論文の公開元へPathological studies on characteristics of macrophages and damage-associated molecular patterns (DAMPs) in thioacetamide-induced rat liver injury
概要
Inflammation is protective and healing reaction for cellular and tissue injury. The inflammatory cells consist mainly of neutrophils, macrophages and lymphocytes. Depending on grades and time of inflammation, the appearance of these cells should be changeable. Generally, neutrophils migrate into injurious lesions at very early stages and then, lymphocytes and macrophages appear by showing various functions; the lymphocytes and macrophages may be related to subacute and chronic stages of inflammation. Injured areas with inflammatory cells heal via reparative fibrosis.
Recently, during the inflammation, the functions of macrophages have been payed attention. Macrophages are classified as M1 (classically activated) and M2 (alternatively activated) (Stein, et al., 1992; Duffield, et al., 2005). M1-macrophages are induced by interferon-γ (IFN-γ) and secrete pro-inflammatory cytokines to promote inflammation and injury (Martinez, et al., 2008). In contrast, M2-macrophages are activated by interleukin-4 (IL-4), and functionally associated with resolution of inflammation and tissue remodeling, as well as fibrosis. M1- and M2-macrophages are recognized using immunohistochemical methods with antibodies specific to antigens (Gratchev, et al., 2006; Sica, et al., 2013); in rats, CD68 antibody is used for M1-macrophages, whereas M2-macrophages express CD163. Since CD68 is a glycoprotein on lysosomal membranes, and therefore, the increase of CD68 expression means increasing lysosomal activity (Zhu, et al., 2012). CD163 is a scavenger receptor playing in clearance of hemoglobin, and macrophages expressing CD163 is important for homeostasis via tissue regeneration. CD163 is expressed not only by M2-macrophages in pathological lesions, but also by tissue-resident macrophage (such as Kupffer cells in the liver) in normal condition (Fabriek, et al., 2005; Polfliet, et al., 2006). Generally, M1-macrophages appear earlier than M2-macrophages. Such M1/M2-macrophage polarization is a key to understand the pathogenesis of progression of inflammation, of which information may be used for therapeutic target of inflammatory conditions (Mantovani, et al., 2004; Barros, et al., 2013; Wijesundera, et al., 2014).
Recently, damage-associated molecular patterns (DAMPs) have been considered as triggers to induce inflammation; the DAMPs are the low-molecular weight molecules and play as endogenous danger signals that are released from injured or necrotic cells (Maher, et al., 2009). In normal condition, on the contrast, DAMPs play physiologic roles intracellularly to keep homeostasis. For example, high-mobility group box 1 (HMGB1), one of the popular DAMPs, binds to DNA as non-histone molecule and regulate transcription for physiological functions in cells. Once DAMPs are released into the extracellular, DAMPs activate innate immune system as a ligand for Toll-like receptors (TLRs), nucleotide binding oligomerization domain-like receptors (NLRs) and the receptor for advanced glycation end-products (RAGE); after then, activated receptors induce inflammatory-related cytokines through inflammatory cells or affected cells, finally leading to progress of inflammation accompanied with inflammatory cells (Maher, et al., 2009; Tang, et al., 2012).
DAMPs have been well-studied in experimentally-induced ischemic/reperfusion disease (Yu, et al., 2010). Under ischemic condition, since parenchymal cells cannot be supplied enough oxygen, endoplasmic reticulum stress and unbalance of ions take place in the cells, resulting in cell necrosis. After reperfusion, furthermore, cell necrosis occurs because of the oxidative stress due to reactive oxygen species (ROSs) produced via sudden supply of oxygen. Through the necrosis process, DAMPs released from necrotic cells spread through re-perfused blood flow and induce inflammation by stimulating receptors such as TLRs. The cell necrosis process has been considered to occur in brain ischemia, myocardial infarction, renal infarction, and organ transplantation as clinical cases. However, the pathological roles of DAMPS remain to be investigated.
