mice before the depletion of the macrophages, as previously described, but there was no significant difference in the liver fibrosis of Mindin −/− and WT mice after the depletion (Fig. 5 f). In addition, Mindin −/− mice had a significantly higher liver indices than WT mice, but the spleen indices of the two groups were similar after the depletion of the macrophages (Figure S6b). Because phagocytosis is a crucial aspect of the phenotypic switch to restorative macrophages, we assessed the hepatic phagocytic capacity of mice with CCl 4 -induced liver fibrosis using flow cytometry. Consistent with previous findings ( 21 , 22 ), Mindin deficiency significantly reduced the phagocytic capacity of liver macrophages, assessed using the engulfment of latex beads (Fig. 5 g). Furthermore, we sorted the liver macrophages of mice with fibrosis using flow cytometry and performed RNA sequencing (Fig. 5 h). Differential gene expression analysis showed that six genes were upregulated and 12 were downregulated in macrophages from Mindin −/− mice, compared to WT mice (Figure S6c). The absence of Mindin from the list of differentially expressed genes showed that the macrophages did not express Mindin, which is consistent with a hepatic parenchymal cell origin. KEGG pathway analysis reconfirmed the significance of Mindin-mediated phagocytosis in the progression of liver fibrosis (Fig. 5 i). To further explore the potential association, we prepared bone marrow-derived macrophages (BMDMs) and incubated them with E. coli. Phagocytosis was then assessed using flow cytometry. The data indicated that BMDMs from Mindin −/− mice exhibited weaker phenotypic switch after phagocytosis compared to WT mice (Figure S6d and e). In addition, we added fluorescent latex beads to RAW264.7 cells and observed their phagocytosis (Figure S6f). It was found that MMP expression increased in RAW264.7 cells after phagocytosis (Figure S6g). Taken together, these findings show that Mindin promotes the phagocytosis of macrophages, the secretion of MMPs to degrade collagen, and helps alleviate liver fibrosis. The Mindin/CD11b axis promotes phagocytosis and the restorative phenotype of macrophages to accelerate the regression of liver fibrosis The integrin Mac-1 (CD11b/CD18), a member of the β2 integrin family, recognizes a wide range of ligands ( 23 , 24 ), including Mindin protein ( 22 ). We studied CCl 4 -induced fibrosis in CD11b-deficient mice and found that the serum Mindin concentration was significantly lower in CD11b-deficient mice than in WT mice (Fig. 6 a, S7a and b). This result provides evidence of an interaction between Mindin and the CD11b receptor during the progression of liver fibrosis. Therefore, we next hybridized CD11b −/− and Mindin −/− mice and successfully generated Mindin/CD11b-DKO mice (Figure S7c). We hypothesized that Mindin affects phagocytosis by macrophages through binding to its receptor CD11b. The flow cytometry analysis showed that the absence of either Mindin or CD11b significantly reduced phagocytosis by macrophages and that the DKO further reduced this, as assessed by the engulfment of latex beads (Fig. 6 b and S7d). Considering that phagocytosis can influence macrophage phenotype switch, we next checked this change during mouse liver fibrosis using flow cytometry. The results revealed that either Mindin −/− or CD11b −/− mice induced a phenotypic change of Ly6C low to Ly6C high, and that was more pronounced in DKO mice (Fig. 6 c and S7e). In addition, we found that the mRNA expression of the genetic markers of restorative macrophages IGF-1, Mertk, and CD36 was significantly lower in DKO than WT mouse livers (Figs. 6 d and S7f). Consistent with restorative macrophages contributing to the regression of hepatic fibrosis through MMP-mediated matrix degradation, we found that both the mRNA and protein expression levels of MMP-9 were significantly lower in the DKO mice than in WT mice with CCl 4 -induced liver fibrosis (Fig. 6 e and f). Furthermore, we induced liver fibrosis by CCl 4 administration in Mindin −/− , CD11b −/− , DKO, and WT mice, and found that either Mindin or CD11b knockout increased the liver fibrosis, and DKO markedly exacerbated this fibrosis (Fig. 6 g and h). Discussion In the present study, we have provided evidence for the crucial role of Mindin in the coordination of the macrophage-driven resolution of liver fibrosis. Classically, hepatic macrophages are classified according to their F4/80 expression as either high F4/80-expressing resident Kupffer cells or low F4/80-expressing bone marrow-derived macrophages that differentiate from circulating monocytes. However, relying solely on the level of F4/80 expression to characterize macrophage heterogeneity in the liver is insufficient, because mature monocyte-derived macrophages also show high F4/80 expression( 25 ). In addition, macrophage subsets that are relevant to liver disease can be categorized into inflammatory or restorative macrophages ( 5 ). The former promote liver fibrosis by activating HSCs in chronic liver injury and the latter are involved in matrix degradation through the expression of MMPs, thereby contributing to the resolution of liver injury and fibrosis. Owing to the heterogeneity of the macrophages, we carefully analyzed the Mindin-mediated recruitment of macrophages to the livers of mice with either CCl 4 - or TAA-induced liver fibrosis. We found that the population of differential macrophages in WT mice exhibited high F4/80 expression, along with increased macrophage phenotype switch from Ly6C high to Ly6C low, and unexpectedly, the augmentation of this macrophage population did not increase the activation of HSCs. Nevertheless, the livers showed higher MMP expression, and the depletion of the macrophage population markedly exacerbated the liver fibrosis. These data suggest that there was a larger population of restorative macrophages, which may play an important role in the regression of hepatic fibrosis. Phagocytosis, a primary characteristic of macrophages. It has been reported that the phagocytosis of apoptotic hepatocytes by macrophages promotes the transition from an Ly6C hi to an Ly6C lo macrophage subset ( 6 ). The RNA-seq of macrophages obtained from Mindin −/− and WT mice with liver fibrosis suggested that phagocytosis plays an important role in the process of liver fibrosis. Therefore, we concluded that Mindin-mediated phagocytosis promotes the phenotypic transition toward restorative macrophages in mice, leading to the resolution of fibrosis. In terms of the downstream effects of increased phagocytosis, it has been shown that the processing of apoptotic cells activates a STAT3-IL10-IL6 autocrine-paracrine loop( 10 ). Additionally, our previous work has investigated the phagocytosis-promoting effects of Mindin. The data indicate that Mindin promotes macrophage phagocytosis through Syk activation and NF-κB p65 translocation( 22 ). Therefore, intracellular responses that lead to enhanced phagocytosis in macrophages were not evaluated in this study, which is a limitation of this study. Integrins are transmembrane proteins that form heterodimers using α and β subunits. We have previously reported that the F-spondin fragment of Mindin binds to the αM-I structural domain of CD11b/CD18 ( 22 ), one of the integrin receptors, which is predominantly expressed in myeloid cells. Therefore, we hypothesized that Mindin acts by binding to CD11b on recruited liver macrophages. The absence of either Mindin or CD11b in mice can aggravate their liver fibrosis, and if both components of the Mindin/CD11b axis fail to function at the same time, the liver fibrosis is much worse. In other words, during the process of liver fibrosis, mouse hepatocyte-derived Mindin increases the migration of hepatic macrophages, and enhances macrophage phagocytosis by binding to the CD11b receptor, promoting the transition from an inflammatory to a restorative macrophage phenotype, and upregulates the MMP-9, which promotes collagen degradation and a reduction in tissue fibrosis, and thereby improves the liver homeostasis. Macrophages bridge the processes of tissue necrosis and repair in many diseases. In principle, hepatic macrophages represent an attractive target for novel therapies for liver disease. Here, we have revealed the role of the Mindin/CD11b axis in the regulation of macrophage phagocytosis and liver homeostasis. By studying the role of Mindin in liver fibrosis in mice, we can hypothesize that Mindin deficiency or dysfunction in humans may exacerbate liver fibrosis in certain populations. Therefore, detection of Mindin levels in serum may be a potential biomarker for early diagnosis or monitoring the progression of liver fibrosis. Furthermore, given the role of Mindin in the regulation of immune responses, it is important to note that liver fibrosis is often accompanied by chronic inflammation and persistent immune system activation. Therefore, targeting the Mindin/CD11b axis, we could potentially