称号及び氏名

博士（獣医学）

学位授与の日付

平成３０年３月３１日

論Pathological study on liver homeostasis and hepatotoxicity

文

名PERVIN MUNMUN

based on macrophage functions and autophagy in rats
（マクロファージ機能とオートファジーに基づいたラットの肝恒常性と肝毒性に関する病理学的研究）

論文審査委員

主査

山手丈至

副査

中村洋一

副査

杉浦喜久弥

論文要旨

Introduction
Liver is a crucial organ in the body, consisting of parenchymal hepatocytes andnon-parenchymal cells (Kupffer cells, hepatic stellate cells (HSCs), cholangiocytes andendothelial cells). The relationship between these parenchymal and non-parenchymal
hepatic cells is important for the maintenance of liver functions and homeostasis.
Hepatic macrophages are considered a key player for initiating and shaping of immune
responses in liver through phagocytosis, cytokine production and antigen presentation.
Hepatic macrophages mainly include Kupffer cells; however, liver harbours hepaticmacrophages with different immunophenotypes such as exudative macrophages andantigen presenting cells. In pathological condition, hepatic macrophages appear as M1
macrophages (classically activated macrophages) and M2 macrophages (alternativelyactivated macrophages) based on the type 1 or 2 helper T-cell polarization.
Interestingly, depending on microenvironmental conditions, M1 macrophages could
shift into M2 phenotypes or vice versa. Therefore, it is interesting to investigate which
type of macrophages may contribute to physiological and pathophysiological
adaptations including hepatotoxicity. Additionally, liver has a basal level of autophagy,
which is an evolutionarily conserved and strictly regulated lysosomal pathway for
intracellular degradation. Autophagy is characterized by the formation ofdouble-membrane vesicle termed autophagosome, demonstrable with LC3B(microtubule-associated protein 1 light chain 3 beta, an essential component ofautophagosome) immunohistochemistry. Autophagy contributes to hepatic homeostasis
and is also activated in a variety of liver diseases including hepatotoxicity.
To investigate the functional properties of hepatic macrophages and autophagy
in liver homeostasis and hepatotoxicity, a series of studies were carried out. These
functional properties were mainly histopathologically analyzed using rats under low
dose of lipopolysaccharide (LPS) or clodronate (CLD) treatment. The obtained data inthese studies would provide important information on heterogeneous macrophagefunctions and autophagy participating in liver homeostasis and pathology.
Chapter 1: Characterization of rat hepatic macrophages participating in liver
homeostasis
Section I: Effects of empty liposomes on hepatic macrophage populations in rats
To identify the transient effects of empty liposomes on hepatic macrophages,
F344 rats were injected with liposome (10 ml/kg BW, iv). Serum and liver samples
were collected one day after injection. The numbers of hepatic macrophages reacting
to CD163, CD68 and MHC class II were significantly increased in liposome-treatedrats. Although there were no changes in liver histoarchitecture, hepatocytes showed
increased proliferating activity with increased gene profiles related to cell cycle.
Interestingly, AST and ALT levels were significantly decreased, and mRNA expressionof MCP-1, IL-1β and TGF-β1 were significantly increased. It was found that hepatic
macrophages activated by liposomes influence liver homeostasis.
SectionII:

Immunophenotypes of depleting and repopulating hepatic
macrophages in rats treated with CLD
To characterize the depleting and repopulating hepatic macrophages, F344 rats
were injected with liposomal CLD (50 mg/kg BW, iv). Serum and liver samples werecollected on post-injection (PI) 1, 3, 5, 7, 9 and 12 days. Hepatic macrophages
detectable by different antibodies were decreased in varying degrees after CLD
injection. CD163+ Kupffer cells were the most susceptible to CLD, which were almost
completely depleted on PI days 1-12. CD68+ macrophages were transiently reduced on
PI day 1 and then, recovered gradually until PI day 12. MHC class II+ macrophages
were moderately decreased during the observation periods. Histologically, no marked
changes were seen in the liver, excluding the increased proliferating activity ofhepatocytes on PI day 1. AST, ALP, ALT, total bilirubin and γ-GTP levels weresignificantly elevated and factors for macrophage induction/activation (MCP-1, CSF-1,IL-6 and IL-4) were increased transiently exclusively on PI day 1, whereas
anti-inflammatory factors (IL-10 and TGF-β1) remain significantly decreased under
hepatic macrophage depletion. These findings showed the importance of hepaticmacrophages in liver homeostasis.
Chapter 2: Characterization of hepatic macrophages and autophagy under LPS
treatment in rats
Section I: Effects of low dose of LPS on hepatic macrophages and regulatory
inflammatory factors in CLD-treated rats
To clarify pathophysiological effects of low dose of LPS on hepaticmacrophages and regulatory inflammatory factors, F344 rats were pretreated with CLD(50 mg/kg BW, iv) (CLD+LPS rats) or vehicle liposome (Lipo) (Lipo+LPS rats) 24h
before LPS injection (0.1 mg/kg BW, ip) or saline. Serum and liver were collected on
LPS PI 0, 6, 12, 24, 48 and 72h. LPS injection did not alter histoarchitectures of theliver, and changes of serum hepatic enzymes were not seen. Macrophages reacting toCD163, CD68 and MHC class II were stimulated to LPS in Lipo+LPS rats and effects
on repopulating Kupffer cells were not seen in CLD+LPS rats. Inflammatory-relatedfactors (IL-1β, IL-6, TNF-α and MCP-1) were dramatically increased at 6h after LPS
exposure in Lipo+LPS rats, which was attenuated by macrophage depletion in
CLD+LPS rats. On the contrary, anti-inflammatory factors (IL-4 and CSF-1) weresignificantly elevated in CLD+LPS rats, but did not change in Lipo+LPS rats.
Activation of hepatic macrophages by low level of LPS could provide a significant
insight into the development of macrophage-based hepatotoxicity
Section II: Characterization of cell specific autophagy in liver and hepatic
macrophages under treatment with low dose of LPS in CLD-treatedrats
Using the above model, LPS-induced autophagy in hepatocytes andnon-parenchymal cells were analysed. Localization of LC3B (autophagy marker) was
seen at basal level in hepatocytes, Kupffer cells, HSCs and cholangiocytes in normal
rat livers. LPS administration resulted in a markedly increased LC3B expression inhepatocytes of Lipo+LPS and CLD+LPS rats at PI 6h and 12h. In comparisons between
these groups, LC3B-positive hepatocytes were markedly lower in CLD+LPS rats.
However, LC3B reactivity was increased in GFAP-expressing HSCs in both groups
after LPS treatment. LPS injection also induced autophagy in CD163+ Kupffer cells, aswell as CD68 + and MHC class II+ macrophages in Lipo+LPS rats. At mRNA level,LC3B and TLR-2 were significantly increased at PI 6h or 12h in Lipo+LPS rats, whilethese factors was significantly lower in CLD+LPS rats. Low dose of LPS could be used
as a potent autophagy inducer in rat livers.
Chapter 3: Roles of hepatic macrophages and autophagy in thioacetamine(TAA)-induced hepatotoxicity in LPS-pretreated rats
Section I: Time dependent protection of LPS in TAA-induced acute liver injury in