The liver plays important roles in maintenance of the health. Main functions of the liver are metabolism of nutrition including glucose, vitamin, lipid and minerals, secretory of bile, and detoxification of external chemicals (Kumar, et al., 2010). In the detoxification, the liver utilizes enzymes for metabolism of substances, such as cytochrome P450 families, flavin-containing monooxygenases and esterase (Chieli and Malvaldi, 1984). Depending on intake dose, chemicals such as drugs and agrochemicals may induce hepatotoxicity. Therefore, toxicologically, hepatotoxicity, which may be induced by chemicals, is important for analyses for safety evaluation during the development. The chemically-induced hepatotoxicity can be caused by chemical itself or its activated metabolites which are formed by liver drug enzymes (Chen, et al., 2015). Chemical hepatotoxicity can cause hepatocyte injury/necrosis accompanied by inflammatory cells such as macrophages; therefore, it has been reported that macrophages should influence inflammation grade depending on M1-/M2-polarization (Yamate, et al., 2000; Ide, et al., 2003; Mori, et al., 2009). Because there are human patients with liver failure which may be induced by chemicals and hepatitis viruses and some of the patients develop cirrhosis and liver tumors at chronic stages, it is important to understand the mechanisms of chemically-induced hepatotoxicity.
In chemically-induced liver injury, little is known about roles of DAMPs and relation of DAMPs with inflammatory cells such as macrophages. Thioacetamide (TAA) has been used as hepatotoxicant to induce hepatotoxicity. TAA can be metabolized into activated metabolites, TAA-S-oxide (TASO) and TAA-S-dioxide (TASO2) due to CYP450 effects (Koen, et al, 2013; Sarma, et al., 2012). TASO and TASO2 can make adducts with high-weight molecules in hepatocytes and then, these adducts cause oxidative mitochondrial stress, resulting in hepatocyte injury/necrosis exclusively in the perivenular areas (Chilakapati, et al., 2005; Hajovsky, et al., 2012). After injury by TAA, inflammatory cells, mainly macrophages, infiltrate in the affected areas (Wijesundera, et al., 2014). To investigate the detailed progression mechanism of inflammation based on participation of macrophages and DAMPs, a series of this thesis were carried out as follows;
In Chapter 1, to investigate macrophage appearance on TAA-induced liver injury, the author studied immunohistochemically macrophage properties using a panel of antibodies recognizing different macrophage subtypes. In Section 1 of Chapter 1, the author analyzed M1/M2-macrophage polarization based on CD68 or CD163 expression and M1/M2-macrophag-related cytokines which may induce and influence macrophage polarization. It was found that M1-macrophages participate in the liver damage and simultaneously M2-macrophages appear in relation to subsequent tissue remodeling. In Section 2 of Chapter 1, to assess the influence of macrophages, the author analyzed TAA-induced live lesions under depletion of macrophages caused by injection of dexamethasone. It was found that M1/M2-macrophages appearing in TAA-induced liver lesions were decreased, resulting in amelioration of reparative fibrosis. In Section 3 of Chapter 1, the author investigated appearance of macrophages in liver lesions by short- term-repeated TAA injections. Interestingly, the repeated injections of TAA reduced liver lesions, accompanied by decreased number of M1-/M2-macrophages. The results of Chapter 1 indicated that macrophages may have functions to modify liver lesions caused by TAA injection.
In Chapter 2, to investigate the relationships between inflammation and DAMPs, the author analyzed the expression and distribution of DAMPs. In Section 1 of Chapter 2, mRNA expressions of DAMPs (such as HMGB1, HMGB2 and S100A4) and receptors (TLR2, 4) increased in rat livers after TAA injection at 300 mg/kg body weight. Out of DAMPs, the author focused on HMGB1. HMGB1 expression was translocated from intranuclei to cytoplasm of hepatocytes. However, the TAA dose used caused so severe liver lesions that it was difficult to analyze effects of DAMPs using anti-HMGB1 antibody neutralization experiment (Chapter 3). Therefore, in Section 2 of Chapter 2, the author analyzed liver lesions by low dose TAA injection (50 mg/kg body weight); similarly, HMGB1 translocation from intranuclei to cytoplasm was confirmed, in addition to appearance of M1-/M2-macrophages.
In Chapter 3, to determine effects of HMGB1, TAA-induced liver lesions by injection of low dose TAA (50 mg/kg body weight; Section 2 of Chapter 2) were evaluated by using anti-HMGB1 antibody neutralization. It was found that the neutralization reduced liver failure induced by TAA.
In conclusion, the present studies showed that liver lesions by TAA were influenced by functions of M1-/M2-macrophage polarization; the influence may depend on inflammatory stages, or be related to appearance grades (presence/depletion) of such macrophages. The appearance of inflammatory cells (such as macrophages) may be induced by DAMPs, particularly HMGB1 in hepatocytes; translocation from intranuclei to cytoplasm of HMGB1 may be important event in injured hepatocytes. The data obtained in the present study would be useful to understand the pathogenesis of hepatotoxicity.
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