inhibit excessive inflammation and promote the breakdown of fibrosis, thereby slowing the progression of the disease. Meanwhile, enhancing the reparative function of macrophages by utilizing the Mindin/CD11b axis may help to improve the ability of liver self-repair. Finally, translating these findings into clinical applications will require extensive preclinical studies and clinical trials. It will be crucial to assess the safety, efficacy, and potential side effects of modulating the Mindin/CD11b axis in patients with liver fibrosis before it can be considered for clinical use. Conclusions In conclusion, in the present study, we have demonstrated the crucial role of Mindin in the coordination of the macrophage-driven resolution of liver fibrosis. The Mindin/CD11b axis drives the regression of liver fibrosis through an increase in phagocytosis by macrophages. These findings provide new molecular targets for the upregulation of the macrophage-mediated regression of liver fibrosis. Abbreviations CCl 4 , carbon tetrachloride; TAA, thioacetamide; DKO, double-knockout; ECM, extracellular matrix; HSC, hepatic stellate cell; MMP, matrix metalloproteinase; tissue inhibitor of metalloproteinases, TIMPs; Ly6C, lymphocyte antigen 6 complex locus C; DAMP, damage-associated molecular pattern; FS, F-spondin; H&E, hematoxylin–eosin; SR, Sirius red; ISH, in situ hybridization; NPC, non-parenchymal cell; ALT, alanine aminotransferase; AST, aspartate aminotransferase; Col1a1, collagen type I alpha 1; α-sma, α-smooth muscle actin; rMindin, recombinant Mindin; NK, natural kill; DC, dendritic cell; GO, Gene Ontology; RT-qPCR, real-time reverse transcription polymerase chain reaction; KEGG, Kyoto Encyclopedia of Genes and Genomes; bone marrow-derived macrophages, BMDMs; Mertk, MER proto-oncogene tyrosine kinase. Declarations Data availability All data generated or analysed during this study are included in this article [and its supplementary information files]. Funding This work was supported by the National Natural Science Foundation of China (No. 81970460) and the Natural Science Foundation Program of Fujian Province (No. 2023J011598). Contributions HYD, ZXL and OYXM performed the major experiments; LY, CXS, LLY, HYN, XGJ and LJH assisted the animal experiments; HYD assisted draft of the manuscript; and JA provided the advises. GB revised the manuscript and supervised this study. All authors have read and approved the final submitted manuscript. Ethics declarations Conflict of interests None of the authors has a conflict of interest. Ethical approval All the protocols used in the animal experiments were approved by the Committee for Animal Research of Xiamen University (No. XMULAC20190127). References Sun M, Kisseleva T. Reversibility of liver fibrosis. Clin Res Hepatol Gastroenterol 2015;39 Suppl 1:S60-63. Benyon RC, Iredale JP. Is liver fibrosis reversible? Gut 2000;46:443-446. Hernandez-Gea V, Friedman SL. Pathogenesis of liver fibrosis. Annu Rev Pathol 2011;6:425-456. Consolo M, Amoroso A, Spandidos DA, Mazzarino MC. Matrix metalloproteinases and their inhibitors as markers of inflammation and fibrosis in chronic liver disease (Review). Int J Mol Med 2009;24:143-152. Varol C, Mildner A, Jung S. Macrophages: development and tissue specialization. Annu Rev Immunol 2015;33:643-675. Kisseleva T, Brenner D. Molecular and cellular mechanisms of liver fibrosis and its regression. Nat Rev Gastroenterol Hepatol 2021;18:151-166. Guillot A, Tacke F. Liver Macrophages: Old Dogmas and New Insights. Hepatol Commun 2019;3:730-743. Ramachandran P, Pellicoro A, Vernon MA, Boulter L, Aucott RL, Ali A, Hartland SN, et al. Differential Ly-6C expression identifies the recruited macrophage phenotype, which orchestrates the regression of murine liver fibrosis. Proc Natl Acad Sci U S A 2012;109:E3186-3195. Wang M, You Q, Lor K, Chen F, Gao B, Ju C. Chronic alcohol ingestion modulates hepatic macrophage populations and functions in mice. J Leukoc Biol 2014;96:657-665. Campana L, Starkey Lewis PJ, Pellicoro A, Aucott RL, Man J, O'Duibhir E, Mok SE, et al. The STAT3-IL-10-IL-6 Pathway Is a Novel Regulator of Macrophage Efferocytosis and Phenotypic Conversion in Sterile Liver Injury. J Immunol 2018;200:1169-1187. Lodder J, Denaës T, Chobert MN, Wan J, El-Benna J, Pawlotsky JM, Lotersztajn S, et al. Macrophage autophagy protects against liver fibrosis in mice. Autophagy 2015;11:1280-1292. Campana L, Esser H, Huch M, Forbes S. Liver regeneration and inflammation: from fundamental science to clinical applications. Nat Rev Mol Cell Biol 2021;22:608-624. Jia W, Li H, He YW. The extracellular matrix protein mindin serves as an integrin ligand and is critical for inflammatory cell recruitment. Blood 2005;106:3854-3859. Bian ZY, Wei X, Deng S, Tang QZ, Feng J, Zhang Y, Liu C, et al. Disruption of mindin exacerbates cardiac hypertrophy and fibrosis. J Mol Med (Berl) 2012;90:895-910. Yang K, Li W, Bai T, Xiao Y, Yu W, Luo P, Cheng F. Mindin deficiency alleviates renal fibrosis through inhibiting NF-κB and TGF-β/Smad pathways. J Cell Mol Med 2020;24:5740-5750. Rana I, Kataria S, Tan TL, Hajam EY, Kashyap DK, Saha D, Ajnabi J, et al. Mindin (SPON2) Is Essential for Cutaneous Fibrogenesis in a Mouse Model of Systemic Sclerosis. J Invest Dermatol 2023;143:699-710.e610. Cheng XS, Huo YN, Fan YY, Xiao CX, Ouyang XM, Liang LY, Lin Y, et al. Mindin serves as a tumour suppressor gene during colon cancer progression through MAPK/ERK signalling pathway in mice. J Cell Mol Med 2020;24:8391-8404. Hammerich L, Tacke F. Hepatic inflammatory responses in liver fibrosis. Nat Rev Gastroenterol Hepatol 2023;20:633-646. Pradere JP, Kluwe J, De Minicis S, Jiao JJ, Gwak GY, Dapito DH, Jang MK, et al. Hepatic macrophages but not dendritic cells contribute to liver fibrosis by promoting the survival of activated hepatic stellate cells in mice. Hepatology 2013;58:1461-1473. Tacke F. Targeting hepatic macrophages to treat liver diseases. J Hepatol 2017;66:1300-1312. He YW, Li H, Zhang J, Hsu CL, Lin E, Zhang N, Guo J, et al. The extracellular matrix protein mindin is a pattern-recognition molecule for microbial pathogens. Nat Immunol 2004;5:88-97. Liu YS, Wang LF, Cheng XS, Huo YN, Ouyang XM, Liang LY, Lin Y, et al. The pattern-recognition molecule mindin binds integrin Mac-1 to promote macrophage phagocytosis via Syk activation and NF-κB p65 translocation. J Cell Mol Med 2019;23:3402-3416. Lamers C, Plüss CJ, Ricklin D. The Promiscuous Profile of Complement Receptor 3 in Ligand Binding, Immune Modulation, and Pathophysiology. Front Immunol 2021;12:662164. Lim K, Hyun YM, Lambert-Emo K, Topham DJ, Kim M. Visualization of integrin Mac-1 in vivo. J Immunol Methods 2015;426:120-127. Ju C, Tacke F. Hepatic macrophages in homeostasis and liver diseases: from pathogenesis to novel therapeutic strategies. Cell Mol Immunol 2016;13:316-327. Supplementary Files HEPID2401239SupplementaryMaterials.docx floatimage1.jpeg Graphical abstract Cite Share Download PDF Status: Published Journal Publication published 05 Apr, 2025 Read the published version in Hepatology International → Version 1 posted Reviewers agreed at journal 24 Nov, 2024 Reviewers invited by journal 23 Nov, 2024 Editor assigned by journal 23 Nov, 2024 First submitted to journal 22 Nov, 2024 Editorial decision: Major Revisions Needed 25 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5155041","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":381814903,"identity":"bd01c51f-70a5-4107-959c-99231c97e075","order_by":0,"name":"Yong dong Huang","email":"","orcid":"","institution":"Zhongshan Hospital Xiamen University","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"dong","lastName":"Huang","suffix":""},{"id":381814904,"identity":"f2cdb1d6-fbbf-420f-b67b-dc441164fb07","order_by":1,"name":"Xian ling Zhao","email":"","orcid":"","institution":"Zhongshan Hospital Xiamen University","correspondingAuthor":false,"prefix":"","firstName":"Xian","middleName":"ling","lastName":"Zhao","suffix":""},{"id":381814905,"identity":"84a055b9-849a-4c41-8040-60b791649268","order_by":2,"name":"Xiao mei Ou yang","email":"","orcid":"","institution":"Zhongshan Hospital Xiamen University","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"mei Ou","lastName":"yang","suffix":""},{"id":381814906,"identity":"3b369431-8329-451c-a4a1-cd0797aa20c0","order_by":3,"name":"Ying Lin","email":"","orcid":"","institution":"Zhongshan Hospital Xiamen University","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Lin","suffix":""},{"id":381814907,"identity":"9eba4223-a110-4220-a382-053bd32b5faf","order_by":4,"name":"Xiao shen Cheng","email":"","orcid":"","institution":"Zhongshan Hospital Xiamen University","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"shen","lastName":"Cheng","suffix":""},{"id":381814908,"identity":"d9454bca-3090-4e81-a0fd-9aaa26fca375","order_by":5,"name":"Lai ying liang","email":"","orcid":"","institution":"Zhongshan Hospital Xiamen