rats
To investigate the effects of low dose of LPS in TAA-induced hepatotoxicity asa protective phenomenon, F344 rats were pretreated with LPS (0.1 mg/kg BW, ip) at 2,6, 12 and 24h before TAA injection (100 mg/kg BW, ip). Serum and liver werecollected one day after TAA injection. Histopathologically, TAA injection produced
centrilobular necrosis in liver. LPS-pretreated TAA-injected rats at 2h and 6h showed
little effect on the hepatic lesions, but the rats at 12h and 24h showed a markedly
reduced hepatic lesions. AST, ALP, ALT and total bilirubin values tended to bedecreased in LPS-pretreated TAA-injected rats at 12h or 24h. LPS pretreatment
showed hepatoprotection against TAA-induced liver lesions depending on time points
before TAA injection, particularly at 12h or 24h.
Section II: LPS-mediated cytoprotection in LPS-pretreated rat liver lesions
induced by TAA in terms of hepatic macrophages and autophagy
To investigate cytoprotective roles of hepatic macrophages and autophagy inTAA-induced hepatotoxicity under LPS treatment, F344 rats were pretreated with LPS(0.1 mg/kg BW, ip) at 2h and 24h before TAA injection (100 mg/kg BW, ip). Serumand liver were collected on TAA PI 0, 1, 2, and 3 days. Histopathologically, LPS
pretreatment at 2h did not reduce the hepatic lesions, whereas the pretreatment at 24hmarkedly reduced hepatic injury, along with decreased serum hepatic enzymes levels.
CD68+ , CD163 + and MHC class II+ macrophages were significantly increased after
TAA administration, regardless of LPS pretreatment at 2h or 24h. Interestingly, MCP-1,IL-1β and IL-4 were significantly decreased on day 1 in LPS-pretreated TAA-injured
livers at 24h, but were not changed in LPS-pretreated TAA-injured livers at 2h. The
autophagy was markedly increased in livers of LPS pretreatment at 2h or 24h. Thesefindings provide information that LPS pretreatment need at least 24h for preparing themicroenvironments for cytoprotection against TAA-induced liver injury. Activatedmacrophages and autophagy may participate in the hepatoprotective mechanism.
Conclusions
Based on the obtained findings, the following conclusions are drawn;
1. Empty liposomes activated hepatic macrophages with different immunophenotypes(mainly, CD68, CD163 and MHC class II), without histopathological changes.
2. CLD injection successfully depleted hepatic macrophages with different
immunophenotypes in rat livers. Particularly, CD163 + Kupffer cells were the mostsusceptible to CLD.
3. Low dose of LPS treatment (0.1 mg/kg BW) activated hepatic macrophages without
liverlesions.

Furthermore,

theLPS

administration

effected

microenvironments via the increased production of inflammatory mediators.

liver

4. Low dose of LPS treatment induced autophagy in hepatocytes andnon-parenchymal cells such as hepatic macrophages and HSCs. Additionally,
LPS-activated hepatic macrophages influenced autophagy functions in liver.
5. LPS pretreatment protected development of TAA-induced acute liver lesions
depending on time points before TAA injection. Particularly, LPS pretreatment at
24h greatly protected TAA-induced hepatotoxicity, whereas LPS pretreatment at 2h
did not show such levels.
6. Hepatic macrophages with different immunopohenotypes and activated autophagy
might participate in the protective phenomenon against TAA-induced
hepatotoxicity.
7. Therefore, the present findings would provide useful information on the propertiesof hepatic macrophages and autophagy in liver homeostasis and hepatotoxicity,leading to possible insights underlying effective therapeutic strategies against liverlesions.