University","correspondingAuthor":false,"prefix":"","firstName":"Lai","middleName":"ying","lastName":"liang","suffix":""},{"id":381814909,"identity":"3d21573b-651b-4fca-930f-c613771c5c5a","order_by":6,"name":"Ya ni Huo","email":"","orcid":"","institution":"Zhongshan Hospital Xiamen University","correspondingAuthor":false,"prefix":"","firstName":"Ya","middleName":"ni","lastName":"Huo","suffix":""},{"id":381814910,"identity":"f46f72d5-b6f1-4f70-8ac6-6bc273db1c09","order_by":7,"name":"Gui jing Xie","email":"","orcid":"","institution":"Zhongshan Hospital Xiamen University","correspondingAuthor":false,"prefix":"","firstName":"Gui","middleName":"jing","lastName":"Xie","suffix":""},{"id":381814911,"identity":"051b5eb8-0404-4674-8cb8-40d3b8314ace","order_by":8,"name":"Jun hui Lin","email":"","orcid":"","institution":"Zhongshan Hospital Xiamen University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"hui","lastName":"Lin","suffix":""},{"id":381814912,"identity":"4190dd75-6f30-4cc6-bbd6-e78599166e17","order_by":9,"name":"Amarsanaa Jazag","email":"","orcid":"","institution":"Otoch Manramba University","correspondingAuthor":false,"prefix":"","firstName":"Amarsanaa","middleName":"","lastName":"Jazag","suffix":""},{"id":381814913,"identity":"abb4aa23-a7df-44c4-a5d5-975d7e5013ca","order_by":10,"name":"Bayasi Guleng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxUlEQVRIiWNgGAWjYBACAwhpA+HxkKAljWQtDIdJ0GLOfsbw44+C84lrZyQwPnjbxiBvTkiLZU+OsTSPwW1jsxsJzIZz2xgMdzYQctiBHANpBoPbckAtbNK8bQwJBgcIaTn/xvjnD4NzPEAt7L+J03Ijx0yCx+AA2BZmorRYznhWZs1jkGxsduZhs+SccxKGGwhpMedP3nzzxx+7xG3Hkw9+eFNmI0/QFgYGDmjUMDA2AAkJguqBgP0BMapGwSgYBaNgJAMAVEQ+A7IVZqMAAAAASUVORK5CYII=","orcid":"","institution":"Zhongshan Hospital Xiamen University","correspondingAuthor":true,"prefix":"","firstName":"Bayasi","middleName":"","lastName":"Guleng","suffix":""}],"badges":[],"createdAt":"2024-09-26 02:26:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5155041/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5155041/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12072-025-10813-7","type":"published","date":"2025-04-05T15:58:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":69888031,"identity":"0d060f8d-16de-4e07-85e9-1f104cf4a0ff","added_by":"auto","created_at":"2024-11-26 10:00:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1458365,"visible":true,"origin":"","legend":"\u003cp\u003eMindin expression is upregulated in fibrotic liver. (a) Comparison of gene expression heat maps between CCl\u003csub\u003e4\u003c/sub\u003e-treated WT and control mice (n=3 per group). (b) Mindin mRNA expression in the livers of mice with fibrosis induced by CCl\u003csub\u003e4\u003c/sub\u003e or TAA. (c) Serum Mindin concentration of mice with CCl\u003csub\u003e4\u003c/sub\u003e-induced and TAA-induced liver fibrosis. (d) Cellular distribution of Mindin, analyzed using RNAscope \u003cem\u003ein situ\u003c/em\u003e hybridization. (e) mRNA expression and (f) protein expression of Mindin in isolated mice hepatocytes and NPCs. Data are mean ± SD; n=5–7 per group; scale bars, 100 µm; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u0026nbsp;\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5155041/v1/f73efbc9914b31ebb06f97df.png"},{"id":69888035,"identity":"ae7f0d70-6bbf-4691-9be9-728058a713f0","added_by":"auto","created_at":"2024-11-26 10:00:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2417608,"visible":true,"origin":"","legend":"\u003cp\u003eMindin deficiency aggravates liver fibrosis. Degree of fibrosis, evaluated using H\u0026amp;E staining, Sirius red staining, and Masson’s trichrome staining after (a) Oil/CCl\u003csub\u003e4\u003c/sub\u003e or (b) Saline/TAA injection in WT and Mindin\u003csup\u003e−/−\u003c/sup\u003e mice. The upper panel shows representative images of stained sections and the lower panel shows a quantitative analysis of the staining. (c) Collagen types in WT and Mindin\u003csup\u003e−/−\u003c/sup\u003e mice with CCl\u003csub\u003e4\u003c/sub\u003e and TAA-induced liver fibrosis. Under the polarized microscope, type Ⅰ collagen is red or yellow and type Ⅲ collagen is green. Left side: representative stained images; right side: quantitative analysis of the staining. (d) Collagen accumulation, assessed using hydroxyproline content, in mouse livers. (e) Serum ALT and AST activities in WT and Mindin\u003csup\u003e−/−\u003c/sup\u003e mice with CCl\u003csub\u003e4\u003c/sub\u003e and (f) TAA-induced liver fibrosis. Data are mean ± SD; n=5–7 per group; scale bars, 100 µm; *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5155041/v1/0c698e04e631c59919ca6a3a.png"},{"id":69889790,"identity":"8101a89a-c170-44d2-ba6e-8e382008389d","added_by":"auto","created_at":"2024-11-26 10:16:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":510162,"visible":true,"origin":"","legend":"\u003cp\u003eMindin does not play a direct role in the activation of hepatic stellate cells. (a) SPON2, Col1a1, α-sma, and TGF-β mRNA expression in LX2 cells transiently overexpressing Mindin for 48 h. (b) Characterization of HSCs using desmin immunofluorescence. (c) Schematic illustration of the strategy for hydroxyproline content assessment in treated activated HSCs. Hydroxyproline content of medium conditioned by culture in HSCs from both (d) WT and (e) Mindin\u003csup\u003e−/−\u003c/sup\u003e mice, measured after stimulation with rMindin protein. Data are mean ± SD; n=3–4 per group; scale bars, 100 µm; NS, \u003cem\u003eP \u003c/em\u003e\u0026gt; 0.05, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5155041/v1/44aa055a6b8db65f39458303.png"},{"id":69888039,"identity":"5f36f85b-a9fc-4493-9296-207b1de983a4","added_by":"auto","created_at":"2024-11-26 10:00:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2739880,"visible":true,"origin":"","legend":"\u003cp\u003eMindin promotes the migration of macrophages into the liver during liver fibrosis. Hepatic macrophage numbers in Mindin\u003csup\u003e−/−\u003c/sup\u003e and WT mice with (a) CCl\u003csub\u003e4\u003c/sub\u003e-induced and (b) TAA-induced liver fibrosis, evaluated using flow cytometry. Left side: representative images of flow-cytometric analysis; right side: quantitative analysis of hepatic macrophages. (c) Bone marrow-derived macrophage counts in CCl\u003csub\u003e4\u003c/sub\u003e-treated mice, assessed using flow cytometry. Left side: representative images of flow-cytometric analysis; right side: quantitative analysis of bone marrow-derived macrophages. (d) Blood macrophage numbers in mice administered CCl\u003csub\u003e4\u003c/sub\u003e, assessed using flow cytometry. Left side: representative images of flow-cytometric analysis; right side: quantitative analysis of blood macrophages. (e) Results of the GO analysis of livers from WT and Mindin\u003csup\u003e−/−\u003c/sup\u003e mice with CCl\u003csub\u003e4\u003c/sub\u003e-induced liver fibrosis. (f) Schematic diagram of the structure of the transwell chamber: RAW264.7 cells in the upper chamber, DMEM containing 0.10 μg/ml rMindin protein in the lower chamber. (g) Representative transwell migration images, obtained 48 hours after stimulation with rMindin. Data are mean ± SD; n=4–8 per group; *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.\u0026nbsp;\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5155041/v1/b723e4e3a85c4ad930394afd.png"},{"id":69888045,"identity":"6d9cec21-92ec-47ad-9205-6760b3ae6fec","added_by":"auto","created_at":"2024-11-26 10:00:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2346748,"visible":true,"origin":"","legend":"\u003cp\u003eMindin promotes the phenotypic switch to restorative macrophages and promotes phagocytosis in liver fibrosis. (a) Ly6C\u003csup\u003ehi\u003c/sup\u003e and Ly6C\u003csup\u003elo\u003c/sup\u003e macrophage numbers, assessed using flow cytometry. The upper panel shows representative images of flow-cytometric analysis and the lower panel shows a quantitative analysis of Ly6C\u003csup\u003ehi\u003c/sup\u003e and Ly6C\u003csup\u003elo\u003c/sup\u003e macrophages. (b) CCl\u003csub\u003e4\u003c/sub\u003e-induced expression of MMP-9 and MMP-12 mRNA in the livers of WT and Mindin\u003csup\u003e−/−\u003c/sup\u003e mice. (c) Representative images of CCl\u003csub\u003e4\u003c/sub\u003e-induced fibrosis in mice 24 hours after MMPSense 680 injection. (d) Schematic of macrophage clearance in C57BL/6 mice with liver fibrosis, induced using intraperitoneal injections of scavenger (clodronate liposomes \u003cem\u003evs\u003c/em\u003e. control liposomes). CCl\u003csub\u003e4\u003c/sub\u003e was injected twice weekly for 7 weeks and macrophage scavenger was injected biweekly. Tissue samples were harvested 3 days after the final CCl\u003csub\u003e4\u003c/sub\u003e injection. (e) Representative images of livers after hepatic macrophage clearance. (f) Degree of fibrosis in WT and Mindin\u003csup\u003e−/−\u003c/sup\u003e mouse livers, with or without macrophage removal, assessed using Sirius red staining. (g) Hepatic phagocytic capacity in mice with CCl\u003csub\u003e4\u003c/sub\u003e-induced liver fibrosis, assessed using flow cytometry. (h) Schematic showing the flow sorting of macrophages obtained from mice with liver fibrosis and their RNA-seq. (i) Results of the KEGG pathway analysis of the differentially expressed genes between WT and Mindin\u003csup\u003e−/−\u003c/sup\u003e mice with CCl\u003csub\u003e4\u003c/sub\u003e-induced fibrosis. Data are mean ± SD; n=4–7 per group; scale bars, 100 µm; NS, \u003cem\u003eP \u003c/em\u003e\u0026gt; 0.05, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. CLs, clodronate liposomes.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5155041/v1/ad7947791455b812db60cdb3.png"},{"id":69888038,"identity":"395cca53-37da-4d40-9b8d-0e54c5ca6cc1","added_by":"auto","created_at":"2024-11-26 10:00:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2358107,"visible":true,"origin":"","legend":"\u003cp\u003eThe Mindin/CD11b axis promotes phagocytosis and the restorative phenotype of macrophages to accelerate the regression of liver fibrosis. (a) Serum Mindin concentrations in WT and CD11b\u003csup\u003e−/−\u003c/sup\u003e mice with and without fibrosis. (b) Hepatic phagocytic capacity of WT, Mindin\u003csup\u003e−/−\u003c/sup\u003e, CD11b\u003csup\u003e−/−\u003c/sup\u003e, and DKO mice with CCl\u003csub\u003e4\u003c/sub\u003e-induced liver fibrosis, assessed using flow cytometry. (c) Quantification of Ly6C\u003csup\u003ehi\u003c/sup\u003e and Ly6C\u003csup\u003elo\u003c/sup\u003e macrophage numbers, assessed using flow cytometry. (d) Expression of restorative macrophage signature genes in mice with CCl\u003csub\u003e4\u003c/sub\u003e-induced fibrosis (Igf1, Mertk, and CD36), assessed using RT-qPCR. (e) MMP-9 mRNA expression in the livers of WT, Mindin\u003csup\u003e−/−\u003c/sup\u003e, CD11b\u003csup\u003e−/−\u003c/sup\u003e, and DKO mice. (f) Representative images of liver sections immunohistochemically stained for MMP-9 from WT, Mindin\u003csup\u003e−/−\u003c/sup\u003e, CD11b\u003csup\u003e−/−\u003c/sup\u003e, and DKO mice with CCl\u003csub\u003e4\u003c/sub\u003e-induced fibrosis. Degree of fibrosis in WT, Mindin\u003csup\u003e−/−\u003c/sup\u003e, CD11b\u003csup\u003e−/−\u003c/sup\u003e, and DKO mice after oil or CCl\u003csub\u003e4\u003c/sub\u003e injection, assessed using (g) the hydroxyproline content and (h) Sirius red staining. Left side: representative stained images; right side: quantitative analysis of the staining. Data are mean ± SD; n=4–10 per group; scale bars, 100 µm; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5155041/v1/c5e73a800f8c6f1c2bbe3550.png"},{"id":80082090,"identity":"b051f305-2c09-4231-8dc1-39456a9fe58e","added_by":"auto","created_at":"2025-04-07 16:06:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":14489868,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5155041/v1/66fcded8-8064-4c5c-9852-0d3137124880.pdf"},{"id":69889166,"identity":"7fd3e38c-93a6-4be8-a8b8-938d9da36152","added_by":"auto","created_at":"2024-11-26 10:08:36","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6487800,"visible":true,"origin":"","legend":"","description":"","filename":"HEPID2401239SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-5155041/v1/59cc989e2b8e02afadef111a.docx"},{"id":69889150,"identity":"e9dab507-265c-40f7-85e1-451ba1d2916c","added_by":"auto","created_at":"2024-11-26 10:08:36","extension":"jpeg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":751975,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5155041/v1/cbdf6dfac6e95bc63e69df9d.jpeg"}],"financialInterests":"","formattedTitle":"Mindin orchestrates the macrophage-mediated resolution of liver fibrosis in mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLiver fibrosis, which is characterized by the accumulation of extracellular matrix (ECM), represents a reversible wound-healing response to acute or chronic injury (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). This can progress to cirrhosis and hepatocellular carcinoma, which are accompanied by severe impaired liver function. Therefore, a better understanding of the physiologic and pathologic processes involved in fibrosis should assist with the development of new therapies for liver fibrosis.\u003c/p\u003e \u003cp\u003eThe ECM is a highly dynamic network that plays essential roles in maintaining normal homeostasis. The dysregulation of ECM composition and structure results in the development and progression of multiple diseases, including liver fibrosis (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The activation of hepatic stellate cells (HSCs) enhances the migration and deposition of ECM components (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The expression of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) in the liver plays crucial roles in both fibrogenesis and fibrinolysis(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe liver is a central immunological organ that contains large numbers of innate immune cells, and particularly macrophages. Macrophages play a crucial role in liver homeostasis and have been proposed to be central in the pathogenesis of chronic liver injury(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). They play dual roles in the process of liver fibrosis, because they can promote fibrogenesis, but also support the regression of fibrosis (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). According to the expression of a surface marker, lymphocyte antigen 6 complex locus C (Ly6C), hepatic macrophages can be classified as Ly6C-high and Ly6C-low types(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Ly6C\u003csup\u003ehi\u003c/sup\u003e macrophages are recruited from the circulation and are critical for inflammation and fibrogenesis, whereas Ly6C\u003csup\u003elo\u003c/sup\u003e macrophages are responsible for the suppression of inflammation and mediating the resolution of fibrosis (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Specific molecular signals in pathological microenvironment promote the regression of liver fibrosis by triggering the transition of macrophages from an inflammatory to a restorative phenotype. A reduction in damage-associated molecular patterns expression (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), phagocytosis (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), and autophagy (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) regulate the phenotypic switch of the macrophages. Restorative macrophages are the primary source of MMPs, which support both the degradation of ECM and the resolution of fibrosis (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe ECM protein Mindin/SPON2, serves as a novel integrin ligand and is critical for the recruitment of inflammatory cells, especially neutrophils and macrophages (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Mice lacking Mindin exhibit impaired resolution of bacterial infections, and Mindin-deficient macrophages fail to respond to substantial microbial stimulation. In addition, Mindin plays an important role in multiple fibrotic diseases, such as cardiac fibrosis (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), renal fibrosis (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), and cutaneous fibrogenesis (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). However, there have been few studies of the role of Mindin in liver homeostasis, and therefore its role in liver fibrosis has not been characterized.\u003c/p\u003e \u003cp\u003eIn this study, we aimed to characterize the role of Mindin in liver fibrosis using two mouse models: repeated CCl\u003csub\u003e4\u003c/sub\u003e and TAA administration. We found that hepatocyte-derived Mindin causes macrophage migration and promotes the phenotypic switching of macrophages by binding to CD11b receptors. This upregulates the production of the collagenase MMP-9, which promotes the regression of hepatic fibrosis. The findings demonstrate that Mindin facilitates the regression of liver fibrosis, which may inform the search for therapies for chronic liver disease.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMice\u003c/h2\u003e \u003cp\u003eMindin\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were generated using the CRISPR-Cas9 system in collaboration with the State Key Laboratory of Cellular Stress Biology at Xiamen University, China. CD11b\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were purchased from the Jackson Laboratory, USA (B6.129S4-Itgamtm1Myd/J, Strain ID: B005273). Mindin\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were crossed and bred with CD11b\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice to obtain homozygous DKO mice.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eModels of liver fibrosis\u003c/h3\u003e\n\u003cp\u003eFor the CCl\u003csub\u003e4\u003c/sub\u003e-induced model of liver fibrosis, 6\u0026ndash;8-week-old mice underwent intraperitoneal (i.p.) injection with carbon tetrachloride (diluted 1:4 in olive oil) or vehicle (olive oil) at a dose of 2.5 mL/kg twice a week for 7 weeks. For the TAA model, liver fibrosis was induced by i.p. injections of TAA in saline (200 mg/kg) or vehicle (saline) twice weekly for 6 weeks. The mice were euthanized 72 hours after the final i.p. injection.