審査結果の要旨

肝臓は、栄養代謝に加え、医薬品などの化学物質の解毒を行う重要な器官である。化学物質による肝毒性は、主に、化学物質の肝細胞に対する直接的な細胞傷害作用や、肝代謝酵素により生じる化学物質の活性型代謝物に起因する肝毒性発現機序に基づき、その評価が行われている。しかし、肝臓には、実質細胞である肝細胞の他に、非実質細胞としてのクッパー細胞、肝星細胞、血管内皮細胞や胆管上皮細胞などがあり、特に、クッパー細胞などのマクロファージ系の細胞は肝構成細胞の 20~25%を占め、肝恒常性や肝毒性に何らかの影響を与えていると考えられる。しかし、肝マクロファージの機能特性に基づいた肝毒性の発現機序の解析はほとんど行われていない。また、オートファジーは、細胞内老廃物や細胞内侵入微生物を処理する機構として知られているが、肝毒性におけるその役割については未だ十分に研究されていない。このような肝マクロファージやオートファジーは、グラム陰性細菌が産生するリポポリサッカライド（LPS）の影響を受けることが知られている。
そこで、本研究では、ラットを用いて、第 1 章においては、肝マクロファージを活性化あるいは、枯渇させることで肝恒常性にどのような影響が生じるかを解析し、第 2 章では、低用量の LPS が肝マクロファージやオートファジーに及ぼす作用を肝機能との関連で調べている。さらに、第 3 章では、低用量 LPS前処置における肝毒性物質であるチオアセトアミド（TAA）により誘発される肝細胞傷害の発現機序を、肝マクロファージとオートファジーとの関連で追究している。この一連の研究に基づいて、肝恒常性や肝毒性に及ぼす肝マクロファージとオートファジーの病態生理学的な役割の重要性を明らかにしている。
第 1 章では、肝マクロファージに貪食され易いリポソームをラットに投与し、出現する肝マクロファージの免疫組織化学的な特性と肝機能に与える影響を解析している。その結果、リポソーム投与により、CD163 発現 M2 型マクロファージ（主にクッパー細胞）に加え、CD68 と MHC クラス II 発現 M1 型マクロファージが増加・活性化し、さらに AST や ALT などの肝逸脱酵素が低下することが分かった。一方、リポソーム封入クロドロネート投与による CD163 発現 M2型 マ ク ロ フ ァ ー ジ の 枯 渇 状 態 で は 、 肝 細 胞 傷 害 が 生 じ て い な い に も 拘 わ らず
AST や ALT 値が上昇することが分かった。すなわち、肝マクロファージの活性化あるいは、枯渇状態により肝機能に変化が生じることを示している。以上より、多様な抗原を発現する肝マクロファージが肝恒常性に係ること、そして肝機能を評価する際には肝マクロファージの状態を把握しておく必要性があることを提示している。
第 2 章では、肝マクロファージとオートファジー機能に与える LPS の影響を解析している。LPS 投与実験では通常は大量投与が行われ、その際には肝細胞傷害が惹起されることが知られているが、この実験では、腸内細菌叢から産生される微量な LPS が門脈を介して肝臓に流入し肝機能に影響を与えている可能性を想定し、低用量の LPS をラットに投与している。その結果、LPS の低用量（0.1 mg/kg 体重、腹腔内）投与では肝機能障害は生じていないが、CD68 と MHC
クラス II 発現 M1 型マクロファージと CD163 発現 M2 型マクロファージが増加し、かつ IL-1β や TNF-α などの炎症性因子が上昇することが分かった。さらに、
LPS 投与により、LC3B 発現のオートファゴゾームが、肝細胞のみならず、M2型マクロファージや肝星細胞においても誘導されることを明らかにした。一方、クロドロネート投与による CD163 発現クッパー細胞枯渇条件下での低用量 LPSの投与では、上記の炎症性因子の産生や肝細胞内のオートファゴゾームの誘導が抑制されることが分かった。すなわち、用いた低用量 LPS は肝マクロファージを活性化し、その影響下でオートファジーが誘起されることを示している。
第 3 章では、ラットを用いて TAA 誘発肝毒性に及ぼす低用量 LPS 投与の影響を追究している。LPS を投与していない TAA 単独投与群では小葉中心性肝細胞凝固壊死が生じ、肝逸脱酵素が上昇していたが、TAA 投与前 2、6、12、24 時間のポイントで LPS を事前投与しておくと、時間依存性に TAA により惹起される肝機能障害が軽減されることが分かった。その機序としては、第 2 章で示したように LPS 投与により活性化された肝マクロファージやオートファジーが、TAA 誘発肝細胞傷害の軽減に係ることを示している。特に、CD68 と MHC クラ
ス II 発現 M1 型マクロファージによる傷害組織の残渣処理や、CD163 発現 M2型マクロファージによる組織修復機能の効果的な作用が係っている可能性を考察している。
本研究は、肝マクロファージの機能が肝恒常性に深く係っていることを明らかにするとともに、LPS はその用量によっては、肝マクロファージやオートファジーを活性化し、肝毒性発現を抑制するとするユニークな知見を見出した。
これらの成果は、獣医学や医学、特に病態生理学や毒性病理学分野の基礎研究の新たな展開に資するものと考える。よって、本論文の審査ならびに最終試験の結果と併せて、博士（獣医学）の学位を授与することを適当と認める。