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using Prism v.9 software (GraphPad, Boston, MA, USA). One- and two-way ANOVA were used for comparisons of multiple groups and Student\u0026rsquo;s unpaired \u003cem\u003et\u003c/em\u003e-test was used for comparisons of two groups. All data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to represent statistical significance.\u003c/p\u003e \u003cp\u003eMore detailed information is provided in the Supplemental Digital Content.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMindin expression is upregulated in fibrotic liver\u003c/h2\u003e \u003cp\u003eTo investigate the relationship between Mindin and liver fibrosis, we initially established a mouse model of liver fibrosis through repeated injections of CCl\u003csub\u003e4\u003c/sub\u003e and characterized its expression (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea). The successful induction of liver fibrosis was first confirmed using H\u0026amp;E, Sirius red, and Masson\u0026rsquo;s trichrome staining of liver sections (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb). Next, we performed transcriptome sequencing analysis of liver tissue samples from the fixation locus. Volcano plot analysis showed that the expression of 565 genes was downregulated and that of 1,670 genes was upregulated in fibrotic livers (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ec). Given that a key component of the pathology of liver fibrosis is the excessive production of ECM proteins, and the top 50 differentially expressed genes were screened for a connection with the ECM. Heat maps show that Mindin expression was high in fibrotic liver tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Furthermore, both the mRNA and protein expression levels of Mindin were high in the CCl\u003csub\u003e4\u003c/sub\u003e- and TAA-induced mouse models of liver fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb and c).\u003c/p\u003e \u003cp\u003eTo characterize the distribution of Mindin in the fibrotic liver, we used RNAscope \u003cem\u003ein situ\u003c/em\u003e hybridization (ISH), and found Mindin RNA expression principally in hepatocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed and S1d). Furthermore, hepatocytes and non-parenchymal cells (NPCs) were isolated from the livers of mice with CCl\u003csub\u003e4\u003c/sub\u003e- and TAA-induced fibrosis. Consistent with the above finding, both the mRNA and protein expression of Mindin was principally in hepatocytes, rather than NPCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee and f).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMindin deficiency aggravates liver fibrosis\u003c/h2\u003e \u003cp\u003eTo define the role of Mindin in liver fibrosis, we used the CRISPR-Cas9 system to generate Mindin\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) and confirmed their genotype by sequencing (Figure S2a). Next, liver fibrosis was induced using CCl\u003csub\u003e4\u003c/sub\u003e and TAA injection in WT and Mindin-deficient mice (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and b, H\u0026amp;E, Sirius red, and Masson\u0026rsquo;s trichrome staining revealed that the area of fibrosis was significantly larger in the Mindin\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. Moreover, collagen type detection in Sirius red-stained sections using a polarizing microscope revealed that there were significantly more type I collagen fibers in Mindin\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice with CCl\u003csub\u003e4\u003c/sub\u003e-induced liver fibrosis than in the WT mice, whereas there were significantly more type I and type Ⅲ collagen fibers in Mindin\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice with TAA-induced liver fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). In addition, assessment of the hepatic hydroxyproline content showed significantly more collagen deposition in Mindin\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice than in WT mice following the induction of liver fibrosis by CCl\u003csub\u003e4\u003c/sub\u003e or TAA administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Consistent with this, the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were higher in Mindin\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice with fibrosis induced by CCl\u003csub\u003e4\u003c/sub\u003e or TAA than in WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee and f). The mice with liver fibrosis showed more significant weight loss than the control mice. However, there were no significant differences in the body masses of the WT and Mindin\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, whether they were controls or had liver fibrosis (Figure S2b and c). Similarly, there were no significant differences in the liver or spleen indices between the two groups (Figure S2d and e). Taking these findings together, Mindin deficiency exacerbates liver fibrosis in mice.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMindin does not play a direct role in the activation of hepatic stellate cells\u003c/h3\u003e\n\u003cp\u003eBecause persistently activated HSCs synthesize the ECM that is involved in the progression of liver fibrosis, we hypothesized that Mindin might slow this process. To test this hypothesis, we established transient Mindin-overexpressing HSC LX2 cells. However, we found no significant differences in the mRNA expression of collagen 1a1, α-sma, or TGF-β in the Mindin-overexpressing and control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). To further evaluate the effect of Mindin in HSCs, we isolated activated HSCs from WT and Mindin\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice with CCl\u003csub\u003e4\u003c/sub\u003e-induced fibrosis and characterized them using desmin immunofluorescence staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) and flow cytometry analysis (Figure S3a and b). The activation of the HSCs was stimulated by recombinant Mindin (rMindin) in a time- and dose-dependent manner. Nevertheless, the collagen concentrations of the conditioned medium, assessed by measuring the hydroxyproline content, did not show significant differences after incubation in the presence or absence of rMindin (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec\u0026ndash;e). These results indicate that Mindin does not play a role in the direct activation of HSCs.\u003c/p\u003e\n\u003ch3\u003eMindin promotes the migration of macrophages into the liver during fibrosis\u003c/h3\u003e\n\u003cp\u003eInflammation plays a pivotal role in liver fibrosis and the interactions between immune cells and activated HSCs can promote fibrogenesis or the regression of fibrosis. To determine whether Mindin is involved in the progression of liver fibrosis through the orchestration of the immune status, we isolated immune cells from the NPC fraction of the digested mouse livers and analyzed these using flow cytometry. The results showed that the percentages and cell counts of the total Live/Dead-CD45\u0026thinsp;+\u0026thinsp;CD11b\u0026thinsp;+\u0026thinsp;F4/80\u0026thinsp;+\u0026thinsp;macrophages were significantly higher in the livers of mice with CCl\u003csub\u003e4\u003c/sub\u003e-induced liver fibrosis than in controls, whereas there were significantly fewer macrophages in Mindin\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice than in WT mice with fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Consistent with this, the percentages and numbers of macrophages in Mindin\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were significantly lower than in WT mice with TAA-induced liver fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eSubsequently, we analyzed CD4\u0026thinsp;+\u0026thinsp;T cells, CD8\u0026thinsp;+\u0026thinsp;T cells, B cells, natural killer cells, and dendritic cells in the livers of mice with CCl\u003csub\u003e4\u003c/sub\u003e-induced fibrosis and controls using flow cytometry. The results showed no significant differences in the percentages or numbers of these cells between WT and Mindin\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Figure S4A and B). Because inflammatory responses drive the activation of HSCs and promote survival (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), we conducted α-sma IHC staining of the mouse livers, which is a marker of the activation of HSCs. Data have shown significantly greater HSC infiltration in mice administered CCl\u003csub\u003e4\u003c/sub\u003e or TAA. However, no notable difference was observed in the livers of WT mice, compared to Mindin\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Figure S5a and b).\u003c/p\u003e \u003cp\u003eWe next counted the viable CD45\u0026thinsp;+\u0026thinsp;Ly6G\u0026thinsp;\u0026minus;\u0026thinsp;CD11b\u0026thinsp;+\u0026thinsp;F4/80\u0026thinsp;+\u0026thinsp;bone marrow-derived macrophages and did not find significant differences between WT or Mindin\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, irrespective of whether they had CCl\u003csub\u003e4\u003c/sub\u003e-induced fibrosis (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec and S5C). These data indicate that the knockout of Mindin in mice does not affect the differentiation of bone marrow-derived macrophages. In addition, we identified circulating live CD45\u0026thinsp;+\u0026thinsp;CD11b\u0026thinsp;+\u0026thinsp;Ly6G\u0026thinsp;\u0026minus;\u0026thinsp;F4/80\u0026thinsp;+\u0026thinsp;cells using flow cytometry (Figure S5D), and found a significantly higher proportion of these macrophages in Mindin\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice than in WT mice in the presence of CCl\u003csub\u003e4\u003c/sub\u003e-induced liver fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Therefore, we hypothesized that the retention of macrophages in the bloodstream results in less entry into the liver, which may be attributable to an impairment in macrophage migration in Mindin-deficient mice, and is consistent with previous findings (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition, GO analysis of the mouse livers illustrated the pivotal role of Mindin in the orchestration of macrophage function (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). We conducted an \u003cem\u003ein vitro\u003c/em\u003e experiment to further explore the effect of Mindin on macrophage migration using Transwell culture. We found that the migration of RAW264.7 cells was significantly greater when they were incubated in medium containing rMindin than in medium lacking this (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef and g). Thus, Mindin promotes the migration of macrophages.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMindin promotes the phenotypic switch to restorative macrophages and promotes phagocytosis in liver fibrosis\u003c/h2\u003e \u003cp\u003eHaving shown that Mindin regulates immunity in liver fibrosis, we next explored the potential mechanisms involved. Hepatic macrophages are a remarkably heterogeneous population of immune cells that play a dual role in liver fibrosis (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Therefore, we analyzed the differential macrophages in more detail, and found that Mindin\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice had larger number of Ly6C\u003csup\u003ehi\u003c/sup\u003e macrophages and fewer Ly6C\u003csup\u003elo\u003c/sup\u003e macrophages in their livers than WT mice in the presence of CCl\u003csub\u003e4\u003c/sub\u003e-induced fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eLy6C\u003csup\u003elo\u003c/sup\u003e macrophages have an important role in matrix degradation by secreting MMPs, which are anti-fibrotic. We employed RT-qPCR analysis to measure the expression of relevant MMPs in the livers of mice with CCl\u003csub\u003e4\u003c/sub\u003e-induced fibrosis. We found significantly lower MMP-9 and MMP-12 expression in Mindin-deficient mice than in WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). To confirm these findings, we injected mice with CCl\u003csub\u003e4\u003c/sub\u003e-induced fibrosis with MMPSense 680, a fluorescent pan-MMP substrate, to enable us to assess the activity of liver MMPs in mice by live animal imaging. This showed lower MMP activity in Mindin\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice than in WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). We then depleted the macrophages using intraperitoneal injections of scavengers during the progression of liver fibrosis and assessed the efficiency of clearance by flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed and S6a). Depletion of the macrophages caused the surfaces of the livers of the mice to become rough and irregular, consistent with worsening cirrhosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Moreover, Sirius red staining confirmed greater fibrosis in the livers after the clearance of the macrophages. There was more severe fibrosis in Mindin\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice than in WT mice before the depletion of the macrophages, as previously described, but there was no significant difference in the liver fibrosis of Mindin\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e and WT mice after the depletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). In addition, Mindin\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice had a significantly higher liver indices than WT mice, but the spleen indices of the two groups were similar after the depletion of the macrophages (Figure S6b).\u003c/p\u003e \u003cp\u003eBecause phagocytosis is a crucial aspect of the phenotypic switch to restorative macrophages, we assessed the hepatic phagocytic capacity of mice with CCl\u003csub\u003e4\u003c/sub\u003e-induced liver fibrosis using flow cytometry. Consistent with previous findings (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), Mindin deficiency significantly reduced the phagocytic capacity of liver macrophages, assessed using the engulfment of latex beads (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). Furthermore, we sorted the liver macrophages of mice with fibrosis using flow cytometry and performed RNA sequencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh). Differential gene expression analysis showed that six genes were upregulated and 12 were downregulated in macrophages from Mindin\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, compared to WT mice (Figure S6c). The absence of Mindin from the list of differentially expressed genes showed that the macrophages did not express Mindin, which is consistent with a hepatic parenchymal cell origin. KEGG pathway analysis reconfirmed the significance of Mindin-mediated phagocytosis in the progression of liver fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei). To further explore the potential association, we prepared bone marrow-derived macrophages (BMDMs) and incubated them with E. coli. Phagocytosis was then assessed using flow cytometry. The data indicated that BMDMs from Mindin\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice exhibited weaker phenotypic switch after phagocytosis compared to WT mice (Figure S6d and e). In addition, we added fluorescent latex beads to RAW264.7 cells and observed their phagocytosis (Figure S6f). It was found that MMP expression increased in RAW264.7 cells after phagocytosis (Figure S6g). Taken together, these findings show that Mindin promotes the phagocytosis of macrophages, the secretion of MMPs to degrade collagen, and helps alleviate liver fibrosis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe Mindin/CD11b axis promotes phagocytosis and the restorative phenotype of macrophages to accelerate the regression of liver fibrosis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe integrin Mac-1 (CD11b/CD18), a member of the β2 integrin family, recognizes a wide range of ligands (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), including Mindin protein (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). We studied CCl\u003csub\u003e4\u003c/sub\u003e-induced fibrosis in CD11b-deficient mice and found that the serum Mindin concentration was significantly lower in CD11b-deficient mice than in WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, S7a and b). This result provides evidence of an interaction between Mindin and the CD11b receptor during the progression of liver fibrosis. Therefore, we next hybridized CD11b\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e and Mindin\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice and successfully generated Mindin/CD11b-DKO mice (Figure S7c). We hypothesized that Mindin affects phagocytosis by macrophages through binding to its receptor CD11b. The flow cytometry analysis showed that the absence of either Mindin or CD11b significantly reduced phagocytosis by macrophages and that the DKO further reduced this, as assessed by the engulfment of latex beads (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb and S7d). Considering that phagocytosis can influence macrophage phenotype switch, we next checked this change during mouse liver fibrosis using flow cytometry. The results revealed that either Mindin\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e or CD11b\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice induced a phenotypic change of Ly6C low to Ly6C high, and that was more pronounced in DKO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec and S7e). In addition, we found that the mRNA expression of the genetic markers of restorative macrophages IGF-1, Mertk, and CD36 was significantly lower in DKO than WT mouse livers (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed and S7f).\u003c/p\u003e \u003cp\u003eConsistent with restorative macrophages contributing to the regression of hepatic fibrosis through MMP-mediated matrix degradation, we found that both the mRNA and protein expression levels of MMP-9 were significantly lower in the DKO mice than in WT mice with CCl\u003csub\u003e4\u003c/sub\u003e-induced liver fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee and f). Furthermore, we induced liver fibrosis by CCl\u003csub\u003e4\u003c/sub\u003e administration in Mindin\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e, CD11b\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e, DKO, and WT mice, and found that either Mindin or CD11b knockout increased the liver fibrosis, and DKO markedly exacerbated this fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg and h).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we have provided evidence for the crucial role of Mindin in the coordination of the macrophage-driven resolution of liver fibrosis. Classically, hepatic macrophages are classified according to their F4/80 expression as either high F4/80-expressing resident Kupffer cells or low F4/80-expressing bone marrow-derived macrophages that differentiate from circulating monocytes. However, relying solely on the level of F4/80 expression to characterize macrophage heterogeneity in the liver is insufficient, because mature monocyte-derived macrophages also show high F4/80 expression(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). In addition, macrophage subsets that are relevant to liver disease can be categorized into inflammatory or restorative macrophages (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). The former promote liver fibrosis by activating HSCs in chronic liver injury and the latter are involved in matrix degradation through the expression of MMPs, thereby contributing to the resolution of liver injury and fibrosis. Owing to the heterogeneity of the macrophages, we carefully analyzed the Mindin-mediated recruitment of macrophages to the livers of mice with either CCl\u003csub\u003e4\u003c/sub\u003e- or TAA-induced liver fibrosis. We found that the population of differential macrophages in WT mice exhibited high F4/80 expression, along with increased macrophage phenotype switch from Ly6C high to Ly6C low, and unexpectedly, the augmentation of this macrophage population did not increase the activation of HSCs. Nevertheless, the livers showed higher MMP expression, and the depletion of the macrophage population markedly exacerbated the liver fibrosis. These data suggest that there was a larger population of restorative macrophages, which may play an important role in the regression of hepatic fibrosis.\u003c/p\u003e \u003cp\u003ePhagocytosis, a primary characteristic of macrophages. It has been reported that the phagocytosis of apoptotic hepatocytes by macrophages promotes the transition from an Ly6C\u003csup\u003ehi\u003c/sup\u003e to an Ly6C\u003csup\u003elo\u003c/sup\u003e macrophage subset (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). The RNA-seq of macrophages obtained from Mindin\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e and WT mice with liver fibrosis suggested that phagocytosis plays an important role in the process of liver fibrosis. Therefore, we concluded that Mindin-mediated phagocytosis promotes the phenotypic transition toward restorative macrophages in mice, leading to the resolution of fibrosis. In terms of the downstream effects of increased phagocytosis, it has been shown that the processing of apoptotic cells activates a STAT3-IL10-IL6 autocrine-paracrine loop(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Additionally, our previous work has investigated the phagocytosis-promoting effects of Mindin. The data indicate that Mindin promotes macrophage phagocytosis through Syk activation and NF-κB p65 translocation(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Therefore, intracellular responses that lead to enhanced phagocytosis in macrophages were not evaluated in this study, which is a limitation of this study.\u003c/p\u003e \u003cp\u003eIntegrins are transmembrane proteins that form heterodimers using α and β subunits. We have previously reported that the F-spondin fragment of Mindin binds to the αM-I structural domain of CD11b/CD18 (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), one of the integrin receptors, which is predominantly expressed in myeloid cells. Therefore, we hypothesized that Mindin acts by binding to CD11b on recruited liver macrophages. The absence of either Mindin or CD11b in mice can aggravate their liver fibrosis, and if both components of the Mindin/CD11b axis fail to function at the same time, the liver fibrosis is much worse. In other words, during the process of liver fibrosis, mouse hepatocyte-derived Mindin increases the migration of hepatic macrophages, and enhances macrophage phagocytosis by binding to the CD11b receptor, promoting the transition from an inflammatory to a restorative macrophage phenotype, and upregulates the MMP-9, which promotes collagen degradation and a reduction in tissue fibrosis, and thereby improves the liver homeostasis.\u003c/p\u003e \u003cp\u003eMacrophages bridge the processes of tissue necrosis and repair in many diseases. In principle, hepatic macrophages represent an attractive target for novel therapies for liver disease. Here, we have revealed the role of the Mindin/CD11b axis in the regulation of macrophage phagocytosis and liver homeostasis. By studying the role of Mindin in liver fibrosis in mice, we can hypothesize that Mindin deficiency or dysfunction in humans may exacerbate liver fibrosis in certain populations. Therefore, detection of Mindin levels in serum may be a potential biomarker for early diagnosis or monitoring the progression of liver fibrosis. Furthermore, given the role of Mindin in the regulation of immune responses, it is important to note that liver fibrosis is often accompanied by chronic inflammation and persistent immune system activation. Therefore, targeting the Mindin/CD11b axis, we could potentially inhibit excessive inflammation and promote the breakdown of fibrosis, thereby slowing the progression of the disease. Meanwhile, enhancing the reparative function of macrophages by utilizing the Mindin/CD11b axis may help to improve the ability of liver self-repair. Finally, translating these findings into clinical applications will require extensive preclinical studies and clinical trials. It will be crucial to assess the safety, efficacy, and potential side effects of modulating the Mindin/CD11b axis in patients with liver fibrosis before it can be considered for clinical use.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, in the present study, we have demonstrated the crucial role of Mindin in the coordination of the macrophage-driven resolution of liver fibrosis. The Mindin/CD11b axis drives the regression of liver fibrosis through an increase in phagocytosis by macrophages. These findings provide new molecular targets for the upregulation of the macrophage-mediated regression of liver fibrosis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCCl\u003csub\u003e4\u003c/sub\u003e, carbon tetrachloride; TAA, thioacetamide; DKO, double-knockout; ECM, extracellular matrix; HSC, hepatic stellate cell; MMP, matrix metalloproteinase; tissue inhibitor of metalloproteinases, TIMPs; Ly6C, lymphocyte antigen 6 complex locus C; DAMP, damage-associated molecular pattern; FS, F-spondin; H\u0026amp;E, hematoxylin\u0026ndash;eosin; SR, Sirius red; ISH, in situ hybridization; NPC, non-parenchymal cell; ALT, alanine aminotransferase; AST, aspartate aminotransferase; Col1a1, collagen type I alpha 1; \u0026alpha;-sma, \u0026alpha;-smooth muscle actin; rMindin, recombinant Mindin; NK, natural kill; DC, dendritic cell; GO, Gene Ontology; RT-qPCR, real-time reverse transcription polymerase chain reaction; KEGG, Kyoto Encyclopedia of Genes and Genomes; bone marrow-derived macrophages, BMDMs; Mertk, MER proto-oncogene tyrosine kinase.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this article [and its supplementary information files].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (No. 81970460) and the Natural Science Foundation Program of Fujian Province (No. 2023J011598).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHYD, ZXL and OYXM performed the major experiments; LY, CXS, LLY, HYN, XGJ and LJH assisted the animal experiments; HYD assisted draft of the manuscript; and JA provided the advises. GB revised the manuscript and supervised this study. All authors have read and approved the final submitted manuscript.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone of the authors has a conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the protocols used in the animal experiments were approved by the Committee for Animal Research of Xiamen University (No. XMULAC20190127).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSun M, Kisseleva T. Reversibility of liver fibrosis. Clin Res Hepatol Gastroenterol 2015;39 Suppl 1:S60-63.\u003c/li\u003e\n\u003cli\u003eBenyon RC, Iredale JP. Is liver fibrosis reversible? Gut 2000;46:443-446.\u003c/li\u003e\n\u003cli\u003eHernandez-Gea V, Friedman SL. Pathogenesis of liver fibrosis. Annu Rev Pathol 2011;6:425-456.\u003c/li\u003e\n\u003cli\u003eConsolo M, Amoroso A, Spandidos DA, Mazzarino MC. Matrix metalloproteinases and their inhibitors as markers of inflammation and fibrosis in chronic liver disease (Review). Int J Mol Med 2009;24:143-152.\u003c/li\u003e\n\u003cli\u003eVarol C, Mildner A, Jung S. Macrophages: development and tissue specialization. Annu Rev Immunol 2015;33:643-675.\u003c/li\u003e\n\u003cli\u003eKisseleva T, Brenner D. Molecular and cellular mechanisms of liver fibrosis and its regression. Nat Rev Gastroenterol Hepatol 2021;18:151-166.\u003c/li\u003e\n\u003cli\u003eGuillot A, Tacke F. Liver Macrophages: Old Dogmas and New Insights. Hepatol Commun 2019;3:730-743.\u003c/li\u003e\n\u003cli\u003eRamachandran P, Pellicoro A, Vernon MA, Boulter L, Aucott RL, Ali A, Hartland SN, et al. Differential Ly-6C expression identifies the recruited macrophage phenotype, which orchestrates the regression of murine liver fibrosis. Proc Natl Acad Sci U S A 2012;109:E3186-3195.\u003c/li\u003e\n\u003cli\u003eWang M, You Q, Lor K, Chen F, Gao B, Ju C. Chronic alcohol ingestion modulates hepatic macrophage populations and functions in mice. J Leukoc Biol 2014;96:657-665.\u003c/li\u003e\n\u003cli\u003eCampana L, Starkey Lewis PJ, Pellicoro A, Aucott RL, Man J, O\u0026apos;Duibhir E, Mok SE, et al. The STAT3-IL-10-IL-6 Pathway Is a Novel Regulator of Macrophage Efferocytosis and Phenotypic Conversion in Sterile Liver Injury. J Immunol 2018;200:1169-1187.\u003c/li\u003e\n\u003cli\u003eLodder J, Dena\u0026euml;s T, Chobert MN, Wan J, El-Benna J, Pawlotsky JM, Lotersztajn S, et al. Macrophage autophagy protects against liver fibrosis in mice. Autophagy 2015;11:1280-1292.\u003c/li\u003e\n\u003cli\u003eCampana L, Esser H, Huch M, Forbes S. Liver regeneration and inflammation: from fundamental science to clinical applications. Nat Rev Mol Cell Biol 2021;22:608-624.\u003c/li\u003e\n\u003cli\u003eJia W, Li H, He YW. The extracellular matrix protein mindin serves as an integrin ligand and is critical for inflammatory cell recruitment. Blood 2005;106:3854-3859.\u003c/li\u003e\n\u003cli\u003eBian ZY, Wei X, Deng S, Tang QZ, Feng J, Zhang Y, Liu C, et al. Disruption of mindin exacerbates cardiac hypertrophy and fibrosis. J Mol Med (Berl) 2012;90:895-910.\u003c/li\u003e\n\u003cli\u003eYang K, Li W, Bai T, Xiao Y, Yu W, Luo P, Cheng F. Mindin deficiency alleviates renal fibrosis through inhibiting NF-\u0026kappa;B and TGF-\u0026beta;/Smad pathways. J Cell Mol Med 2020;24:5740-5750.\u003c/li\u003e\n\u003cli\u003eRana I, Kataria S, Tan TL, Hajam EY, Kashyap DK, Saha D, Ajnabi J, et al. Mindin (SPON2) Is Essential for Cutaneous Fibrogenesis in a Mouse Model of Systemic Sclerosis. J Invest Dermatol 2023;143:699-710.e610.\u003c/li\u003e\n\u003cli\u003eCheng XS, Huo YN, Fan YY, Xiao CX, Ouyang XM, Liang LY, Lin Y, et al. Mindin serves as a tumour suppressor gene during colon cancer progression through MAPK/ERK signalling pathway in mice. J Cell Mol Med 2020;24:8391-8404.\u003c/li\u003e\n\u003cli\u003eHammerich L, Tacke F. Hepatic inflammatory responses in liver fibrosis. Nat Rev Gastroenterol Hepatol 2023;20:633-646.\u003c/li\u003e\n\u003cli\u003ePradere JP, Kluwe J, De Minicis S, Jiao JJ, Gwak GY, Dapito DH, Jang MK, et al. Hepatic macrophages but not dendritic cells contribute to liver fibrosis by promoting the survival of activated hepatic stellate cells in mice. Hepatology 2013;58:1461-1473.\u003c/li\u003e\n\u003cli\u003eTacke F. Targeting hepatic macrophages to treat liver diseases. J Hepatol 2017;66:1300-1312.\u003c/li\u003e\n\u003cli\u003eHe YW, Li H, Zhang J, Hsu CL, Lin E, Zhang N, Guo J, et al. The extracellular matrix protein mindin is a pattern-recognition molecule for microbial pathogens. Nat Immunol 2004;5:88-97.\u003c/li\u003e\n\u003cli\u003eLiu YS, Wang LF, Cheng XS, Huo YN, Ouyang XM, Liang LY, Lin Y, et al. The pattern-recognition molecule mindin binds integrin Mac-1 to promote macrophage phagocytosis via Syk activation and NF-\u0026kappa;B p65 translocation. J Cell Mol Med 2019;23:3402-3416.\u003c/li\u003e\n\u003cli\u003eLamers C, Pl\u0026uuml;ss CJ, Ricklin D. The Promiscuous Profile of Complement Receptor 3 in Ligand Binding, Immune Modulation, and Pathophysiology. Front Immunol 2021;12:662164.\u003c/li\u003e\n\u003cli\u003eLim K, Hyun YM, Lambert-Emo K, Topham DJ, Kim M. Visualization of integrin Mac-1 in vivo. J Immunol Methods 2015;426:120-127.\u003c/li\u003e\n\u003cli\u003eJu C, Tacke F. Hepatic macrophages in homeostasis and liver diseases: from pathogenesis to novel therapeutic strategies. Cell Mol Immunol 2016;13:316-327.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"hepatology-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hepi","sideBox":"Learn more about [Hepatology International](https://www.springer.com/journal/12072)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/hepi/default.aspx","title":"Hepatology International","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"extracellular matrix, hepatic stellate cell, flow cytometry, migration, Ly6C, MMP, phagocytosis, phenotypic switch, restorative macrophages, CD11b, DKO","lastPublishedDoi":"10.21203/rs.3.rs-5155041/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5155041/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground \u0026amp; Aims:\u003c/h2\u003e \u003cp\u003eLiver disease that progresses to cirrhosis is an enormous health problem worldwide. The extracellular matrix protein Mindin is known to have immune functions, but its role in liver homeostasis remains largely unexplored. We aimed to characterize the role of Mindin in the regulation of liver fibrosis.\u003c/p\u003e\u003ch2\u003eApproach \u0026amp; Results:\u003c/h2\u003e \u003cp\u003eMindin was upregulated in mice with carbon tetrachloride (CCl\u003csub\u003e4\u003c/sub\u003e) or thioacetamide (TAA)-induced liver fibrosis, and was primarily expressed in hepatocytes. Global Mindin knockout mice were generated, which were susceptible to liver fibrosis. Notably, Mindin failed to activate hepatic stellate cells directly; however, it played a role in promoting the recruitment and phagocytosis of macrophages, and caused a phenotypic switch toward restorative macrophages during liver fibrosis. Furthermore, Mindin was found to bind to the αM-I domain of CD11b/CD18 heterodimeric receptors. To further explore this mechanism, we created Mindin and CD11b double-knockout (DKO) mice. In DKO mice, phagocytosis was further reduced, and liver fibrosis was markedly exacerbated.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eMindin promotes the resolution of liver fibrosis and the Mindin/CD11b axis might represent a novel target for the macrophage-mediated regression of liver fibrosis.\u003c/p\u003e\u003ch2\u003eGraphical abstract:\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Mindin orchestrates the macrophage-mediated resolution of liver fibrosis in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-26 10:00:31","doi":"10.21203/rs.3.rs-5155041/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-11-24T06:35:56+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-24T00:48:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-23T06:26:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Hepatology International","date":"2024-11-22T21:54:29+00:00","index":"","fulltext":""},{"type":"decision","content":"Major Revisions Needed","date":"2024-10-26T01:36:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"hepatology-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hepi","sideBox":"Learn more about [Hepatology International](https://www.springer.com/journal/12072)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/hepi/default.aspx","title":"Hepatology International","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b10492e6-7571-491d-bf64-b1fffd5109bc","owner":[],"postedDate":"November 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-04-07T16:03:04+00:00","versionOfRecord":{"articleIdentity":"rs-5155041","link":"https://doi.org/10.1007/s12072-025-10813-7","journal":{"identity":"hepatology-international","isVorOnly":false,"title":"Hepatology International"},"publishedOn":"2025-04-05 15:58:02","publishedOnDateReadable":"April 5th, 2025"},"versionCreatedAt":"2024-11-26 10:00:31","video":"","vorDoi":"10.1007/s12072-025-10813-7","vorDoiUrl":"https://doi.org/10.1007/s12072-025-10813-7","workflowStages":[]},"version":"v1","identity":"rs-5155041","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5155041","identity":"rs-5155041","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}